Antitumor Effect And Regulatory Mechanism Of TMEM166 In HCC

Objective: Primary hepatocellular carcinoma(HCC)is a common malignancy,and molecular targeted therapy has been shown to significantly improve the prognosis of patients with advanced HCC.The discovery of new tumor regulatory molecules is crucial to the in-depth analysis of the pathogenesis of HCC and the search for effective therapeutic targets.Endoplasmic reticulum transmembrane protein 166(TMEM166),has an important antitumor function in human gastric cancer,neuroblastoma and other tumors,mainly by regulating autophagy and apoptosis.However,the function and mechanism of TMEM166 in HCC is still unclear.Besides,studies have shown that numerous micro RNAs are up-regulated in HCC,and whether the down-regulation of TMEM166 in HCC is regulated by micro RNA,researching of which will contribute to the clarification of the pathogenesis of HCC.This study will explore the function and relative mechanism of TMEM166 in HCC,and preliminarily analyze the regulatory mechanism of TMEM166 expression in HCC from the perspective of micro RNA.Methods: In this study,firstly,bioinformatics method was used to analyze the correlation between the expression and clinical significance of TMEM166 in TCGA database in HCC.Samples of 30 HCC patients were collected and the expression of TMEM166 in HCC tissues and para-carcinoma tissues was detected and verified by real-time fluorescent quantitative PCR(RT-q PCR),immunohistochemistry,and western blotting,and the expression profile was confirmed in normal liver cell lines and HCC cell lines.Secondly,TMEM166 was overexpressed in HCC cell lines(Huh7,Hccl-M3,QGY-7703),and its effects on the proliferation,migration and invasion of HCC cells were detected by MTT,cell scratching and Transwell respectively.DAPI staining and flow cytometry were used to detect the effects on apoptosis and cell cycle of liver cancer cells.Thirdly,western blotting was used to detect the effects on expression of apoptosis,cyclically related proteins p53,BAX,BCL-2,p21 and expression of epithelial mesenchymal transformation(EMT)related proteins e-cadherin,n-cadherin and Vimentin.Finally,the micro RNA targeted TMEM166 was predict by bioinformatics,screened from by RT-q PCR from 10 pairs of tissue samples randomly selected,then the true targeted micro RNA was determined by dual luciferase assay.Results: TCGA data analysis showed that the expression of TMEM166 in human HCC tissues was significantly lower than that in normal tissues,the expression of TMEM166 was negatively correlated with the stages of HCC progression within a certain range,and the low expression of TMEM166 was significantly correlated with shorter overall survival and shorter disease-free survival.The results of patient samples showed that the expression levels of TMEM166 m RNA and protein in liver cancer tissues were significantly lower than those in para-cancer tissues(P<0.001).The expression level of TMEM166 in Hccl-M3 cells and QGY-7703 cells was significantly lower than that in normal liver cells L02(P<0.001).TMEM166 overexpression significantly inhibited the proliferation of HCC cells(P<0.01),migration(P<0.05)and invasion(P<0.01);and caused early apoptosis and cell cycle arrest in HCC cells(P<0.05).Overexpression of TMEM166 induced p53 up-regulated,induced the pro-apoptotic protein BAX levels to significantly increase,and the anti-apoptotic protein BCL-2 levels to significantly decrease,giving rise to apoptosis.At the same time,the expression level of cell cycle inhibitor p21 was significantly increased and cell cycle arrest was induced.A significant increase in EMT-related protein E‐cadherin and a significant decrease in N‐cadherin and Vimentin led to EMT supression.Bioinformatics prediction combined with the screening results of RT-q PCR showed that mi R-107 、 mi R-103a-3p and mi R-1250-3p were up-regulated in the tumor tissues of HCC patients.Dual luciferase detection showed that the real targeted micro RNA was mi R-103a-3p、mi R-1250-3p.Conclusion: Our study is the first to comprehensively analyze the role of TMEM166 in HCC.Low expression of TMEM166 is associated with the development and poor prognosis of HCC.TMEM166 is siginificantly downregulated in HCC tissues and cell lines comparing with normal tissue and liver cells.TMEM166 has a significant anti-tumor activity on HCC cells and can remarkbally inhibit the growth,metastasis and invasion of tumor cells by inducing apoptosis,cell cycle arrest and EMT inhibition.mi R-103a-3p and mi R-1250-3p are the upstream target mi RNAs of TMEM166 in HCC.The up-regulation of mi R-103a-3p and mi R-1250-3p in HCC induces the down-regulation of TMEM166,which may be involved in the tumorigenesis or progression of HCC.TMEM166 is a potential target for the treatment of HCC.