| Background and ObjectiveGastric carcinoma is one of the most common gastrointestinal malignancies in Asians. As we all known, the traditional treatments of malignant tumor include surgical resection, radiotherapy and chemotherapy. Surgical resection is the most effective treatment for gastric cancer and the efficancy of chemotherapy remains limited. And the specificity of these treatments is low and thepostoperative recurrence and mortality rate remain high. An important factor in treatment failure is the invasiveness of the tumor cells causing metastasis. In recent years, molecularly gene therapy for treatment in gastric cancer has been given great concern. Therefore, identification of novel targets for gene therapy is major goals in this field.ANXA2, a member of annexin family, is excessively expressed and plays important roles in tumor development. The excessive expression of ANXA2was reported to enhance the malignancy of numerous cancers in digestion system. To investigate how ANXA2is involved with the cell malignancy of gastric cancer and evaluate its potential for gastric cancer gene therapy, in this study, we inhibited ANXA2expression in SGC-7901cells via siRNA method and analyzed the effects of ANXA2on the cell cycle, motility, apoptosis, and the expression of ANXA2related genes.Method1. Based on the sequences of ANXA2searched 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 pU6H1to construct recombinant plasmids.2. Optimize transfection conditions of the calcium phosphate transfection method, then transfect the recombinant plasmids into the SGC-7901cells.3. Use Hoechst33258, MitoView633staining and flow cytometry assay to detect the effects of down-regulation of ANXA2to cell apoptosis and cell cycle.4. Use Tanswell champer assay to estimate the effect of down-regulation of ANXA2to the motility of SGC-7901cells.5. Use gene colocolization expression assay to detect the interaction between FAK. and ANXA2. 6. The mRNA and protein level of ANXA2related genes (S100A10, tPA, β-actin) were measured by PT-PCR and Western blot at72h post transefection in order to research the relationship among these genes.Result1. We designed four different ANXA2-targeting siRNAs and a control siRNA, then the recombinants were constructed successfully; only the best interference ANXA2vector and the control vector were selected to perform this experiment.2. Calcium phosphate transfection method was used to transfect the recombinant plasmids into SGC-7901cells, about80%GFP expression at72h post-transfection. RT-PCR assay and Western blot assay were used to detect the inhibition efficiency. The result showed that the expression of ANXA2was significantly inhibited by the designated siRNA.3. To elucidate the cell proliferation of gastric cancer, cell cycle and apoptosis were detected using flow cytometry, and Hoechst33342, MitoViewTM633staining. We found that Inhibition of ANXA2expression caused cell proliferation decreased significantly with the Gl time arrest, and apoptosis enhanced without significance.4. We used Trans Well Champer assay to detect the motility of gastric cnacer, and we found that after knocking down the expression of ANXA2, the motility of SGC-7901cells was inhibited significantly.5. Gene colocolization expression assay was used to detect the relationship between ANXA2and FAK, we found that FAK and ANXA2are colocolization in cell membrane, nucleus and cytoplasm of SGC-7901cells.6. The results of RT-PCR and Western blot showed when the expression of ANXA2was inhibited, the expression of tPA and S100A10were significantly dereased, but the expression of β-actin was not affected.Conclusion To investigate how ANXA2is involved with the cell malignancy of gastric cancerand evaluate its potential for gastric cancer gene therapy, down-regulation of ANXA2expression, which resulted in a marked impairment of cell migration ability, significantsuppression of cell proliferation but not enhancement of apoptosis in SGC-7901cells.The results of RT-PCR and Western blot showed when the expression of ANXA2wasinhibited, the expression of tPA and S100A10were significantly dereased, but theexpression of β-actin was not affected. These findings demonstrate that ANXA2 expression may enhance cell motility for the invasion and metastasis of cancers and the unlimited proliferation of cancer cells. The expression of ANXA2is closely related with the motility of gastric carcinoma, after knowking down of ANXA2, the motility was significantly inhibited, which suggest that inhibiting the expression of ANXA2can inhibit the malignancy of gastric carcinoma.Combing our previous data together, our results indicate that expression of ANXA2is closely related to maintain the malignancy of cancer cell, and has potential to be considered as a target for the gene therapy of gastric carcinoma.
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