Annexin A4: Its Expression In Hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is one of the malignant tumors threatening human health, which has become the second leading cause of mortality in China. Hepatocarcinogenesis is considered as a complex, multi-steps process, in which multi-gene be involed in. Therefore, the mechanism of HCC is still unclear. There are so many protein molecules taking changes with their expression level during the process of carcinogenesis, which suggest that they are play a important role in hepatocarcinogenesis. Annexins are a family of Ca2+-regulated phospholipid-binding and membrane-binding proteins. It participates in the regulation of membrane organization and membrane traffic. It also regulates cellular ion (Ca2+) currents across membranes and Ca2+ concentrations. According to the studies, the expression of annexins has changed in many tumors, which suggests that annexins family might invole in tumorigenesis. In addition, Annexin A4, a member of Annexin family, has been found to have changes in expression levels in Pancreatic cancer and renal cell carcinoma. In this study, we detected the expression of Annexin A4 in a series HCC cell lines with different metastatic potential. We try to investigate how such Annexin A4 play a role in HCC cell functions, such as cell cycle, proliferation, apoptosis and adhesion in vitro, and then further analysis of its relevant mechanisms. Part 1 The expression of Annexin A4 in human HCC cell lines with different metastatic potentialObjective: To study the expression of Annexin A4 in human hepatocellular carcinoma cell lines with different metastatic potential.Method: Western Blot was used to detect the expression of Annexin A4 in a series HCC cell lines with different metastatic potential.Results: The results of Western Blot showed that Annexin A4 were low expressed in BEL-7402 and SMMC-7721, but highly expressed in 97 series hepatocellular carcinoma cell lines.Conclusions: The expression of Annexin A4 was reduced in low metastatic potential hepatocellular carcinoma cell lines, but increased in high metastatic potential cell lines. Part 2 The effect of Annexin A4 RNAi on biological characteristics in the hepatocellular carcinoma cellObjective: To investigate the effect of Annexin A4 RNA interference on biological characteristics in the hepatocellular carcinoma cell.Method: Double strain siRNAs were synthesized by chemical method. We transfected it into MHCC97H cell line by liposome method. The expression of ANXA4 mRNA and protein levels were detected by quantitative RT-PCR and Western blot. Distributions of cell cycle and cell apoptosis were assessed by flow cytometry. Tumor cell proliferation was tested by CCK-8 method. Cell adhension ability was measured by adhension test.Results: The expression of ANXA4 mRNA and protein were decreased over 75% when compared with the negative and blank control groups. Cell proliferation and adhension in ANXA4 siRNA groups were significantly decreased by 19.06% and 23.19%. However, there were no changes in cell apoptosis and cell cycle.Conclusions: Annexin A4 expression is positively correlated with proliferation and adhesion ability in human MHCC97H cell line, but it has no correlation with cell cycle and cell apoptosis. Part 3 The effect of Annexin A4 RNAi on the expression of cell adhension moleculesObjective: To investigate the expression of adhesion molecules after RNAi Annexin A4 gene.Method: MHCC97H was transfected by Annexin A4 siRNA using liposome method, then, extracted the total RNA and reverse transcripted into cDNA, last, Realtime-qPCR detected the expression of adhesion molecules integrinα5, integrinβ1, E-cadherin, ICAM1, CD44 and OPN.Results: The results of Realtime-qPCR show that the expressions of integrinα5, integrinβ1, E-cadherin were decreased by 3.23 times, 1.53 times and 1.26 times respectively, but there were no changes in ICAM1, CD44 and OPN.Conclusions: The expression of integrinα5 was significantly reduced after RNAi Annexin A4 gene, while the adhesion ability of MHCC97H was decreased significantly, which suggested that ANXA4 genes involved in cell adhesion by integrinα5. Conclusion1. Annexin A4 was highly expressed in high metastatic potential hepatocellular carcinoma cell lines, but lowly expressed in low metastatic potential cell lines.2. Annexin A4 expression is positively correlated with proliferation and adhesion ability in human MHCC97H cell line, but has no correlation with cell cycle and cell apoptosis.3. The expression of integrinα5 is significantly reduced after RNAi Annexin A4 gene, while the adhesion ability of MHCC97H was decreased significantly, which suggested that ANXA4 genes involved in cell adhesion by integrinα5.Novelty of this project1. We use specificity siRNA silencing ANXA4 gene to analysis ANXA4 function in Hepatocellular carcinoma cell lines2. We use Realtime-qPCR to investigate the expression of adhesion molecules after RNAi Annexin A4 gene treatment.The potential application of this work1. Annexin A4 may play a potential and valuable biomarker and to be therapeutic target in HCC.2. Our study provides experimental data to further research molelurlar mechanism in which Annexin A4 expression linked to HCC development and metastasis.