MicroRNA-639 Targets ZEB1 To Suppresses The Migration And Invasion In Hepatocellular Carcinoma Cells

Objective:Hepatocellular carcinoma(HCC) is one of the most common primary tumors. The incidence rate of HCC is the fifith and mortaligy rate is the second in the world. When HCC developed to advanced stage, the hepatocellular carcinoma cell is easy to metastasize distally, that is why it is difficult to cure. Micro RNA(mi RNA) is the endogenous small non coding RNA,which can recognize and bind to the 3’UTR of m RNA and act as posttrascription regulator by mediating the degradation of m RNA or inhibitting protein translation. In HCC, the expression profiles of many mi RNAs are changed. Based on our previous work, we demonstrated that the expression of mi RNA-639(mi R-639) was downregulated and it inhibits the growth and proliferation of hepatoma cells, thus mi R-639 playing as a tumor supressor in HCC. In order to investigate how mi R-639 exerts its biological mechanism, we explored the effect of mi R-639 on migration and invasion in HCC cells, and screened it’s target gene. Combining with the biological process of mi R-639 and its target gene ZEB1(Zinc finger E-box-binding homeobox 1), we deeply discussed the molecular biological mechanisms about how mi R-639 regulated migration and invasion ability in liver cancer cell and the possible regulatory pathways. We hope our work could prove theoretical basis for research on the regulation mechanism of metastasis of hepatocellular carcinoma.Method: Transwell migration and invasion assay to measure the influence of mi R-639 on migration and invasion ability of HCC cell lines QGY-7703 and Hep G2. Next software Target Scan, mi RBase Targets and Pic Tar were used to examine the targeted m RNA, and select ZEB1 as the research object. We insert the wild type or mutant 3’UTR of ZEB1 into EGFP reporter vector. Then the fluorescent reporter experiment was carried out to determine the relationship of mi R-639 and ZEB1. Furthermore q RT-PCR and Western Blot assays were use to verify the target relationship from m RNA and protein level. Next, we constructed the overexpression and knock down plasmid of ZEB1 and confirmed that ZEB1 promote the migration and invasion ability of QGY-7703 Hep G2 cells through the Transwell migration and invasion assays. Moreover the rescue experiment was used to confirm whether thefunction of mi R-639 on migration and invasion are depended on ZEB1 in HCC cells. EMT pathway is a popular mechanism to explan migration and invasion, so the expression changes of EMT marker E-cadherin, Vimentin and ICAM-1 was tested by Western blot when mi R-639 and ZEB1 were overpessed or knockdown, to investigate the mechanism of mi R-639 and ZEB1 on migration and invasion in HCC.Results: Overexpression of mi R-639 inhibited the migration and invasion, while knockdown mi R-639 promoted the migration and invasion in HCC cells. Bioinformatics methods found that mi R-639 targeting 3’ UTR region of ZEB1 m RNA. The fluorescence reporter experiments, q RT-PCR and Western blot experiments proved that ZEB1 was a direct target gene of mi R-639 and negatively regulated by mi R-639. The rescue experiment further comfirmed that the inhibitory effect of mi R-639 on migration and invasion could be rescued by ZEB1. Finally Western blot results showed that mi R-639 could up regulate the expression of E-cadherin, and down-regulate the expression of Vimentin and ICAM-1, inplying that mi R-639 inhibited the EMT conversion of hepatoma cells. Meanwhile, overexpression of ZEB1 could promote EMT by inhibiting the expression of E-cadherin and up regulating the expression of Vimentin and ICAM-1. The reuslts indicated the opposite effection of mi R-639 and ZEB1 in EMT pathway.Conclusions: For our knowledge, this is first time to find that mi R-639 inhibited migration and invasion abilities of hepatocellular carcinoma cells by negatively regulating ZEB1 gene. mi R-639 binds ZEB1 3’ UTR to suppress its expression and functions. Thus, we conclude that mi R-639 inhibites the migration and invasion through repressing the EMT process in HCC cells by targetting the 3’UTR region of ZEB1 m RNA and negatively regulating ZEB1 expression.