The Study on the Effects of FAK Expression to the Behaviors of Human Gastric Carcinoma Cells

【摘要】：Gastric cancer is the second most common cause of cancer-related death in the world. It is often asymptomatic or causes only nonspecific symptoms in its early stages. By the time symptoms occur, the cancer has often reached an advanced stage, one of the main reasons for its poor prognosis. Even patients who present in the most favorable condition and undergo curative surgical resection often die of recurrent disease. Currently, surgery is still the main theatment, in which the application of chemotherapy and radiotherapy is also combined. However, these theatments result in serious poisonous side effects and high metastasis and recurrence. With the development of cellular and molecular biology, as a new therapy method, gene therapy has become prominent, and it has achieved considerable progresses on cancer diagnosis, therapy and prognosis. RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing technique, which has a great significance in gene function research, and has received much consideration. Two types of small RNA molecules:microRNA (miRNA) and small interfering RNA (siRNA) are central to RNA interference. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase involved in cancer cell survival, proliferation, apoptosis, and metastasis. To discover what roles FAK plays to regulate the malignant behaviors of gastric cancer cells, we studied its effects on gastric cancer proliferation, apoptosis and motility after FAK expression was inhibited in SGC-7901 cells (human gastric carcinoma cells) using RNAi. In addition, we detected Annexin A2 mRNA and protein levels after down-regulation of FAK, and to find if there is a relationship between them. It is concluded that this research can be used as the references for gastric cancer gene therapy research projects. Methods:1. Based on FAK variants mRNA sequences supplied by Genbank database, four siRNAs targeting to FAK variants were synthesized and constructed to recombinant plasmids pU6H1-GFP. Scramble control sequence (SCR) of human genome was also cloned to plasmid pU6H1-GFP.2. Recombinant plasmids pU6Hl-GFP-siFAK and pU6H1-GFP-siRNASCR were introduced into the human gastric cell, SGC-7901, using the optimized method. And six groups (NC, siRNASCR, siFAK-1, siFAK-2, siFAK-3 and siFAK-4) were included.3. FAK mRNA and protein levels were evaluated by semi-quantitative RT-PCR and western blotting at 24 h,48 h,72 h and 96 h post thansfection.4. The cell proliferation and motility were determined with MTT and wound healing assay after FAK down-regulation. Moreover, cell apoptosis at 96 h post transfection was detected by Hoechst 33258 staining and MitoViewTM 633 mitochondrial dye.5. Cancer related gene Annexin A2 mRNA and protein levels were evaluated by semi-quantitative RT-PCR and western blotting at 72 h after down-regulation of FAK. Results:1. The exogenous siRNAs were inserted into plasmid pU6Hl-GFP and siRNAs recombinants were constructed successfully, which were verified through enzyme digestion and gene sequencing.2. Tranfection condition was optimized and high efficiency was obtained. Following parameters were indicated as the optimal transfection conditions:60%-70% cultured cell cover ratio before transfection,8μg plasmid per 30 mm dish,30 mins complex formation time,16-18 h incubation time. 3. FAK mRNA and protein level were detected by semi-quantitative RT-PCR and western blotting. And the results showed, compared to NC group, FAK mRNA and protein expression were significantly down-regulated for every siFAK group at each time point post transfection (p0.05). The mRNA level inhibition was achieved maximal at 72 h post transfection with time dependent. Moreover, siFAK-2 and siFAK-3 decreased more significantly. However, siRNASCR indicated no significance with NC at each time point post transfection (p0.05).4. In MTT, and wound healing assay, tranfected with siFAK groups showed suppressed proliferation and motility rate (p0.05), and the inhibition efficiency was positively correlated with FAK knockdown rate. Evaluated apoptosis ability was observed in FAK silencing group at 96 h post transfection. The proliferation, apoptosis and motility ability of siRNASCR group indicated no significance with NC group (p0.05).5. Annexin A2 mRNA and protein levels were increased after down-regulation of FAK. Conclusion:Using RNAi, knockdown of FAK level in cultured SGC-7901 can suppress its proliferation and motility rate, and increase its apoptosis ability in vitro. However, there was no significant influence was indicated on proliferation, apoptosis and motility for siRNASCR.It is concluded that the expression of FAK in SGC-7901 is closely associated with the cell malignant behaviors, and siRNA can inhibit the FAK expression efficiently in SGC-7901 cells. Therefore, FAK can be used as gene therapy target and plasmid pU6Hl-GFP as safe clinical vector for gastric cancer gene therapy. The results indicate that FAK may serve as a potential therapeutic target for gantric cancer therapy.