The Mechanism Of MiR-452 Promoting Stem-like Triats Of Hepatocellular Carcinoma Cells

PartⅠ The expression level and its clinical significance of miR-452 in stem-like cells and tissues of hepatocellularcarcinomaObjectivesThe expression level of miR-452 differed in various malignancies. The goal of our study was to explore the expression of miR-452 in HCC stem-like cells. Further, we also analyzed the clinical significance in 180 HCC patients.MethodsWe enriched stem-like HCC cells by serial passages of hepatospheres with doxorubicin and sorafenib. Stem-like characteristics including theproportations of CD44, CD 133, CD90, EpCAM positive HCC cells, stemness-related gene expression profiling, tumorigenicity were detected. QPCR was used to detect the expression of miR-452 in hepatospheres. The expression of miR-452 in HCC tissue was detected by in situ hybridization.ResultsThe expression of miR-452 in the chemoresistant hepatospheres was significantly elevated than the differentiated HCC cells.The proportations of CD44、CD133、CD90、 EpCAM positive HCC cells were also elevated. The capability of tumorigenicity of chemoresistant hepatosphere HCC cells markedly strengthened. The ISH results showed that the miR-452 was also overexpressed in HCC tissues than adjacent normal tissues in HCC patients. Moreover, high miR-452 expression was positively correlated with poor patient survival and advanced TNM stages.Kaplan Meier curves showed that miR-452 overexpression was also associated with poor overall survival in HCC patients.ConclusionsSerial passages of the spheres with doxorubicin and sorafenib could enrich CSC populations in vitro, and miR-452 was up-regulated in chemoresistant HCC hepatospheres. MiR-452 was overexpressed in HCC cell lines and tissues. Finally, miR-452 overexpression indicated poor overall survival in HCC patients.PartⅡ The mechanism of miR-452 promoting stem-like triats of hepatocellular carcinoma cellsObjectivesIn our first part, we found that miR-452 was up-regulated in stem-like HCC cells. Thus, the goal of this part in our study was to explore the molecular mechanisms related to the miR-452 overexpression in regulating CSCs in HCC.MethodsFor miR-452 up-regulation and down-regulation, HCC cells were labeled with luciferase using a lentiviral-based approach. Stem-like characteristics including the capabilities of chemo-resistance, sternness-related gene expression profiling, self-renewal, tumorigenicity and metastasis formation were detected. Luciferase reporter assay and western blot were used to demonstrate the target gene of miR-452. TOPFlash and FOPFlash reporter assay were used to detect the activitaty of Wnt/β-catenin signaling pathway. The immunofluorescent analysis and CO-IP were used to identify the interaction between target gene and β-catenin or TCF4 in the nucleus.ResultsMiR-452 significantly promoted stem-like characteristics including the capabilities of chemo-resistance, sternness-related gene expression profiling, self-renewal, tumorigenicity and metastasis formation in vitro and in vivo. Further, Sox7 was identified as the direct target of miR-452. We also found that the restoration of SOX7 could partially, but significantly eliminate the effects of miR-452. Finally, we demonstrated that SOX7 could physically bind with β-catenin and TCF4 in the nucleus and then inhibit the activity of wnt/β-catenin signaling pathway.ConclusionsWe identified that miR-452 promoted stem-like traits of HCC. Sox7 was found to be a direct and functionally relevant target of miR-452 in HCC. Finally, we demonstrated that miR-452 promoted stem-like traits of hepatocellular carcinoma by inhibiting Sox7 involving wnt/β-catenin signaling pathway.