The Role And Mechanism Of MiR-150 In The Cell Proliferation And Metastasis Of Hepatocellular Carcinoma

[Background] Hepatocellular carcinoma(HCC) is one of the most common malignancy worldwide. It is the third leading cause of cancer-related death in the world and the second most common cause of that in China. Recently, although the surgical technique, modern imaging techniques and non-surgical local treatment technology have advanced considerably and the therapeutic effect of HCC has improved, the prognosis of these patients is still not ideal. The main reasons may be associated with the difficulty of early diagnosis, and the recurrence and metastasis of primary tumors. Therefore, it is necessary to further study the underlying molecular mechanism of HCC occurrence and development, thus define some potential biomarkers and seek new effective therapeutic targets. micro RNAs(mi RNA) are small noncoding RNAs which negatively regulate gene expression mainly through direct interaction with the 3’ untranslated region(3’-UTR) of corresponding target messenger RNAs(m RNAs). Many studies indicated that mi RNAs participate in a variety of cell functions, including cell proliferation, apoptosis,differentiation, metabolism and regulating the endocrine system, etc. Abnormal expression of micro RNAs is associated with a variety of diseases including tumor. Current researches of mi RNAs in cancer cover the cell proliferation, apoptosis, differentiation, drug resistance, invasion and metastasis. Among them, mi R-150 plays an important role in the development process of tumor. Studies indicated that mi R-150 may be a tumor suppressor in colon cancer, pancreatic cancer and ovarian cancer. While mi R-150 could promote the progress of tumor in gastric cancer and breast cancer. Therefore, mi R-150 may play a different role in different tumor types. Research has shown that the expression of mi R-150 was lower in the CD133+ liver cancer stem cells by comparing the data of gene chip of CD133+ and CD133- primary hepatocellular carcinoma subpopulations. Another study have found that mi R-150 was downregulated in the venous metastases of HCC by comparing the mi RNA expression differences in nontumorous livers, primary HCCs, and venous metastases in the same livers from HCC patients. Thus, mi R-150 may participated in the development of HCC. However, whether mi R-150 participate in the regulation of HCC carcinogenesis? What is its specific function and mechanism in HCC? All of these need further research.[Objective] 1. To detect the expression of mi R-150 in HCC tissues and corresponding adjacent tissues and discuss the relationship between mi R-150 expression and patient clinical pathological characteristics and the prognosis of patients. 2. To investigate the effect of mi R-150 on HCC cells proliferation, invasion and metastasis ability. 3. To explore the underlying molecular mechanisms of mi R-150 regulating HCC cells malignant phenotype.[Methods] 1. The expression levels of mi R-150 in 84 HCC tissues and adjacent noncancerous liver tissues were quantitated by q RT-PCR, and then the relationships between mi R-150 expressions and various clinicopathological characteristics and prognosis of patients with HCC were explored.2. A lentiviral plasmid expressing mi R-150(Lenti-mi R-150) or a lentiviral control plasmid(Lenti-mi R-NC) was transfected into the HCC cell lines to stably overexpress mi R-150 or negative control, respectively. The CCK-8 assay, colony formation assay, and tumorigenesis assay were used to analyze the effect of mi R-150 on HCC cell proliferation ability. Transwell assay and the in vivo metastasis assay were used to analyze the effect of mi R-150 on HCC cell invasion and migration ability. 3. Bioinformatics tools(Target Scan, mi Randa, and mi RWalk) were used to predict putative target genes of mi R-150. Among the candidates, GAB1 was chosen for further analysis. The luciferase reporter assay, q PCR and western blot experiments were used to verify the target of mi R-150. Detection of GAB1 expression in HCC tissue was carried out to analyze its correlation with mi R-150. Using RNAi technology to silence GAB1 expression in HCC cells, and investigate its effect on HCC cell proliferation, invasion and metastasis ability. Recovery experiments were also done to confirm the function of GAB1. At last, western blot and immunohistochemistry were used to detect the relationship between mi R-150 and GAB1, phospho-ERK1/2 and EMT marker proteins expression levels.[Results] 1. mi R-150 expression was significantly lower in HCC tissues than in adjacent noncancerous tissues. Low mi R-150 expression was significantly correlated with tumor size, venous invasion and metastasis. Further study indicated that patients with low mi R-150 expression had a shorter overall survival time than those with high mi R-150 expression, and that low mi R-150 expression was an independent prognostic factor of survival of patients with HCC 2. mi R-150 was significantly lower in HCC cells compared to the normal human hepatocyte cell line HL-7702. MHCC97-H and SMMC-7721 cells, which have relatively lower mi R-150 expression, were selected for follow-up experiments. To investigate the role of mi R-150 in HCC progression, we transfected the HCC cell lines MHCC97-H and SMMC-7721 with a lentiviral plasmid expressing mi R-150(Lenti-mi R-150) or a lentiviral control plasmid(Lenti-mi R-NC) to stably overexpress mi R-150 or negative control,respectively. mi R-150 overexpression significantly inhibited the proliferation, clony formation and tumor growth of HCC cells compared to the Lenti-mi R-NC-expressing cells in vitro and in vivo, respectively. 3. Transwell experiments confirmed that overexpression of mi R-150 significantly inhibited the invasion and migration of HCC cells. Overexpression of mi R-150 significantly inhibited the lung metastasis of HCC cells in vivo, which is consistent with the in vitro experimental results. 4. Bioinformatics software predicted that GAB1 may be a target of mi R-150. Dual luciferase reporter assay showed that mi R-150 significantly decreased the relative luciferase activity of the GAB1-WT-3’-UTR reporter in both MHCC97-H and SMMC-7721 cells(P<0.05), whereas the activity of the GAB1-MUT-3’-UTR reporter was not affected by mi R-150. In addition, q RT-PCR showed that mi R-150 overexpression significantly decreased GAB1 m RNA expression in HCC cells. Western blot analysis revealed that mi R-150 overexpression markedly lowered the levels of GAB1 protein in HCC cells. Further experiment indicated that GAB1 m RNA expression was significantly higher in HCC tissues than in adjacent noncancerous tissues. Spearman’s correlation analysis indicated a significant inverse correlation between mi R-150 and GAB1 expression in the 20 HCC tissues examined. All of these results indicated that GAB1 is the direct target of mi R-150. 5. GAB1 knockdown significantly inhibited the proliferation, migration and invasion of HCC cells in a manner similar to mi R-150 overexpression in these cells. Furthermore, the inhibitory effects of mi R-150 on HCC cells were partially counteracted by restoring GAB1 expression. 6. mi R-150 overexpression significantly decreased GAB1 protein expression and ERK1/2 phosphorylation compared to the control but did not alter total ERK1/2 protein levels. Moreover, mi R-150 overexpression enhanced the expression of an epithelial marker(E-cadherin) and reduced the expression of mesenchymal markers(N-cadherin and Vimentin), indicating that mi R-150 overexpression inhibited EMT in HCC cell lines. Further, we further verified that overexpression of mi R-150 inhibited GAB1 proteinexpression and ERK1/2 phosphorylation in vivo.[Conclusions] 1. mi R-150 was significantly downregulated in HCC tissues and that decreased mi R-150 expression was associated with worse clinicopathological characteristics and a poor prognosis. 2. Overexpression of mi R-150 inhibited the proliferation, migration and invasion of HCC cell lines in vitro and in vivo. 3. GAB1 was a direct target of mi R-150. mi R-150 directly targeted the GAB1-ERK1/2 axis to suppress cell proliferation, migration and invasion of HCC cells, and there was an inverse correlation between mi R-150 and GAB1 levels in HCC tissues. These results suggest that the mi R-150-GAB1-ERK1/2 axis might be a potential target for the HCC therapy.