小RNA干扰STAT3基因表达对胃癌细胞增殖和侵袭的影响

Effect of RNA interference targeting STAT3 on the proliferation and invasion of gastric cancer cell

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 a expression vector of short hairpin RNA targeting STAT3 gene and observe its effects on cell growth and invasion ability of human gastric cancer cell line BGC 823 in vitro. METHODS： The recombinant plasmid pSilencer- STAT3 was constructed by combination of double-strand DNA containing STAT3 with vector pSilencer that underwent restriction digest. Then these recombinant plasmids were transfected by liposome into BGC 823 cells. The expression of STAT3 mRNA and protein were detected by RT PCR and Western blot. MTT assay was applied to assess cell proliferation. The invasion ability of BGC 823 cells was determined by transwells assay. RESULTS： Re striction enzyme digesting assay and DNA sequencing analysis was confirmed successful construction of recombinant plasmid pSilencer STAT3. Compared with the non-transfected group and pSilencer neospecial group, pSilence-STAT3 inhibited the expression of STAT3 mRNAinBGC-823 cells （F=39.424,P=0.000）. Theex pression of STAT3 protein was also knocked down by pSilencer STAT3 siRNA （F= 31. 911, P= 0.001）. The proliferation of the BGC-823 cells was suppressed significantly （47.3 %） after transfection of pSilencer STAT3 recombinant plasmid, which was markedly higher than that of the pSilencer-neospecial vector control group （8.1% , F=40.835,P =0.000）. The migration of the recombinant plasmid transfected cells was significantly suppressed compared with that of cells transfected with pSilencer-neospecial vector or non-transfected （75±10,111±10,104±9,F =11.311,P= 0.009）. CONCLUSION：Recombinant plasmid pSilencer-STAT3 can significantly reduce STAT3 gene expression in human gastric cancer cell BGC 823 and inhibit the proliferation and invasion ability.