The Expression And Significance Of MicroRNA Biosynthesis Related Genes And ECT2in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the five most common cancers in the world, and the mortality rate of patients with HCC ranks the top three among all diseases. Though there are many treatments including surgical resection, chemotherapy, radiation therapy, local treatment, and liver transplantation, the long-term prognosis of the patients undergoing such treatment is still poor, with reported5-year survival rates ranging from about30%to50%and with high recurrence. It is necessary to study the molecular mechanism of HCC development, find out more effective ways of HCC early detection and explore effective interventions and treatments in the metastasis and recurrence of HCC.In this way, earlier detection, better prognosis and longer survival might be further achieved.MicroRNAs(miRNAs) play an essential role in gene expression regulators, which are often deregulated in many types of cancers. Mature miRNA leading to direct mRNA cleavage, regulation and inhibition of the target gene expression at the transcriptional level. MiRNAs have been reported to be involved in the modulation of diverse cellular processes. An increasing number of literatues showed that there are abnormal expression of miRNAs in tumors. The importance of miRNAs in cancer is highlighted by the observation that50%of miRNAs genes are located in cancer associated genomic regions or fragile sites.Our previous study found that microRNA expression profiles associated with metastasis of HCC, and we established a molecular prediction tag which composed of20miRNAs. Based on the study of gene expression profiles, we further found epithelial cell transforming sequence2(ECT2) is a candidate gene related HCC metastasis which is a guanine nucleotide exchange factor for Rho family GTPase.In this subject, we further study on the tole of microRNA biosynthesis genes and the preliminary result gene ECT2in HCC development to find out the evidence in the prognostic markers and screening therapeutic targets Part A The Expression and Significance of MicroRNA Biosynthesis Related Genes in Hepatocellular CarcinomaPurpose:To investigate the relationship between the expression of miRNA biosynthesis related genes and the malignant phenotype of HCCMethods:The expression levels and prognostic significance of miRNA biosynthesis related genes mRNA in HCC with paried adjacent nontumorous tissues were assessed by qRT-PCR. The immunohistochemical analysis was performed to detect the expressions and the prognostic significance of Dicer in another independent HCC cohort.Results:The results of RT-PCR showed that the expressions of Drosha, DGCR8, DBR1, ADAR, Exportin5in HCC tissues were up-regulated compared with adjacent tissues (P<0.0001), and the expression of Dicer, AG02, GEMIN3, GEMIN4in HCC tissues were down-regulated compared with adjacent tissues (P<0.0001). The overall survival rate(OS) of patients with high DBR1expression was significantly higher than patients with low expression in HCC tissues (p=0.0453). The overall survival rate(OS) of patients with high Exportin5expression was significantly higher than patients with low expression in HCC tissues (p=0.0294). The overall survival rate(OS) of patients with high DGCR8expression was significantly higher than patients with low expression in adjacent tissues (p=0.0111). Immunohistochemistry result reviewed that Dicer protein mainly expressing in the cytoplasm of adjacent tissues, and the expressions in adjacent tissues and tumor tissues were significantly different (p=0.0024).Conclusion:The lower expression of Dicer may plays an important role in HCC genesis. It may exist a regulatory mechanism in HCC cells that can induce the down regulation of Dicer enzyme and leading to the decline of related miRNA. There may be a negative feedback in miRNA biogenesis, which can stabilize the expression of miRNA. DBR1, Exportin5and DGCR8intervention can serve as a potential intervention target for HCC treatment. Part B The Expression and Significance of ECT2in Hepatocellular CarcinomaPurpose:To investigate the relationship between the expression of ECT2and the malignant phenotype of HCCMethods:The expression and the prognostic significance of ECT2was evaluated in HCC, adjacent nontumorous and normal liver tissues by qRT-PCR, immunohistochemistry. The expression of ECT2was evaluated in four human HCC cell lines (7721, HepG2, MHCC-97L and MHCC-97H) with various metastatic potentials and two human nontransformed hepatic cell lines, Chang Liver and L02by qRT-PCR. The immunohistochemical analysis was performed to detect the expression and the prognostic significance of ECT2in another independent HCC cohort.Results:The results of RT-PCR showed that the expressions of ECT2in HCC tissues and adjacent tissues were up-regulated compared with normal liver tissues (p<0.0001; p=0.0216), and the expression of ECT2in HCC tissues were up-regulated compared with adjacent tissues (P<0.0001). The mRNA expression levels of ECT2in four established HCC cell lines were found to be significantly increased in comparison to nontransformed hepatic cell lines (Chang liver and L02cell lines) detected by real-time PCR(p=0.0189).The mRNA expression levels of ECT2in high-metastatic HCC cell lines (MHCC-97H) were much higher than those of the three low-metastatic HCC cell lines (7721, HepG2, MHCC-97L)(p=0.0294). Immunohistochemistry result reviewed that ECT2protein mainly expressing in the cytoplasm and nucleus of HCC tissues, and the expressions in tumor tissues and adjacent tissues were significantly different (p=0.0013).Conclusion:ECT2in HCC cell lines and HCC tissues were highly expressing that may plays an important role in the malignant behavior of HCC.