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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     DOI: 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
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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 Authors: Kezhen Li   
Online First Date: 04 May 2017    Issue Date: 01 June 2017
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0518-7     OR     http://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.
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