ANXA2 Action Research On Human Colorectal Cancer In Regulating Cell Behavior

Objective:Gene knockout is better than gene knockdown methods, because some protein may be in trace of circumstances, namely to complete its special physiological function. Gene knockdown cannot completely remove target protein expression background, and the experimental results obtained with uncertainty. ANXA2belongs to a super family of closely related calcium-and membrane-binding proteins. The family for a class of calcium phosphatide-binding protein, they are involved in a variety of organism physiological and pathological process. At present, Annexins have12members in human and vertebrate orthologues, collectively referred to as ANXA. Existing research data shows ANXA2’s expression was closely related to the process of colorectal cancer and others many kinds of tumor, and participated in the many kinds of disease physiological process, e.g. cardiovascular and cerebrovascular diseases. And, colorectal cancer is the human very common malignant tumor. The ANXA2in cancer occurrence, development research is not enough, the existing research materials are mostly employing RNAi technology or gene knockdown, so that the results of the study has some uncertainty. So, in order to reveal exactly ANXA2in tumor especially colorectal cancer development process’s function, the ANXA2-/-Caco2cell line was generated, with wild type Caco2cells (ANXA2+/+Caco2) for comparison, in order to invest the function the ANXA2gene expression in the tumor proliferation, motility and apoptosis.Methods:1. Based on the sequences of ANXA2searched from GenBank database, we select ANXA2gene exon6as the target using clustalx1.83software, then construct the pPNT/ANXA2/EGFP recombinant plasmids, then linearized by Not-I.2. Linearized recombinant was introduced into Caco2cells by using a Bio-Rad Xcell-Electroporator. Using limited dilution method, we cultured the single cell in96well plates. We employ the exogenous gene EGFP (enhanced green fluorescent protein, here as reporter gene), neoR (G418resistance gene, here as a cell genetic screening gene), inserted into the Caco2cell genome ANXA2gene exon6, in specific PCR and Western blot appraisal knockout success. So, the ANXA2-/-Caco2cell line was generated successfully.3. MTT assay was to investigate the influence of knockout treatment for ANXA2on cell proliferation.4. Wound healing assay was to estimate the effect of down-regulation of ANXA2on the cell motility of Caco2cells.5. Hoechst33258staining, MitoViewTM633staining and DAPI staining were performed to measure the effects of gene knockout of ANXA2on cell apoptosis.Results:1. Construct the pPNT/ANXA2/EGFP recombinant plasmids successfully, then linearized by Not1.2. Using the method of electricity transfection method, limit dilution method and resistance screening, we established a ANXA2-/-Caco2cell lines.3. In MTT, Hoechst33258staining, wound healing assay and MitoViewTM633staining and DAPI staining, the results show that ANXA2gene knockout cells’cell proliferation was lower (p<0.05); cell migration ability appeared inhibition (p<0.05); cell apoptosis rate was significantly difference (p<0.05) in ANXA2-/-Caco2cells than in ANXA2+/+Caco2cells.Conclusion:We studied the function of ANXA2in cancer development process for the first time in ANXA2gene knock out of Caco2cells (ANXA2-/-Caco2), this study suggested ANXA2’s expression in human colorectal cancer development process plays an important role, which promoted the development of malignant tumor. So, knockout of the ANXA2for tumor prevention, diagnosis and treatment has potential application value.