Membrane Associated Protein A2 Expression Studies On Human Hepatoma Cell Biological Behavior

Hepotacarcinoma cell (HCC) is one of the most common malignant types of tumors in our country with high morbidity and mortality efficiency. Currently, the treatment for HCC mainly contains traditional surgery, chemotherapy and radiotherapy. However, the overall survival has undergone little improvement. The major cause of the lethal progression of this type of cancer is the malignant proliferation and metastasis. With the development of cellular and molecular biology, gene therapy, one new treatment, has become prominent and achieved considerable progresses on HCC diagnosis, therapy and prognosis. RNA interference (RNAi),which has been known as one of the ten great breakthroughs by Science in 2002, has great applied potential in gene founction and gene therapy research. The mechanism for RNAi is the post-transcriptional gene siliencing mediated by double stranded RNA (dsRNA).Therefore, it is to explore genes related to hepatocellular vicious transformation and dig out pathogenesis of HCC generation and progression that will greatly help to break through the bottleneck of HCC therapy. The changed expression of ANXA2, which has played important roles in cell vicious transformation, is greatly associated with many types of Human tumors proliferation, invasiveness and metastasis. Moreover, ANXA2 level is up-regulated in HCC and it may be used as serum mark for HCC diagnosis. In this paper, the recombinant plasmid pU6H1-GFP-siANXA2 were introduced into SMMC-7721 cells with optimized calcium phosphate transfection method by RNAi. ANXA2 expression and the effects on HCC proliferation, apoptosis and motility were investigated.It is concluded that this research can be used as the references for HCC gene therapy research projects.Method:1.Based on ANXA2 variants mRNA sequences supplied by GeneBank database, four siRNAs targeting to ANXA2 variants were synthesized and constructed to recombinant plasmids pU6H1-GFP. Scramble control sequence (SCR) of human genome was also cloned to plasmid pU6Hl-GFP 2.Recombinant plasmid pU6Hl-GFP-siANXA2 and pU6H1-GFP-siRNASCR were introduced into the human hepatocarcinoma cell,SMMC-7721,using the optimized method. And six groups(NC, siRNASCR, siA2-1,siA2-2, siA2-3 and siA2-4)were included.3.ANXA2 mRNA and protein levels were evaluated by using semi-quantitative RT-PCR and western blotting at 24hrs,48hrs.72hrs and 96hrs post thansfection.4.The cell proliferation and motility were determined with MTT and wound healing assay after ANXA2 downregulation.Moreover, cell apoptosis morphology at 96hrs post transfection was observed by Hoechast33258 staining.Result:1.The exogenous siRNAs were inserted into plasmid pU6Hl-GFP and siRNAs recombinants were successfully constructed, which were verified through enzyme digestion and gene sequencing.2. Following parameters were indicated as the optimal transfection conditions: 50%-60% cultured cell cover ratio before transfection,8μg, plasmid per 30mm dish,20mins-30mins complex formation time,6hrs incubation time. The absence/attendance of serum and antibiotics showed no significant influence. Serum was indispensable for this method.3.The semi-quantitative RT-PCR and western blotting results indicated, Four siRNAs suppressed ANXA2 expression effectively with time dependence (p<0.05) at 24hrs,48hrs,72hrs and 96hrs post transfection.The maximal mRNA inhibition efficiency was achieved at 72hrs post transfection and protein inhibition peak lasts from 72hrs to 96hrs post transfection.Moreover, siA2-1 and siA2-2 group decreased more significantly. The results of immunocytochemistry also enhanced the above conclusion.However, siRNAscR displayed no significance with NC in ANXA2 expression (p>0.05). 4.In MTT, Hochast33258 staining and wound healing assay, SMMC-7721 proliferation and motility ability were inhibited after pU6H 1-GFP-siANXA2 transfection (p<0.05), and the inhibitory efficiency was positively correlated with ANXA2 knockdown level.The maximal inhibitory effciency was achieved at 72hrs and 96hrs post transfetion (p<0.01)and SMMC-7721 showed typical morphological characterastics. However, siRNAscR group indicated no significance with NC group (p>0.05).Conclusion:It is concluded that the over-expression of ANXA2 in HCC is closely associated with the cell malignant behaviors, and siRNA can inhibit the ANXA2 expression efficiently in SMMC-7721 cells.Therefore, ANXA2 can be used as gene therapy target and plasmid pU6Hl-GFP as safe clinical vector for HCC gene therapy. The results may set up great foundation for HCC gene therapy.