Annexin A2 On Human Gastric Cancer Cell Biological Behavior

Background and Objective:Gastric carcinoma is a common malignant tumor in digestive tract. Delayed diagnosis, recurrence and metastasis post surgery operation are still the leading causes of patient death. So it is necessary to explore pathogenesis of gastric carcinoma generation and progression, which is important to overcome the difficulties of gastric carcinoma therapy. Molecular targeted therapy has created a new stage for the status of gastric cancer patients, which always have short time of survival and poor therapeutic effects of chemotherapy and radiotherapy. RNA interference has showed a good application prospect in tumour gene therapy with its high efficiency and specificity. ANXA2 has shown to be dysregulated in many tumour diseases, the expression levels of which are closely related to the oncogenesis, metastasis, clinical stage and prognosis of tumour, and there is no exception to gastric carcinoma. In this study, we designed the siRNA targeting the mRNA of ANXA2 and expression vector using RNAi technology to study the effects of ANXA2 gene silencing on the proliferation, motility and apoptosis of SGC-7901 cells. The relationship between ANXA2 expression and biological behaviors of SGC-7901 cells was analysised to investigate the possible mechanism. All the results and the data collected provide evidence for the further research of ANXA2 mechanism in tumours and anti-gastric cancer.Method:1. Based on the sequences of ANXA2 searched from GenBank database, select the effective interference targets using online software, then siRNA sequences and missense control sequence were synthesized and cloned into the vector pU6H1 to construct recombinant plasmids.2. Optimized transfection conditions of the calcium phosphate transfection method, then the recombinant plasmids were transfected into the SGC-7901 cells.3. The ANXA2 mRNA and protein levels were analysised by RT-PCR and western blot following siRNA treatment for ANXA2 at 24h, 48h,72h and 96h to identify the inhibition efficiency.4. MTT assay was to investigate the influence of siRNA treatment for ANXA2 on cell proliferation.5. Hoechst33258 staining, MitoViewTM 633 staining and flow cytometry assay were performed to measure the effects of down-regulation of ANXA2 on cell apoptosis.6. Wound healing assay was to estimate the effect of down-regulation of ANXA2 on the cell motility of SGC-7901 cells.7. The FAK mRNA and protein levels were analysised by RT-PCR and western blot to study the effect of depletion of ANXA2 on the FAK expression.Result:1. We designed four effective ANXA2-targeting siRNAs, and the recombinants were constructed successfully.2. The optimal transfection conditions as follows:60%-80%cell confluence before transfection,6μg plasmid per 30mm dish,20min-30min complex formation time, 16h incubation time, and in which high transfection efficiency was achieved.3. The RT-PCR and western blot results suggested that the mRNA and protein levels of ANXA2 were significantly suppressed post transfection.4. In MTT, Hoechst33258 staining, MitoViewTM 633 staining, flow cytometry assay and wound healing assay, the abilities of proliferation and motility of SGC-7901 cells were inhibited markedly in which SGC-7901 cells were depleted of ANXA2 with siRNA. A series of typical morphological changes occurred to ANXA2-depleted cells were detected; as well as apoptotic analysis showed that the apoptosis rate increased. RNAi-mediated depletion of ANXA2 had no effect on the total expression of FAK.Conclusion:Transfecting SGC-7901 cells with the siRNA that targets human ANXA2 led to significant downregulation of ANXA2 expression, which resulted in a marked delay of cell migration, significant suppression of cell proliferation and enhancement of cell apoptosis in SGC-7901 cell lines. Thus we can come to the conclusion that the expression of ANXA2 is closely related to the biological behaviors of SGC-7901 cells; ANXA2 might be a potential target for gastric carcinoma gene therapy or drug therapy.