Anxa2 Expression Levels In Human Colorectal Cancer Cell Proliferation And Movement Affect The Study

Colorectal carcinoma is one of malignant tumors that terribly endangers human life wordwide. Recently,the incidence of the disease tends to rise year by year because of the change of people's life style and the irrationality of dietary pattern.At present,the surgery is the most primary and effective treatment of the disease,but the survival rate in five years doesn't enhance significantly all the time.The development of molecular biology brings a new hope to people from tumor gene therapy.The key step of gene therapy is the establishment of target gene.Like other cancers,the oncogenesis of colorectal carcinoma is also a complicated pathological process involved with many factors,genes and stages.As the member of annexin family,a Ca²⁺ binding protein family,ANXA2 gene localizes on human chromosome 15q21-q22,and contains 13 extrons and 12 introns.The ANXA2 protein is involved with a serial of vital biological activities including membrane formation, membrane transportation,exocytosis,endocytosis,cell proliferation,signal transduction,cell differentiation and apoptosis.Recently,study data showed that the up-regulation of ANXA2 protein level in most cancer tissues is closely related to oncogenesis,cancer development,invasion and metastasis.RNAi is a phenomenon of genetic interference mediated by double strand RNA which can degrade mRNA specifically and effectively and cause post-transcriptional gene silencing.Now it has emerged as a powerful tool for gene function research,and provided with great applicable foreground in the tumor gene therapy.In this study,we employ RNAi-mediated suppression of ANXA2 gene expression to evaluate its effects on Caco-2 cells growth,proliferation and apoptosis,and establish its potency to be used as a target for the gene therapy of colorectal carcinoma.Objective:Our investigation attempts to study the effects of the siRNA mediated gene silencing of ANXA2 on the biological behaviors of colorectal carcinoma cells and the roles of ANXA2 gene in colorectal carcinogenesis and development,and provide the experimental evidences for the gene therapy of colorectal carcinoma through ANXA2 silencing.Methods:(1) Design four siRNA squences and one negative control sequence for four target sites on ANXA2 mRNA based on the gene information from GeneBank,then construct the pU6H1-GFP-siANXA2 recombinants. (2) Transfect the recombinants into Caco-2 cells using Lipofectamine^TM 2000,and set up negative control group and wild type group meanwhile.The transfection efficiency and changes of cell morphology were observed at 24h and 48h post transfection.(3) The levels of ANXA2 mRNA and protein were detected by RT-PCR and western blot/immunocytochemistry at different time points post transfection.(5) The rates of the proliferation,apoptosis and migration of Caco-2 cells were detected using MTT method,hoechst 33258 staining method and Dunn Chemotaxis Chamber assay at different time points post transfection.Results:(1) DNA sequencing confirmed that the ANXA2-siRNA expression vectors have been constructed successfullly.(2) After transfection,wild type and negative control Caco-2 cells grown well,but ANXA2-siRNA transfected cells did not.No significant difference between negative control and wild type cells could be found.The transfection efficiency is about 40%-50%at 24h post transfection.(3) The RT-PCR assay showed that the four siRNAs,especially ANXA2-siRNA1 could inhibit the expression of ANXA2 effectively.Over 70%ANXA2 expression was inhibited at 72h post transfection.(4) The results of immunocytochemistry and western blot showed that ANXA2-siRNA1 significantlly inhibited the ANXA2 protein expression in Caco-2 ceils at 72h post transfection.The outcome tally with RT-PCR in essence.(5) The four pU6H1-GFP-siANXA2 recombinants obviously inhibited Caco-2 cell proliferation. The highest inhibition rate was 66%.(6) The apoptotic Caco-2 cells were observed post transfection with a time dependent apoptosis rate. Significant difference between ANXA2-siRNA1 group and control group at each time point post transfection could be found(p＜0.01).(7) The migration rates of the ANXA2-siRNA transfected cells were siginificantly inhibited (p＜0.01).Conclusion:ANXA2 gene is closely associated with Caco-2 cell growth,proliferation,apoptosis and motility.ANXA2 expression knockdown could inhibit the proliferation and migration of Caco-2, and enhance Caco-2 apoptosis.ANXA2 gene silencing is of applicable potential for the gene therapy of colorectal carcinoma.