miR-93 regulates epithelial-to-mesenchymal transition process in metastatic colorectal cancer by targeting EphA4
Sheng Gao1,2*, Bo jiang1,2,*, Haiyi Liu1,2, Shenghuai Hou1,2,Liming Wu3,4,5, Zhe Yang3,4,5, Junpeng Shen6, Lin Zhou3,4,5, Shu-Sen Zheng3,4,5 and Wenqi Bai1,2
Authors’ Affiliation: 1Department of Colorectal Cancer, Shanxi Cancer Hospital and Institute, Taiyuan 030013, China. 2Affiliated Cancer Hospital of Shanxi Medical University, Taiyuan 030013, China.. 3Key Laboratory of Combined Multi-organ Transplantation, Ministry of Public Health. 4Key Laboratory of Organ Transplantation, Zhejiang Province.5Division of Hepatobiliary and Pancreatic Surgery, Department of Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China.6Department of General Surgery, Licheng County People’s Hospital, Licheng 647600, China.
Correspondence to: Sheng Gao, Department of Colorectal Cancer, Shanxi Cancer Hospital and Institute, No.3 Xinchun Road, Taiyuan 030013, China,, P.R.China. Phone: 86-351-4651225; Fax: 86-351-4651883. E-mail address: gaoshengmail@163.com.
Abstract
Background: Regulating epithelial-to-mesenchymal transition (EMT) in cancer cells has been widely considered as an approach to combat cancer progression and therapeutic resistance, whereas a limited number of broadly comprehensive investigations of microRNAs involved in metastatic colorectal cancer (mCRC) have been conducted. In this study, we investigated the roles and mechanisms of EMT process in mCRC.
Materials and Methods: We used in situ hybridization and quantitative reverse transcriptase polymerase chain reaction to measure expression of miR-93 in colorectal tissues, nontumor tissues and metastatic liver tissues. CRC cell lines were transduced with lentiviruses that expressed miR-93, its inhibitor sequence targeted miR-93 or a scrambled sequence (control); proliferation, metastasis, invasion and colony formation were analysed. We analysed growth of CRC cells that overexpress miR-93 or its inhibitor in severe combined immune-deficient mice. Western blot, and luciferase reporter assays were used to measure expression and activity of Eph tyrosine kinase receptor (EphA4) and related signalling molecules.
Results: In this study, we demonstrated that miR-93 regulated the epithelial-mesenchymal transition (EMT) process by targeting EphA4 in metastasis colorectal cancer (mCRC). We examined the fact that CRC tissues and metastatic liver tissues had increased levels of miR-93 compared with the nontumor tissues and cells, by which we identified miR-93 can regulate EMT process. In addition, overexpression of miR-93 increased proliferation of CRC cells, metastasis, invasion and colony formation in vitro, whereas miR-93 depletion reduced these parameters. In severe combined immune-deficient mice, overexpression of miR-93 by CRC cells increased liver metastasis and overexpression of the miR-93 inhibitor reduced it. By further study the role of miR-93 in EMT and tumor metastasis, we identified by microarray analysis that the direct and functional target genes of miR-93 was EphA4. Knockdown of EphA4 phenocopied the effect of miR-93 and ectopic expression of EphA4 restored the effect of miR-93 on proliferation, migration, invasion and liver metastasis in CRC cells.
Conclusions: Our findings for the first time revealed that miR-93 regulates the epithelial-mesenchymal transition (EMT) process by targeting EphA4 to affect liver metastasis in colorectal cancer (CRC). Mechanistically, miR-93 inhibited tumor metastasis by directly targeting EphA4 which is a crucial factor in regulating EMT. Collectively, this study provide new insights into exploring the therapeutic potential of miR-93 which is able to regulate EMT process to affect tumor metastasis, and warrant further study in clinical settings.