论文部分内容阅读
目的:构建针对STAT3基因的短发夹状小干扰RNA重组质粒,探讨应用RNAi技术沉默STAT3基因对胃癌细胞BGC-823增殖和侵袭的影响。方法:应用DNA重组技术,构建针对STAT3基因的干扰RNA重组质粒(pSilencer-STAT3),经脂质体转染BGC-823胃癌细胞,逆转录聚合酶链反应和Western blot免疫印迹法检测STAT3的表达水平;MTT法检测RNA干扰后细胞增殖情况;Transwell细胞侵袭实验检测细胞侵袭能力的变化。结果:酶切鉴定和测序证实了重组质粒构建成功。与未转染和转染阴性对照质粒组相比,pSilencer-STAT 3转染组胃癌细胞BGC-823中STAT 3mRNA和STAT 3蛋白的表达水平均明显降低,F=39.424,P=0.000和F=31.911,P=0.001。pSilencer-STAT3转染组胃癌细胞BGC-823的生长受到明显抑制,抑制率达47.3%,与阴性对照质粒组的8.1%抑制率相比,差异具有统计学意义,F=40.835,P=0.000。侵袭实验显示,pSilencer-STAT3转染组胃癌细胞BGC-823穿膜细胞数,显著低于阴性对照质粒组和未转染组(75±10,111±10,104±9,F=11.311,P=0.009)。结论:构建的pSilenc-er-STAT3重组质粒能有效地抑制STAT3基因在人胃癌细胞BGC-823中的表达,抑制细胞的增殖和侵袭。
OBJECTIVE: To construct short hairpin RNA interference plasmids targeting STAT3 gene and investigate the effect of STAT3 gene silencing RNAi on the proliferation and invasion of gastric cancer cell line BGC-823. METHODS: The recombinant plasmid pSilencer-STAT3 targeting STAT3 gene was constructed by DNA recombinant technology. BGC-823 gastric cancer cells were transfected by lipofectamine 2000. Reverse transcription polymerase chain reaction and Western blot were used to detect the expression of STAT3 The cell proliferation was detected by MTT assay and the invasion ability was detected by Transwell cell invasion assay. Results: Enzyme digestion and sequencing confirmed that the recombinant plasmid was successfully constructed. STAT3 mRNA and STAT3 protein expression in BGC-823 cells transfected with pSilencer-STAT3 were significantly lower than those in untransfected and transfected negative control plasmids, F = 39.424, P = 0.000 and F = 31.911, P = 0.001. The growth of BGC-823 cells was significantly inhibited in pSilencer-STAT3 transfected group, and the inhibition rate was 47.3%. Compared with the negative control plasmid group, the difference was statistically significant (F = 40.835, P = 0.000). The results of invasion assay showed that the number of transmembrane cells in gastric cancer cell line BGC-823 transfected with pSilencer-STAT3 was significantly lower than that in negative control plasmid group and non-transfected cells (75 ± 10,111 ± 10,104 ± 9, F = 11.311, P = 0.009). CONCLUSION: The constructed pSilenc-er-STAT3 recombinant plasmid can effectively inhibit the expression of STAT3 gene in human gastric cancer cell line BGC-823 and inhibit the proliferation and invasion of cells.