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目的构建靶向抑制脆性组氨酸三联体(FHIT)基因的短发夹双链RNA(shRNA)真核表达质粒,观察FHIT抑制后对胃癌细胞株BGC-823增殖和凋亡的影响。方法构建FHIT基因特异性的小RNA干扰质粒PGPU6/GFP/Neo-shRNA1、PGPU6/GFP/Neo-shRNA2,利用脂质体LipofactamineTM2000转染胃癌细胞BGC-823,实验分为未转染组、阴性对照组(转染PGPU6/GFP/Neo-shNC组)及PGPU6/GFP/Neo-shRNA1转染组和PGPU6/GFP/Neo-shRNA2转染组,G418筛选得到稳定表达株,Real-time PCR检测在mRNA水平干扰质粒对FHIT的抑制效应,MTT法、流式细胞术观察转染前后细胞生长特性的变化。结果与阴性对照组相比,转染shRNA-FHIT重组质粒的BGC-823细胞FHITmRNA表达明显下降(P<0.05)。与未转染组和阴性对照组相比,转染干扰质粒组细胞增殖活性增强,凋亡率减低、生长周期出现S期和G2/M期的比例上调(均P<0.05)。结论成功将重组shR-NA-FHIT表达载体转染BGC-823细胞,并筛选出稳定低表达FHIT的细胞。shRNA-FHIT可以促进胃癌细胞增殖、降低凋亡率,并削弱G0/G1期阻滞,为进一步研究FHIT基因在肿瘤中的作用机制奠定了实验基础。
Objective To construct a short hairpin double stranded RNA (shRNA) eukaryotic expression plasmid targeting FHIT gene and study the effect of FHIT on proliferation and apoptosis of gastric cancer cell line BGC-823. Methods The small interfering plasmids PGPU6 / GFP / Neo-shRNA1, PGPU6 / GFP / Neo-shRNA2 of FHIT gene were constructed and transfected into BGC-823 gastric cancer cells by LipofactamineTM2000. The experiment was divided into untransfected group and negative control The cells were transfected with PGPU6 / GFP / Neo-shNC group, PGPU6 / GFP / Neo-shRNA1 transfected group and PGPU6 / GFP / Neo-shRNA2 transfected group. The stable expression strains were screened by G418. The inhibitory effect of horizontal interference plasmids on FHIT, MTT assay and flow cytometry were used to observe the changes of cell growth characteristics before and after transfection. Results Compared with the negative control group, the expression of FHIT mRNA in BGC-823 cells transfected with shRNA-FHIT recombinant plasmid was significantly decreased (P <0.05). Compared with untransfected group and negative control group, the cell proliferation activity was enhanced and the apoptosis rate was decreased in the transfected plasmid group. The proportion of S phase and G2 / M phase in the growth cycle increased (all P <0.05). Conclusion The recombinant shR-NA-FHIT expression vector was successfully transfected into BGC-823 cells and the cells stably expressing FHIT were screened out. shRNA-FHIT can promote the proliferation of gastric cancer cells, reduce the apoptosis rate, and weaken the G0 / G1 phase arrest, which lays the foundation for further research on the mechanism of FHIT gene in the tumor.