MiR-200a inhibits nasopharyngeal carcinoma cell growth, migration and invasion by regulating Wnt/beta-catenin signal pathway

Abstract

MicroRNAs (miRNAs) are a class of endogenous, small non-protein coding RNA molecules which are recognized as crucial post-transcriptional regulators of gene expression. Emerging evidence has shown that miRNAs play an important role in various human diseases including cancer. Nasopharyngeal carcinoma (NPC) is a malignancy arising from the epithelial cells lining the nasopharynx and is mainly endemic in southern China and Southeast Asia, including Guangdong and Hong Kong. Environmental factors, genetic susceptibility, as well as Epstein-Barr virus (EBV) infection have been reported to be associated with the etiology of NPC, however, the molecular basis of NPC pathogenesis has not been well defined and the prognosis of patients is still poor. In light of this, it is important to develop effective treatment approaches. In this study, we investigated the role of human miR-200a in NPC carcinogenesis. We discovered that miR-200a was significantly down-regulated in NPC C666-1 cells. By loss-of-function and gain-of-function studies, we observed that over-expression of miR-200a inhibited C666-1 cell growth, migration and invasion, while knock-down of miR-200a generated opposite results. To understand the mechanisms of these effects, we used online miRNA target prediction programs (TargetScan, PicTar and miRDB) to analyze miR-200a targets in humans. Among hundreds of targets predicted, several genes, including ZEB2, CTNNB1, MMP16, protocadherin and CTNND2 have been proposed to take part in NPC carcinogenesis or as probable functional targets of miR-200 family. We are particularly interested in beta-catenin encoding CTNNB1, a key mediator of canonical signaling in the Wnt/beta-catenin pathway. Previous studies showed that nuclear beta-catenin levels were increased in 92% of NPC tumors, suggesting that nuclear beta-catenin is an important component of NPC development. We therefore measured the protein expression levels of beta-catenin in response to the changes of miR-200a expression in C666-1 cells. We found that the protein expression levels of beta-catenin were down-regulated or up-regulated by transfection of miR-200a mimics or inhibitors respectively. To validate whether miR-200a directly recognizes the 3\#8242;-UTRs of CTNNB1 mRNA, we cloned the fragments containing predicted target sites downstream of the pGL3 luciferase reporter gene to generate pGL3-CTNNB1 vector. The vector was then co-transfected with miR-200a mimics or control into C666-1 cells. We found that luciferase activity in C666-1 cells co-transfected with miR-200a and pGL3-CTNNB1 vector was decreased when compared with the control groups. These data showed that CTNNB1 is one of the functional targets of miR-200a. Hence, miR-200a might serve as a regulatory factor of NPC carcinogenesis as well as a potential therapeutic candidate for miRNA-based cancer gene therapy.