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Polygonatum odoratum lectin induces apoptosis and autophagy by regulation of microRNA-1290 and microRNA-15a-3p in human lung adenocarcinoma A549 cells
Accepted Manuscript Title: Polygonatum odoratum lectin induces apoptosis and autophagy by regulation of microRNA-1290 and microRNA-15a-3p in human lung adenocarcinoma A549 cells Author: Lei Wu Tao Liu Yan Xiao Xing Li Yanan Zhu Yan Zhao Jinku Bao Chuanfang Wu PII: DOI: Reference:
S0141-8130(15)30107-0 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.11.014 BIOMAC 5519
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
25-7-2015 4-11-2015 5-11-2015
Please cite this article as: L. Wu, T. Liu, Y. Xiao, X. Li, Y. Zhu, Y. Zhao, J. Bao, C. Wu, Polygonatum odoratum lectin induces apoptosis and autophagy by regulation of microRNA-1290 and microRNA-15a-3p in human lung adenocarcinoma A549 cells, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights (for review)
Highlights
► miR-1290 is down-regulated in POL treated A549 cells. ► GSK-3β is a target of miR-1290.
miR-15a-3p resulted in A549 cells apoptosis and autophagy.
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► miR-15a-3p is up-regulated after POL treatment and over-expression of
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► miR-15a-3p-ROS-p53 pathway is involved in POL-induced apoptosis and
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autophagy in A549 cells
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*Manuscript Click here to view linked References
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Polygonatum
odoratum
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autophagy
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microRNA-15a-3p in human lung adenocarcinoma A549
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cells
regulation
induces
of
apoptosis
and
microRNA-1290
and
ip t
by
lectin
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Lei Wu, Tao Liu, Yan Xiao, Xing Li, Yanan Zhu, Yan Zhao, Jinku Bao* and
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Chuanfang Wu*
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School of Life Sciences and Key Laboratory of Bio-resources and Eco-environment,
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Ministry of Education, State Key Laboratory of Biotherapy and Cancer Center, West
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China Hospital, Sichuan University, and Collaborative Innovation Center for
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Biotherapy, Chengdu, 610064, China
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*Correspondence: J. K. Bao, School of Life Sciences and Key Laboratory of Bio-resources, Ministry of
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Education,
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[email protected]
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C. F. Wu, School of Life Sciences and Key Laboratory of Bio-resources, Ministry of Education, Sichuan
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University, Chengdu, China. Tel./fax: +86-28-85415171; E-mail: [email protected]
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Lei Wu and Tao Liu contributed equally to this work.
Sichuan
University,
Chengdu,
China.
Tel./fax:
+86-28-85415171;
Email:
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Abstract Polygonatum odoratum lectin (POL), a mannose-binding specific Galanthus
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nivalis agglutinin (GNA)-related lectin has been reported with remarkable
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anti-proliferative and apoptosis-inducing effects against several tumor cells. Our
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previous research revealed that POL can induce apoptosis and autophagy in A549
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cells. However, whether microRNAs (miRNAs) are involved in POL-induced
34
apoptosis and autophagy in A549 cells has not been investigated. The
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study was to evaluate whether miRNAs were involved in POL-induced apoptosis and
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autophagy in A549 cells. In the present study, we performed microarray analysis on
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A549 cells to identify altered miRNAs after POL treatment. We found that miR-1290
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was down-regulated after POL treatment and down-regulated miR-1290 amplifies
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POL-induced apoptosis in A549 cells. Moreover, we revealed that glycogen synthase
40
kinase-3β (GSK3β) was a direct target of miR-1290 and POL treatment could result in
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Wnt pathway down regulation. We also found that miR-15a-3p was up-regulated after
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POL treatment and over-expression of miR-15a-3p resulted in A549 cells apoptosis
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and autophagy. In addition, we confirmed that a miR-15a-3p mediated ROS-p53
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pathway was involved in POL-induced apoptosis and autophagy in A549 cells. Taken
45
together, these data provide evidence that POL induces A549 cells apoptosis and
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autophagy by regulation of miR-1290 and miR-15a-3p.
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Keywords: Polygonatum odoratum lectin; Apoptosis; Autophagy; miR-1290;
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miR-15a-3p 2 Page 3 of 37
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1. Introduction Plant lectins are a class of highly diverse, non-immune origin and
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carbohydrate-binding proteins [1]. Plant lectins have been reported to possess
53
antitumor activities via targeting programmed cell death (PCD), including apoptosis
54
and autophagy [2]. Apoptosis is a cell-intrinsic mechanism for suicide that regulated
55
by numerous cellular signaling pathways and autophagy is a conserved eukaryotic
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cellular degradative process that helps in maintaining the cellular metabolism and
57
homeostasis [3]. Several major plant lectins families including Galanthus nivalis
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agglutinin (GNA) related lectins, legume lectins and type II ribosome-inactivating
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proteins (RIP) have been reported to induce apoptosis and autophagy in many types of
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cancer cells [2].
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MicroRNAs (miRNAs) are small non-coding RNAs of about 18-24 nucleotides in
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length that negatively regulate gene expression by binding to the complementary
63
sequences in the 3′-untranslated regions (3′UTR) of the target mRNAs [4]. In the past
64
decade, it is becoming clear that miRNAs have essential roles in the regulation of
65
apoptotic and autophagic pathways [5]. For example, miRNAs including
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miR-15a-miR-16-1 cluster and let-7 family can positively modulate apoptosis and
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promote cancer cell death [6]. In addition, miR-30a and miR-376b have been reported
68
involved in the autophagic pathways by directly targeting Beclin 1 and Atg4C [7].
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Our previous studies have demonstrated that Polygonatum odoratum lectin (POL)
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displayed remarkable anti-proliferative and apoptosis or autophagy inducing activities
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toward several cancer cells, such as murine fiborsarcoma L929 cells, human 3 Page 4 of 37
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melanoma A375 cells and A549 cells [8-10]. However, whether miRNAs participated
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in POL-induced apoptosis and autophagy in A549 cells remains to be investigated.
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2. Materials and methods
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2.1. Reagents and Cell Culture POL was purified as previously described [10]. Fetal bovine serum (FBS) was
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purchased from Gibco BRL (Grand Island, NY, USA). 3,3-diaminobenzidine
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tetrahydrochloride (DAB), monodansylcadaverine (MDC), N-acetyl-cysteine (NAC),
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2′,7′-dichlorofluorescein diacetate (DCF-DA) and pifithrin-α were purchased from
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Sigma Chemical (St. Louis, MO, USA). Human lung carcinoma A549 cells, human
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embryonic kidney (HEK) 293T cells, large cell lung cancer H460 cells and lung
102
adenocarcinoma H1299 cells were purchased from American Type Culture Collection
103
(Manassas, VA, USA). A549 cells were cultured in RPMI 1640 medium; HEK293T
104
cells, H460 cells and H1299 cells were cultured in DMEM medium with high glucose,
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respectively. Both media contained 100 U/ml penicillin, 100 μg/ml streptomycin and
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10% FBS, and the cells were cultured at 37 °C in a 5% CO2 atmosphere.
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2.2. MiRNA microarray analysis
A549 cells were dispensed in 6-well flat bottom microtiter plates at a density of
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5 × 104 cells/ml. After 24 h incubation, they were treated with 23 μg/ml POL or equal
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volume PBS for 24 h [8]. Then, the total RNA was isolated using the Trizol reagent
112
(Invitrogen) and sent to CapitalBio, Beijing, for miRNA microarray analysis. One
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microgram of RNA was used as a template for DNA preparations and hybridized to
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Agilent 8*60K miRNA arrays (Agilent Technologies, Human miRNA Microarray
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Release 19.0). cDNA was labeled with Cy5-dUTP and a reference control (Stratagene)
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was labeled with Cy3-dUTP using the Agilent (Agilent Technologies) low RNA input
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linear amplification kit and hybridized overnight at 65°C to Agilent 8*60K miRNA
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arrays (Agilent Technologies). Arrays were washed and scanned using an Agilent
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scanner (Agilent Technologies). The results were compared with those from the
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control group; fold changes (abs) > 2 and P < 0.05 were considered significant.
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2.3. Quantitative real-time PCR
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Total RNA was extracted from cells using Trizol reagent according to the
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manufacturer's instructions. Integrity of RNA was verified by electrophoresis, using
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2.2 M formaldehyde, 1.5% agarose gel stained with ethidium bromide. First-strand
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cDNA was obtained using the Reverse Transcription System (Promega), Oligo (dT)
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and stem-loop Reverse Transcription primer for miRNA. Quantitative real-time PCR
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(qPCR) was performed using SYBR Premier Dimer Eraser™ (TaKaRa, Dalian, China)
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on a LightCycler 480 (Roche, Basel, Switzerland), and relative quantification (2-ΔΔCt)
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method was used to analyze the data. GAPDH mRNA was used as reference for
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mRNA quantification and miRNAs expression levels were normalized to U6 rRNA.
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The primers information used for qRT-PCR was detailed in Table S1.
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2.4. Cell viability detection
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Cells were seeded into 96-well cultured plate at a density of 1 × 104 cell/well.
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After 24 h incubation, cells were transfected with or without Negative control (NC)
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mimics, miR-1290 mimics, NC inhibitor, miR-1290 inhibitor, miR-15a-3p mimics 6 Page 7 of 37
and miR-15a-3p inhibitor with Lipofectamine 2000 (Invitrogen) and subsequently
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treated with different concentrations of POL or NAC for the indicated time periods.
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NC mimics, miR-1290 mimics, NC inhibitor, miR-1290 inhibitor, miR-15a-3p
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mimics and miR-15a-3p inhibitor were purchased from GenePharma (Shanghai,
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China) and their sequence information were detailed in Table S2. Cell viability was
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measured by the CCK-8 method as previously described [11].
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2.5. Apoptosis assay
The A549 cells were treated with PBS or different concentrations of POL for the
147
indicated time periods. Cell morphology was imaged by phase contrast microscopy
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(Leica, Wetzlar, Germany) as well as fluorescence microscopy (Olympus, Tokyo,
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Japan) with DAPI staining. After POL treatment or miRNA transfection, cells were
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harvested and stained with Annexin V-FITC and propidium iodide (PI) according to
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the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA).
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2.6. Autophagy assays
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After incubation with POL for the fixed times, the cells were cultured with 0.05 mM
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MDC at 37 °C for 60 min. The cellular fluorescent changes were observed under
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fluorescence microscope (Olympus, Tokyo). The fluorescence intensity of cells was
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analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ).
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2.7. Luciferase reporter assay 7 Page 8 of 37
To measure the effect of miR-1290 on β-catenin expression in A549 cells,
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TOPflash β-catenin luciferase reporter (Addgene, Inc., Cambridge MA) or control
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FOPflash luciferase reporter (0.2 μg) was co-transfected with NC mimics, miR-1290
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mimics, NC inhibitor and miR-1290 inhibitor, along with 5 ng of Renilla luciferase
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reporter plasmid using X-tremeGENE (Invitrogen). The transfection was performed
165
according to the manufacturer's protocol. Transfected cells were incubated for 48 h
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and assayed for relative luciferase activity using dual-luciferase reporter system
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(Promega). The full length 3' UTR of GSK3β was amplified from the genomic DNA
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of HEK293T cells and cloned between the Renilla luciferase coding sequence and the
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poly(A) site of the psiCHECK-2 plasmid (Promega, Madison, WI, USA) using
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XhoI/NotI sites to produce psiCHE-GSK3β 3′UTR-wt. The primers used for the
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amplification of 3' UTR of GSK3β were detailed in Table S2. To generate the GSK3β
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3' UTR miR-1290 target site mutant, PCR-directed mutagenesis was performed using
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the QuikChange site-directed mutagenesis kit (Stratagene) according to the
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manufacturer's instructions (designated as psiCHE-GSK3β 3′UTR-mut). HEK293T
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cells were plated in 48-well plates at a density of 6 × 104 cells per well. After 24 h,
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cells were transfected with 50 ng psiCHECK-2 recombination vector and NC mimics
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or miR-1290 mimics. Firefly and Renilla luciferase activities were assayed with the
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Dual-Glo luciferase system (Promega) and measured with Synergy™ 2 multi-mode
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readers (BioTek, Vermont, USA) at 48 h after transfection.
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2.8. Western blot analysis 8 Page 9 of 37
A549 cells were treated with different concentrations of POL for the indicated
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time periods, and then both adherent and floating cells were collected. Western blot
184
analysis of cell lysates was performed as previously described [12]. Rabbit polyclonal
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antibodies against Bax, Bid, Bcl-xL, Bcl-2, pro-caspase3, caspase-3, pro-caspase9,
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caspase-9, β-catenin, cyclin D1, LC3, Beclin 1, p53, p-p53 (Ser 392), β-Actin, mouse
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polyclonal
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(HRP)-conjugated secondary antibodies were purchased from Santa Cruz
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Biotechnology. Rabbit polyclonal antibodies including GSK-3β and c-myc were from
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Abcam. Proteins were visualized using HRP-conjugated anti-rabbit or anti-mouse IgG
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and DAB as the HRP substrate.
against
cytochrome
and
horseradish
peroxidase
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2.9. Determination of intracellular ROS
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After treatment with POL or miRNA transfection for 24h, A549 cells were
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incubated with 10μM 2’,7’-dichlorofluorescein diacetate (DCFH-DA) for 60 min at
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37℃. The fluorescence intensity of A549 cells was measured by flow cytometry.
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2.10. Analysis of mitochondrial membrane potential The mitochondrial-specific cationic dye JC-1 (Invitrogen), which undergoes
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potential-dependent accumulation in the mitochondria, was used to detect the effect of
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POL and miR-15a-3p on mitochondrial potential. Briefly, cells were plated at a
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seeding density of 2 × 105 cells/well in a 12-well plate. After 24 h of treatment with
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POL (23 μg/ml) or transfected with NC mimics, miR-15a-3p mimics, NC inhibitor 9 Page 10 of 37
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and miR-15a-3p inhibitor, cells were incubated with 5 μM JC-1 for 30 min in dark at
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room temperature. Fluorescence was monitored with the Multimode Plate Reader at
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excitation wavelengths of (540 nm) and emission wavelength (590 nm).
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2.11. Statistical analyses
All the presented data and results were confirmed in at least three independent
210
experiments and are expressed as means ± SD. Student's t-test and one-way ANOVA
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were used for the statistical analyses, employing SPSS version 18.0 software.
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P < 0.05 was considered statistically significant.
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3. Results and discussion
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3.1. Effect of POL on miRNA expression in A549 cells and confirmation of microarray
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results by quantitative RT-PCR In our previous study, we found POL could simultaneously induce apoptosis and
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autophagy in A549 cells [8]. To determine the miRNA expression profile in A549
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cells with POL treatment, we screened and validated the altered miRNAs after POL
232
treatment. As shown in Fig. 1A, the array results of miRNA expression patterns in
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treated and untreated POL cells were distinguished from each other. Compared to the
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control group, there were 35 up-regulated miRNAs and 39 down-regulated miRNA in
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POL-treated A549 cells (the altered miRNAs information were detailed in Table S3).
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To validate the miRNA microarray results, four differentially expressed miRNAs were
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selected to perform the expression analysis with qRT-PCR. The selected miRNAs
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included one down-regulated miRNAs (hsa-miR-1290) and three up-regulated
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(hsa-miR-15a-3p, hsa-miR-3138 and hsa-miR-3940-5p). We found the expression
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alteration of these 4 miRNAs observed in qRT-PCR was consistent with that of
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microarray (Fig. 1B). In addition, we found that POL also inhibited proliferation of
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H460 cells and H1299 cells (Fig. S1). Subsequently, we performed qRT-PCR analysis
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to determine whether POL treatment altered hsa-miR-1290, hsa-miR-15a-3p,
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hsa-miR-3138 and hsa-miR-3940-5p expression in H460 cells and H1299 cells. In Fig.
245
S1, POL treatment also altered hsa-miR-1290, hsa-miR-15a-3p, hsa-miR-3138 and
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hsa-miR-3940-5p expression in these cells, suggesting these miRNAs may also
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involve in POL-induced cell death of H460 cells and H1299 cells.
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3.2. Down-regulated miR-1290 amplifies POL-induced apoptosis Given that miR-1290 was down-regulated after POL treatment, it is of interest to
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investigate its role in A549 cells. We first transfected miR-1290 mimics
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(over-expression of miR-1290) and inhibitor (knockdown of miR-1290) and
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examined its effect on cellular proliferation of A549 cells. The CCK-8 assay showed
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that the proliferation of A549 cells was not affected after miR-1290 over-expression
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12 or 24 hours (Fig. 2A) and this is consistent with a previous study [13].
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Subsequently, we examined the effect of miR-1290 inhibition on A549 cells
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proliferation. Intriguingly, the treatment of miR-1290 inhibitor also did not affect the
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proliferation rates of A549 cells compared with cells transfected with NC inhibitor in
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12 or 24 hours (Fig 2A). Then, we investigated whether POL-induced cell death can
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be affected by over-expressing or down-expressing of miR-1290. The A549 cells were
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transfected with miR-1290 mimics followed by treatment with 23 μg/ml POL for 12
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and 24 h. As expected, over-expression of miR-1290 significantly reduced
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POL-induced cell death while knockdown of miR-1290 markedly enhanced the
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POL-induced cell death (Fig. 2B).
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To test whether miR-1290 regulates POL induced apoptosis, we transfected A549
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cells with NC mimics, miR-1290 mimics, NC inhibitor and miR-1290 inhibitor
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followed by treatment with 23 μg/ml POL for 24 h, and the morphologic varieties
268
were detected under phase contrast microscopy. As shown in Fig. 2C, POL induced
269
typical apoptotic features, over-expression of miR-1290 in A549 cells reversed 12 Page 13 of 37
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POL-induced cell death while knockdown of miR-1290 amplified POL-induced
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apoptosis. Besides, Annexin V-FITC/PI assay also demonstrated that knockdown of
272
miR-1290 amplified POL-induced apoptosis (Fig. 2D). Western blot analysis was then performed to further confirm whether the
274
apoptosis pathway was affected in A549 cells with increased and decreased miR-1290
275
expression. We noted that POL induced cleavage of caspase-3 and caspase-9 could be
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rescued
277
down-regulation of pro-apoptotic Bax as well as up-regulation of anti-apoptotic Bcl-2
278
and Bcl-xL proteins was observed after miR-1290 over-expression (Fig. 2E). All these
279
results indicate that over-expression of miR-1290 can protect A549 cells from
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POL-induced apoptosis and down-regulate miR-1290 may amplify POL-induced
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apoptosis in A549 cells.
miR-1290
mimics
transfection
in
A549
cells.
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Meanwhile, we performed transfection assays to assess whether miR-1290 can
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affect autophagy in A549 cells. It is found that the autophagy ratio of A549 cells was
284
not affected after miR-1290 over-expression or knockdown (Fig.S2).
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MiR-1290 has been proven to be overexpressed in several different types of
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human cancers, such as colon cancer, pancreatic cancer and esophageal squamous cell
287
carcinoma, suggesting it may function as an oncogenic miRNA [13-15]. Since
288
miR-1290 was down-regulated miRNA after POL treatment, we first investigated its
289
role in A549 cells. However, both over-expression and knockdown of miR-1290 did
290
not affect the proliferation of A549 cells. Thus, we checked whether over-expression
291
or knockdown of miR-1290 can affect POL-induced apoptosis and found
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over-expression of miR-1290 suppressed POL induced apoptosis in A549 cells.
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Previous study has shown that miR-1290 over-expression activated the Akt and
294
NF-κB pathways that promoted cell proliferation and inhibited cell apoptosis in colon
295
cancer cells [13]. Therefore, it is reasonable to speculate that miR-1290 might exert
296
the anti-apoptotic role by upregulating of AKT and NF-κB activities and thus suppress
297
POL-induced apoptosis. However, we found knockdown of miR-1290 did not change
298
the viability of A549 cells, suggesting it may have a complex role in A549 cell. Thus,
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further studies are needed to understand the influence of miR-1290 on tumor
300
proliferation.
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3.3. POL and miR-1290 regulate Wnt/β-catenin pathway WNT/β-catenin pathway is frequently activated in various cancers [16]. Mazieres
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et al. found that activation of Wnt/β-catenin signaling is important for both initiation
305
and progression of lung cancer [17, 18]. To determine whether POL regulates the
306
Wnt/β-catenin pathway, we first performed western blot to examine Wnt/β-catenin
307
pathway-related protein levels in A549 cells after POL treatment. We found GSK3β
308
was markedly elevated in a time-dependent manner after POL administration (Fig.
309
3A). In addition, a significant time-dependent reduction of β-catenin and its
310
downstream factors including c-myc and cyclin D1 was observed in POL treated cells
311
compared to the control groups. Previous study showed that over-expression of
312
miR-1290 could activate the Wnt pathway in colon cancer cells [13]. However,
313
whether miR-1290 can activate the Wnt pathway in A549 cells is still unclear. To
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determine this, TOPflash reporter was used. As shown in Fig. 3B, TOPflash reporter
315
was strongly activated by miR-1290 mimics and inactivated by miR-1290 inhibitor.
316
Moreover, western blot data showed that the treatment of A549 cells with miR-1290
317
mimics resulted in down-regulation of GSK3β and up-regulation of β-catenin, c-myc
318
and cyclin D1 proteins levels (Fig. 3C). In contrast, miR-1290 inhibitor could increase
319
the expression levels of GSK3β and decrease the expression levels of β-catenin and its
320
downstream targets in A549 cells. Collectively, these results indicate that both POL
321
and miR-1290 can regulate Wnt/β-catenin pathway.
322
3.4. GSK3β is a target of miR-1290
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To identify the molecular target of miR-1290, we searched potential miR-1290
324
targets predicted by TargetScan database (http://www.targetscan.org, Release 6.2
325
Version). The genes involved in Wnt/β-catenin signaling pathway got more attention,
326
because we found both POL and miR-1290 could regulate Wnt/β-catenin pathway.
327
Consequently, GSK3β, a key component of Wnt signaling pathway, was predicted by
328
TargetScan database as a prime target of miR-1290 (Fig. 4A).
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To confirm this prediction, a luciferase reporter assay was conducted to assess
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whether miR-1290 directly targets the 3' UTR of GSK3β. HEK293T cells were
331
co-transfected with wild-type reporter, mutant reporter, miR-1290 mimics and NC
332
mimics. As shown in Fig. 4B, compared with NC group, co-transfection of miR-1290
333
mimics with the GSK3β 3′UTR wild-type reporter (psiCHE-GSK3β 3′UTR-wt)
334
resulted in a significant decrease in luciferase activity. No appreciable inhibitory
335
effect was observed when miR-1290 mimics or NC mimics co-transfected with the
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empty vector or the mutant reporter (psiCHE-GSK3β 3′UTR-mut), indicate that the
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GSK3β is a direct target of miR-1290. To confirm whether miR-1290 can regulate GSK3β expression, GSK3β mRNA
339
and protein quantities were measured in A549 cells transiently transfected with NC
340
mimics, miR-1290 mimics, NC inhibitor and miR-1290 inhibitor respectively. As
341
shown in Fig. 4C, the mRNA levels of GSK3β were not influenced after transfection
342
for 24h. However, the GSK3β protein expression was inhibited by miR-1290 mimics
343
in A549 cells and miR-1290 inhibitor increased the protein level of GSK3β (Fig. 4D).
344
Taken together, these data support that GSK3β is a direct target of miR-1290.
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In a previous study, Wu et al. reported that over-expression of miR-1290 can
346
activate the Wnt pathway and increase the levels of reprogramming-related
347
transcription factors c-myc and Nanog in colon cancer cells [13]. Consistent with
348
these observations, our results demonstrated that miR-1290 could regulate Wnt
349
pathway by targeting GSK3β in A549 cells. Of note, GSK3β is a key component of
350
Wnt/β-catenin signaling, and decreased GSK3β can increase the accumulation of
351
β-catenin in cytoplasm. Our results demonstrated that miR-1290 over-expression
352
elevated β-catenin and its downstream targets Cyclin D1 and c-myc expression in
353
A549 cells.
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3.5. MiR-15a-3p induces apoptosis and autophagy in A549 cells
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Since miR-15a-3p was up-regulated after POL treatment in A549 cells, we
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hypothesized that miR-15a-3p might have a pro-apoptotic role in A549 cells. To 16 Page 17 of 37
verify our hypothesis, we transfected A549 cells with NC mimics, miR-15a-3p
359
mimics, NC inhibitor and miR-15a-3p inhibitor for 24h, and the cell viability was
360
evaluated by CCK-8 assay. Over-expression of miR-15a-3p significantly decreased
361
the proliferation rates of A549 cells compared with cells transfected with NC mimics
362
in 24 hours (Fig. 5A). We next determined whether miR-15a-3p mediated decreased
363
cell viability was implicated in apoptosis. As shown in Fig. 5B, miR-15a-3p mimics
364
transfection resulted in a decrease in cell density compared with NC mimics
365
transfection group, and over-expression of miR-15a-3p induced typical apoptotic
366
features in A549 cells. Apoptosis was evaluated by the Annexin V-FITC/PI assay. In
367
Fig. 5C, miR-15a-3p mimics transfection markedly induced the increase of apoptotic
368
cells proportion. Moreover, western blot analysis showed that procaspase-3 and
369
procaspase-9 were decreased while cleaved caspase-3 and caspase-9 were increased
370
after miR-15a-3p mimics transfection, indicates miR-15a-3p can activate caspase-3
371
and caspase-9 (Fig. 5D). It has been well known that Bcl-2 is a key target of miR-15a.
372
Thus, we investigated whether miR-15a-3p can also affect the Bcl-2 and Bcl-xL
373
proteins expression by western blot analysis. We found Bcl-2 and Bcl-xL levels were
374
decreased in A549 cells transfected with miR-15a-3p mimics compared with NC
375
mimics, NC inhibitor and miR-15a-3p inhibitor.
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Meanwhile, typical characteristics of autophagy were also observed in
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miR-15a-3p mimics transfected A549 cells. The formation of autophagic vacuoles
378
(AVOs) was assessed by staining the cells with MDC. As shown in Fig. 5E,
379
autophagic vacuoles were formed in A549 cells that featured by increased MDC 17 Page 18 of 37
fluorescent intensity, indicating the presence of autophagic cell death. The formation
381
of autophagic vacuoles was further confirmed by measurement of Beclin1 and LC3
382
levels in transfected cells. As shown in Fig. 5D, a significant increase expression of
383
Beclin 1 and LC3-II proteins was observed in miR-15a-3p mimics transfected cells in
384
comparison to the control cells. All these results indicate that miR-15a-3p induces
385
both apoptosis and autophagy in A549 cells.
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MiR-15a-3p is a novel member of the pro-apoptotic miRNA cluster [19]. A
387
previous study reported that miR-15a-3p has a pro-apoptotic role in human cells by its
388
ability to activate Caspase-3/7, to reduce cell viability and to inhibit the expression of
389
Bcl-xL in HeLa and AsPc-1 cells [19]. In this study, we confirmed the pro-apoptotic
390
role of miR-15a-3p in A549 cells by its ability to activate Caspase-3/9 and to inhibit
391
the expression of Bcl-2 and Bcl-xL. Moreover, we found over-expression of
392
miR-15a-3p also resulted in A549 cells autophagy, suggesting it may be considered
393
for both apoptosis and autophagy modulating therapies in cancers.
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3.6. MiR-15a-3p regulates ROS generation and induces p53 activation in A549 cells
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Mitochondria are the major source of ROS production, and our previous study
397
have demonstrated that ROS was implicated in POL-induced both apoptosis and
398
autophagy in A549 cells [8, 20]. Therefore, we investigated whether miR-15a-3p can
399
affect mitochondria ROS production using flow cytometry. The data showed that ROS
400
was accumulated in miR-15a-3p mimics transfected A549 cells (Fig. 6A). The
401
mitochondrial membrane potential disruption is always associated with ROS 18 Page 19 of 37
formation [21]. Next we examined the mitochondrial membrane potential by using the
403
cationic dye JC-1. In Fig .6B, after POL treatment or miR-15a-3p mimics transfection,
404
the mitochondrial membrane potential was found to be interrupted, as evidenced by
405
the migration of JC-1 dye from the mitochondria into the cytoplasm of treated cells,
406
and the subsequent reduction in the mitochondrial red fluorescence signals. Notably,
407
pretreatment with a ROS scavenger NAC could reverse POL or miR-15a-3p-induced
408
cytotoxicity, and suppress miR-15a-3p-induced apoptosis and autophagy (Fig. 6C).
409
These results indicate that miR-15a-3p can regulate mitochondria ROS generation
410
which was involved in miR-15a-3p-induced apoptosis and autophagy.
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To assess whether p53 was activated in miR-15a-3p-transfected cells, the levels
412
of p53 and p-p53 were determined by western blot analysis. As shown in Fig. 6D, the
413
level of p53 was not obviously changed but the level of p-p53 (ser 392) was markedly
414
enhanced after miR-15a-3p mimics transfection. Furthermore, pretreatment with NAC
415
remarkably inhibited the activation of p53. To investigate whether the p53 is involved
416
in miR-15a-3p-induced A549 cells apoptosis and autophagy, p53 inhibitor pifithrin α
417
was used. In Fig. 6E, pretreatment with pifithrin α suppressed miR-15a-3p-induced
418
apoptosis and autophagy in A549 cells, indicates that miR-15a-3p-ROS-p53 pathway
419
is involved in POL-induced apoptosis and autophagy in A549 cells.
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In our previous study, we found another typical GNA-related lectin P. cyrtonema
421
lectin (PCL) could induce apoptosis and autophagy in human melanoma A375 cells
422
via mitochondrial-mediated ROS-p38-p53 pathway [22]. Similar with these results,
423
we demonstrated that the ROS-mediated p53 activation was involved in the POL 19 Page 20 of 37
induced apoptosis and autophagy in A549 cells. However, previous studies also
425
demonstrated that some plant lections could induce apoptosis in a p53 independent
426
manner. For instance, mistletoe lectin was reported to induce apoptosis independent
427
on p53 activation [23, 24]. Concanavalin A was reported to induce apoptosis in
428
p53-null cells and shows no effects on normal cells [25]. Therefore, the precise
429
function of p53 in plant lectins induced cancer cells apoptosis and autophagy remains
430
further explored.
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4. Conclusion In summary, we report for the first time that miR-1290 and miR-15a-3p are
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involved in POL induced A549 cells apoptosis and autophagy. In addition, we provide
449
evidence that GSK3β is a target of miR-1290. Importantly, we further confirm that a
450
miR-15a-3p mediated ROS-p53 pathway is involved in POL-induced apoptosis and
451
autophagy in A549 cells. A proposed model for the roles of miRNAs in POL-induced
452
apoptosis and autophagy is presented in Fig. 7. These findings would possibly open a
453
new venue for plant lectins as a potential antineoplastic agent in treatment of different
454
human cancer in future.
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Acknowledgements
This work was supported in part by National Natural Science Foundation of
458
China (No. 31300674, No. 81173093, No. 30970643, No. 81373311 and No.
459
J1103518).
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Figure legends
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Fig. 1. Effect of POL on miRNA expression in A549 cells.
470
(A) Hierarchical clustering of the differentially expressed miRNA in A549 cells after
471
POL treatment. There were 35 upregulated miRNAs and 39 downregulated miRNAs
472
in POL-treated A549 cells compared with control group.
473
(B) Real-time PCR was performed to confirm the miRNA array results. It showed that
474
hsa-miR-1290
475
hsa-miR-3138 and hsa-miR-3940-5p was upregulated after POL treatment. (means ±
476
SD, n = 3).
after
POL
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downregulated
treatment.
Hsa-miR-15a-3p,
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Fig. 2. Down-regulated miR-1290 amplifies POL-induced apoptosis in A549 cells.
480
(A) The A549 cells transfected with NC mimics, miR-1290 mimics, NC inhibitor and
481
miR-1290 inhibitor for indicated time and examined its effect on cellular proliferation.
482
The CCK-8 assay showed that the proliferation of A549 cells was not affected after
483
miR-1290 over-expression or knockdown in 12 and 24 h. (means ± SD, n = 3) (ns: no
484
significance vs. NC inhibitor group).
485
(B) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC
486
inhibitor and miR-1290 inhibitor for 24 h followed by stimulation with or without 23
487
μg/ml POL for indicated time, the cell growth inhibition ratio was measured by the
488
CCK-8 assay. (means ± SD, n = 3) (**p < 0.01 vs. Control group, #p < 0.05 vs. NC
489
mimics+POL group, ##p < 0.01 vs. POL group). Knockdown of miR-1290 markedly
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22 Page 23 of 37
enhanced the POL-induced cell death and miR-1290 mimics significant reduced POL
491
induced cell death. Control: cells without transfection or POL treatment.
492
(C) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC
493
inhibitor and miR-1290 inhibitor, followed by treatment with or without 23 μg/ml
494
POL for 24 h, the morphologic varieties were detected under phase contrast
495
microscopy.
496
(D) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC
497
inhibitor and miR-1290 inhibitor, followed by treatment with or without 23 μg/ml
498
POL for 24 h, POL-induced apoptosis was analyzed by flow cytometry. After
499
miR-1290 mimics transfection, the percentage of early (c: 15.64 ± 3.3%, d: 10.14 ±
500
2.6%) and late (c: 9.35 ± 2.4%, d: 6.84 ± 3.1%) apoptotic cells induced by POL were
501
reduced. Knockdown of miR-1290 markedly enhanced the POL-induced early (e:
502
12.95 ± 1.4%, f: 24.48 ± 3.6%) and late (e: 11.83 ± 3.4%, f: 12.50 ± 2.1%) apoptotic
503
cells death.
504
(E) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC inhibitor
505
and miR-1290 inhibitor for 24 h followed by stimulation with or without 23 μg/ml
506
POL for 24h, the pro-caspase-3,caspase-3, pro-caspase-9, caspase-9, Bax, Bcl-2 and
507
Bcl-xL levels were examined by western blot analysis.
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508 509 510
Fig. 3. POL and miR-1290 regulate the Wnt/β-catenin pathway.
511
(A) The A549 cells were treated with 23 μg/ml POL for indicated time periods, 23 Page 24 of 37
followed by western blot analysis for detection of GSK3β, β-catenin, c-myc and
513
cyclin D1 levels. β-Actin was used as an equal loading control.
514
(B) TOPFflash/FOPflash luciferase assays on A549 cells after NC mimics, miR-1290
515
mimics, NC inhibitor and miR-1290 inhibitor transfection for 48 h. (means ± SD, n =
516
3). (**p < 0.01 vs. NC mimics group, #p < 0.05 vs. NC inhibitor group).
517
(C) Western blot was performed to determine the protein level of GSK3β, β-catenin,
518
c-myc and cyclin D1 in A549 cells after transfection with NC mimics, miR-1290
519
mimics, NC inhibitor and miR-1290 inhibitor for 48 h. β-Actin served as a loading
520
control.
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Fig. 4. miR-1290 directly targeted 3′UTR of GSK3β.
524
(A) The predicted binding site of miR-1290 in the 3′UTR of GSK3β by targetscan.
525
(B) The psiCHECK2, psiCHE-GSK3β 3′UTR wt or psiCHE-GSK3β 3′UTR mut
526
vector was co-transfected with miR-1290 mimics or NC mimics into HEK293T cells,
527
and normalized Renilla luciferase activity was determined. Luciferase report assay
528
data showed that co-transfection of HEK293T cells with miR-1290 and wild type
529
GSK3β 3′UTR led to a remarkable decrease in luciferase activity. (means ± SD, n =
530
3). (**p < 0.01 vs. NC mimics group).
531
(C) Expression level of GSK3β mRNA was measured by qPCR in A549 cells
532
transiently transfected with NC mimics, miR-1290 mimics, NC inhibitor and
533
miR-1290 inhibitor for 24h. (means ± SD, n = 3).
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(D) Detection of GSK3β in transfected A549 cells. Western blot analysis
535
demonstrated that GSK3β was downregulated in miR-1290 over-expression cells after
536
24 h post-transfection. β-Actin was used as a loading control.
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537 538
Fig. 5. Over-expression of miR-15a-3p induces A549 cells apoptosis and autophagy.
540
(A) The A549 cells were treated with 23 μg/ml POL or transfected with NC mimics,
541
miR-15a-3p mimics, NC inhibitor and miR-15a-3p inhibitor for 24 h and examined its
542
effect on cellular proliferation. Values are expressed as means ± SD. ∗∗p < 0.01 vs.
543
NC mimics group, ns: no significance vs. NC inhibitor group.
M
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539
544
(B) Cells were transfected with or without NC mimics, miR-15a-3p mimics, NC
546
inhibitor and miR-15a-3p inhibitor for 24 h, the morphologic varieties were detected
547
under phase contrast microscopy.
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(C) The A549 cells were transfected with NC mimics, miR-15a-3p mimics, NC
550
inhibitor and miR-15a-3p inhibitor for 24 h, transfection-induced apoptosis was
551
analyzed by flow cytometry. After miR-15a-3p mimics transfection, the percentage of
552
early (b: 1.51 ± 0.4%, c: 12.31 ± 2.4%) and late (b: 1.65 ± 0.3%, c: 22.64 ± 3.6%)
553
apoptotic cells were increased.
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549
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(D) The A549 cells were transfected with NC mimics, miR-15a-3p mimics, NC 25 Page 26 of 37
556
inhibitor and miR-15a-3p inhibitor for 24 h, the Bcl-2, Bcl-xL, procaspase-3,
557
caspase-3, procaspase-9, caspase-9, Beclin 1 and LC3 levels were examined by
558
western blot analysis. β-Actin was used as a loading control.
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(E) The A549 cells were transfected with NC mimics, miR-15a-3p mimics, NC
561
inhibitor and miR-15a-3p inhibitor for 24 h, the morphologic changes were observed
562
under a fluorescence microscope by MDC staining (a). The MDC fluorescent
563
intensity was analyzed by flow cytometry (b). Values are expressed as means ± SD.
564
∗∗p < 0.01 vs. NC mimics group.
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Fig. 6. miR-15a-3p induces ROS generation and p53 activation.
567
(A) The A549 cells were treated with 23 μg/ml POL or transfected with NC mimics,
568
miR-15a-3p mimics, NC inhibitor and miR-15a-3p inhibitor for 24 h and the DCF
569
fluorescent intensity was measured by flow cytometry. Values are expressed as means
570
± SD. (**p < 0.01 vs. Control group, ##p < 0.01 vs. NC mimics group).
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(B) The A549 cells were treated with 23 μg/ml POL or transfected with NC mimics,
573
miR-15a-3p mimics, NC inhibitor and miR-15a-3p inhibitor for 24 h and JC-1 dye
574
incubation for the final 20 min. After POL treatment or miR-15a-3p mimics
575
transfection, the mitochondrial membrane potential was found to be interrupted.
576 577
(C) Cells were treated with 23 μg/ml POL or transfected with miR-15a-3p mimics for 26 Page 27 of 37
578
24h in the presence or absence of 2 mM NAC, the cell viablity (a), early as well as
579
late apoptosis ratio (b) and MDC fluorescent intensity (c) were analyzed by flow
580
cytometry. (**p < 0.01 vs. Control group).
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(D) Cells were treated with 23 μg/ml POL or transfected with miR-15a-3p mimics for
583
the indicated time periods in the presence or absence of 2 mM NAC, followed by
584
Western blot analysis for detection of p53 and p-p53 (Ser 392) levels. β-actin was
585
used as an equal loading control.
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an
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(E) Cells were treated with 23 μg/ml POL or transfected with miR-15a-3p mimics for
588
24h in the presence or absence of pifithrin α (pif), the apoptosis ratio (a) and MDC
589
fluorescent intensity (b) were analyzed by flow cytometry. (**p < 0.01 vs. Control
590
group).
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Fig. 7. The cartoon illustrating the roles of miRNAs in POL-induced apoptosis and
594
autophagy.
595
Microarray analysis was performed on A549 cells to identify altered miRNAs after
596
POL treatment. MiR-1290 was down-regulated after POL treatment and GSK3β was a
597
target of miR-1290. MiR-15a-3p was up-regulated after POL treatment and
598
over-expression of miR-15a-3p resulted in A549 cells apoptosis and autophagy via
599
ROS-p53 pathway.
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S0141-8130(15)30107-0 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.11.014 BIOMAC 5519
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
25-7-2015 4-11-2015 5-11-2015
Please cite this article as: L. Wu, T. Liu, Y. Xiao, X. Li, Y. Zhu, Y. Zhao, J. Bao, C. Wu, Polygonatum odoratum lectin induces apoptosis and autophagy by regulation of microRNA-1290 and microRNA-15a-3p in human lung adenocarcinoma A549 cells, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.11.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights (for review)
Highlights
► miR-1290 is down-regulated in POL treated A549 cells. ► GSK-3β is a target of miR-1290.
miR-15a-3p resulted in A549 cells apoptosis and autophagy.
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► miR-15a-3p is up-regulated after POL treatment and over-expression of
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► miR-15a-3p-ROS-p53 pathway is involved in POL-induced apoptosis and
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autophagy in A549 cells
Page 1 of 37
*Manuscript Click here to view linked References
1
Polygonatum
odoratum
2
autophagy
3
microRNA-15a-3p in human lung adenocarcinoma A549
4
cells
regulation
induces
of
apoptosis
and
microRNA-1290
and
ip t
by
lectin
5
Lei Wu, Tao Liu, Yan Xiao, Xing Li, Yanan Zhu, Yan Zhao, Jinku Bao* and
7
Chuanfang Wu*
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8
School of Life Sciences and Key Laboratory of Bio-resources and Eco-environment,
10
Ministry of Education, State Key Laboratory of Biotherapy and Cancer Center, West
11
China Hospital, Sichuan University, and Collaborative Innovation Center for
12
Biotherapy, Chengdu, 610064, China
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*Correspondence: J. K. Bao, School of Life Sciences and Key Laboratory of Bio-resources, Ministry of
23
Education,
24
[email protected]
25
C. F. Wu, School of Life Sciences and Key Laboratory of Bio-resources, Ministry of Education, Sichuan
26
University, Chengdu, China. Tel./fax: +86-28-85415171; E-mail: [email protected]
27
Lei Wu and Tao Liu contributed equally to this work.
Sichuan
University,
Chengdu,
China.
Tel./fax:
+86-28-85415171;
Email:
1 Page 2 of 37
28
Abstract Polygonatum odoratum lectin (POL), a mannose-binding specific Galanthus
30
nivalis agglutinin (GNA)-related lectin has been reported with remarkable
31
anti-proliferative and apoptosis-inducing effects against several tumor cells. Our
32
previous research revealed that POL can induce apoptosis and autophagy in A549
33
cells. However, whether microRNAs (miRNAs) are involved in POL-induced
34
apoptosis and autophagy in A549 cells has not been investigated. The
35
study was to evaluate whether miRNAs were involved in POL-induced apoptosis and
36
autophagy in A549 cells. In the present study, we performed microarray analysis on
37
A549 cells to identify altered miRNAs after POL treatment. We found that miR-1290
38
was down-regulated after POL treatment and down-regulated miR-1290 amplifies
39
POL-induced apoptosis in A549 cells. Moreover, we revealed that glycogen synthase
40
kinase-3β (GSK3β) was a direct target of miR-1290 and POL treatment could result in
41
Wnt pathway down regulation. We also found that miR-15a-3p was up-regulated after
42
POL treatment and over-expression of miR-15a-3p resulted in A549 cells apoptosis
43
and autophagy. In addition, we confirmed that a miR-15a-3p mediated ROS-p53
44
pathway was involved in POL-induced apoptosis and autophagy in A549 cells. Taken
45
together, these data provide evidence that POL induces A549 cells apoptosis and
46
autophagy by regulation of miR-1290 and miR-15a-3p.
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Keywords: Polygonatum odoratum lectin; Apoptosis; Autophagy; miR-1290;
49
miR-15a-3p 2 Page 3 of 37
50
1. Introduction Plant lectins are a class of highly diverse, non-immune origin and
52
carbohydrate-binding proteins [1]. Plant lectins have been reported to possess
53
antitumor activities via targeting programmed cell death (PCD), including apoptosis
54
and autophagy [2]. Apoptosis is a cell-intrinsic mechanism for suicide that regulated
55
by numerous cellular signaling pathways and autophagy is a conserved eukaryotic
56
cellular degradative process that helps in maintaining the cellular metabolism and
57
homeostasis [3]. Several major plant lectins families including Galanthus nivalis
58
agglutinin (GNA) related lectins, legume lectins and type II ribosome-inactivating
59
proteins (RIP) have been reported to induce apoptosis and autophagy in many types of
60
cancer cells [2].
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MicroRNAs (miRNAs) are small non-coding RNAs of about 18-24 nucleotides in
62
length that negatively regulate gene expression by binding to the complementary
63
sequences in the 3′-untranslated regions (3′UTR) of the target mRNAs [4]. In the past
64
decade, it is becoming clear that miRNAs have essential roles in the regulation of
65
apoptotic and autophagic pathways [5]. For example, miRNAs including
66
miR-15a-miR-16-1 cluster and let-7 family can positively modulate apoptosis and
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promote cancer cell death [6]. In addition, miR-30a and miR-376b have been reported
68
involved in the autophagic pathways by directly targeting Beclin 1 and Atg4C [7].
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Our previous studies have demonstrated that Polygonatum odoratum lectin (POL)
70
displayed remarkable anti-proliferative and apoptosis or autophagy inducing activities
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toward several cancer cells, such as murine fiborsarcoma L929 cells, human 3 Page 4 of 37
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melanoma A375 cells and A549 cells [8-10]. However, whether miRNAs participated
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in POL-induced apoptosis and autophagy in A549 cells remains to be investigated.
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2. Materials and methods
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2.1. Reagents and Cell Culture POL was purified as previously described [10]. Fetal bovine serum (FBS) was
97
purchased from Gibco BRL (Grand Island, NY, USA). 3,3-diaminobenzidine
98
tetrahydrochloride (DAB), monodansylcadaverine (MDC), N-acetyl-cysteine (NAC),
99
2′,7′-dichlorofluorescein diacetate (DCF-DA) and pifithrin-α were purchased from
100
Sigma Chemical (St. Louis, MO, USA). Human lung carcinoma A549 cells, human
101
embryonic kidney (HEK) 293T cells, large cell lung cancer H460 cells and lung
102
adenocarcinoma H1299 cells were purchased from American Type Culture Collection
103
(Manassas, VA, USA). A549 cells were cultured in RPMI 1640 medium; HEK293T
104
cells, H460 cells and H1299 cells were cultured in DMEM medium with high glucose,
105
respectively. Both media contained 100 U/ml penicillin, 100 μg/ml streptomycin and
106
10% FBS, and the cells were cultured at 37 °C in a 5% CO2 atmosphere.
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2.2. MiRNA microarray analysis
A549 cells were dispensed in 6-well flat bottom microtiter plates at a density of
110
5 × 104 cells/ml. After 24 h incubation, they were treated with 23 μg/ml POL or equal
111
volume PBS for 24 h [8]. Then, the total RNA was isolated using the Trizol reagent
112
(Invitrogen) and sent to CapitalBio, Beijing, for miRNA microarray analysis. One
113
microgram of RNA was used as a template for DNA preparations and hybridized to
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Agilent 8*60K miRNA arrays (Agilent Technologies, Human miRNA Microarray
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Release 19.0). cDNA was labeled with Cy5-dUTP and a reference control (Stratagene)
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was labeled with Cy3-dUTP using the Agilent (Agilent Technologies) low RNA input
117
linear amplification kit and hybridized overnight at 65°C to Agilent 8*60K miRNA
118
arrays (Agilent Technologies). Arrays were washed and scanned using an Agilent
119
scanner (Agilent Technologies). The results were compared with those from the
120
control group; fold changes (abs) > 2 and P < 0.05 were considered significant.
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2.3. Quantitative real-time PCR
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Total RNA was extracted from cells using Trizol reagent according to the
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manufacturer's instructions. Integrity of RNA was verified by electrophoresis, using
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2.2 M formaldehyde, 1.5% agarose gel stained with ethidium bromide. First-strand
126
cDNA was obtained using the Reverse Transcription System (Promega), Oligo (dT)
127
and stem-loop Reverse Transcription primer for miRNA. Quantitative real-time PCR
128
(qPCR) was performed using SYBR Premier Dimer Eraser™ (TaKaRa, Dalian, China)
129
on a LightCycler 480 (Roche, Basel, Switzerland), and relative quantification (2-ΔΔCt)
130
method was used to analyze the data. GAPDH mRNA was used as reference for
131
mRNA quantification and miRNAs expression levels were normalized to U6 rRNA.
132
The primers information used for qRT-PCR was detailed in Table S1.
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2.4. Cell viability detection
135
Cells were seeded into 96-well cultured plate at a density of 1 × 104 cell/well.
136
After 24 h incubation, cells were transfected with or without Negative control (NC)
137
mimics, miR-1290 mimics, NC inhibitor, miR-1290 inhibitor, miR-15a-3p mimics 6 Page 7 of 37
and miR-15a-3p inhibitor with Lipofectamine 2000 (Invitrogen) and subsequently
139
treated with different concentrations of POL or NAC for the indicated time periods.
140
NC mimics, miR-1290 mimics, NC inhibitor, miR-1290 inhibitor, miR-15a-3p
141
mimics and miR-15a-3p inhibitor were purchased from GenePharma (Shanghai,
142
China) and their sequence information were detailed in Table S2. Cell viability was
143
measured by the CCK-8 method as previously described [11].
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2.5. Apoptosis assay
The A549 cells were treated with PBS or different concentrations of POL for the
147
indicated time periods. Cell morphology was imaged by phase contrast microscopy
148
(Leica, Wetzlar, Germany) as well as fluorescence microscopy (Olympus, Tokyo,
149
Japan) with DAPI staining. After POL treatment or miRNA transfection, cells were
150
harvested and stained with Annexin V-FITC and propidium iodide (PI) according to
151
the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA).
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2.6. Autophagy assays
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After incubation with POL for the fixed times, the cells were cultured with 0.05 mM
155
MDC at 37 °C for 60 min. The cellular fluorescent changes were observed under
156
fluorescence microscope (Olympus, Tokyo). The fluorescence intensity of cells was
157
analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ).
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2.7. Luciferase reporter assay 7 Page 8 of 37
To measure the effect of miR-1290 on β-catenin expression in A549 cells,
161
TOPflash β-catenin luciferase reporter (Addgene, Inc., Cambridge MA) or control
162
FOPflash luciferase reporter (0.2 μg) was co-transfected with NC mimics, miR-1290
163
mimics, NC inhibitor and miR-1290 inhibitor, along with 5 ng of Renilla luciferase
164
reporter plasmid using X-tremeGENE (Invitrogen). The transfection was performed
165
according to the manufacturer's protocol. Transfected cells were incubated for 48 h
166
and assayed for relative luciferase activity using dual-luciferase reporter system
167
(Promega). The full length 3' UTR of GSK3β was amplified from the genomic DNA
168
of HEK293T cells and cloned between the Renilla luciferase coding sequence and the
169
poly(A) site of the psiCHECK-2 plasmid (Promega, Madison, WI, USA) using
170
XhoI/NotI sites to produce psiCHE-GSK3β 3′UTR-wt. The primers used for the
171
amplification of 3' UTR of GSK3β were detailed in Table S2. To generate the GSK3β
172
3' UTR miR-1290 target site mutant, PCR-directed mutagenesis was performed using
173
the QuikChange site-directed mutagenesis kit (Stratagene) according to the
174
manufacturer's instructions (designated as psiCHE-GSK3β 3′UTR-mut). HEK293T
175
cells were plated in 48-well plates at a density of 6 × 104 cells per well. After 24 h,
176
cells were transfected with 50 ng psiCHECK-2 recombination vector and NC mimics
177
or miR-1290 mimics. Firefly and Renilla luciferase activities were assayed with the
178
Dual-Glo luciferase system (Promega) and measured with Synergy™ 2 multi-mode
179
readers (BioTek, Vermont, USA) at 48 h after transfection.
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2.8. Western blot analysis 8 Page 9 of 37
A549 cells were treated with different concentrations of POL for the indicated
183
time periods, and then both adherent and floating cells were collected. Western blot
184
analysis of cell lysates was performed as previously described [12]. Rabbit polyclonal
185
antibodies against Bax, Bid, Bcl-xL, Bcl-2, pro-caspase3, caspase-3, pro-caspase9,
186
caspase-9, β-catenin, cyclin D1, LC3, Beclin 1, p53, p-p53 (Ser 392), β-Actin, mouse
187
polyclonal
188
(HRP)-conjugated secondary antibodies were purchased from Santa Cruz
189
Biotechnology. Rabbit polyclonal antibodies including GSK-3β and c-myc were from
190
Abcam. Proteins were visualized using HRP-conjugated anti-rabbit or anti-mouse IgG
191
and DAB as the HRP substrate.
against
cytochrome
and
horseradish
peroxidase
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2.9. Determination of intracellular ROS
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After treatment with POL or miRNA transfection for 24h, A549 cells were
195
incubated with 10μM 2’,7’-dichlorofluorescein diacetate (DCFH-DA) for 60 min at
196
37℃. The fluorescence intensity of A549 cells was measured by flow cytometry.
198 199
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2.10. Analysis of mitochondrial membrane potential The mitochondrial-specific cationic dye JC-1 (Invitrogen), which undergoes
200
potential-dependent accumulation in the mitochondria, was used to detect the effect of
201
POL and miR-15a-3p on mitochondrial potential. Briefly, cells were plated at a
202
seeding density of 2 × 105 cells/well in a 12-well plate. After 24 h of treatment with
203
POL (23 μg/ml) or transfected with NC mimics, miR-15a-3p mimics, NC inhibitor 9 Page 10 of 37
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and miR-15a-3p inhibitor, cells were incubated with 5 μM JC-1 for 30 min in dark at
205
room temperature. Fluorescence was monitored with the Multimode Plate Reader at
206
excitation wavelengths of (540 nm) and emission wavelength (590 nm).
208
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2.11. Statistical analyses
All the presented data and results were confirmed in at least three independent
210
experiments and are expressed as means ± SD. Student's t-test and one-way ANOVA
211
were used for the statistical analyses, employing SPSS version 18.0 software.
212
P < 0.05 was considered statistically significant.
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3. Results and discussion
227
3.1. Effect of POL on miRNA expression in A549 cells and confirmation of microarray
228
results by quantitative RT-PCR In our previous study, we found POL could simultaneously induce apoptosis and
230
autophagy in A549 cells [8]. To determine the miRNA expression profile in A549
231
cells with POL treatment, we screened and validated the altered miRNAs after POL
232
treatment. As shown in Fig. 1A, the array results of miRNA expression patterns in
233
treated and untreated POL cells were distinguished from each other. Compared to the
234
control group, there were 35 up-regulated miRNAs and 39 down-regulated miRNA in
235
POL-treated A549 cells (the altered miRNAs information were detailed in Table S3).
236
To validate the miRNA microarray results, four differentially expressed miRNAs were
237
selected to perform the expression analysis with qRT-PCR. The selected miRNAs
238
included one down-regulated miRNAs (hsa-miR-1290) and three up-regulated
239
(hsa-miR-15a-3p, hsa-miR-3138 and hsa-miR-3940-5p). We found the expression
240
alteration of these 4 miRNAs observed in qRT-PCR was consistent with that of
241
microarray (Fig. 1B). In addition, we found that POL also inhibited proliferation of
242
H460 cells and H1299 cells (Fig. S1). Subsequently, we performed qRT-PCR analysis
243
to determine whether POL treatment altered hsa-miR-1290, hsa-miR-15a-3p,
244
hsa-miR-3138 and hsa-miR-3940-5p expression in H460 cells and H1299 cells. In Fig.
245
S1, POL treatment also altered hsa-miR-1290, hsa-miR-15a-3p, hsa-miR-3138 and
246
hsa-miR-3940-5p expression in these cells, suggesting these miRNAs may also
247
involve in POL-induced cell death of H460 cells and H1299 cells.
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248 249
3.2. Down-regulated miR-1290 amplifies POL-induced apoptosis Given that miR-1290 was down-regulated after POL treatment, it is of interest to
251
investigate its role in A549 cells. We first transfected miR-1290 mimics
252
(over-expression of miR-1290) and inhibitor (knockdown of miR-1290) and
253
examined its effect on cellular proliferation of A549 cells. The CCK-8 assay showed
254
that the proliferation of A549 cells was not affected after miR-1290 over-expression
255
12 or 24 hours (Fig. 2A) and this is consistent with a previous study [13].
256
Subsequently, we examined the effect of miR-1290 inhibition on A549 cells
257
proliferation. Intriguingly, the treatment of miR-1290 inhibitor also did not affect the
258
proliferation rates of A549 cells compared with cells transfected with NC inhibitor in
259
12 or 24 hours (Fig 2A). Then, we investigated whether POL-induced cell death can
260
be affected by over-expressing or down-expressing of miR-1290. The A549 cells were
261
transfected with miR-1290 mimics followed by treatment with 23 μg/ml POL for 12
262
and 24 h. As expected, over-expression of miR-1290 significantly reduced
263
POL-induced cell death while knockdown of miR-1290 markedly enhanced the
264
POL-induced cell death (Fig. 2B).
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To test whether miR-1290 regulates POL induced apoptosis, we transfected A549
266
cells with NC mimics, miR-1290 mimics, NC inhibitor and miR-1290 inhibitor
267
followed by treatment with 23 μg/ml POL for 24 h, and the morphologic varieties
268
were detected under phase contrast microscopy. As shown in Fig. 2C, POL induced
269
typical apoptotic features, over-expression of miR-1290 in A549 cells reversed 12 Page 13 of 37
270
POL-induced cell death while knockdown of miR-1290 amplified POL-induced
271
apoptosis. Besides, Annexin V-FITC/PI assay also demonstrated that knockdown of
272
miR-1290 amplified POL-induced apoptosis (Fig. 2D). Western blot analysis was then performed to further confirm whether the
274
apoptosis pathway was affected in A549 cells with increased and decreased miR-1290
275
expression. We noted that POL induced cleavage of caspase-3 and caspase-9 could be
276
rescued
277
down-regulation of pro-apoptotic Bax as well as up-regulation of anti-apoptotic Bcl-2
278
and Bcl-xL proteins was observed after miR-1290 over-expression (Fig. 2E). All these
279
results indicate that over-expression of miR-1290 can protect A549 cells from
280
POL-induced apoptosis and down-regulate miR-1290 may amplify POL-induced
281
apoptosis in A549 cells.
miR-1290
mimics
transfection
in
A549
cells.
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Meanwhile, we performed transfection assays to assess whether miR-1290 can
283
affect autophagy in A549 cells. It is found that the autophagy ratio of A549 cells was
284
not affected after miR-1290 over-expression or knockdown (Fig.S2).
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MiR-1290 has been proven to be overexpressed in several different types of
286
human cancers, such as colon cancer, pancreatic cancer and esophageal squamous cell
287
carcinoma, suggesting it may function as an oncogenic miRNA [13-15]. Since
288
miR-1290 was down-regulated miRNA after POL treatment, we first investigated its
289
role in A549 cells. However, both over-expression and knockdown of miR-1290 did
290
not affect the proliferation of A549 cells. Thus, we checked whether over-expression
291
or knockdown of miR-1290 can affect POL-induced apoptosis and found
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over-expression of miR-1290 suppressed POL induced apoptosis in A549 cells.
293
Previous study has shown that miR-1290 over-expression activated the Akt and
294
NF-κB pathways that promoted cell proliferation and inhibited cell apoptosis in colon
295
cancer cells [13]. Therefore, it is reasonable to speculate that miR-1290 might exert
296
the anti-apoptotic role by upregulating of AKT and NF-κB activities and thus suppress
297
POL-induced apoptosis. However, we found knockdown of miR-1290 did not change
298
the viability of A549 cells, suggesting it may have a complex role in A549 cell. Thus,
299
further studies are needed to understand the influence of miR-1290 on tumor
300
proliferation.
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3.3. POL and miR-1290 regulate Wnt/β-catenin pathway WNT/β-catenin pathway is frequently activated in various cancers [16]. Mazieres
304
et al. found that activation of Wnt/β-catenin signaling is important for both initiation
305
and progression of lung cancer [17, 18]. To determine whether POL regulates the
306
Wnt/β-catenin pathway, we first performed western blot to examine Wnt/β-catenin
307
pathway-related protein levels in A549 cells after POL treatment. We found GSK3β
308
was markedly elevated in a time-dependent manner after POL administration (Fig.
309
3A). In addition, a significant time-dependent reduction of β-catenin and its
310
downstream factors including c-myc and cyclin D1 was observed in POL treated cells
311
compared to the control groups. Previous study showed that over-expression of
312
miR-1290 could activate the Wnt pathway in colon cancer cells [13]. However,
313
whether miR-1290 can activate the Wnt pathway in A549 cells is still unclear. To
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determine this, TOPflash reporter was used. As shown in Fig. 3B, TOPflash reporter
315
was strongly activated by miR-1290 mimics and inactivated by miR-1290 inhibitor.
316
Moreover, western blot data showed that the treatment of A549 cells with miR-1290
317
mimics resulted in down-regulation of GSK3β and up-regulation of β-catenin, c-myc
318
and cyclin D1 proteins levels (Fig. 3C). In contrast, miR-1290 inhibitor could increase
319
the expression levels of GSK3β and decrease the expression levels of β-catenin and its
320
downstream targets in A549 cells. Collectively, these results indicate that both POL
321
and miR-1290 can regulate Wnt/β-catenin pathway.
322
3.4. GSK3β is a target of miR-1290
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To identify the molecular target of miR-1290, we searched potential miR-1290
324
targets predicted by TargetScan database (http://www.targetscan.org, Release 6.2
325
Version). The genes involved in Wnt/β-catenin signaling pathway got more attention,
326
because we found both POL and miR-1290 could regulate Wnt/β-catenin pathway.
327
Consequently, GSK3β, a key component of Wnt signaling pathway, was predicted by
328
TargetScan database as a prime target of miR-1290 (Fig. 4A).
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To confirm this prediction, a luciferase reporter assay was conducted to assess
330
whether miR-1290 directly targets the 3' UTR of GSK3β. HEK293T cells were
331
co-transfected with wild-type reporter, mutant reporter, miR-1290 mimics and NC
332
mimics. As shown in Fig. 4B, compared with NC group, co-transfection of miR-1290
333
mimics with the GSK3β 3′UTR wild-type reporter (psiCHE-GSK3β 3′UTR-wt)
334
resulted in a significant decrease in luciferase activity. No appreciable inhibitory
335
effect was observed when miR-1290 mimics or NC mimics co-transfected with the
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336
empty vector or the mutant reporter (psiCHE-GSK3β 3′UTR-mut), indicate that the
337
GSK3β is a direct target of miR-1290. To confirm whether miR-1290 can regulate GSK3β expression, GSK3β mRNA
339
and protein quantities were measured in A549 cells transiently transfected with NC
340
mimics, miR-1290 mimics, NC inhibitor and miR-1290 inhibitor respectively. As
341
shown in Fig. 4C, the mRNA levels of GSK3β were not influenced after transfection
342
for 24h. However, the GSK3β protein expression was inhibited by miR-1290 mimics
343
in A549 cells and miR-1290 inhibitor increased the protein level of GSK3β (Fig. 4D).
344
Taken together, these data support that GSK3β is a direct target of miR-1290.
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In a previous study, Wu et al. reported that over-expression of miR-1290 can
346
activate the Wnt pathway and increase the levels of reprogramming-related
347
transcription factors c-myc and Nanog in colon cancer cells [13]. Consistent with
348
these observations, our results demonstrated that miR-1290 could regulate Wnt
349
pathway by targeting GSK3β in A549 cells. Of note, GSK3β is a key component of
350
Wnt/β-catenin signaling, and decreased GSK3β can increase the accumulation of
351
β-catenin in cytoplasm. Our results demonstrated that miR-1290 over-expression
352
elevated β-catenin and its downstream targets Cyclin D1 and c-myc expression in
353
A549 cells.
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3.5. MiR-15a-3p induces apoptosis and autophagy in A549 cells
356
Since miR-15a-3p was up-regulated after POL treatment in A549 cells, we
357
hypothesized that miR-15a-3p might have a pro-apoptotic role in A549 cells. To 16 Page 17 of 37
verify our hypothesis, we transfected A549 cells with NC mimics, miR-15a-3p
359
mimics, NC inhibitor and miR-15a-3p inhibitor for 24h, and the cell viability was
360
evaluated by CCK-8 assay. Over-expression of miR-15a-3p significantly decreased
361
the proliferation rates of A549 cells compared with cells transfected with NC mimics
362
in 24 hours (Fig. 5A). We next determined whether miR-15a-3p mediated decreased
363
cell viability was implicated in apoptosis. As shown in Fig. 5B, miR-15a-3p mimics
364
transfection resulted in a decrease in cell density compared with NC mimics
365
transfection group, and over-expression of miR-15a-3p induced typical apoptotic
366
features in A549 cells. Apoptosis was evaluated by the Annexin V-FITC/PI assay. In
367
Fig. 5C, miR-15a-3p mimics transfection markedly induced the increase of apoptotic
368
cells proportion. Moreover, western blot analysis showed that procaspase-3 and
369
procaspase-9 were decreased while cleaved caspase-3 and caspase-9 were increased
370
after miR-15a-3p mimics transfection, indicates miR-15a-3p can activate caspase-3
371
and caspase-9 (Fig. 5D). It has been well known that Bcl-2 is a key target of miR-15a.
372
Thus, we investigated whether miR-15a-3p can also affect the Bcl-2 and Bcl-xL
373
proteins expression by western blot analysis. We found Bcl-2 and Bcl-xL levels were
374
decreased in A549 cells transfected with miR-15a-3p mimics compared with NC
375
mimics, NC inhibitor and miR-15a-3p inhibitor.
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376
Meanwhile, typical characteristics of autophagy were also observed in
377
miR-15a-3p mimics transfected A549 cells. The formation of autophagic vacuoles
378
(AVOs) was assessed by staining the cells with MDC. As shown in Fig. 5E,
379
autophagic vacuoles were formed in A549 cells that featured by increased MDC 17 Page 18 of 37
fluorescent intensity, indicating the presence of autophagic cell death. The formation
381
of autophagic vacuoles was further confirmed by measurement of Beclin1 and LC3
382
levels in transfected cells. As shown in Fig. 5D, a significant increase expression of
383
Beclin 1 and LC3-II proteins was observed in miR-15a-3p mimics transfected cells in
384
comparison to the control cells. All these results indicate that miR-15a-3p induces
385
both apoptosis and autophagy in A549 cells.
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MiR-15a-3p is a novel member of the pro-apoptotic miRNA cluster [19]. A
387
previous study reported that miR-15a-3p has a pro-apoptotic role in human cells by its
388
ability to activate Caspase-3/7, to reduce cell viability and to inhibit the expression of
389
Bcl-xL in HeLa and AsPc-1 cells [19]. In this study, we confirmed the pro-apoptotic
390
role of miR-15a-3p in A549 cells by its ability to activate Caspase-3/9 and to inhibit
391
the expression of Bcl-2 and Bcl-xL. Moreover, we found over-expression of
392
miR-15a-3p also resulted in A549 cells autophagy, suggesting it may be considered
393
for both apoptosis and autophagy modulating therapies in cancers.
396
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3.6. MiR-15a-3p regulates ROS generation and induces p53 activation in A549 cells
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386
Mitochondria are the major source of ROS production, and our previous study
397
have demonstrated that ROS was implicated in POL-induced both apoptosis and
398
autophagy in A549 cells [8, 20]. Therefore, we investigated whether miR-15a-3p can
399
affect mitochondria ROS production using flow cytometry. The data showed that ROS
400
was accumulated in miR-15a-3p mimics transfected A549 cells (Fig. 6A). The
401
mitochondrial membrane potential disruption is always associated with ROS 18 Page 19 of 37
formation [21]. Next we examined the mitochondrial membrane potential by using the
403
cationic dye JC-1. In Fig .6B, after POL treatment or miR-15a-3p mimics transfection,
404
the mitochondrial membrane potential was found to be interrupted, as evidenced by
405
the migration of JC-1 dye from the mitochondria into the cytoplasm of treated cells,
406
and the subsequent reduction in the mitochondrial red fluorescence signals. Notably,
407
pretreatment with a ROS scavenger NAC could reverse POL or miR-15a-3p-induced
408
cytotoxicity, and suppress miR-15a-3p-induced apoptosis and autophagy (Fig. 6C).
409
These results indicate that miR-15a-3p can regulate mitochondria ROS generation
410
which was involved in miR-15a-3p-induced apoptosis and autophagy.
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To assess whether p53 was activated in miR-15a-3p-transfected cells, the levels
412
of p53 and p-p53 were determined by western blot analysis. As shown in Fig. 6D, the
413
level of p53 was not obviously changed but the level of p-p53 (ser 392) was markedly
414
enhanced after miR-15a-3p mimics transfection. Furthermore, pretreatment with NAC
415
remarkably inhibited the activation of p53. To investigate whether the p53 is involved
416
in miR-15a-3p-induced A549 cells apoptosis and autophagy, p53 inhibitor pifithrin α
417
was used. In Fig. 6E, pretreatment with pifithrin α suppressed miR-15a-3p-induced
418
apoptosis and autophagy in A549 cells, indicates that miR-15a-3p-ROS-p53 pathway
419
is involved in POL-induced apoptosis and autophagy in A549 cells.
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420
In our previous study, we found another typical GNA-related lectin P. cyrtonema
421
lectin (PCL) could induce apoptosis and autophagy in human melanoma A375 cells
422
via mitochondrial-mediated ROS-p38-p53 pathway [22]. Similar with these results,
423
we demonstrated that the ROS-mediated p53 activation was involved in the POL 19 Page 20 of 37
induced apoptosis and autophagy in A549 cells. However, previous studies also
425
demonstrated that some plant lections could induce apoptosis in a p53 independent
426
manner. For instance, mistletoe lectin was reported to induce apoptosis independent
427
on p53 activation [23, 24]. Concanavalin A was reported to induce apoptosis in
428
p53-null cells and shows no effects on normal cells [25]. Therefore, the precise
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function of p53 in plant lectins induced cancer cells apoptosis and autophagy remains
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further explored.
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4. Conclusion In summary, we report for the first time that miR-1290 and miR-15a-3p are
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involved in POL induced A549 cells apoptosis and autophagy. In addition, we provide
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evidence that GSK3β is a target of miR-1290. Importantly, we further confirm that a
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miR-15a-3p mediated ROS-p53 pathway is involved in POL-induced apoptosis and
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autophagy in A549 cells. A proposed model for the roles of miRNAs in POL-induced
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apoptosis and autophagy is presented in Fig. 7. These findings would possibly open a
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new venue for plant lectins as a potential antineoplastic agent in treatment of different
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human cancer in future.
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Acknowledgements
This work was supported in part by National Natural Science Foundation of
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China (No. 31300674, No. 81173093, No. 30970643, No. 81373311 and No.
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J1103518).
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Figure legends
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Fig. 1. Effect of POL on miRNA expression in A549 cells.
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(A) Hierarchical clustering of the differentially expressed miRNA in A549 cells after
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POL treatment. There were 35 upregulated miRNAs and 39 downregulated miRNAs
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in POL-treated A549 cells compared with control group.
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(B) Real-time PCR was performed to confirm the miRNA array results. It showed that
474
hsa-miR-1290
475
hsa-miR-3138 and hsa-miR-3940-5p was upregulated after POL treatment. (means ±
476
SD, n = 3).
after
POL
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downregulated
treatment.
Hsa-miR-15a-3p,
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Fig. 2. Down-regulated miR-1290 amplifies POL-induced apoptosis in A549 cells.
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(A) The A549 cells transfected with NC mimics, miR-1290 mimics, NC inhibitor and
481
miR-1290 inhibitor for indicated time and examined its effect on cellular proliferation.
482
The CCK-8 assay showed that the proliferation of A549 cells was not affected after
483
miR-1290 over-expression or knockdown in 12 and 24 h. (means ± SD, n = 3) (ns: no
484
significance vs. NC inhibitor group).
485
(B) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC
486
inhibitor and miR-1290 inhibitor for 24 h followed by stimulation with or without 23
487
μg/ml POL for indicated time, the cell growth inhibition ratio was measured by the
488
CCK-8 assay. (means ± SD, n = 3) (**p < 0.01 vs. Control group, #p < 0.05 vs. NC
489
mimics+POL group, ##p < 0.01 vs. POL group). Knockdown of miR-1290 markedly
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22 Page 23 of 37
enhanced the POL-induced cell death and miR-1290 mimics significant reduced POL
491
induced cell death. Control: cells without transfection or POL treatment.
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(C) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC
493
inhibitor and miR-1290 inhibitor, followed by treatment with or without 23 μg/ml
494
POL for 24 h, the morphologic varieties were detected under phase contrast
495
microscopy.
496
(D) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC
497
inhibitor and miR-1290 inhibitor, followed by treatment with or without 23 μg/ml
498
POL for 24 h, POL-induced apoptosis was analyzed by flow cytometry. After
499
miR-1290 mimics transfection, the percentage of early (c: 15.64 ± 3.3%, d: 10.14 ±
500
2.6%) and late (c: 9.35 ± 2.4%, d: 6.84 ± 3.1%) apoptotic cells induced by POL were
501
reduced. Knockdown of miR-1290 markedly enhanced the POL-induced early (e:
502
12.95 ± 1.4%, f: 24.48 ± 3.6%) and late (e: 11.83 ± 3.4%, f: 12.50 ± 2.1%) apoptotic
503
cells death.
504
(E) The A549 cells were transfected with NC mimics, miR-1290 mimics, NC inhibitor
505
and miR-1290 inhibitor for 24 h followed by stimulation with or without 23 μg/ml
506
POL for 24h, the pro-caspase-3,caspase-3, pro-caspase-9, caspase-9, Bax, Bcl-2 and
507
Bcl-xL levels were examined by western blot analysis.
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Fig. 3. POL and miR-1290 regulate the Wnt/β-catenin pathway.
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(A) The A549 cells were treated with 23 μg/ml POL for indicated time periods, 23 Page 24 of 37
followed by western blot analysis for detection of GSK3β, β-catenin, c-myc and
513
cyclin D1 levels. β-Actin was used as an equal loading control.
514
(B) TOPFflash/FOPflash luciferase assays on A549 cells after NC mimics, miR-1290
515
mimics, NC inhibitor and miR-1290 inhibitor transfection for 48 h. (means ± SD, n =
516
3). (**p < 0.01 vs. NC mimics group, #p < 0.05 vs. NC inhibitor group).
517
(C) Western blot was performed to determine the protein level of GSK3β, β-catenin,
518
c-myc and cyclin D1 in A549 cells after transfection with NC mimics, miR-1290
519
mimics, NC inhibitor and miR-1290 inhibitor for 48 h. β-Actin served as a loading
520
control.
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Fig. 4. miR-1290 directly targeted 3′UTR of GSK3β.
524
(A) The predicted binding site of miR-1290 in the 3′UTR of GSK3β by targetscan.
525
(B) The psiCHECK2, psiCHE-GSK3β 3′UTR wt or psiCHE-GSK3β 3′UTR mut
526
vector was co-transfected with miR-1290 mimics or NC mimics into HEK293T cells,
527
and normalized Renilla luciferase activity was determined. Luciferase report assay
528
data showed that co-transfection of HEK293T cells with miR-1290 and wild type
529
GSK3β 3′UTR led to a remarkable decrease in luciferase activity. (means ± SD, n =
530
3). (**p < 0.01 vs. NC mimics group).
531
(C) Expression level of GSK3β mRNA was measured by qPCR in A549 cells
532
transiently transfected with NC mimics, miR-1290 mimics, NC inhibitor and
533
miR-1290 inhibitor for 24h. (means ± SD, n = 3).
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(D) Detection of GSK3β in transfected A549 cells. Western blot analysis
535
demonstrated that GSK3β was downregulated in miR-1290 over-expression cells after
536
24 h post-transfection. β-Actin was used as a loading control.
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Fig. 5. Over-expression of miR-15a-3p induces A549 cells apoptosis and autophagy.
540
(A) The A549 cells were treated with 23 μg/ml POL or transfected with NC mimics,
541
miR-15a-3p mimics, NC inhibitor and miR-15a-3p inhibitor for 24 h and examined its
542
effect on cellular proliferation. Values are expressed as means ± SD. ∗∗p < 0.01 vs.
543
NC mimics group, ns: no significance vs. NC inhibitor group.
M
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539
544
(B) Cells were transfected with or without NC mimics, miR-15a-3p mimics, NC
546
inhibitor and miR-15a-3p inhibitor for 24 h, the morphologic varieties were detected
547
under phase contrast microscopy.
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ed
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(C) The A549 cells were transfected with NC mimics, miR-15a-3p mimics, NC
550
inhibitor and miR-15a-3p inhibitor for 24 h, transfection-induced apoptosis was
551
analyzed by flow cytometry. After miR-15a-3p mimics transfection, the percentage of
552
early (b: 1.51 ± 0.4%, c: 12.31 ± 2.4%) and late (b: 1.65 ± 0.3%, c: 22.64 ± 3.6%)
553
apoptotic cells were increased.
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549
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(D) The A549 cells were transfected with NC mimics, miR-15a-3p mimics, NC 25 Page 26 of 37
556
inhibitor and miR-15a-3p inhibitor for 24 h, the Bcl-2, Bcl-xL, procaspase-3,
557
caspase-3, procaspase-9, caspase-9, Beclin 1 and LC3 levels were examined by
558
western blot analysis. β-Actin was used as a loading control.
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(E) The A549 cells were transfected with NC mimics, miR-15a-3p mimics, NC
561
inhibitor and miR-15a-3p inhibitor for 24 h, the morphologic changes were observed
562
under a fluorescence microscope by MDC staining (a). The MDC fluorescent
563
intensity was analyzed by flow cytometry (b). Values are expressed as means ± SD.
564
∗∗p < 0.01 vs. NC mimics group.
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Fig. 6. miR-15a-3p induces ROS generation and p53 activation.
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(A) The A549 cells were treated with 23 μg/ml POL or transfected with NC mimics,
568
miR-15a-3p mimics, NC inhibitor and miR-15a-3p inhibitor for 24 h and the DCF
569
fluorescent intensity was measured by flow cytometry. Values are expressed as means
570
± SD. (**p < 0.01 vs. Control group, ##p < 0.01 vs. NC mimics group).
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(B) The A549 cells were treated with 23 μg/ml POL or transfected with NC mimics,
573
miR-15a-3p mimics, NC inhibitor and miR-15a-3p inhibitor for 24 h and JC-1 dye
574
incubation for the final 20 min. After POL treatment or miR-15a-3p mimics
575
transfection, the mitochondrial membrane potential was found to be interrupted.
576 577
(C) Cells were treated with 23 μg/ml POL or transfected with miR-15a-3p mimics for 26 Page 27 of 37
578
24h in the presence or absence of 2 mM NAC, the cell viablity (a), early as well as
579
late apoptosis ratio (b) and MDC fluorescent intensity (c) were analyzed by flow
580
cytometry. (**p < 0.01 vs. Control group).
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(D) Cells were treated with 23 μg/ml POL or transfected with miR-15a-3p mimics for
583
the indicated time periods in the presence or absence of 2 mM NAC, followed by
584
Western blot analysis for detection of p53 and p-p53 (Ser 392) levels. β-actin was
585
used as an equal loading control.
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an
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(E) Cells were treated with 23 μg/ml POL or transfected with miR-15a-3p mimics for
588
24h in the presence or absence of pifithrin α (pif), the apoptosis ratio (a) and MDC
589
fluorescent intensity (b) were analyzed by flow cytometry. (**p < 0.01 vs. Control
590
group).
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Fig. 7. The cartoon illustrating the roles of miRNAs in POL-induced apoptosis and
594
autophagy.
595
Microarray analysis was performed on A549 cells to identify altered miRNAs after
596
POL treatment. MiR-1290 was down-regulated after POL treatment and GSK3β was a
597
target of miR-1290. MiR-15a-3p was up-regulated after POL treatment and
598
over-expression of miR-15a-3p resulted in A549 cells apoptosis and autophagy via
599
ROS-p53 pathway.
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