- Email: [email protected]
Oncogene LSD1 is epigenetically suppressed by miR-137 overexpression in human non-small cell lung cancer
Accepted Manuscript Oncogene LSD1 is epigenetically suppressed by miR-137 overexpression in human non-small cell lung cancer Xin Zhang, Xiujuan Zhang, Bo Yu, Rongpeng Hu, Lanxiang Hao PII:
S0300-9084(17)30039-1
DOI:
10.1016/j.biochi.2017.02.010
Reference:
BIOCHI 5150
To appear in:
Biochimie
Received Date: 28 December 2016 Accepted Date: 16 February 2017
Please cite this article as: X. Zhang, X. Zhang, B. Yu, R. Hu, L. Hao, Oncogene LSD1 is epigenetically suppressed by miR-137 overexpression in human non-small cell lung cancer, Biochimie (2017), doi: 10.1016/j.biochi.2017.02.010. 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.
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Title: Oncogene LSD1 is epigenetically suppressed by miR-137 overexpression in human non-small cell lung cancer Xin Zhang1,#, Xiujuan Zhang1,#, Bo Yu1,*, Rongpeng Hu2, Lanxiang Hao3
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1: Department of Respiration, Liaocheng People’s Hospital, Liaocheng, 252000, China 2: Department of internal medicine, Liaocheng Infection Hospital, Liaocheng, 252000, China
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3: Department of Endocrinology, Yancheng City No.1 People’s Hospital, Yancheng,
#: Those authors contribute equally
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224001, China
Short title: LSD1 inhibited by miR-137 in NSCLC
*: Corresponding Author
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Bo Yu, M.D. 67 W. Dongchang Road
Department of Respiration
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Liaocheng People’s Hospital Liaocheng, 252000, China
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Phone: +86-15906356663 Email: [email protected]
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Abstract Purpose: We examined the epigenetic regulation of microRNA-137 (miR-137) on lysinespecific demethylase 1 (KDM1A, or LSD1) induced oncogenic effects in NSCLC.
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Methods: NSCLC cell lines, A549 and H460 cells were transfected with a mammalian LSD1 overexpression plasmid. It’s effects on endogenous KDM1A gene and LSD1 protein expressions were examined by qRT-PCR and western blot assays. NSCLC
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proliferation and migration were also examined by MTT proliferation and woundscratch assays, respectively. In LSD1-overexpeseed NSCLC cells, lentiviral
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transfection was conducted to upregulated miR-137 expression. The subsequent effects of miR-137 upregulation on LSD1-mediated cancer regulations were also examined. In addition, key components of histone deacetylases-associated signaling pathways, including EZH2, HDAC1 and HDAC2 were also examined by western blot in LSD1-
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and miR-137-mediated NSCLC cells.
Results: Mammalian LSD1 overexpression plasmid was efficient in upregulating KDM1A gene and LSD1 protein in A549 and H460 cells. It also exerted oncogenic effects in
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NSCLC by promoting cancer proliferation and migration. MiR-137 was inversely correlated with LSD1 in NSCLC, as lentivirus-mediated miR-137 upregulation suppressed
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KDM1A / LSD1 productions and inhibited proliferation or migration in LSD1overexpressed A549 and H460 cells. Further western blot analysis demonstrated EZH2, HDAC1 and HDAC2 were activated by LSD1, but inhibited by miR-137 in NSCLC. Conclusion: Oncogenic effects of LSD1 were reversely regulated by its upstream epigenetic modulator miR-137 in NSCLC. The interaction between LSD1 and miR-137 may very well involve the regulation on histone deacetylases-associated signaling pathways.
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Keywords: lung cancer; NSCLC; LSD1; miR-137; biochemistry; western blot
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1. Introduction Lung cancer is one of the most malignant human cancers for both male and female patients. In the United States, lung cancer is among the most common causes of
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cancer deaths, with nearly quarter million new cases of lung cancers are diagnosed and
more than 150,000 patients died of lung cancer ever year [1]. In some of the developing countries, including African nations and China, lung cancer has become the leading cause
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of cancer deaths among female patients, largely due to the massive scale of air pollution [2, 3]. Among various types of lung cancers, non-small cell lung cancer (NSCLC) is the
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major subtype, accounting for approximately 85% of all lung cancers [4]. Over the past twenty or thirty years, while great strides have been achieved in early diagnosis and targeted immunotherapy / chemotherapy for patients with NSCLC [5-8], the underling genetic mechanisms contributing to NSCLC ontogenesis, maturation metastasis and
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apoptosis are still largely unknown.
Histone methylation has long been considered an important mechanism in human cancer carcinogenesis [9, 10]. Recently, a newly discovered member of
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histone demethylases, lysine-specific demethylase 1 (LSD1), was found to specifically demethylate mono- and di-methylated histone H3 at lysine 4, suggesting that histone
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methylation was completely reversible [11]. Ever since, reports had been emerged showing LSD1 was overwhelmingly acting as an oncogenic factor in various types of human cancers [12, 13]. Specifically in NSCLC, a recent report by Lv and colleagues demonstrated that LSD1 was highly expressed in human NSCLC tumors, and overexpressing LSD1 had oncogenic effects whereas inhibiting LSD1 had suppressive effects on NSCLC in vitro development [14]. However, it is still unknown whether any
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upstream modulators were present in NSCLC to initiate the upregulation or downregulation of LSD1 to exert regulatory effects upon cancer development. MicroRNAs (miRNAs) are families of evolutionally conserved non-coding small
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RNAs that attach to the 3’ un-translated region (3’ UTR) of downstream target genes to post-transcriptionally suppress gene and protein productions, thus modulating various aspects of biological processes in both animal and human [15-18]. In human cancers,
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epigenetic regulation of miRNAs has been demonstrated to play important roles in cancer carcinogenesis, maturation and cell death, as well as holding promises in developing new
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therapeutic targets for cancer treatment [16, 19, 20]. Among many of the cancerregulating human mature miRNAs, microRNA-137 (miR-137) has been shown to be aberrantly expressed in various types of carcinoma tissues, as well as playing significantly roles in modulate cancer proliferation, migration, cell-division or apoptosis
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[21-24]. In human NSCLC, miR-137 was demonstrated to be downregulated in tumorous lung tissues and upregulating miR-137 was shown to inhibit NSCLC development [22]. Intriguingly, it was recently demonstrated that, miR-137 may inversely regulate its
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downstream target gene of KDM1A in neuronal cells or neuroblastoma [25, 26]. However, it is not clear whether miR-137 may regulate KDM1A (or LSD1) to have
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functional impact in NSCLC.
2. Materials and methods 2.1. Ethic approval
The approvals for conducting this study were jointly granted by the Ethic Committees at all participating hospitals, including Liaocheng City People’s Hospital &
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Liaocheng Infection Hospital at Liaocheng, Shandong Province, and Yancheng City No.1 People’s Hospital at Yancheng, Jiangsu Province in China. All experimental paradigms
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were designed and performed in accordance with the Declaration of Helsinki.
2.2. NSCLC cell lines
Two of the NSCLC cell lines, A549 and H460, were purchased from China
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Center for Type Culture Collection and Cell Bank of the Chinese Academy of Sciences in Shanghai, China. Cells were maintained in 12-well tissue-culture plates (BD, USA) in
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RPMI-1640 medium (ThermoFisher Scientific, USA), supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific, USA), penicillin (50 U/mL) and streptomycin (50 µg/mL) (ThermoFisher, USA), in a humidified tissue-culture chamber
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circulated with 5% CO2 at 37 ̊C.
2.3. LSD1 overexpression assay
Complementary DNA (cDNA) of human KDM1A gene (which encodes LSD1
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protein) was cloned and amplified from a human cDNA library, and then sub-cloned into a recombinant mammalian expression plasmid pcDNA3.1 (Promega, USA) using
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the NheI and BamH1 restriction enzymes, resulting in a human KDM1A (or LSD1) expression plasmid, pcDBA/LSD1. An empty pcDNA3.1 plasmid, pcDNA/NS, was used as control in this study. In 12-well plate culture (50,000 cells per well), A549 and H460 cells were transfected with pcDNA/NS or pcDNA/LSD1 by Lipofectamine 3000 transfection reagent for 48 h, followed by G418 (200 ng/mL, ThermoFisher Scientific, USA) and hygromycin (200 ng/mL, ThermoFisher Scientific, USA) selection treatment
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for another 48 h. Transfection efficiency was then verified by qRT-PCR and western
2.4. RNA extraction and quantitative real-time PCR
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blot assays.
Total RNA was extracted from A549 and H460 cells using a miRCURY™ RNA isolation kit (Exiqon, MA) according to manufacturer’ recommendation. A Nano Drop
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ND-3000 spectrophotometer (NanoDrop Technologies, USA) was used to check RNA
quality, and a TaqMan microRNA reverse transcription kit (Applied Biosystems, USA)
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was then performed to synthesize complimentary DNA (cDNA), all according to manufacturers’ recommendations. Quantitative real-time PCR (qRT-PCR) to detect KDM1A and miR-137 expressions was carried out using a miScript SYBR Green PCR Kit (Qiagen USA) on an ABI PRISM 7900HT Sequence Detection System (Applied
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Biosystems, USA) according to manufacturer’ recommendation. GAPDH and U6 small nuclear RNA (snRNA) expressions were served as loading controls for KDM1A and
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miR-137, respectively.
2.5. Western blot assay
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In culture, A549 and H460 cells were washed by phosphate-buffered saline
(PBS, ThermoFisher Scientific, USA) for three times, and then treated with Tris-based lysis buffer (pH=6.8, ThermoFisher Scientific, USA). For each sample, 30 µg of total protein was separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, ThermoFisher Scientific, USA) and transferred to nitrocellulose membranes. Protein detection was carried out by incubating nitrocellulose membranes
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with several primary antibodies, including anti-LSD1 rabbit monoclonal antibody (1:500, Sigma-Aldrich, USA), anti-EZH2 rabbit polyclonal antibody, (1:250, SigmaAldrich, USA), anti-HDAC1 mouse IgG1 monoclonal antibody (1:200, Sigma-Aldrich,
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USA), anti-HDAC2 rabbit polyclonal antibody (1:200, Abcam, USA) and anti-betaactin rabbit polyclonal antibody (1:10,000, Sigma-Aldrich, USA) overnight at 4 °C,
followed by incubating membranes with horseradish peroxidase-conjugated secondary
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antibody (Sigma-Aldrich, USA) for 2 h at room temperature. Blot visualization was carried out using an enhanced chemiluminesence system (Amersham Biosciences,
2.6. Cancer proliferation assay
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USA) along with ImageLab software (Bio-Rad, USA).
In vitro growth of NSCLC cells was characterized using a Vybrant MTT
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proliferation kit (Life Technologies, USA) according to manufacturer’ recommendation. Briefly, A549 and H460 cells were re-plated in 96-well plate (2,000 cells / well) in culture medium, and allowed to proliferate for 5 days. At interval of every 24 h, the
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culture medium of tested wells was mixed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazoliumbromide (MTT) solution (100 µg/mL, 15 µL) for 4 h, followed by
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another 4 h treatment of HCl-SDS to solubilize the formazan product. After that, 96-well plate was examined by a Model 680 Microplate Reader (Bio-Rad, USA) and the absorbance was detected at optical density (O.D.) of 570 nm.
2.7. Cancer migration assay
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In vitro migration of NSCLC cells was characterized using a wound-scratch assay described previously [14]. Briefly, A549 and H460 cells were re-plated in 6-well plates and allowed to grow till 80% ~90% confluence. Then, a sterile cell scrapper was used to
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introduce a wound line across the diameter of the well. After removing floating debris
from the wells, NSCLC cells were allowed to grow for 48 h. At times of 0h, 6h, 12h, 24h and 48h, cancer-cell-covered surface areas were measured at each well, and normalized
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2.8. MiR-137 overexpression assay
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against the areas measured at 0 h.
ShMIMIC Human Lentiviral microRNA hsa-miR-137 mimics, miR-137, and its nonspecific lentiviral mimics, miR-NS, were purchased from GE Dharmacon (GE Dharmacon, Shanghai, China). In NSCLC culture, A549 and H460 cells were transduced
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with miR-137 or miR-NS, using Lipofectamine 3000 and polybrene (8 µg/mL, SigmaAldrich, USA) for 48 h (multiplicity of infection = 20~30), followed by selection treatment of blasticidin (1 mg/mL) for another 72 h. After that, healthy cell colonies
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were handpicked and transferred to new 12-well plate to be continuously cultured til
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confluence. After three passages, transduction efficiency was verified by qRT-PCR.
2.9. Cancer viability assay In NSCLC culture, A549 were collected and centrifuged at 5,000 xg for 5 mins.
The supernatant was discarded. Cell pellets were re-suspended in RPMI-1640 medium with the addition of 0.2% trypan blue (Sigma-Aldrich, USA) for 30 min at 37°C. Dead and live NSCLC cells were counted by a Countess Automated Cell Counter
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(ThermoFisher Scientific, USA) according to manufacturer’s recommendation. Relative viability was measured as the percentage of live A549 cells among all counted A549
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cells.
2.10. Statistical analysis
In this study, all experiments were independently repeated for at least three
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times. Data was presented as mean +/- standard errors. Statistical analysis was performed using an unpaired two-tail student’s t-test on Windows Office Excel
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Software (Microsoft, USA). Significant difference was determined if P < 0.05.
3. Results
3.1. KDM1A overexpression upregulated LSD1 and inhibited NSCLC proliferation and
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migration
A recent report demonstrated that LSD1 played an oncogenic role in NSCLC by promoting cancer growth and migration [14]. However, in that study, LSD1
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overexpression plasmid was tagged with FLAG antibody, which may result in unexpected artifacts in biological studies. In this study, we constructed a mammalian
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overexpression plasmid, pcDNA/LSD1, which contains the whole DNA sequence of human KDM1A gene. In culture of NSCLC cells, we transfected A549 and H460 cells with pcDNA/LSD1 or pcDNA/NS (an empty overexpression plasmid). After transfection was stabilized, qRT-PCR demonstrated that, in both A549 and H460 cells, endogenous KDM1A mRNA levels were significantly upregulated by pcDNA/LSD1 transfection (Figure 1A, * P < 0.05). In addition, western blot assay confirmed that, LSD1 proteins
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were also significantly upregulated by pcDNA/LSD1 transfection in A549 and H460 cells (Figure 1B). The effect of overexpressing KDM1A on NSCLC in vitro development was
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further evaluated by proliferation and migration assays. In a MTT proliferation assay, A549 and H460 cells were re-plated in 96-well plate and allowed to grow for 5
consecutive days. Daily cancer cell proliferation was characterized by MTT-reaction
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induced fluorescent signals detected at optical density (O.D.) of 570 nm. It showed that, two to five days after cells were re-plated in 96-well plate, cancer proliferations of both
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A549 and H460 cells were significantly upregulated by pcDNA/LSD1 transfection (Figure 1C, * P < 0.05). In addition, through a wound-scratch assay, we discovered that cancer migrations in A549 and H460 cells were also significantly promoted by pcDNA/LSD1 transfection 6 to 48 h after wound creation (Figure 1D, * P < 0.05).
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Thus, our results clearly demonstrated that using a mammalian overexpression assay to overexpress DKM1A gene in NSCLC cells was efficient in endogenously upregulating both DKM1A gene and LSD1 protein, as well as promoting cancer
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proliferation and migration.
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3.2. MicroRNA-137 inhibits LSD1 in NSCLC While LSD1 was characterized as an active tumor modulator in NSCLC [14],
little is known about its upstream regulation. As indicated in studies in neural stem cells and neuroblastoma [25, 26], human mature microRNA-137 (miR-137) is speculated to be the upstream regulator of LSD1, as it may bind to the complimentary DNA sequence on 3-UTR of human KDM1A gene (Figure 2A).
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To examine whether KDM1A (or LSD1) was actually modulated by miR-137 in NSCLC, we transduced lentiviral miR-137 mimics (miR-137) into LSD1-overexpressed A549 and H460 cells (transfected with pcDNA/LSD1). The control A549 and H460 cells
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were transduced with a non-specific lentiviral mimics, miR-NS. After lentiviral
transduction was stable, analysis of qRT-PCR demonstrated that endogenous miR-137 expressions were significantly upregulated in NSCLC cells through lentiviral
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transduction of miR-137 mimics (Figure 2B, * P < 0.05).
We also assessed the endogenous KDM1A mRNA levels. The result of qRT-
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PCR showed that KDM1A gene was significantly downregulated in NSCLC cells by lentiviral transduction of miR-137 mimics (Figure 2C, * P < 0.05). In addition, western blot assay showed that LSD1 protein levels were also significantly downregulated by
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lentiviral transduction of miR-137 mimics (Figure 2D-E, * P < 0.05).
3.3. MicroRNA-137 reversed oncogenic effects of LSD1 in NSCLC We then asked whether upregulating miR-137 would have functional roles in
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LSD1-induced oncogenic regulation in NSCLC. Firstly, since we performed two times of gene modification in NSCLC cells, we would like to know whether it might induce
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cell death. Through a cancer viability assay (Figure 3A), we compared NSCLC viability between A549 cells without transfection or transduction (Figure 3A, condition 1), and A549 cells transfected with pcDNA/AS (Figure 3A, condition 2), or transfected with pcDNA/LSD1 (Figure 3A, condition 3), or transfected with pcDNA/LSD1 and transduced with miR-NS (Figure 3A, condition 4), or transfected with pcDNA/LSD1 and transduced with miR-137 (Figure 3A, condition 5). It showed that, under all conditions,
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A549 cells were fairly healthy and no significant cell death was detected (Figure 3A, ∆ P > 0.05). In LSD1-overexpressed A549 or H460 cells, we examined cancer cell in vitro
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proliferations after they were transduced with either miR-NS or miR-137-mimics
lentiviruses. The result of 5-day MTT proliferation assay showed that, in both A549 and H460 cells, miR-137 upregulation significantly suppressed NSCLC proliferations
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(Figure 3B, * P < 0.05). In addition, through a wound-scratch assay, we found that
NSCLC migrations were also significantly inhibited by miR-137 upregulation (Figure
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3C, * P < 0.05). Therefore, our results clearly demonstrated that miR-137 upregulation (or overexpression) reversed the oncogenic effects of LSD1 on NSCLC proliferation and migration.
pathways in NSCLC
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3.4. MiR-137 and LSD1 oppositely regulated histone deacetylases-associated signaling
Finally in this study, we asked whether histone deacetylases-associated signaling
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pathways, including proteins of EZH2, HDAC1 and HDAC2, were involved in the regulation of LSD1 / miR-137 in NSCLC. We firstly used western blot analysis to
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examine EZH2, HDAC1 and HDAC2 protein expressions in A549 cells transfected with mammalian overexpression plasmid. It showed that, between control A549 cells (nontransfected, non-transduced), and A549 cells transfected with pcDNA/NS, there was no change in the expression levels of EZH2, HDAC1 or HDAC2 proteins (Figure 4, columns 1 vs. 2). On the other hand, while A549 cells were transfected with
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pcDNA/LSD1 to upregulate KDM1A, EZH2, HDAC1 and HDAC2 proteins were significantly upregulated (Figure 4, columns 2 vs. 3). Then, while LSD1-overexpressed A549 cells were further transduced with
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lentiviruses, western blot analysis demonstrated that, EZH2, HDAC1 and HDAC2
proteins were unaltered by miR-NS transductions in A549 cells (Figure 4, columns 4 vs. 3). However, in miR-137-upregulated A549 cells (those transduced with miR-137),
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EZH2, HDAC1 and HDAC2 protein expressions were considerably reduced (Figure 4, columns 5 vs. 4).
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Therefore, our results suggest that histone deacetylases-associated signaling pathways were activated (or upregulated) by LSD1-overexpression, but inhibited (or downregulated) by subsequent miR-137 upregulation, in NSCLC.
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4. Discussions
In a recent report, it was demonstrated that epigenetic regulation of LSD1 plays critical role in regulating NSCLC development and predicting prognosis among NSCLC
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patients, including both NSCLC and small cell lung cancer [14]. However in that report, Lv and colleagues used a FLAG-tagged fusion protein to exogenously overexpress LSD1
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protein in NSCLC cells, at that time not knowing whether KDM1A, the encoding gene of LSD1, would also be involved in functional regulation in NSCLC [14]. Therefore, as the first step of our study, we constructed a mammalian KDM1A overexpression plasmid and transfected it into A549 and H460 cells. The subsequent qRT-PCR and western blot analyses confirmed the success of transfection, showing that endogenous KDM1A gene, as well as LSD1 protein, was significantly upregulate in NSCLC cells. Most importantly,
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the oncogenic mechanisms of LSD1 were confirmed by our biochemical assays, demonstrating that NSCLC proliferation and migration were markedly promoted by overexpressing KDM1A gene in A549 and H460 cells.
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As the next step of our study, we sought the upstream initiator of KDM1A / LSD1 in NSCLC. In studies of neuroblastoma and neural stem cells, it was demonstrated that miR-137 was the upstream regulator of LSD1 [25, 26]. Based on this knowledge, we
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transduced LSD1-overexpressed A549 and H460 cells with lentivirus to upregulate miR137. And we found that miR-137 upregulation could suppress both KDM1A gene and
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LSD1 protein in NSCLC cells, thus confirming that miR-137 may directly and inversely regulate KDM1A / LSD1 in NSCLC. Furthermore, we used functional assays to examine the effect of upregulating miR-137 in LSD1-overexpressed A549 and H460 cells. We found that, miR-137 upregulation had opposite effects as LSD1 overexpression, or tumor
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suppressive effects, in NSCLC by suppressing cancer proliferation and migration. Thus, all those data confirmed that miR-137 is an upstream regulator and reversely modulates KDM1A / LSD1 expression and oncogenic effects in NSCLC. To our knowledge, this is
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the first-ever report to show the correlation of miR-137 and KDM1A / LSD1 in NSCLC. Also in our study, we explored the associated signaling pathways involved in
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LSD1 / MIR-137 regulation in NSCLC. Through western blot analysis, we discovered that EZH2, HDAC1 and HDAC1 proteins were upregulated by KDM1A / LSD1, whereas downregulated by miR-137, in NSCLC. EZH2, acting as acts as a histone lysine methyltransferase, was found to be upregulated in human NSCLC carcinomas and correlated with poor prognosis of NSCLC patients [27-29], similar to the expression pattern and prognostic characteristics of LSD1 in NSCLC. However, our report was the
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first one to show that EZH2 was directly regulated by KDM1A / LSD1 in NSCLC. It would be interesting to learn, possibly through future studies, what the signaling pathways are involved in linking KDM1A / LSD1 toward EZH2 in NSCLC. As for
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LSD1-induced HDAC1/2 upregulation, since LSD1 and HDAC1/2 were often found be co-expressed in an LSD1/CoREST1/HDAC complex in chromatin to synergistically
regulate cancer development [30, 31], it is possible that LSD1 may upregulate HDAC1/2
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(or other histone deacetylases) through the form of chromatin complex, to regulate cancer development in NSCLC. On the other hand, the inhibitory effect of miR-137 on EZH2 or
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HDAC1/2 may well through the downstream regulation of LSD1.
Figure legends
Figure 1. LSD1 overexpression was oncogenic in NSCLC. A549 and H460 cells were
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transfected with a mammalian KDM1A overexpression plasmid, pcDNA/LSD1, or an empty control plasmid, pcDNA/NS. (A) After transfection was stabilized, qRT-PCR was carried out to compare endogenous DKM1A mRNA levels between A549 and H460 cells
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transfected with pcDNA/LSD1 and those transfected with pcDNA/NS (* P < 0.05). (B) A549 and H460 cells were lysed and western blot assay was carried out to compare
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LSD1 protein levels between A549 and H460 cells transfected with pcDNA/LSD1 and those transfected with pcDNA/NS. (C) Transfected A549 and H460 cells were re-plated in 96-well plate and allowed to grow for 5 days. In vitro proliferation of NSCLC cells was characterized using a MTT proliferation assay. Daily measurement of absorbance was determined at optical density (O.D) of 570 nm (* P < 0.05). (C) Transfected A549
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and H460 cells were also examined by a wound-scratch assay to assess their migration (* P < 0.05).
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Figure 2. MiR-137 inhibits LSD1 in NSCLC. (A) Complimentary binding was shown
between human mature miR-137, hsa-miR-137, and 3’-UTR of human KDM1A gene.
(B) In LSD1-overexpresed A549 and H460 cells (transfected with pcDNA/LSD1), they
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were transduced with a lentiviral human miR-137 mimics, miR-137, or a non-specific
lentiviral mimics, miR-NS. QRT-PCR was carried out to compare endogenous miR-137
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gene levels between A549 and H460 cells transduced with miR-137 mimics and those transduced with miR-NS (* P < 0.05). (C) QRT-PCR was also carried out to compare endogenous KDM1A mRNA levels between A549 and H460 cells transduced with miR137 mimics and those transduced with miR-NS (* P < 0.05). (D) After lentiviral
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transduction, A549 and H460 cells were lysed. Western blot assay was carried out to examine LSD1 protein levels between A549 and H460 cells transduced with miR-137 mimics and those transduced with miR-NS. (E) Quantification on LSD1 protein
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expression levels (LSD1 / β-actin) was performed for (D) (* P < 0.05).
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Figure 3. MiR-137 suppressed LSD1-induced oncogenic effects in NSCLC. (A) In A549 cells, cancer cell viability was compared between those without transfection or transduction (condition 1), and those transfected with pcDNA/AS (condition 2), or transfected with pcDNA/LSD1 (condition 3), or transfected with pcDNA/LSD1 and transduced with miR-NS (condition 4), or transfected with pcDNA/LSD1 and transduced with miR-137 (condition 5) (∆ P > 0.05). (B) In A549 and H460 cells transfected with
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pcDNA/LSD1 and transduced with lentiviruses of miR-NS or miR-137 mimics, a MTT cancer proliferation assay was carried out for 5 days (* P < 0.05). (C) Those A549 and H460 cells were also examined by a wound-scratch assay to assess their migration (* P <
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0.05).
Figure 4. MicroRNA-137 and LSD1 modulated EZH2 and HDAC in NSCLC. Western
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blot assay was carried out to assess protein levels of EZH2, HDAC1 and HDAC2 in
A549 cells without transfection or transduction (column 1), or A549 cells transfected
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with pcDNA/AS (column 2), or A549 cells transfected with pcDNA/LSD1 (column 3), or A549 cells transfected with pcDNA/LSD1 and transduced with miR-NS (column 4), or A549 cells transfected with pcDNA/LSD1 and transduced with miR-137 (column 5).
Acknowledgements
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Conflict of interest: None.
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We thank Dr. Xianliang Wang for his critical review on the manuscript.
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LSD1 promoted NSCLC proliferation and migration. MiR-137 was inversely correlated with LSD1 in NSCLC. MiR-137 upregulation suppressed KDM1A / LSD1 productions in NSCLC.
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MiR-137 upregulation inhibited proliferation and migration in NSCLC
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EZH2, HDAC1 and HDAC2 were activated by LSD1, but inhibited by miR-137 in NSCLC.
S0300-9084(17)30039-1
DOI:
10.1016/j.biochi.2017.02.010
Reference:
BIOCHI 5150
To appear in:
Biochimie
Received Date: 28 December 2016 Accepted Date: 16 February 2017
Please cite this article as: X. Zhang, X. Zhang, B. Yu, R. Hu, L. Hao, Oncogene LSD1 is epigenetically suppressed by miR-137 overexpression in human non-small cell lung cancer, Biochimie (2017), doi: 10.1016/j.biochi.2017.02.010. 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.
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Title: Oncogene LSD1 is epigenetically suppressed by miR-137 overexpression in human non-small cell lung cancer Xin Zhang1,#, Xiujuan Zhang1,#, Bo Yu1,*, Rongpeng Hu2, Lanxiang Hao3
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1: Department of Respiration, Liaocheng People’s Hospital, Liaocheng, 252000, China 2: Department of internal medicine, Liaocheng Infection Hospital, Liaocheng, 252000, China
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3: Department of Endocrinology, Yancheng City No.1 People’s Hospital, Yancheng,
#: Those authors contribute equally
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224001, China
Short title: LSD1 inhibited by miR-137 in NSCLC
*: Corresponding Author
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Bo Yu, M.D. 67 W. Dongchang Road
Department of Respiration
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Liaocheng People’s Hospital Liaocheng, 252000, China
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Phone: +86-15906356663 Email: [email protected]
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Abstract Purpose: We examined the epigenetic regulation of microRNA-137 (miR-137) on lysinespecific demethylase 1 (KDM1A, or LSD1) induced oncogenic effects in NSCLC.
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Methods: NSCLC cell lines, A549 and H460 cells were transfected with a mammalian LSD1 overexpression plasmid. It’s effects on endogenous KDM1A gene and LSD1 protein expressions were examined by qRT-PCR and western blot assays. NSCLC
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proliferation and migration were also examined by MTT proliferation and woundscratch assays, respectively. In LSD1-overexpeseed NSCLC cells, lentiviral
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transfection was conducted to upregulated miR-137 expression. The subsequent effects of miR-137 upregulation on LSD1-mediated cancer regulations were also examined. In addition, key components of histone deacetylases-associated signaling pathways, including EZH2, HDAC1 and HDAC2 were also examined by western blot in LSD1-
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and miR-137-mediated NSCLC cells.
Results: Mammalian LSD1 overexpression plasmid was efficient in upregulating KDM1A gene and LSD1 protein in A549 and H460 cells. It also exerted oncogenic effects in
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NSCLC by promoting cancer proliferation and migration. MiR-137 was inversely correlated with LSD1 in NSCLC, as lentivirus-mediated miR-137 upregulation suppressed
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KDM1A / LSD1 productions and inhibited proliferation or migration in LSD1overexpressed A549 and H460 cells. Further western blot analysis demonstrated EZH2, HDAC1 and HDAC2 were activated by LSD1, but inhibited by miR-137 in NSCLC. Conclusion: Oncogenic effects of LSD1 were reversely regulated by its upstream epigenetic modulator miR-137 in NSCLC. The interaction between LSD1 and miR-137 may very well involve the regulation on histone deacetylases-associated signaling pathways.
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Keywords: lung cancer; NSCLC; LSD1; miR-137; biochemistry; western blot
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1. Introduction Lung cancer is one of the most malignant human cancers for both male and female patients. In the United States, lung cancer is among the most common causes of
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cancer deaths, with nearly quarter million new cases of lung cancers are diagnosed and
more than 150,000 patients died of lung cancer ever year [1]. In some of the developing countries, including African nations and China, lung cancer has become the leading cause
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of cancer deaths among female patients, largely due to the massive scale of air pollution [2, 3]. Among various types of lung cancers, non-small cell lung cancer (NSCLC) is the
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major subtype, accounting for approximately 85% of all lung cancers [4]. Over the past twenty or thirty years, while great strides have been achieved in early diagnosis and targeted immunotherapy / chemotherapy for patients with NSCLC [5-8], the underling genetic mechanisms contributing to NSCLC ontogenesis, maturation metastasis and
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apoptosis are still largely unknown.
Histone methylation has long been considered an important mechanism in human cancer carcinogenesis [9, 10]. Recently, a newly discovered member of
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histone demethylases, lysine-specific demethylase 1 (LSD1), was found to specifically demethylate mono- and di-methylated histone H3 at lysine 4, suggesting that histone
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methylation was completely reversible [11]. Ever since, reports had been emerged showing LSD1 was overwhelmingly acting as an oncogenic factor in various types of human cancers [12, 13]. Specifically in NSCLC, a recent report by Lv and colleagues demonstrated that LSD1 was highly expressed in human NSCLC tumors, and overexpressing LSD1 had oncogenic effects whereas inhibiting LSD1 had suppressive effects on NSCLC in vitro development [14]. However, it is still unknown whether any
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upstream modulators were present in NSCLC to initiate the upregulation or downregulation of LSD1 to exert regulatory effects upon cancer development. MicroRNAs (miRNAs) are families of evolutionally conserved non-coding small
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RNAs that attach to the 3’ un-translated region (3’ UTR) of downstream target genes to post-transcriptionally suppress gene and protein productions, thus modulating various aspects of biological processes in both animal and human [15-18]. In human cancers,
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epigenetic regulation of miRNAs has been demonstrated to play important roles in cancer carcinogenesis, maturation and cell death, as well as holding promises in developing new
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therapeutic targets for cancer treatment [16, 19, 20]. Among many of the cancerregulating human mature miRNAs, microRNA-137 (miR-137) has been shown to be aberrantly expressed in various types of carcinoma tissues, as well as playing significantly roles in modulate cancer proliferation, migration, cell-division or apoptosis
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[21-24]. In human NSCLC, miR-137 was demonstrated to be downregulated in tumorous lung tissues and upregulating miR-137 was shown to inhibit NSCLC development [22]. Intriguingly, it was recently demonstrated that, miR-137 may inversely regulate its
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downstream target gene of KDM1A in neuronal cells or neuroblastoma [25, 26]. However, it is not clear whether miR-137 may regulate KDM1A (or LSD1) to have
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functional impact in NSCLC.
2. Materials and methods 2.1. Ethic approval
The approvals for conducting this study were jointly granted by the Ethic Committees at all participating hospitals, including Liaocheng City People’s Hospital &
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Liaocheng Infection Hospital at Liaocheng, Shandong Province, and Yancheng City No.1 People’s Hospital at Yancheng, Jiangsu Province in China. All experimental paradigms
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were designed and performed in accordance with the Declaration of Helsinki.
2.2. NSCLC cell lines
Two of the NSCLC cell lines, A549 and H460, were purchased from China
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Center for Type Culture Collection and Cell Bank of the Chinese Academy of Sciences in Shanghai, China. Cells were maintained in 12-well tissue-culture plates (BD, USA) in
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RPMI-1640 medium (ThermoFisher Scientific, USA), supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific, USA), penicillin (50 U/mL) and streptomycin (50 µg/mL) (ThermoFisher, USA), in a humidified tissue-culture chamber
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circulated with 5% CO2 at 37 ̊C.
2.3. LSD1 overexpression assay
Complementary DNA (cDNA) of human KDM1A gene (which encodes LSD1
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protein) was cloned and amplified from a human cDNA library, and then sub-cloned into a recombinant mammalian expression plasmid pcDNA3.1 (Promega, USA) using
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the NheI and BamH1 restriction enzymes, resulting in a human KDM1A (or LSD1) expression plasmid, pcDBA/LSD1. An empty pcDNA3.1 plasmid, pcDNA/NS, was used as control in this study. In 12-well plate culture (50,000 cells per well), A549 and H460 cells were transfected with pcDNA/NS or pcDNA/LSD1 by Lipofectamine 3000 transfection reagent for 48 h, followed by G418 (200 ng/mL, ThermoFisher Scientific, USA) and hygromycin (200 ng/mL, ThermoFisher Scientific, USA) selection treatment
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for another 48 h. Transfection efficiency was then verified by qRT-PCR and western
2.4. RNA extraction and quantitative real-time PCR
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blot assays.
Total RNA was extracted from A549 and H460 cells using a miRCURY™ RNA isolation kit (Exiqon, MA) according to manufacturer’ recommendation. A Nano Drop
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ND-3000 spectrophotometer (NanoDrop Technologies, USA) was used to check RNA
quality, and a TaqMan microRNA reverse transcription kit (Applied Biosystems, USA)
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was then performed to synthesize complimentary DNA (cDNA), all according to manufacturers’ recommendations. Quantitative real-time PCR (qRT-PCR) to detect KDM1A and miR-137 expressions was carried out using a miScript SYBR Green PCR Kit (Qiagen USA) on an ABI PRISM 7900HT Sequence Detection System (Applied
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Biosystems, USA) according to manufacturer’ recommendation. GAPDH and U6 small nuclear RNA (snRNA) expressions were served as loading controls for KDM1A and
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miR-137, respectively.
2.5. Western blot assay
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In culture, A549 and H460 cells were washed by phosphate-buffered saline
(PBS, ThermoFisher Scientific, USA) for three times, and then treated with Tris-based lysis buffer (pH=6.8, ThermoFisher Scientific, USA). For each sample, 30 µg of total protein was separated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, ThermoFisher Scientific, USA) and transferred to nitrocellulose membranes. Protein detection was carried out by incubating nitrocellulose membranes
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with several primary antibodies, including anti-LSD1 rabbit monoclonal antibody (1:500, Sigma-Aldrich, USA), anti-EZH2 rabbit polyclonal antibody, (1:250, SigmaAldrich, USA), anti-HDAC1 mouse IgG1 monoclonal antibody (1:200, Sigma-Aldrich,
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USA), anti-HDAC2 rabbit polyclonal antibody (1:200, Abcam, USA) and anti-betaactin rabbit polyclonal antibody (1:10,000, Sigma-Aldrich, USA) overnight at 4 °C,
followed by incubating membranes with horseradish peroxidase-conjugated secondary
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antibody (Sigma-Aldrich, USA) for 2 h at room temperature. Blot visualization was carried out using an enhanced chemiluminesence system (Amersham Biosciences,
2.6. Cancer proliferation assay
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USA) along with ImageLab software (Bio-Rad, USA).
In vitro growth of NSCLC cells was characterized using a Vybrant MTT
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proliferation kit (Life Technologies, USA) according to manufacturer’ recommendation. Briefly, A549 and H460 cells were re-plated in 96-well plate (2,000 cells / well) in culture medium, and allowed to proliferate for 5 days. At interval of every 24 h, the
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culture medium of tested wells was mixed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyl-tetrazoliumbromide (MTT) solution (100 µg/mL, 15 µL) for 4 h, followed by
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another 4 h treatment of HCl-SDS to solubilize the formazan product. After that, 96-well plate was examined by a Model 680 Microplate Reader (Bio-Rad, USA) and the absorbance was detected at optical density (O.D.) of 570 nm.
2.7. Cancer migration assay
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In vitro migration of NSCLC cells was characterized using a wound-scratch assay described previously [14]. Briefly, A549 and H460 cells were re-plated in 6-well plates and allowed to grow till 80% ~90% confluence. Then, a sterile cell scrapper was used to
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introduce a wound line across the diameter of the well. After removing floating debris
from the wells, NSCLC cells were allowed to grow for 48 h. At times of 0h, 6h, 12h, 24h and 48h, cancer-cell-covered surface areas were measured at each well, and normalized
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2.8. MiR-137 overexpression assay
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against the areas measured at 0 h.
ShMIMIC Human Lentiviral microRNA hsa-miR-137 mimics, miR-137, and its nonspecific lentiviral mimics, miR-NS, were purchased from GE Dharmacon (GE Dharmacon, Shanghai, China). In NSCLC culture, A549 and H460 cells were transduced
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with miR-137 or miR-NS, using Lipofectamine 3000 and polybrene (8 µg/mL, SigmaAldrich, USA) for 48 h (multiplicity of infection = 20~30), followed by selection treatment of blasticidin (1 mg/mL) for another 72 h. After that, healthy cell colonies
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were handpicked and transferred to new 12-well plate to be continuously cultured til
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confluence. After three passages, transduction efficiency was verified by qRT-PCR.
2.9. Cancer viability assay In NSCLC culture, A549 were collected and centrifuged at 5,000 xg for 5 mins.
The supernatant was discarded. Cell pellets were re-suspended in RPMI-1640 medium with the addition of 0.2% trypan blue (Sigma-Aldrich, USA) for 30 min at 37°C. Dead and live NSCLC cells were counted by a Countess Automated Cell Counter
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(ThermoFisher Scientific, USA) according to manufacturer’s recommendation. Relative viability was measured as the percentage of live A549 cells among all counted A549
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cells.
2.10. Statistical analysis
In this study, all experiments were independently repeated for at least three
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times. Data was presented as mean +/- standard errors. Statistical analysis was performed using an unpaired two-tail student’s t-test on Windows Office Excel
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Software (Microsoft, USA). Significant difference was determined if P < 0.05.
3. Results
3.1. KDM1A overexpression upregulated LSD1 and inhibited NSCLC proliferation and
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migration
A recent report demonstrated that LSD1 played an oncogenic role in NSCLC by promoting cancer growth and migration [14]. However, in that study, LSD1
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overexpression plasmid was tagged with FLAG antibody, which may result in unexpected artifacts in biological studies. In this study, we constructed a mammalian
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overexpression plasmid, pcDNA/LSD1, which contains the whole DNA sequence of human KDM1A gene. In culture of NSCLC cells, we transfected A549 and H460 cells with pcDNA/LSD1 or pcDNA/NS (an empty overexpression plasmid). After transfection was stabilized, qRT-PCR demonstrated that, in both A549 and H460 cells, endogenous KDM1A mRNA levels were significantly upregulated by pcDNA/LSD1 transfection (Figure 1A, * P < 0.05). In addition, western blot assay confirmed that, LSD1 proteins
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were also significantly upregulated by pcDNA/LSD1 transfection in A549 and H460 cells (Figure 1B). The effect of overexpressing KDM1A on NSCLC in vitro development was
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further evaluated by proliferation and migration assays. In a MTT proliferation assay, A549 and H460 cells were re-plated in 96-well plate and allowed to grow for 5
consecutive days. Daily cancer cell proliferation was characterized by MTT-reaction
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induced fluorescent signals detected at optical density (O.D.) of 570 nm. It showed that, two to five days after cells were re-plated in 96-well plate, cancer proliferations of both
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A549 and H460 cells were significantly upregulated by pcDNA/LSD1 transfection (Figure 1C, * P < 0.05). In addition, through a wound-scratch assay, we discovered that cancer migrations in A549 and H460 cells were also significantly promoted by pcDNA/LSD1 transfection 6 to 48 h after wound creation (Figure 1D, * P < 0.05).
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Thus, our results clearly demonstrated that using a mammalian overexpression assay to overexpress DKM1A gene in NSCLC cells was efficient in endogenously upregulating both DKM1A gene and LSD1 protein, as well as promoting cancer
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proliferation and migration.
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3.2. MicroRNA-137 inhibits LSD1 in NSCLC While LSD1 was characterized as an active tumor modulator in NSCLC [14],
little is known about its upstream regulation. As indicated in studies in neural stem cells and neuroblastoma [25, 26], human mature microRNA-137 (miR-137) is speculated to be the upstream regulator of LSD1, as it may bind to the complimentary DNA sequence on 3-UTR of human KDM1A gene (Figure 2A).
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To examine whether KDM1A (or LSD1) was actually modulated by miR-137 in NSCLC, we transduced lentiviral miR-137 mimics (miR-137) into LSD1-overexpressed A549 and H460 cells (transfected with pcDNA/LSD1). The control A549 and H460 cells
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were transduced with a non-specific lentiviral mimics, miR-NS. After lentiviral
transduction was stable, analysis of qRT-PCR demonstrated that endogenous miR-137 expressions were significantly upregulated in NSCLC cells through lentiviral
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transduction of miR-137 mimics (Figure 2B, * P < 0.05).
We also assessed the endogenous KDM1A mRNA levels. The result of qRT-
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PCR showed that KDM1A gene was significantly downregulated in NSCLC cells by lentiviral transduction of miR-137 mimics (Figure 2C, * P < 0.05). In addition, western blot assay showed that LSD1 protein levels were also significantly downregulated by
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lentiviral transduction of miR-137 mimics (Figure 2D-E, * P < 0.05).
3.3. MicroRNA-137 reversed oncogenic effects of LSD1 in NSCLC We then asked whether upregulating miR-137 would have functional roles in
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LSD1-induced oncogenic regulation in NSCLC. Firstly, since we performed two times of gene modification in NSCLC cells, we would like to know whether it might induce
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cell death. Through a cancer viability assay (Figure 3A), we compared NSCLC viability between A549 cells without transfection or transduction (Figure 3A, condition 1), and A549 cells transfected with pcDNA/AS (Figure 3A, condition 2), or transfected with pcDNA/LSD1 (Figure 3A, condition 3), or transfected with pcDNA/LSD1 and transduced with miR-NS (Figure 3A, condition 4), or transfected with pcDNA/LSD1 and transduced with miR-137 (Figure 3A, condition 5). It showed that, under all conditions,
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A549 cells were fairly healthy and no significant cell death was detected (Figure 3A, ∆ P > 0.05). In LSD1-overexpressed A549 or H460 cells, we examined cancer cell in vitro
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proliferations after they were transduced with either miR-NS or miR-137-mimics
lentiviruses. The result of 5-day MTT proliferation assay showed that, in both A549 and H460 cells, miR-137 upregulation significantly suppressed NSCLC proliferations
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(Figure 3B, * P < 0.05). In addition, through a wound-scratch assay, we found that
NSCLC migrations were also significantly inhibited by miR-137 upregulation (Figure
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3C, * P < 0.05). Therefore, our results clearly demonstrated that miR-137 upregulation (or overexpression) reversed the oncogenic effects of LSD1 on NSCLC proliferation and migration.
pathways in NSCLC
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3.4. MiR-137 and LSD1 oppositely regulated histone deacetylases-associated signaling
Finally in this study, we asked whether histone deacetylases-associated signaling
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pathways, including proteins of EZH2, HDAC1 and HDAC2, were involved in the regulation of LSD1 / miR-137 in NSCLC. We firstly used western blot analysis to
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examine EZH2, HDAC1 and HDAC2 protein expressions in A549 cells transfected with mammalian overexpression plasmid. It showed that, between control A549 cells (nontransfected, non-transduced), and A549 cells transfected with pcDNA/NS, there was no change in the expression levels of EZH2, HDAC1 or HDAC2 proteins (Figure 4, columns 1 vs. 2). On the other hand, while A549 cells were transfected with
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pcDNA/LSD1 to upregulate KDM1A, EZH2, HDAC1 and HDAC2 proteins were significantly upregulated (Figure 4, columns 2 vs. 3). Then, while LSD1-overexpressed A549 cells were further transduced with
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lentiviruses, western blot analysis demonstrated that, EZH2, HDAC1 and HDAC2
proteins were unaltered by miR-NS transductions in A549 cells (Figure 4, columns 4 vs. 3). However, in miR-137-upregulated A549 cells (those transduced with miR-137),
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EZH2, HDAC1 and HDAC2 protein expressions were considerably reduced (Figure 4, columns 5 vs. 4).
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Therefore, our results suggest that histone deacetylases-associated signaling pathways were activated (or upregulated) by LSD1-overexpression, but inhibited (or downregulated) by subsequent miR-137 upregulation, in NSCLC.
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4. Discussions
In a recent report, it was demonstrated that epigenetic regulation of LSD1 plays critical role in regulating NSCLC development and predicting prognosis among NSCLC
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patients, including both NSCLC and small cell lung cancer [14]. However in that report, Lv and colleagues used a FLAG-tagged fusion protein to exogenously overexpress LSD1
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protein in NSCLC cells, at that time not knowing whether KDM1A, the encoding gene of LSD1, would also be involved in functional regulation in NSCLC [14]. Therefore, as the first step of our study, we constructed a mammalian KDM1A overexpression plasmid and transfected it into A549 and H460 cells. The subsequent qRT-PCR and western blot analyses confirmed the success of transfection, showing that endogenous KDM1A gene, as well as LSD1 protein, was significantly upregulate in NSCLC cells. Most importantly,
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the oncogenic mechanisms of LSD1 were confirmed by our biochemical assays, demonstrating that NSCLC proliferation and migration were markedly promoted by overexpressing KDM1A gene in A549 and H460 cells.
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As the next step of our study, we sought the upstream initiator of KDM1A / LSD1 in NSCLC. In studies of neuroblastoma and neural stem cells, it was demonstrated that miR-137 was the upstream regulator of LSD1 [25, 26]. Based on this knowledge, we
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transduced LSD1-overexpressed A549 and H460 cells with lentivirus to upregulate miR137. And we found that miR-137 upregulation could suppress both KDM1A gene and
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LSD1 protein in NSCLC cells, thus confirming that miR-137 may directly and inversely regulate KDM1A / LSD1 in NSCLC. Furthermore, we used functional assays to examine the effect of upregulating miR-137 in LSD1-overexpressed A549 and H460 cells. We found that, miR-137 upregulation had opposite effects as LSD1 overexpression, or tumor
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suppressive effects, in NSCLC by suppressing cancer proliferation and migration. Thus, all those data confirmed that miR-137 is an upstream regulator and reversely modulates KDM1A / LSD1 expression and oncogenic effects in NSCLC. To our knowledge, this is
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the first-ever report to show the correlation of miR-137 and KDM1A / LSD1 in NSCLC. Also in our study, we explored the associated signaling pathways involved in
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LSD1 / MIR-137 regulation in NSCLC. Through western blot analysis, we discovered that EZH2, HDAC1 and HDAC1 proteins were upregulated by KDM1A / LSD1, whereas downregulated by miR-137, in NSCLC. EZH2, acting as acts as a histone lysine methyltransferase, was found to be upregulated in human NSCLC carcinomas and correlated with poor prognosis of NSCLC patients [27-29], similar to the expression pattern and prognostic characteristics of LSD1 in NSCLC. However, our report was the
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first one to show that EZH2 was directly regulated by KDM1A / LSD1 in NSCLC. It would be interesting to learn, possibly through future studies, what the signaling pathways are involved in linking KDM1A / LSD1 toward EZH2 in NSCLC. As for
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LSD1-induced HDAC1/2 upregulation, since LSD1 and HDAC1/2 were often found be co-expressed in an LSD1/CoREST1/HDAC complex in chromatin to synergistically
regulate cancer development [30, 31], it is possible that LSD1 may upregulate HDAC1/2
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(or other histone deacetylases) through the form of chromatin complex, to regulate cancer development in NSCLC. On the other hand, the inhibitory effect of miR-137 on EZH2 or
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HDAC1/2 may well through the downstream regulation of LSD1.
Figure legends
Figure 1. LSD1 overexpression was oncogenic in NSCLC. A549 and H460 cells were
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transfected with a mammalian KDM1A overexpression plasmid, pcDNA/LSD1, or an empty control plasmid, pcDNA/NS. (A) After transfection was stabilized, qRT-PCR was carried out to compare endogenous DKM1A mRNA levels between A549 and H460 cells
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transfected with pcDNA/LSD1 and those transfected with pcDNA/NS (* P < 0.05). (B) A549 and H460 cells were lysed and western blot assay was carried out to compare
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LSD1 protein levels between A549 and H460 cells transfected with pcDNA/LSD1 and those transfected with pcDNA/NS. (C) Transfected A549 and H460 cells were re-plated in 96-well plate and allowed to grow for 5 days. In vitro proliferation of NSCLC cells was characterized using a MTT proliferation assay. Daily measurement of absorbance was determined at optical density (O.D) of 570 nm (* P < 0.05). (C) Transfected A549
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and H460 cells were also examined by a wound-scratch assay to assess their migration (* P < 0.05).
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Figure 2. MiR-137 inhibits LSD1 in NSCLC. (A) Complimentary binding was shown
between human mature miR-137, hsa-miR-137, and 3’-UTR of human KDM1A gene.
(B) In LSD1-overexpresed A549 and H460 cells (transfected with pcDNA/LSD1), they
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were transduced with a lentiviral human miR-137 mimics, miR-137, or a non-specific
lentiviral mimics, miR-NS. QRT-PCR was carried out to compare endogenous miR-137
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gene levels between A549 and H460 cells transduced with miR-137 mimics and those transduced with miR-NS (* P < 0.05). (C) QRT-PCR was also carried out to compare endogenous KDM1A mRNA levels between A549 and H460 cells transduced with miR137 mimics and those transduced with miR-NS (* P < 0.05). (D) After lentiviral
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transduction, A549 and H460 cells were lysed. Western blot assay was carried out to examine LSD1 protein levels between A549 and H460 cells transduced with miR-137 mimics and those transduced with miR-NS. (E) Quantification on LSD1 protein
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expression levels (LSD1 / β-actin) was performed for (D) (* P < 0.05).
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Figure 3. MiR-137 suppressed LSD1-induced oncogenic effects in NSCLC. (A) In A549 cells, cancer cell viability was compared between those without transfection or transduction (condition 1), and those transfected with pcDNA/AS (condition 2), or transfected with pcDNA/LSD1 (condition 3), or transfected with pcDNA/LSD1 and transduced with miR-NS (condition 4), or transfected with pcDNA/LSD1 and transduced with miR-137 (condition 5) (∆ P > 0.05). (B) In A549 and H460 cells transfected with
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pcDNA/LSD1 and transduced with lentiviruses of miR-NS or miR-137 mimics, a MTT cancer proliferation assay was carried out for 5 days (* P < 0.05). (C) Those A549 and H460 cells were also examined by a wound-scratch assay to assess their migration (* P <
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0.05).
Figure 4. MicroRNA-137 and LSD1 modulated EZH2 and HDAC in NSCLC. Western
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blot assay was carried out to assess protein levels of EZH2, HDAC1 and HDAC2 in
A549 cells without transfection or transduction (column 1), or A549 cells transfected
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with pcDNA/AS (column 2), or A549 cells transfected with pcDNA/LSD1 (column 3), or A549 cells transfected with pcDNA/LSD1 and transduced with miR-NS (column 4), or A549 cells transfected with pcDNA/LSD1 and transduced with miR-137 (column 5).
Acknowledgements
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Conflict of interest: None.
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We thank Dr. Xianliang Wang for his critical review on the manuscript.
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LSD1 promoted NSCLC proliferation and migration. MiR-137 was inversely correlated with LSD1 in NSCLC. MiR-137 upregulation suppressed KDM1A / LSD1 productions in NSCLC.
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MiR-137 upregulation inhibited proliferation and migration in NSCLC
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EZH2, HDAC1 and HDAC2 were activated by LSD1, but inhibited by miR-137 in NSCLC.