MiR-130a Regulates The Proliferation And Metastasis Of HCC Cells Through Targeting ZEB1/ZEB2

Objective:In this study,we aimed to determine the targeted relationship between miR-130 a and ZEB1/ZEB2,to analyze the expression levels and correlation of miR-130 a and ZEB1/ZEB2 in HCC cell lines and tissue samples.We investigated the effects of miR-130 a on the proliferation and metastasis of HCC cells in vivo and in vitro.Methods:1.Real time PCR was performed to detect the expression levels of miR-130 a and ZEB1/ZEB2 in three different HCC cell lines(Hep G2,Hep3 B,SMMC-7721),and a normal liver epithelium cell line(L02).HCC tissues(n=36)and pair-matched noncancerous tissues were collected from patients with HCC.Real time PCR were performed to evaluate the expression levels of miR-130 a and ZEB1/ZEB2.The correlation of their expression were analyzed.2.The expression of miR-130 a could be up-regulated and down-regulated by miRNA mimics and inhibitor respectively.The expression of ZEB1/ZEB2 m RNA and protein were detected by Real-time PCR and western blot.At the same time,dual luciferase reporter assay was used to clarify the transcriptional regulation effects of miR-130 a on ZEB1/ZEB2.3.To investigate the effects of miR-130 a on malignant phenotype of HCC cells,miR-130 a mimics and inhibitor were transfected into HCC cells.The effects of miR-130 a on proliferation and metastasis of HCC cells were examined using MTT assay,colony formation assay,wound healing assay,and transwell chamber experiments.To observe the effect of miR-130 a on tumorigenesis in vivo,SMMC-7721-miR-130 a stablely expressing miR-130 a was constructed and a subcutaneous HCC model was established.4.To investigate the effect of miR-130 a targeted regulation of ZEB1/ZEB2 on the malignant biological phenotype of HCC cells,we transfected SMMC-7721 cells with miR-130 a mimic and pc DNA3.1/ZEB1 or ZEB2 to increase individually or simultaneously miR-130 a and ZEB1/ZEB2 expression.And then,we detected the proliferation,invasion and migration of HCC cell lines using MTT,colony formation,healing and transwell assays.Results:1.We found that the expression levels of miR-130 a in HCC cell lines SMMC-7721 and Hep3 B were significantly lower than that in normal liver cell line L02,while the expression of ZEB1/ZEB2 m RNA in HCC was significantly enhanced,and there was no significant difference between Hep G2 and L02 cell lines.The analysis of 36 cases of HCC tissues and adjacent normal tissues showed that the expression level of miR-130 a in HCC tissues was significantly lower than that in adjacent tissues(P < 0.01).Similarly,the expression of ZEB1/ZEB2 m RNA increased significantly in tissues.The levels of ZEB1 and ZEB2 were negative correlation with miR-130a(Spearman’s correlation analysis,r =-0.884,P < 0.01 and r =-0.899,P < 0.01).2.Dual luciferase reporter assay confirmed that miR-130 a could directly interact with ZEB1/ZEB2.The m RNA and protein levels of ZEB1 and ZEB2 were markedly decreased by miR-130 a mimic transfection and obviously increased by miR-130 a inhibitor transfection compared with the negative control,respectively.The results demonstrate that ZEB1/ZEB2 are direct targets of miR-130 a and that miR-130 a regulates ZEB1/ZEB2 in the transcriptional level.3.MTT assay showed that up/down-regulation of miR-130 a expression could significantly inhibit or promote the proliferation of HCC cells.Healing and invasion experiments found that up/down-regulation of miR-130 a expression could significantly inhibit or promote HCC cells migration and invasion as well.Tumorigenesis experiment results were consistent with in vitro study and the effect of miR-130 a overexpression was significantly greater than that of control groups.4.We increased the expression of miR-130 a and ZEB1/ZEB2 separately or simultaneously,and found that ZEB1 or ZEB2 alone could not completely reverse the inhibitory effect of miR-130 a on the proliferation and migration of HCC cells.However,overexpression of miR-130 a and ZEB1 or ZEB2 simultaneously can completely block this inhibitory effect of miR-130 a on HCC cells.Conclusion:1.MiR-130 a is lowly expressed in HCC cell lines and tissues,and the expression of ZEB1/ZEB2 is increased.There is obviously negative correlation between miR-130 a and ZEB1/ZEB2.2.MiR-130 a can effectively inhibit the transcription of target gene ZEB1/ZEB2 and cell proliferation,migration,invasion and other malignant biological behaviors in HCC cells.