The miR-599 promotes non-small cell lung cancer cell invasion via SATB2

Accepted Manuscript The miR-599 promotes non-small cell lung cancer cell invasion via SATB2 Wenjun Tian, Guanghai Wang, Yiqing Liu, Zhenglan Huang, Caiqing Zhang, Kang Ning, Cuixiang Yu, Yajuan Shen, Minghui Wang, Yuantang Li, Yong Wang, Bingchang Zhang, Yaoran Zhao PII:

S0006-291X(17)30266-8

DOI:

10.1016/j.bbrc.2017.02.005

Reference:

YBBRC 37253

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 29 January 2017 Revised Date:

1 February 2017

Accepted Date: 2 February 2017

Please cite this article as: W. Tian, G. Wang, Y. Liu, Z. Huang, C. Zhang, K. Ning, C. Yu, Y. Shen, M. Wang, Y. Li, Y. Wang, B. Zhang, Y. Zhao, The miR-599 promotes non-small cell lung cancer cell invasion via SATB2, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.02.005. 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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The miR-599 Promotes Non-Small Cell Lung Cancer Cell

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Invasion via SATB2.

Wenjun Tian1·, Guanghai Wang2*·, Yiqing Liu1·, Zhenglan Huang3·, Caiqing Zhang2·, Kang Ning2· , Cuixiang Yu2·, Yajuan Shen1·, Minghui Wang1·, Yuantang Li1·, Yong Wang 1·,

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Bingchang Zhang1·, Yaoran Zhao1*

University, Jinan,China

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1 Department of Clinical Laboratory, Shandong Provincial Hospital Affiliated to Shandong

2 Department of Respiratory Medicine,Shandong Provincial Qianfoshan hospital, Shandong University,Jinan, China.

3 Key Laboratory of Medical Diagnostics of Ministry of Education, Faculty of Laboratorial

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Medicine, Chongqing Medical University, Chongqing, China.

Address correspondence to: Yaoran Zhao, Department of Central Laboratory, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China.

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Guanghai Wang, Department of Respiratory Medicine,Shandong Provincial Qianfoshan

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hospital Shandong University,Jinan, China.

Key words: miR-599, SATB2, NSCLC

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Abstract MicroRNAs (miRNAs) play important roles in the pathogenesis of many types of cancers by negatively regulating gene expression at posttranscriptional level. Here, we identified that miR-599 is up-regulated in non-small cell lung cancer

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(NSCLC) patients. It promoted NSCLC cell proliferation by negatively regulating SATB2. In NSCLC cell lines, CCK-8 proliferation assay indicated that the cell proliferation is promoted by miR-599 mimics. Transwell assay

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showed that miR-599 mimics promoted the invasion and migration of NSCLC cells. Luciferase assays confirmed that miR-599 directly binds to the

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3'untranslated region of SATB2, and western blotting showed that miR-599 suppresses the expression of SATB2 at the protein level. This study indicates that miR-599 promotes proliferation and invasion of NSCLC cell lines via SATB2. The miR-599 may represent a potential therapeutic target for NSCLC

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treatment.

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Introduction Non-small cell lung cancer is the most common type of lung cancer. About 85% of lung cancers are non-small cell lung cancers (NSCLC) [1]. NSCLC is the one of the most common cause of cancer-related death worldwide[2, 3]. The

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discovery of microRNAs (miRNAs) started a new generation of discovering the molecular mechanisms underlying the carcinogenesis of NSCLC[4, 5]. miRNAs are small non-coding RNAs which inhibit the expression of target

miR-143[7] and miR-186[8]

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genes by translational repression or mRNA degradation[6]. Some miRNAs, e.g. are found to play roles in the mechanisms

Understanding

the

miRNA

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underlying the carcinogenesis of NSCLC by inhibiting their target genes. involved

mechanisms

underlying

the

carcinogenesis of NSCLC will be helpful to improve the treatment of NSCLC. Recently, miR-599 was found to be involved in the carcinogenesis of melanomas [9] and renal cancer [10]. To investigate whether miR-599 play

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roles in NSCLC, we analyzed the expression pattern of miR-599 NSCLC, and found miR-599 was up-regulated in NSCLC patients. This leads to the hypothesis that miR-599 may play roles in NSCLC. Further experiments

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indicated that miR-599 inhibited the proliferation and invasion of NSCLC cell lines. To investigate the downstream molecular mechanisms of miR-599 in

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NSCLC, we predicted its potential target genes according to structure complementation. Among the predicted target genes, special AT-rich sequence-binding protein 2 (SATB2) was found to be the most probable one. SATB2 encodes a DNA-binding protein that specifically binds nuclear matrix attachment regions and is involved in transcriptional regulation and chromatin remodeling[11].The deregulation of SATB2 is involved in many types of diseases, including cancers [11-13]. Further experiments in NSCLC cell lines confirmed that miR-599 directly bound to the 3'untranslated region of SATB2, and suppressed the expression of SATB2 at the protein levels.

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Materials and Methods Patients and Ethics Statement The study was approved by the ethical committee of Shandong Provincial

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Hospital Affiliated to Shandong University. Patients undergoing NSCLC at Shandong Provincial Hospital Affiliated to Shandong University were included. Written informed consents were obtained from all the patients. Lung samples from 351 patients with NSCLC were collected between January 2013 and

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January 2016 at the Shandong Provincial Hospital Affiliated to Shandong University (Table 1). The entire investigation conforms to the principles

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outlined in the Declaration of Helsinki. Real-time PCR

The expression of miRNA and mRNA were determined by the Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) kit (Life Technologies,

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Beijing, China). The qRT-PCR was performed in an QuantStudio® 6 Flex Real-Time PCR System (Life Technologies, Beijing, China) using a SYBR Green kit (TaKaRa, Tokyo, Japan), and the relative changes were quantified.

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The primer for miR-599 is 5’- GUUGUGUCAGUUUAUCAAAC-3’ and 5’CTCCATATCGCACTTTAATCTCTAACT-3’. The primer for SATB2 is 5’and

5’-

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AGGAGTTTGGGAGATGGTAT-3’

ACTGAACCTGACCGTACACCCAGAACACAATAGTCTGAA -3’. miRNA and mRNA Profile Data miRNA and mRNA profiles data of NSCLC samples and normal control samples were collected from GEO database (www.ncbi.nlm.nih.gov/gds, GSE44077, GSE17681, GSE27705 and GSE29250). After quality control, 43 NSCLC samples and 35 normal lung specimen samples were used in miRNA analysis, while 61 NSCLC samples and 71 normal lung specimen samples

ACCEPTED MANUSCRIPT were used in further mRNA analysis. Identification of differentially expressed miRNA and mRNA The identification of differentially expressed miRNA and mRNA in tissues from

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patients with NSCLC were performed with Limma package on R platform using download miRNA and mRNA profiles data as mentioned above. The cutline of

and |lgFC|>1(fold change). miRNA Target Genes Prediction

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significantly differentially expressed miRNA and mRNA is P.value<0.01 (T test)

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Human miRNA target genes prediction uses miRNA sequences that downloaded from the Rfam website (http://www.sanger.ac.uk/Software/Rfam) and satisfy the established criteria[14]. The target genes of miRNAs were predicted using miRanda[15] and TargetScan[16] methods. The predicted target genes supported by both methods were selected for further analysis.

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Cell Culture and Transfection

The human NSCLC cell lines, A549 and A427 was obtained from American

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Type Culture Collection (ATCC, USA). The A549 and A427 cell line was cultured in RPMI 1640 media (Life Technologies, Beijing, China) and

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supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Beijing, China). Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. A549 and A427 cell lines were seeded in 24-we ll plates at 3×105 cells/wells and incubated overnight. Transfection of the miR-599 miRNA mimic, the anti-miR-599, inactive control cel-mir-67 (Life Technologies, Beijing, China), or pMIR-Report vectors was taken using Lipofectamine 2000 transfection reagent (Invitrogen, Beijing, China) with 300 nmol of miRNA or 1µg/ml DNA plasmid, respectively. Total proteins of A549 and A427 cells were isolated at 48 hours after transfection.

ACCEPTED MANUSCRIPT Cell Proliferation Cell proliferations were measured using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). A549 and A427 NSCLC cells were plated in 24-well plates at 3x105 cells/well. Then cells were incubated in 10% CCK-8 which was diluted

determined at 24, 48 and 72 hours after transfection. Cell migration and Invasion

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in normal culture medium at 37oC for color conversion. Proliferation rates were

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Cell invasion and migration were measured using a transwell chamber (Corning, Beijing, China) with and without Matrigel (Invitrogen, Beijing, China).

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For the determination of A549 and A427 NSCLC cells invasion, transwell chambers were placed into 24-well plates, and coated with 30 µl Matrigel, then incubated at 37oC for 40 minutes. In transwell assays with and without Matrigel, A549 and A427 cells were trypsinized and then seeded in chambers at the density of 8x104 cells/well at 48 hours after transfection. These cells were

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cultured in RPMI 1640 medium with 2% serum. Meanwhile 600 µl of 10% FBS-1640 was added to the lower chamber. After 24 hours, migrated A549 and A427 cells were fixed in 100% methanol for 30 minutes. These

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non-migrated A549 and A427 cells were removed by cotton swabs. After that cells on the bottom surface of the membrane were stained with the 0.1%

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crystal violet for 20 minutes. Images of A549 and A427 cells were taken under a phase-contrast microscope. Luciferase assay

A549 and A427 cells were seeded in 24-well plates at 3x105 cells/well and incubated for 24 hours before transfection. In the reporter gene assay, the A549 and A427 cells were co-transfected with 0.6 µg of pGL3-SATB2-3’UTR or pGL3-SATB2-3’UTR Mut plasmid, 0.06 ng of the phRL-SV40 control vector (Promega, Beijing, China), and 100 nM miR-599 or control RNA using

ACCEPTED MANUSCRIPT Lipofectamine 2000 (Invitrogen, Beijing, China). The renilla and firefly luciferase activities were determined with a dual luciferase assay (Promega, Beijing, China) 24 hours after transfection.

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Western Blot Proteins were separated by 12% SDS-PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad, Beijing, China). Membranes was blocked by 5% non-fat milk and incubated with anti-P21 antibody (Abcam, Beijing,

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China) or anti-β-actin antibody (Abcam, Beijing, China). After being washed extensively, the secondary antibody (Abcam, Beijing, China) was then added

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to the system. Finally, Immunoreactive protein bands were detected with the Enhanced Chemiluminescence (ECL) system. Statistical Analysis

Experiments were repeated at least three times. Statistical analyses were

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performed using R. Values were expressed as means ± S.D. Differences between groups were estimated with T-test. The correlation was determined by Pearson correlation coefficient. Statistical analysis was considered to be

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Results

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significant when P. value<0.05.

The miR-599 and its predicted target gene were differentially expressed in NSCLC

The expression pattern of miR-599 was analyzed between NSCLC patients and normal controls using the microarray data from GEO database. The result indicated that miR-599 was significantly up-regulated (lgFC<0 & P-value<0.01) in the 43 NSCLC patients (Figure 1). It implied that miR-599 may play roles in NSCLC. To investigate the potential downstream mechanisms of miR-599 in NSCLC,

we

predicted

its

target

genes

according

to

sequence

ACCEPTED MANUSCRIPT complementation. Among these predicted target genes, SATB2 was found to be correspondingly down-regulated (lgFC>0 & P-value<0.01) in NSCLC patients (Figure 1). Then we investigated the potential relationship between miR-599 and SATB2 at mRNA level in 351 NSCLC patients (Table 1) using

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RT-PCR. The result indicated that the expression of miR-599 is significantly correlated with SATB2 in NSCLC (R2=0.7288, P-value<0.01) (Figure 1 D). The down-regulation of SATB2 is critical for induction of invasion of NSCLC cells [17]. This leads to the hypothesis that miR-599 may play roles in NSCLC by

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inhibiting SATB2. Therefore, we selected miR-599 and SATB2 for further functional investigation in the human NSCLC cells.

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MiR-599 targets and inhibits SATB2 in NSCLC cells

As predicted by miRanda[15] and TargetScan[16], there was complementarity between has-miR-599 and the 3’ untranslated region (UTR) of SATB2. Overexpression of miR-599 reduced the protein levels of SATB2 in NSCLC

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cells with time (Figure. 2 A and B). Conversely, miR-599 inhibitor increased the protein levels of SATB2 in NSCLC cells with time (Figure. 2 A and B). The effect of miR-599 on the translation of SATB2 mRNA into protein was then

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assessed by using a luciferase reporter assay (Figure. 2 C and D). the miR-599 inhibitor significantly enhanced the luciferase activity of the reporter

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gene with the wild type construct but not with the mutant SATB2 3’UTR construct (Figure. 2C), while enforced expression of miR-599 significantly reduced the luciferase activity of the reporter gene with the wild type construct but not with the mutant SATB2 3’UTR construct (Figure. 2D). These evidences indicate that miR-599 directly targeted the 3’UTR region of SATB2 and negatively regulated its expression. miR-599 promotes NSCLC cell proliferation, migration and invasion Since the down-regulation of SATB2 is critical for induction of invasion of

ACCEPTED MANUSCRIPT NSCLC cells [17], we explored the potential impact of miR-599 in NSCLC cell proliferation, migration and invasion in A549 and A427 cell lines. A549 and A427 cells were transfected with miR-599 mimics or inhibitor or inactive control cel-mir-67. CCK-8 proliferation assay indicated that the cell proliferation was

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promoted in both of the miR-599-mimics-transfected NSCLC cell lines compared with inactive control cel-mir-67-transfected cell lines (Figure. 3). Conversely, miR-599 inhibitor significantly inhibited the proliferation of the A549 and A427 cells (Figure. 3). Interestingly, migration and invasion assay

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showed that miR-599 inhibitors significantly inhibited the migration and invasion of A549 and A427 cells compared with the inactive cel-mir-67 control,

A549 and A427 cells (Figure. 4).

Discussion

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whereas miR-599 mimics promoted cell migration and invasion in both of the

In the past decades, miRNAs have been found to be important regulators in

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cancer [18, 19]. miR-599 is a new tumor-related miRNA in melanomas [9] and renal cancer [10]. The relationship between miR-599 and NSCLC has never been reported before. By analyzing the expression pattern of miR-599 and its

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predicted target genes, we found the clues for uncovering the roles of miR-599 in NSCLC, leading to the hypothesis that miR-599 may play roles in NSCLC by

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targeting SATB2.

Thus, our further study intended to investigate the biological function of miR-599 in NSCLC. miR-599 promoted proliferation, migration and invasion of A549 and A427 cells, which suggest that miR-599 plays a critical role in the carcinogenesis of NSCLC. Then we addressed the potential downstream molecular mechanisms of miR-599 in regulating proliferation, migration and invasion of NSCLC cells. In this study, western blots and luciferase assays showed that SATB2 is a target gene of miR-599. miR-599 directly bound to the 3’UTR region of SATB2 and negatively regulated its expression. SATB2

ACCEPTED MANUSCRIPT encodes DNA binding protein that specifically binds nuclear matrix attachment regions. The encoded protein is involved in transcription regulation and chromatin remodeling [20, 21]. SATB2 suppressed cell proliferation in colorectal cancer [21], gastric cancer [22] and colorectal cancer [23]. The

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down-regulation of SATB2 is critical for induction of invasion of NSCLC cells [17]. In this study, we found that miR-599 inhibits SATB2 in NSCLC. The down-regulation of SATB2 seems to be part of important downstream mechanisms of miR-599 in NSCLC. It’s very interesting that miR-599 was ever

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found to play a tumor-suppressive role in hepatocellular carcinoma [24]. It wasn’t the first time to find one miRNA plays different roles in different cancer.

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For example, miR-590 not only plays a tumor-suppressive role in colorectal cancer [25], breast cancer[26], but also plays a oncogenic role in lung adenocarcinoma[27] and T-cell acute lymphoblastic leukaemia[28]. This phenomenon revealed that the functions of miRNA is very complicated in cancer and partially implied the molecular basement of tumor heterogeneity.

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In conclusion, our results have shown that miR-599, which is highly expressed in NSCLC, promotes NSCLC cell proliferation, migration and invasion through directly binding and negatively regulating SATB2. This novel miR-599 / SATB2

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axis may provide new insights into the mechanisms underlying NSCLC, and miR-599 may be a potential therapeutic target to improve the treatment of

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NSCLC in the future.

Acknowledgement This work was supported by grants from the Science and technology development project of Shandong Province(2014GGH218041), Shandong Natural Science Fund(ZR2013HM057).

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Conflict of interest

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Authors have declared that no competing interest exists.

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Figure Legends

Figure 1. The expression of miR-599 and SATB2 in NSCLC patients. (A) The

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predicted structure complementation between miR-599 and SATB2. (B) The expression of miR-599 in NSCLC patients. (C) The expression of SATB2 in NSCLC patients. (D) The Pearson’s correlation between miR-599 and SATB2

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*p<0.05, **p<0.01, and ***p<0.001.

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in 351 NSCLC patients.

Figure 2. miR-599 targets SATB2 in NSCLC cells. (A) Protein of SATB2 significantly decreased with time after transfection with miR-599 mimics in A427; (B) Protein of SATB2 significantly decreased with time after transfection

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with miR-599 mimics in A549 cells; (C) The analysis of the relative luciferase activities of SATB2-WT, SATB2-MUT in A549 and A427 cells after transfection

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with anti-miR-599; (D) Protein of SATB2 increased with time after transfection with miR-599 mimics in A549 and A427 cells. *p<0.05, **p<0.01, and

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***p<0.001.

Figure 3. miR-599 promoted NSCLC cell proliferation. (A) Growth of A427 NSCLC cells was shown after transfection with miR-599 mimics or inhibitor or inactive control. (B) Growth of A549 NSCLC cells was shown after transfection with miR-599 mimics or inhibitor or inactive control. The growth index as assessed at 0, 24, 48 and 72 h;

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Figure 4. miR-599 promoted NSCLC cell migration and invasion. (A) Transwell analysis of NSCLC cells migration after treatment with miR-599 mimics,

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inhibitors or inactive control in A427 cells; (B) Transwell analysis of NSCLC cells migration after treatment with miR-599 mimics, inhibitors or inactive control in A549 cells; (C) Transwell analysis of NSCLC cells invasion after

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treatment with miR-599 mimics, inhibitors or inactive control in A427 cells; (D)

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Transwell analysis of NSCLC cells invasion after treatment with miR-599 mimics, inhibitors or inactive control in A549 cells. *p<0.05, **p<0.01, and

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***p<0.001.

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ACCEPTED MANUSCRIPT Table 1. NSCLC patients’ characteristics.

Patients

Characteristics

(n=351) Gender, n (%) Male Female Age, n (%) <70 ≥70

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233 (66.4) 118 (33.6) 207 (59) 144 (41)

Stage, n (%) III/IV Recurrence

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319 (90.8) 32 (9.2)

Smoking history, n (%) Yes No

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264 (75.2) 87 (24.8)

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ACCEPTED MANUSCRIPT 1. miR-599 is up-regulated in NSCLC. 2. miR-599 promotes the proliferation and invasion of NSCLC cells. 3. miR-599 inhibitors inhibits the proliferation and invasion of NSCLC cells. 4. miR-599 targets 3’ UTR of SATB2 in NSCLC cells.

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5. miR-599 inhibits SATB2 in NSCLC cells.