- Email: [email protected]
WITHDRAWN: miR-543 promotes proliferation and invasion of non-small cell lung cancer cells by inhibiting PTEN
Accepted Manuscript miR-543 promotes proliferation and invasion of non-small cell lung cancer cells by inhibiting PTEN Ping Zhang, Hong-Xing Zhou, Ming-Xia Yang, Yan Wang, Wen-Ming Cao, Ke-Feng Lu, Rong-Qiang Wu PII:
S0006-291X(16)30478-8
DOI:
10.1016/j.bbrc.2016.03.157
Reference:
YBBRC 35589
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 26 March 2016 Accepted Date: 30 March 2016
Please cite this article as: P. Zhang, H.-X. Zhou, M.-X. Yang, Y. Wang, W.-M. Cao, K.-F. Lu, R.-Q. Wu, miR-543 promotes proliferation and invasion of non-small cell lung cancer cells by inhibiting PTEN, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.03.157. 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.
ACCEPTED MANUSCRIPT
miR-543 promotes proliferation and invasion of non-small cell lung cancer cells by inhibiting PTEN.
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Ping Zhang1,#,*,Hong-Xing Zhou1,#, Ming-Xia Yang2, Yan Wang1, Wen-Ming Cao1, Ke-Feng Lu1, Rong-Qiang Wu1, 1. Department of Clinical Laboratory, Affiliated Changzhou Second Hospital of Nanjing Medical University, Changzhou, Jiangsu, China 2. Respiratory Medicine, Affiliated Changzhou Second Hospital of Nanjing Medical University, Changzhou, Jiangsu, China
*Corresponding author: Ping Zhang
Contributed equally to this work.
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#
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Address: 68 Gehu Road in Wujin district, Changzhou, Jiangsu Province, 213164, China. Phone: +8613813658238, Fax: +8651981087709. E-mail: [email protected]
Key words: miR-543, PTEN, NSCLC, gefitinib
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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-543 is up-regulated in gefitinib-resistant non-small
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cell lung cancer (NSCLC) patients comparing gefitinib-sensitive ones. It promotes NSCLC cell proliferation by negatively regulates its target gene PTEN. In NSCLC cell lines, CCK-8 proliferation assay indicated that the cell
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proliferation is promoted by miR-543 mimics. Transwell assay showed that miR-543 mimics promotes the invasion and migration of NSCLC cells.
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Luciferase assays confirmed that miR-543 directly binds to the 3'untranslated region of PTEN, and western blotting showed that miR-543 suppresses the expression of PTEN at the protein level. This study indicates that miR-543 promtes proliferation and invasion of NSCLC cell lines by PTEN. The miR-543 may represent a potential therapeutic target for gefitinib-resistant NSCLC
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intervention.
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Introduction Gefitinib, the first selective inhibitor of epidermal growth factor receptor's (EGFR) tyrosine kinase domain, is used to treat non-small cell lung cancer (NSCLC), which is the one of the most common cause of cancer-related death
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worldwide[1,2]. However, resistance to gefitinib limits its therapeutic effects on NSCLC patients. The discovery of MicroRNAs (miRNAs) opened a new generation for understanding the carcinogenesis of NSCLC[3]. miRNAs are
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small non-coding RNAs which inhibit the expression of target genes by translational repression or mRNA degradation[4]. Some miRNAs, e.g. miR-
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34a[5] and MiR- 451[6] are found to play roles in the mechanisms underlying drug resistance to NSCLC by regulating their target genes. Underlying the miRNA-related molecular mechanisms underlying gefitinib-resistant NSCLC will be helpful to improve NSCLC therapies.
Therefore, we analyzed the expression pattern of miRNA and genes in
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gefitinib-resistant and -sensitive NSCLC, and found miR-543 and its predicted target gene PTEN was deregulated in gefitinib-resistant NSCLC patients comparing to sensitive ones. This leads to the hypothesis that miR-543 may
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play roles in NSCLC by PTEN. Further experiments in NSCLC cell lines confirmed that miR-543 directly bound to the 3'untranslated region of PTEN,
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and suppressed the expression of PTEN at the protein levels. Moreover, modulation of miR-543 expression regulated proliferation and invasion of NSCLC cell lines.
Materials and Methods 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,
ACCEPTED MANUSCRIPT GSE51828, GSE34228). After quality control, 4 Gefitinib-resistant NSCLC samples and 4 Gefitinib-sensitive NSCLC samples were used in further miRNA analysis,
while
104
Gefitinib-resistant
NSCLC
samples
and
104
Gefitinib-sensitive NSCLC samples were used in further mRNA analysis.
The
identification
of
differentially
expressed
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Identification of differentially expressed miRNA and mRNA
miRNA and
mRNA in
durg-resistant patients with NSCLC tissues were performed with Limma
SC
package on R platform using download miRNA and mRNA profiles data as mentioned above. The cutline of significantly differentially expressed miRNA
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and mRNA is P.value<0.01 (T test) and |lgFC|>1(fold change). miRNA Target Genes Prediction
Human miRNA target genes prediction uses miRNA sequences that downloaded from the Rfam website (http://www.sanger.ac.uk/Software/Rfam)
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and satisfy the established criteria[7]. The target genes of miRNAs were predicted using miRanda[8] and TargetScan[9] methods. The predicted target genes supported by both methods were selected for further analysis.
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Cell Culture and Transfection
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The human NSCLC cell lines, A427 and A549 was obtained from American Type Culture Collection (ATCC, USA). The A427 and A549 cell line was cultured in RPMI 1640 media(Life Technologies, Shanghai, China) and supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Shanghai, China). Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. A427 and A549 cell lines were seeded i n 24-well plates at 3× 105 cells/wells and incubated overnight. Transfection of the miR-543 miRNA mimic, the anti-miR-543, inactive control cel-mir-67 (Life Technologies, Shanghai, China), or pMIR-Report vectors was taken using Lipofectamine
ACCEPTED MANUSCRIPT 2000 transfection reagent (Invitrogen, Shanghai, China) with 300 nmol of miRNA or 1µg/ml DNA plasmid, respectively. Total proteins of A427 and A549 cells were isolated at 48 hours after transfection.
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Cell Proliferation Cell proliferations were measured using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). A427 and A549 NSCLC cells were plated in 24-well plates at 3x105 cells/well. Then cells were incubated in 10% CCK-8 which was diluted
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in normal culture medium at 37oC for color conversion. Proliferation rates were
Cell migration and Invasion
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determined at 24, 48 and 72 hours after transfection.
Cell invasion and migration were measured using a transwell chamber (Corning, Shanghai, China) with and without Matrigel (Invitrogen, Shanghai, China). For the determination of A427 and A549 NSCLC cells invasion,
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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, A427 and A549 cells were trypsinized and then seeded in
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chambers at the density of 8x104 cells/well at 48 hours after transfection. These cells were cultured in RPMI 1640 medium with 2% serum. Meanwhile
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600 µl of 10% FBS-1640 was added to the lower chamber. After 24 hours, migrated A427 and A549 cells were fixed in 100% methanol for 30 minutes. These non-migrated A427 and A549 cells were removed by cotton swabs. After that cells on the bottom surface of the membrane were stained with the 0.1% crystal violet for 20 minutes. Images of A427 and A549 cells were taken under a phase-contrast microscope. Luciferase assay A427 and A549 cells were seeded in 24-well plates at 3x105 cells/well and
ACCEPTED MANUSCRIPT incubated for 24 hours before transfection. In the reporter gene assay, the A427 and A549 cells were co-transfected with 0.6 µg of pGL3-PTEN-3’UTR or pGL3-PTEN-3’UTR Mut plasmid, 0.06 ng of the phRL-SV40 control vector (Promega, Shanghai, China), and 100 nM miR-543 or control RNA using
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Lipofectamine 2000 (Invitrogen, Shanghai, China). The renilla and firefly luciferase activities were determined with a dual luciferase assay (Promega, Shanghai, China) 24 hours after transfection.
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Western Blot
Proteins were separated by 12% SDS-PAGE gel and transferred onto
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nitrocellulose membranes (Bio-Rad, Shanghai, China). Membranes was blocked by 5% non-fat milk and incubated with anti-P21 antibody (Abcam, Shanghai, China) or anti-β-actin antibody (Abcam, Shanghai, China). After being washed extensively, the secondary antibody (Abcam, Shanghai, China) was then added to the system. Finally, Immunoreactive protein bands were
Statistical Analysis
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detected with the Enhanced Chemiluminescence (ECL) system.
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Experiments were repeated at least three times. Statistical analyses were performed using R. Values were expressed as means ± S.D. Differences
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between groups were estimated with T-test. Statistical analysis were considered to be significant when P. value<0.05.
Results
The miR-543 and its predicted target gene were differentially expressed in NSCLC The expression patterns of miRNAs and their predicted target genes were analyzed between Gefitinib-resistant and Gefitinib-sensitive patients group. Among these pairs, only miR-543 (lgFC>0 & P-value<0.01) and it’s predicted
ACCEPTED MANUSCRIPT target PTEN (lgFC>0 & P-value<0.01) were found to be deregulated in pairs in Gefitinib-resistant NSCLC. Moreover, miR-543 and PTEN are differentially expressed between human gefitinib-resistant A549 NSCLC cells and gefitinib-sensitive A427 NSCLC cells (Figure 1). This leads to the hypothesis
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that miR-543 may play roles in gefitinib-resistant NSCLC by targeting PTEN. Therefore, we selected miR-543 and PTEN for further functional investigation in the human gefitinib-resistant A549 NSCLC cells and gefitinib-sensitive A427 NSCLC cells.
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MiR-543 targets and inhibits PTEN in NSCLC cells
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As predicted by miRanda[8] and TargetScan[9], there was complementarity between has-miR-543 and the 3’ UTR of PTEN. Overexpression of miR-543 reduced the protein levels of PTEN in NSCLC cells with time (Figure. 2 A and B). Conversely, miR-543 inhibitor increased the protein levels of PTEN in NSCLC cells with time (Figure. 2 A and B). The effect of miR-543 on the
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translation of PTEN mRNA into protein was then assessed by using a luciferase reporter assay (Figure. 2 C and D). the miR-543 inhibitor significantly enhanced the luciferase activity of the reporter gene with the wild
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type construct but not with the mutant PTEN 3’UTR construct (Figure. 2C), while enforced expression of miR-543 significantly reduced the luciferase
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activity of the reporter gene with the wild type construct but not with the mutant PTEN 3’UTR construct (Figure. 2D). These evidences indicate that miR-543 directly targeted the 3’UTR region of PTEN and negatively regulated its expression.
miR-543 promotes NSCLC cell proliferation, migration and invasion We explored the potential impact of miR-543 in NSCLC cell proliferation, migration and invasion in A427 and A549 cell lines. A427 and A549 cells were transfected with miR-543 mimics or inhibitor or inactive control cel-mir-67.
ACCEPTED MANUSCRIPT CCK-8 proliferation assay indicated that the cell proliferation was promoted in both of the miR-543-mimics-transfected NSCLC cell lines compared with inactive control cel-mir-67-transfected cell lines (Figure. 3). Conversely, miR-543 inhibitor significantly inhibited the proliferation of the A427 and A549
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cells (Figure. 3). Interestingly, migration and invasion assay showed that miR-543 inhibitors significantly inhibited the migration and invasion of A427 and A549 cells compared with the inactive cel-mir-67 control, whereas miR-543 mimics promoted cell migration and invasion in both of the A427 and
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A549 cells (Figure. 4).
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Discussion
In the past decades, miRNAs have be identified to be important regulators in cancer [10]. miR-543 is a new tumor-related miRNA in gastric cancers[11] and hepatocellular carcinoma[12]. The relationship between miR-543 and NSCLC has never been reported before. By analyzing the expression pattern of
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miR-543 and its predicted target genes, we found the clues for uncovering the roles of miR-543 in NSCLC, leading to the hypothesis that miR-543 may play roles in NSCLC by targeting PTEN.
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Thus, our further study intended to investigate the biological function of miR-543 in NSCLC. miR-543 promoted proliferation, migration and invasion of
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A427 and A549 cells, which suggest that miR-543 plays a critical role in the carcinogenesis of NSCLC. Then we addressed the potential molecular mechanisms of miR-543 in regulating proliferation, migration and invasion of NSCLC cells. In this study, western blots and luciferase assays showed that PTEN is a target gene of miR-543. miR-543 directly bound to the 3’UTR region of PTEN and negatively regulated its expression. PTEN encodes a putative protein tyrosine phosphatase that suppresses tumor cell growth by antagonizing protein tyrosine kinases and may regulate tumor cell invasion and metastasis through interactions at focal adhesions [13,14]. The
ACCEPTED MANUSCRIPT down-regulation of PTEN seems to be part of important downstream mechanisms of miR-543 in the Gefitinib-resistant mechanisms of NSCLC. In conclusion, our results have shown that miR-543, which is highly expressed in Gefitinib-resistant NSCLC, promotes NSCLC cell proliferation, migration and
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invasion through directly binding and negatively regulating PTEN. This novel miR-543 / PTEN axis may provide new insights into the mechanisms underlying Gefitinib-resistant NSCLC, and miR-543 may be a potential
Conflict of interest
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therapeutic target to improve the treatment of NSCLC in the future.
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Authors have declared that no competing interest exists.
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Figure Legends
Figure 1. The expression of miR-543 and PTEN in gefitinib-resistant and NSCLC
cells.
(A)
Relative
miR-543
levels
in
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gefitinib-sensitive
gefitinib-resistant A549 cells and gefitinib-sensitive A427 cells; (A) Relative
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A427 cells. *p<0.05, **p<0.01, and ***p<0.001.
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PTEN protein levels in gefitinib-resistant A549 cells and gefitinib-sensitive
Figure 2. miR-543 targets PTEN in NSCLC cells. (A) Protein of PTEN significantly decreased with time after transfection with miR-543 mimics in A427; (B) Protein of PTEN significantly decreased with time after transfection
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with miR-543 mimics in A549 cells; (C) The analysis of the relative luciferase activities of PTEN-WT, PTEN-MUT in A427 and A549 cells after transfection
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with anti-miR-543; (D) Protein of PTEN(P21) increased with time after transfection with miR-543 mimics in A427 and A549 cells. *p<0.05, **p<0.01,
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and ***p<0.001.
Figure 3. miR-543 regulates NSCLC cell proliferation. (A) Growth of NSCLC cells was shown after transfection with miR-543 mimics or inhibitor or inactive control in A427 cells. (B) Growth of NSCLC cells was shown after transfection with miR-543 mimics or inhibitor or inactive control in A549 cells. The growth index as assessed at 0, 24, 48 and 72 h;
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Figure 4. miR-543 regulates NSCLC cell migration and invasion. (A) Transwell analysis of NSCLC cells migration after treatment with miR-543 mimics,
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inhibitors or inactive control in A427 cells; (B) Transwell analysis of NSCLC cells migration after treatment with miR-543 mimics, inhibitors or inactive control in A549 cells; (C) Transwell analysis of NSCLC cells invasion after
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treatment with miR-543 mimics, inhibitors or inactive control in A427 cells; (D)
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Transwell analysis of NSCLC cells invasion after treatment with miR-543 mimics, inhibitors or inactive control in A549 cells. *p<0.05, **p<0.01, and
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***p<0.001.
ACCEPTED MANUSCRIPT Reference [1] Y.A. Ghouri, I. Mian, B. Blechacz, Cancer review: Cholangiocarcinoma, J Carcinog 14 (2015) 1. [2] M. Maemondo, A. Inoue, K. Kobayashi, S. Sugawara, S. Oizumi, H. Isobe, A. Gemma, M. Harada, H. Yoshizawa, I. Kinoshita, Y. Fujita, S. Okinaga, H. Hirano, K. Yoshimori, T. Harada, T. Ogura, M. Ando, H. Miyazawa, T. Tanaka, Y. Saijo, K. Hagiwara, S. Morita, T. Nukiwa, G. North-East Japan Study, Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR, N Engl J
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Med 362 (2010) 2380-2388.
[3] Y. Mizuguchi, T. Takizawa, H. Yoshida, E. Uchida, Dysregulated microRNAs in progression of hepatocellular carcinoma: A systematic review, Hepatol Res (2015).
[4] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell 136 (2009) 215-233.
[5] J.Y. Zhou, X. Chen, J. Zhao, Z. Bao, X. Chen, P. Zhang, Z.F. Liu, J.Y. Zhou, MicroRNA-34a overcomes
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HGF-mediated gefitinib resistance in EGFR mutant lung cancer cells partly by targeting MET, Cancer Lett 351 (2014) 265-271.
[6] O. Kovalchuk, J. Filkowski, J. Meservy, Y. Ilnytskyy, V.P. Tryndyak, V.F. Chekhun, I.P. Pogribny,
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Involvement of microRNA-451 in resistance of the MCF-7 breast cancer cells to chemotherapeutic drug doxorubicin, Mol Cancer Ther 7 (2008) 2152-2159. [7] V. Ambros, B. Bartel, D.P. Bartel, C.B. Burge, J.C. Carrington, X. Chen, G. Dreyfuss, S.R. Eddy, S. Griffiths-Jones, M. Marshall, M. Matzke, G. Ruvkun, T. Tuschl, A uniform system for microRNA annotation, RNA 9 (2003) 277-279.
[8] B. John, A.J. Enright, A. Aravin, T. Tuschl, C. Sander, D.S. Marks, Human MicroRNA targets, PLoS Biol 2 (2004) e363.
[9] B.P. Lewis, C.B. Burge, D.P. Bartel, Conserved seed pairing, often flanked by adenosines, indicates
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that thousands of human genes are microRNA targets, Cell 120 (2005) 15-20. [10] J. Miao, S. Wu, Z. Peng, M. Tania, C. Zhang, MicroRNAs in osteosarcoma: diagnostic and therapeutic aspects, Tumour Biol 34 (2013) 2093-2098. [11] J. Li, G. Dong, B. Wang, W. Gao, Q. Yang, miR-543 promotes gastric cancer cell proliferation by targeting SIRT1, Biochem Biophys Res Commun 469 (2016) 15-21.
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[12] L. Yu, L. Zhou, Y. Cheng, L. Sun, J. Fan, J. Liang, M. Guo, N. Liu, L. Zhu, MicroRNA-543 acts as an oncogene by targeting PAQR3 in hepatocellular carcinoma, Am J Cancer Res 4 (2014) 897-906.
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[13] J. Li, C. Yen, D. Liaw, K. Podsypanina, S. Bose, S.I. Wang, J. Puc, C. Miliaresis, L. Rodgers, R. McCombie, S.H. Bigner, B.C. Giovanella, M. Ittmann, B. Tycko, H. Hibshoosh, M.H. Wigler, R. Parsons, PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer, Science 275 (1997) 1943-1947.
[14] C.W. Armstrong, P.J. Maxwell, C.W. Ong, K.M. Redmond, C. McCann, J. Neisen, G.A. Ward, G. Chessari, C. Johnson, N.T. Crawford, M.J. LaBonte, K.M. Prise, T. Robson, M. Salto-Tellez, D.B. Longley, D.J. Waugh, PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy, Oncotarget (2016).
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ACCEPTED MANUSCRIPT 1. miR-543 is highly expressed in gefitinib-resistant NSCLC. 2. miR-543 promotes the proliferation and invasion of NSCLC cells. 3. miR-543 inhibitors inhibits the proliferation and invasion of NSCLC cells. 4. miR-543 targets 3’ UTR of PTEN in NSCLC cells.
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5. miR-543 inhibits PTEN in NSCLC cells.
S0006-291X(16)30478-8
DOI:
10.1016/j.bbrc.2016.03.157
Reference:
YBBRC 35589
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 26 March 2016 Accepted Date: 30 March 2016
Please cite this article as: P. Zhang, H.-X. Zhou, M.-X. Yang, Y. Wang, W.-M. Cao, K.-F. Lu, R.-Q. Wu, miR-543 promotes proliferation and invasion of non-small cell lung cancer cells by inhibiting PTEN, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.03.157. 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.
ACCEPTED MANUSCRIPT
miR-543 promotes proliferation and invasion of non-small cell lung cancer cells by inhibiting PTEN.
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Ping Zhang1,#,*,Hong-Xing Zhou1,#, Ming-Xia Yang2, Yan Wang1, Wen-Ming Cao1, Ke-Feng Lu1, Rong-Qiang Wu1, 1. Department of Clinical Laboratory, Affiliated Changzhou Second Hospital of Nanjing Medical University, Changzhou, Jiangsu, China 2. Respiratory Medicine, Affiliated Changzhou Second Hospital of Nanjing Medical University, Changzhou, Jiangsu, China
*Corresponding author: Ping Zhang
Contributed equally to this work.
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Address: 68 Gehu Road in Wujin district, Changzhou, Jiangsu Province, 213164, China. Phone: +8613813658238, Fax: +8651981087709. E-mail: [email protected]
Key words: miR-543, PTEN, NSCLC, gefitinib
ACCEPTED MANUSCRIPT
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-543 is up-regulated in gefitinib-resistant non-small
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cell lung cancer (NSCLC) patients comparing gefitinib-sensitive ones. It promotes NSCLC cell proliferation by negatively regulates its target gene PTEN. In NSCLC cell lines, CCK-8 proliferation assay indicated that the cell
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proliferation is promoted by miR-543 mimics. Transwell assay showed that miR-543 mimics promotes the invasion and migration of NSCLC cells.
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Luciferase assays confirmed that miR-543 directly binds to the 3'untranslated region of PTEN, and western blotting showed that miR-543 suppresses the expression of PTEN at the protein level. This study indicates that miR-543 promtes proliferation and invasion of NSCLC cell lines by PTEN. The miR-543 may represent a potential therapeutic target for gefitinib-resistant NSCLC
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intervention.
ACCEPTED MANUSCRIPT
Introduction Gefitinib, the first selective inhibitor of epidermal growth factor receptor's (EGFR) tyrosine kinase domain, is used to treat non-small cell lung cancer (NSCLC), which is the one of the most common cause of cancer-related death
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worldwide[1,2]. However, resistance to gefitinib limits its therapeutic effects on NSCLC patients. The discovery of MicroRNAs (miRNAs) opened a new generation for understanding the carcinogenesis of NSCLC[3]. miRNAs are
SC
small non-coding RNAs which inhibit the expression of target genes by translational repression or mRNA degradation[4]. Some miRNAs, e.g. miR-
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34a[5] and MiR- 451[6] are found to play roles in the mechanisms underlying drug resistance to NSCLC by regulating their target genes. Underlying the miRNA-related molecular mechanisms underlying gefitinib-resistant NSCLC will be helpful to improve NSCLC therapies.
Therefore, we analyzed the expression pattern of miRNA and genes in
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gefitinib-resistant and -sensitive NSCLC, and found miR-543 and its predicted target gene PTEN was deregulated in gefitinib-resistant NSCLC patients comparing to sensitive ones. This leads to the hypothesis that miR-543 may
EP
play roles in NSCLC by PTEN. Further experiments in NSCLC cell lines confirmed that miR-543 directly bound to the 3'untranslated region of PTEN,
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and suppressed the expression of PTEN at the protein levels. Moreover, modulation of miR-543 expression regulated proliferation and invasion of NSCLC cell lines.
Materials and Methods 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,
ACCEPTED MANUSCRIPT GSE51828, GSE34228). After quality control, 4 Gefitinib-resistant NSCLC samples and 4 Gefitinib-sensitive NSCLC samples were used in further miRNA analysis,
while
104
Gefitinib-resistant
NSCLC
samples
and
104
Gefitinib-sensitive NSCLC samples were used in further mRNA analysis.
The
identification
of
differentially
expressed
RI PT
Identification of differentially expressed miRNA and mRNA
miRNA and
mRNA in
durg-resistant patients with NSCLC tissues were performed with Limma
SC
package on R platform using download miRNA and mRNA profiles data as mentioned above. The cutline of significantly differentially expressed miRNA
M AN U
and mRNA is P.value<0.01 (T test) and |lgFC|>1(fold change). miRNA Target Genes Prediction
Human miRNA target genes prediction uses miRNA sequences that downloaded from the Rfam website (http://www.sanger.ac.uk/Software/Rfam)
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and satisfy the established criteria[7]. The target genes of miRNAs were predicted using miRanda[8] and TargetScan[9] methods. The predicted target genes supported by both methods were selected for further analysis.
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Cell Culture and Transfection
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The human NSCLC cell lines, A427 and A549 was obtained from American Type Culture Collection (ATCC, USA). The A427 and A549 cell line was cultured in RPMI 1640 media(Life Technologies, Shanghai, China) and supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Shanghai, China). Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C. A427 and A549 cell lines were seeded i n 24-well plates at 3× 105 cells/wells and incubated overnight. Transfection of the miR-543 miRNA mimic, the anti-miR-543, inactive control cel-mir-67 (Life Technologies, Shanghai, China), or pMIR-Report vectors was taken using Lipofectamine
ACCEPTED MANUSCRIPT 2000 transfection reagent (Invitrogen, Shanghai, China) with 300 nmol of miRNA or 1µg/ml DNA plasmid, respectively. Total proteins of A427 and A549 cells were isolated at 48 hours after transfection.
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Cell Proliferation Cell proliferations were measured using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). A427 and A549 NSCLC cells were plated in 24-well plates at 3x105 cells/well. Then cells were incubated in 10% CCK-8 which was diluted
SC
in normal culture medium at 37oC for color conversion. Proliferation rates were
Cell migration and Invasion
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determined at 24, 48 and 72 hours after transfection.
Cell invasion and migration were measured using a transwell chamber (Corning, Shanghai, China) with and without Matrigel (Invitrogen, Shanghai, China). For the determination of A427 and A549 NSCLC cells invasion,
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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, A427 and A549 cells were trypsinized and then seeded in
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chambers at the density of 8x104 cells/well at 48 hours after transfection. These cells were cultured in RPMI 1640 medium with 2% serum. Meanwhile
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600 µl of 10% FBS-1640 was added to the lower chamber. After 24 hours, migrated A427 and A549 cells were fixed in 100% methanol for 30 minutes. These non-migrated A427 and A549 cells were removed by cotton swabs. After that cells on the bottom surface of the membrane were stained with the 0.1% crystal violet for 20 minutes. Images of A427 and A549 cells were taken under a phase-contrast microscope. Luciferase assay A427 and A549 cells were seeded in 24-well plates at 3x105 cells/well and
ACCEPTED MANUSCRIPT incubated for 24 hours before transfection. In the reporter gene assay, the A427 and A549 cells were co-transfected with 0.6 µg of pGL3-PTEN-3’UTR or pGL3-PTEN-3’UTR Mut plasmid, 0.06 ng of the phRL-SV40 control vector (Promega, Shanghai, China), and 100 nM miR-543 or control RNA using
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Lipofectamine 2000 (Invitrogen, Shanghai, China). The renilla and firefly luciferase activities were determined with a dual luciferase assay (Promega, Shanghai, China) 24 hours after transfection.
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Western Blot
Proteins were separated by 12% SDS-PAGE gel and transferred onto
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nitrocellulose membranes (Bio-Rad, Shanghai, China). Membranes was blocked by 5% non-fat milk and incubated with anti-P21 antibody (Abcam, Shanghai, China) or anti-β-actin antibody (Abcam, Shanghai, China). After being washed extensively, the secondary antibody (Abcam, Shanghai, China) was then added to the system. Finally, Immunoreactive protein bands were
Statistical Analysis
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detected with the Enhanced Chemiluminescence (ECL) system.
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Experiments were repeated at least three times. Statistical analyses were performed using R. Values were expressed as means ± S.D. Differences
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between groups were estimated with T-test. Statistical analysis were considered to be significant when P. value<0.05.
Results
The miR-543 and its predicted target gene were differentially expressed in NSCLC The expression patterns of miRNAs and their predicted target genes were analyzed between Gefitinib-resistant and Gefitinib-sensitive patients group. Among these pairs, only miR-543 (lgFC>0 & P-value<0.01) and it’s predicted
ACCEPTED MANUSCRIPT target PTEN (lgFC>0 & P-value<0.01) were found to be deregulated in pairs in Gefitinib-resistant NSCLC. Moreover, miR-543 and PTEN are differentially expressed between human gefitinib-resistant A549 NSCLC cells and gefitinib-sensitive A427 NSCLC cells (Figure 1). This leads to the hypothesis
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that miR-543 may play roles in gefitinib-resistant NSCLC by targeting PTEN. Therefore, we selected miR-543 and PTEN for further functional investigation in the human gefitinib-resistant A549 NSCLC cells and gefitinib-sensitive A427 NSCLC cells.
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MiR-543 targets and inhibits PTEN in NSCLC cells
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As predicted by miRanda[8] and TargetScan[9], there was complementarity between has-miR-543 and the 3’ UTR of PTEN. Overexpression of miR-543 reduced the protein levels of PTEN in NSCLC cells with time (Figure. 2 A and B). Conversely, miR-543 inhibitor increased the protein levels of PTEN in NSCLC cells with time (Figure. 2 A and B). The effect of miR-543 on the
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translation of PTEN mRNA into protein was then assessed by using a luciferase reporter assay (Figure. 2 C and D). the miR-543 inhibitor significantly enhanced the luciferase activity of the reporter gene with the wild
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type construct but not with the mutant PTEN 3’UTR construct (Figure. 2C), while enforced expression of miR-543 significantly reduced the luciferase
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activity of the reporter gene with the wild type construct but not with the mutant PTEN 3’UTR construct (Figure. 2D). These evidences indicate that miR-543 directly targeted the 3’UTR region of PTEN and negatively regulated its expression.
miR-543 promotes NSCLC cell proliferation, migration and invasion We explored the potential impact of miR-543 in NSCLC cell proliferation, migration and invasion in A427 and A549 cell lines. A427 and A549 cells were transfected with miR-543 mimics or inhibitor or inactive control cel-mir-67.
ACCEPTED MANUSCRIPT CCK-8 proliferation assay indicated that the cell proliferation was promoted in both of the miR-543-mimics-transfected NSCLC cell lines compared with inactive control cel-mir-67-transfected cell lines (Figure. 3). Conversely, miR-543 inhibitor significantly inhibited the proliferation of the A427 and A549
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cells (Figure. 3). Interestingly, migration and invasion assay showed that miR-543 inhibitors significantly inhibited the migration and invasion of A427 and A549 cells compared with the inactive cel-mir-67 control, whereas miR-543 mimics promoted cell migration and invasion in both of the A427 and
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A549 cells (Figure. 4).
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Discussion
In the past decades, miRNAs have be identified to be important regulators in cancer [10]. miR-543 is a new tumor-related miRNA in gastric cancers[11] and hepatocellular carcinoma[12]. The relationship between miR-543 and NSCLC has never been reported before. By analyzing the expression pattern of
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miR-543 and its predicted target genes, we found the clues for uncovering the roles of miR-543 in NSCLC, leading to the hypothesis that miR-543 may play roles in NSCLC by targeting PTEN.
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Thus, our further study intended to investigate the biological function of miR-543 in NSCLC. miR-543 promoted proliferation, migration and invasion of
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A427 and A549 cells, which suggest that miR-543 plays a critical role in the carcinogenesis of NSCLC. Then we addressed the potential molecular mechanisms of miR-543 in regulating proliferation, migration and invasion of NSCLC cells. In this study, western blots and luciferase assays showed that PTEN is a target gene of miR-543. miR-543 directly bound to the 3’UTR region of PTEN and negatively regulated its expression. PTEN encodes a putative protein tyrosine phosphatase that suppresses tumor cell growth by antagonizing protein tyrosine kinases and may regulate tumor cell invasion and metastasis through interactions at focal adhesions [13,14]. The
ACCEPTED MANUSCRIPT down-regulation of PTEN seems to be part of important downstream mechanisms of miR-543 in the Gefitinib-resistant mechanisms of NSCLC. In conclusion, our results have shown that miR-543, which is highly expressed in Gefitinib-resistant NSCLC, promotes NSCLC cell proliferation, migration and
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invasion through directly binding and negatively regulating PTEN. This novel miR-543 / PTEN axis may provide new insights into the mechanisms underlying Gefitinib-resistant NSCLC, and miR-543 may be a potential
Conflict of interest
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therapeutic target to improve the treatment of NSCLC in the future.
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Authors have declared that no competing interest exists.
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Figure Legends
Figure 1. The expression of miR-543 and PTEN in gefitinib-resistant and NSCLC
cells.
(A)
Relative
miR-543
levels
in
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gefitinib-sensitive
gefitinib-resistant A549 cells and gefitinib-sensitive A427 cells; (A) Relative
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A427 cells. *p<0.05, **p<0.01, and ***p<0.001.
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PTEN protein levels in gefitinib-resistant A549 cells and gefitinib-sensitive
Figure 2. miR-543 targets PTEN in NSCLC cells. (A) Protein of PTEN significantly decreased with time after transfection with miR-543 mimics in A427; (B) Protein of PTEN significantly decreased with time after transfection
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with miR-543 mimics in A549 cells; (C) The analysis of the relative luciferase activities of PTEN-WT, PTEN-MUT in A427 and A549 cells after transfection
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with anti-miR-543; (D) Protein of PTEN(P21) increased with time after transfection with miR-543 mimics in A427 and A549 cells. *p<0.05, **p<0.01,
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and ***p<0.001.
Figure 3. miR-543 regulates NSCLC cell proliferation. (A) Growth of NSCLC cells was shown after transfection with miR-543 mimics or inhibitor or inactive control in A427 cells. (B) Growth of NSCLC cells was shown after transfection with miR-543 mimics or inhibitor or inactive control in A549 cells. The growth index as assessed at 0, 24, 48 and 72 h;
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Figure 4. miR-543 regulates NSCLC cell migration and invasion. (A) Transwell analysis of NSCLC cells migration after treatment with miR-543 mimics,
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inhibitors or inactive control in A427 cells; (B) Transwell analysis of NSCLC cells migration after treatment with miR-543 mimics, inhibitors or inactive control in A549 cells; (C) Transwell analysis of NSCLC cells invasion after
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treatment with miR-543 mimics, inhibitors or inactive control in A427 cells; (D)
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Transwell analysis of NSCLC cells invasion after treatment with miR-543 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 1. miR-543 is highly expressed in gefitinib-resistant NSCLC. 2. miR-543 promotes the proliferation and invasion of NSCLC cells. 3. miR-543 inhibitors inhibits the proliferation and invasion of NSCLC cells. 4. miR-543 targets 3’ UTR of PTEN in NSCLC cells.
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5. miR-543 inhibits PTEN in NSCLC cells.