The Study On The Role And Mechanism Of Let-7g Inhibiting HCC Proliferation And Invasion By Downregulation Of HMA2

Objectives:Hepatocellular carcinoma (HCC), which with high incidence and mortality, is one of the most common malignant tumors in china. Therefore, it is important to study the development and progression mechanism of HCC for improving curative effects of patients. High-mobility group A2(HMGA2), as a small non-histone chromosomal protein, was highly expressed in many human tumors and associated with poor prognosis, but the role of HMGA2in HCC remained unclear. The microRNAs (miRNA), is a family of mature noncoding small RNA. Let-7g, as a member of miRNA family, by binding to the3’-untranslated regions (3’UTR) of the target mRNA, induces translational repression or mRNA degradation of many genes. However, there is no repots on the regulation of HMGA2by let-7g in HCC. In this study, we first investigated the expression of HMGA2and let-7g in human HCC tissues and cells, the relationship between HMGA2and let-7was also analyzed. Then, we analyzed HMGA2expression with respect to various clinicopathologic factors in HCC patients. By transfecting let-7g mimics into HepG2, we also analyzed the effects of overexpressed let-7g on the expression of HMGA2and the bioactivity of HepG2cell and explored possible downstream mechanism. Methods:(1) Real-time qPCR assay was used to detect the expression level of HMGA2mRNA and let-7g between fresh HCC tissues and paired non-tumor tissues, HCC cell lines and hepatocytes respectively. HMGA2protein expression was assessed by immunohistochemical analysis of paraffin-embedded specimens of HCC and paired specimens of adjacent normal liver tissue. Correlations between HMGA2and clinicopathologic features and prognosis were also tested.(2) Let-7g mimics was designed and synthesized, and transfected into HepG2cells. Real-time qPCR and western blot were used to detect the effects of overexressed let-7g on HMGA2expression. Luciferase reporter assay was performed to detect whether the HMGA2was regulated by let-7g. In vitro, cell proliferation, apoptosis and migration and invasive ability were assessed by MTT assay, flow cytometry and transwell assay respectively.(3) Real-time qPCR and western blot were performed to detect p16expression after transfection of let-7g mimics into HepG2.Results:(1) The expression of HMGA2mRNA was upregulated in HCC tissues and cell lines than in paried non-tumor tissues and hepatocytes respectively. While the expression level of let-7g was higher in non-tumor tissues than in HCC tissues. An inverse correlation was noted between HMGA2mRNA and let-7g in HCC tissues. The positive expression of HMGA2in HCC was higher than in paired specimens of adjacent normal liver tissue. Elevated HMGA2protein expression was significantly correlated with tumor size, vascular invasion and capsule invasion and poor clinical prognosis.(2) With upregulation of endogenous let-7g through transfected let-7g mimics, there was a significant downregulation of HMGA2mRNA and protein expression levels in HepG2cells. The luciferase reporter assay confirmed that HMGA2was a target of let-7g. Overexpression of let-7g inhibited the proliferation, migration, and invasion of HCC cells and increased cell apoptosis.(3) Real-time qPCR and western blot analysis showed that the mRNA and protein levels of p16were significantly increased after the upregulation of let-7g by transfecting let-7g mimics into HepG2cell.Conclusions:(1) HMGA2was significantly upregulated in HCC. Overexpressed HMGA2was associated with tumor size, vascular invasion and capsule invasion and poor prognosis. Overexpression of HMGA2might be associated with HCC development and progress and HMGA2expression status could be served as an independent prognostic factor in HCC.(2) Overexpression of let-7g could inhibit expression of HMGA2mRNA and protein in HepG2cells and HMGA2was a target of let-7g. Overexpressed let-7g could inhibit the proliferation, migration and invasion and induce apoptosis of HepG2cells, indicating that let-7g could serve as a tumor suppressor gene in HCC.(4) let-7g could inhibit proliferation of hepatocellular carcinoma cells by downregulation of HMGA2and upregulation of p16.