Please wait a minute...
Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2017, Vol. 11 Issue (2) : 214-222    https://doi.org/10.1007/s11684-017-0518-7
RESEARCH ARTICLE
Overexpressed miR-9 promotes tumor metastasis via targeting E-cadherin in serous ovarian cancer
Bo Zhou1,2, Hongbin Xu3, Meng Xia1, Chaoyang Sun1, Na Li1, Ensong Guo1, Lili Guo1, Wanying Shan1, Hao Lu1, Yifan Wu1, Yuan Li1, Degui Yang3, Danhui Weng1, Li Meng1, Junbo Hu1, Ding Ma1, Gang Chen1, Kezhen Li1()
1. Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
2. Department of Gynecological Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Wuhan 430071, China
3. Department of Gynecology and Obstetrics, the People’s Hospital of Shenzhen, Shenzhen 518000, China
 Download: PDF(363 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract

MicroRNAs (miRNAs) play critical roles in the development and progression in various cancers. Dysfunctional miR-9 expression remains ambiguous, and no consensus on the metastatic progression of ovarian cancer has been reached. In this study, results from the bioinformatics analysis show that the 3′-UTR of the E-cadherin mRNA was directly regulated by miR-9. Luciferase reporter assay results confirmed that miR-9 could directly target this 3′-UTR. miR-9 and E-cadherin expression in ovarian cancer tissue was quantified by qRT-PCR. Migration and invasion were detected by wound healing and Transwell system assay in SKOV3 and A2780. qRT-PCR and Western blot were performed to detect the epithelial?mesenchymal transition-associated mRNA and proteins. Immunofluorescence technique was used to analyze the expression and subcellular localization of E-cadherin, N-cadherin, and vimentin. The results showed that miR-9 was frequently upregulated in metastatic serous ovarian cancer tissue compared with paired primary ones. Upregulation of miR-9 could downregulate the expression of E-cadherin but upregulate the expression of mesenchymal markers (N-cadherin and vimentin). Overexpression of miR-9 could promote the cell migration and invasion in ovarian cancer, and these processes could be effectively inhibited via miR-9 inhibitor. Thus, our study demonstrates that miR-9 may promote ovarian cancer metastasis via targeting E-cadherin and a novel potential therapeutic approach to control metastasis of ovarian cancer.

Keywords ovarian cancer      metastasis      miR-9      E-cadherin     
Corresponding Author(s): Kezhen Li   
Online First Date: 04 May 2017    Issue Date: 01 June 2017
 Cite this article:   
Bo Zhou,Hongbin Xu,Meng Xia, et al. Overexpressed miR-9 promotes tumor metastasis via targeting E-cadherin in serous ovarian cancer[J]. Front. Med., 2017, 11(2): 214-222.
 URL:  
https://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0518-7
https://academic.hep.com.cn/fmd/EN/Y2017/V11/I2/214
Fig.1  miR-9 was upregulated and correlated with the expression of E-cadherin and vimentin in metastatic ovarian cancer tissue compared with primary tissue. (A) qRT-PCR shows that miR-9 was frequently upregulated in the 25 metastatic site tissue samples compared with their paired primary tissue (P<0.05). (B) Upregulated miR-9 was also detected in five paired patient-derived serous ovarian tumor cell lines. U6 was used as an endogenous control. (C and D) The expression of E-cadherin and vimentin was detected by qRT-PCR at 25 metastatic sites tissue samples compared with their paired primary tissue. The relative expression was normalized to b-actin.
Fig.2  miR-9 directly targets E-cadherin in ovarian cancer cells. (A) Upregulated miR-9 was detected by qRT-PCR using miR-9 mimics in SKOV3 and A2780 cells. (B and C) The relative E-cadherin expression was downregulated after miR-9 mimics transfection for 36 or 48 h as detected by qRT-PCR or Western blot, respectively. (D and E) Relative E-cadherin expression was upregulated after miR-9 inhibitor transfection for 36 or 48 h as detected by qRT-PCR or Western blot, respectively. (F) Potential binding sites of miR-9 in the 3′-UTR of E-cadherin. (G) Plasmid containing wild-type or mutant-type 3′-UTR of E-cadherin was co-transfected in SKOV3 cells with miR-9 mimics. Dual-luciferase reporter assay showed that transfected miR-9 could reduce the luciferase activity in wild-type 3′-UTR (wt-3′-UTR) of E-cadherin but not in mutant-type 3′ UTR (mut-3′ UTR) of E-cadherin. Data are expressed as mean±SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. NS represents no significance.
Fig.3  miR-9 promotes ovarian cancer cell metastasis. (A and B) Phase contrast images of SKOV3 and A2780 cells transfected with miR-9 mimics or inhibitor. Images showed that these cells become scattered and displayed spindle-like or fibroblast morphology. Transfected miR-9 inhibitor could reverse this morphology. (C) Wound healing assay showed that transfected miR-9 mimics promoted SKOV3 cell migration compared with transfection with miR-9 inhibitor or miR-9 NC. Images were taken at 0 and 24 h after wound application. (D) Images and summary of SKOV3 cell invasion and migration assay. Transfected miR-9 mimics enhanced the invasion and migration capabilities of SKOV3 cells compared with those of transfected miR-9 NC cells, which could be rescued by transfection of miR-9 inhibitor. Data are expressed as mean±SD of three independent experiments. *P<0.05, **P<0.01.
Fig.4  miR-9 induces ovarian cancer cells epithelial?mesenchymal transition (EMT). (A and B) A2780 and SKOV3 cells were transfected with miR-9 mimics. The data showed that E-cadherin was downregulated, but mesenchymal markers were upregulated, respectively. (C) IF showed that the intensity of E-cadherin was decreased, while the intensity of N-cadherin was increased after miR-9 mimic transfection for 48 h in A2780 cells. (D) IF showed that the intensity of E-cadherin was decreased, while the intensity of vimentin was increased after miR-9 mimic transfection for 48 h in SKOV3 cells.
1 Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer J Clin 2013; 63(1): 11–30
https://doi.org/10.3322/caac.21166
2 Landen CN Jr, Birrer MJ, Sood AK. Early events in the pathogenesis of epithelial ovarian cancer. J Clin Oncol 2008; 26(6): 995–1005
https://doi.org/10.1200/JCO.2006.07.9970
3 Cho KR, Shih Ie M. Ovarian cancer. Annu Rev Pathol 2009; 4(1): 287–313
https://doi.org/10.1146/annurev.pathol.4.110807.092246
4 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116(2): 281–297
https://doi.org/10.1016/S0092-8674(04)00045-5
5 Slack FJ, Weidhaas JB. MicroRNA in cancer prognosis. N Engl J Med 2008; 359(25): 2720–2722
https://doi.org/10.1056/NEJMe0808667
6 Croce CM, Calin GA. miRNAs, cancer, and stem cell division. Cell 2005; 122(1): 6–7
https://doi.org/10.1016/j.cell.2005.06.036
7 Chen CZ, Li L, Lodish HF, Bartel DP. MicroRNAs modulate hematopoietic lineage differentiation. Science 2004; 303(5654): 83–86
https://doi.org/10.1126/science.1091903
8 Ma L, Young J, Prabhala H, Pan E, Mestdagh P, Muth D, Teruya-Feldstein J, Reinhardt F, Onder TT, Valastyan S, Westermann F, Speleman F, Vandesompele J, Weinberg RA. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 2010; 12(3): 247–256
9 Song Y, Li J, Zhu Y, Dai Y, Zeng T, Liu L, Li J, Wang H, Qin Y, Zeng M, Guan XY, Li Y. MicroRNA-9 promotes tumor metastasis via repressing E-cadherin in esophageal squamous cell carcinoma. Oncotarget 2014; 5(22): 11669–11680
https://doi.org/10.18632/oncotarget.2581
10 Sun Z, Han Q, Zhou N, Wang S, Lu S, Bai C, Zhao RC. MicroRNA-9 enhances migration and invasion through KLF17 in hepatocellular carcinoma. Mol Oncol 2013; 7(5): 884–894
https://doi.org/10.1016/j.molonc.2013.04.007
11 Zhu L, Chen H, Zhou D, Li D, Bai R, Zheng S, Ge W. MicroRNA-9 up-regulation is involved in colorectal cancer metastasis via promoting cell motility. Med Oncol 2012; 29(2): 1037–1043
https://doi.org/10.1007/s12032-011-9975-z
12 Shiiyama R, Fukushima S, Jinnin M, Yamashita J, Miyashita A, Nakahara S, Kogi A, Aoi J, Masuguchi S, Inoue Y, Ihn H. Sensitive detection of melanoma metastasis using circulating microRNA expression profiles. Melanoma Res 2013; 23(5): 366–372
https://doi.org/10.1097/CMR.0b013e328363e485
13 Iorio MV, Visone R, Di Leva G, Donati V, Petrocca F, Casalini P, Taccioli C, Volinia S, Liu CG, Alder H, Calin GA, Menard S, Croce CM. MicroRNA signatures in human ovarian cancer. Cancer Res 2007; 67(18): 8699–8707
https://doi.org/10.1158/0008-5472.CAN-07-1936
14 Laios A, O’Toole S, Flavin R, Martin C, Kelly L, Ring M, Finn SP, Barrett C, Loda M, Gleeson N, D’Arcy T, McGuinness E, Sheils O, Sheppard B, O’ Leary J. Potential role of miR-9 and miR-223 in recurrent ovarian cancer. Mol Cancer 2008; 7(1): 35
https://doi.org/10.1186/1476-4598-7-35
15 Sun C, Li N, Yang Z, Zhou B, He Y, Weng D, Fang Y, Wu P, Chen P, Yang X, Ma D, Zhou J, Chen G. miR-9 regulation of BRCA1 and ovarian cancer sensitivity to cisplatin and PARP inhibition. J Natl Cancer Inst 2013; 105(22): 1750–1758
https://doi.org/10.1093/jnci/djt302
16 Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2002; 2(6): 442–454
https://doi.org/10.1038/nrc822
17 Dai Y, Zhou X. Computational methods for the identification of microRNA targets. Open Access Bioinformatics 2010; 2:29–39
18 Dweep H, Sticht C, Pandey P, Gretz N. miRWalk–database: prediction of possible miRNA binding sites by “walking” the genes of three genomes. J Biomed Inform 2011; 44(5): 839–847
https://doi.org/10.1016/j.jbi.2011.05.002
19 Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2(2): 329–333
https://doi.org/10.1038/nprot.2007.30
20 Valster A, Tran NL, Nakada M, Berens ME, Chan AY, Symons M. Cell migration and invasion assays. Methods 2005; 37(2): 208–215
https://doi.org/10.1016/j.ymeth.2005.08.001
21 Weng D, Song X, Xing H, Ma X, Xia X, Weng Y, Zhou J, Xu G, Meng L, Zhu T, Wang S, Ma D. Implication of the Akt2/survivin pathway as a critical target in paclitaxel treatment in human ovarian cancer cells. Cancer Lett 2009; 273(2): 257–265
https://doi.org/10.1016/j.canlet.2008.08.027
22 Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009; 139(5): 871–890
https://doi.org/10.1016/j.cell.2009.11.007
23 Vergara D, Merlot B, Lucot JP, Collinet P, Vinatier D, Fournier I, Salzet M. Epithelial-mesenchymal transition in ovarian cancer. Cancer Lett 2010; 291(1): 59–66
https://doi.org/10.1016/j.canlet.2009.09.017
24 Hurst DR, Edmonds MD, Welch DR. MetastamiR: the field of metastasis-regulatory microRNA is spreading. Cancer Res 2009; 69(19): 7495–7498
https://doi.org/10.1158/0008-5472.CAN-09-2111
25 Deo M, Yu JY, Chung KH, Tippens M, Turner DL. Detection of mammalian microRNA expression by in situ hybridization with RNA oligonucleotides. Dev Dyn 2006; 235(9): 2538–2548
https://doi.org/10.1002/dvdy.20847
26 Nass D, Rosenwald S, Meiri E, Gilad S, Tabibian-Keissar H, Schlosberg A, Kuker H, Sion-Vardy N, Tobar A, Kharenko O, Sitbon E, Lithwick Yanai G, Elyakim E, Cholakh H, Gibori H, Spector Y, Bentwich Z, Barshack I, Rosenfeld N. miR-92b and miR-9/9* are specifically expressed in brain primary tumors and can be used to differentiate primary from metastatic brain tumors. Brain Pathol 2009; 19(3): 375–383
https://doi.org/10.1111/j.1750-3639.2008.00184.x
27 Luo X, Fan S, Huang W, Zhai S, Ma Z, Li P, Sun SY, Wang X. Downregulation of IRS-1 promotes metastasis of head and neck squamous cell carcinoma. Oncol Rep 2012; 28(2): 659–667
28 Lu MH, Huang CC, Pan MR, Chen HH, Hung WC. Prospero homeobox 1 promotes epithelial-mesenchymal transition in colon cancer cells by inhibiting E-cadherin via miR-9. Clin Cancer Res 2012; 18(23): 6416–6425
https://doi.org/10.1158/1078-0432.CCR-12-0832
29 Gwak JM, Kim HJ, Kim EJ, Chung YR, Yun S, Seo AN, Lee HJ, Park SY. MicroRNA-9 is associated with epithelial-mesenchymal transition, breast cancer stem cell phenotype, and tumor progression in breast cancer. Breast Cancer Res Treat 2014; 147(1): 39–49
https://doi.org/10.1007/s10549-014-3069-5
30 Wilting SM, Snijders PJ, Verlaat W, Jaspers A, van de Wiel MA, van Wieringen WN, Meijer GA, Kenter GG, Yi Y, le Sage C, Agami R, Meijer CJ, Steenbergen RD. Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis. Oncogene 2013; 32(1): 106–116
https://doi.org/10.1038/onc.2012.20
31 Zheng L, Qi T, Yang D, Qi M, Li D, Xiang X, Huang K, Tong Q. MicroRNA-9 suppresses the proliferation, invasion and metastasis of gastric cancer cells through targeting cyclin D1 and Ets1. PLoS One 2013; 8(1): e55719
https://doi.org/10.1371/journal.pone.0055719
32 Omura N, Li CP, Li A, Hong SM, Walter K, Jimeno A, Hidalgo M, Goggins M. Genome-wide profiling of methylated promoters in pancreatic adenocarcinoma. Cancer Biol Ther 2008; 7(7): 1146–1156
https://doi.org/10.4161/cbt.7.7.6208
33 Lehmann U, Hasemeier B, Christgen M, Muller M, Romermann D, Langer F, Kreipe H. Epigenetic inactivation of microRNA gene hsa-mir-9–1 in human breast cancer. J Pathol 2008; 214(1): 17–24
https://doi.org/10.1002/path.2251
34 Inoue T, Iinuma H, Ogawa E, Inaba T, Fukushima R. Clinicopathological and prognostic significance of microRNA-107 and its relationship to DICER1 mRNA expression in gastric cancer. Oncol Rep 2012; 27(6): 1759–1764
35 Qiu Y, Luo X, Kan T, Zhang Y, Yu W, Wei Y, Shen N, Yi B, Jiang X. TGF-β upregulates miR-182 expression to promote gallbladder cancer metastasis by targeting CADM1. Mol Biosyst 2014; 10(3): 679–685
https://doi.org/10.1039/c3mb70479c
36 Yu J, Lei R, Zhuang X, Li X, Li G, Lev S, Segura MF, Zhang X, Hu G. MicroRNA-182 targets SMAD7 to potentiate TGFβ-induced epithelial-mesenchymal transition and metastasis of cancer cells. Nat Commun 2016; 7: 13884
https://doi.org/10.1038/ncomms13884
[1] Yun Zhang, Robert A. Weinberg. Epithelial-to-mesenchymal transition in cancer: complexity and opportunities[J]. Front. Med., 2018, 12(4): 361-373.
[2] Yugang Cheng,Hanxiang Zhan,Lei Wang,Jianwei Xu,Guangyong Zhang,Zongli Zhang,Sanyuan Hu. Analysis of 100 consecutive cases of resectable pancreatic neuroendocrine neoplasms: clinicopathological characteristics and long-term outcomes[J]. Front. Med., 2016, 10(4): 444-450.
[3] Chunxiao Li,Haijuan Wang,Feng Lin,Hui Li,Tao Wen,Haili Qian,Qimin Zhan. Bioinformatic exploration of MTA1-regulated gene networks in colon cancer[J]. Front. Med., 2016, 10(2): 178-182.
[4] Jiangnan Liu,Bin Yi,Zhe Zhang,Yi Cao. CD176 single-chain variable antibody fragment inhibits the adhesion of cancer cells to endothelial cells and hepatocytes[J]. Front. Med., 2016, 10(2): 204-211.
[5] Felice Ho-Ching Tsang,Sandy Leung-Kuen Au,Lai Wei,Dorothy Ngo-Yin Fan,Joyce Man-Fong Lee,Carmen Chak-Lui Wong,Irene Oi-Lin Ng,Chun-Ming Wong. MicroRNA-142-3p and microRNA-142-5p are downregulated in hepatocellular carcinoma and exhibit synergistic effects on cell motility[J]. Front. Med., 2015, 9(3): 331-343.
[6] Lunxiu Qin. Osteopontin is a promoter for hepatocellular carcinoma metastasis: a summary of 10 years of studies[J]. Front Med, 2014, 8(1): 24-32.
[7] Xianxian Li, Hui Xing, Lin Li, Yanli Huang, Min Zhou, Qiong Liu, Xiaomin Qin, Min He. Clinical significance of para-aortic lymph node dissection and prognosis in ovarian cancer[J]. Front Med, 2014, 8(1): 96-100.
[8] Tian Wang, Yan Li, Abidan Tuerhanjiang, Wenwen Wang, Zhangying Wu, Ming Yuan, Shixuan Wang. Correlation of Twist upregulation and senescence bypass during the progression and metastasis of cervical cancer[J]. Front Med, 2014, 8(1): 106-112.
[9] Cunjian Yi, Lei Zhang, Fayun Zhang, Li Li, Shengrong Ling, Xiaowen Wang, Xiangqiong Liu, Wei Liang. Methodologies for the establishment of an orthotopic transplantation model of ovarian cancer in mice[J]. Front Med, 2014, 8(1): 101-105.
[10] Carmen Chak-Lui Wong, Alan Ka-Lun Kai, Irene Oi-Lin Ng. The impact of hypoxia in hepatocellular carcinoma metastasis[J]. Front Med, 2014, 8(1): 33-41.
[11] Lei CHEN, Liang HU, Liang LI, Yuan LIU, Qian-Qian TU, Yan-Xin CHANG, He-Xin YAN, Meng-Chao WU, Hong-Yang WANG, . Dysregulation of &#946;-catenin by hepatitis B virus X protein in HBV-infected human hepatocellular carcinomas[J]. Front. Med., 2010, 4(4): 399-411.
[12] Dong KUANG, Guo-Ping WANG, . Hilar cholangiocarcinoma: Pathology and tumor biology[J]. Front. Med., 2010, 4(4): 371-377.
[13] Ling XU MM, Feng WANG MM, Xuan-Fu XU MD, Wen-Hui MO BM, Rong WAN MD, Chuan-Yong GUO MD, Xing-Peng WANG MD, . Data mining of microarray for differentially expressed genes in liver metastasis from gastric cancer[J]. Front. Med., 2010, 4(2): 247-253.
[14] Liu LIU MD, PhD, Yaogui NING MM, Chen CHEN MD, Daowen WANG MD, PhD, . Effect of atorvastatin on tumor growth and metastasis in a breast cancer cell xenograft model and its mechanism[J]. Front. Med., 2009, 3(4): 443-446.
[15] Bin YANG MS , Xianglin YUAN , Yanmei ZOU , Qingsong XI , Guoxian LONG , Qiang FU , Guangyuan HU MM , . Effects of hypoxia inducible factor-1alpha siRNA on the invasion of human Hela cells and expression of related proteins[J]. Front. Med., 2009, 3(3): 303-308.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed