转化生长因子-β1基因shRNA表达质粒的构建及其对结肠癌细胞SW480侵袭能力的影响

Construction of transformation growth factor-β1-shRNA expression plasmid and its effect on invasion ability of human colon cancer SW480 cells

目的构建转化生长因子-β1(Transforming growth factor-β1,TGF-β1)基因短发夹RNA(Short hairpin RNA,shRNA)表达质粒,并观察其对结肠癌细胞SW480侵袭能力的影响。方法设计并合成2对靶向TGF-β1基因的RNA干扰序列和1对阴性对照序列,并构建重组表达质粒pshRNA-TGF-β1a、pshRNA-TGF-β1b和pshRNA-TGF-β1c(阴性对照),以脂质体介导转染SW480细胞,荧光显微镜观察转染情况;RT-PCR和Western blot检测转染细胞中TGF-β1基因mRNA的转录水平和蛋白的表达水平;Transwell侵袭试验检测沉默TGF-β1基因表达对细胞侵袭能力的影响。结果 TGF-β1基因shRNA重组表达质粒经酶切鉴定和测序分析证明构建正确。与空白对照组相比,3种质粒转染的细胞均有较强的荧光表达;pshRNA-TGF-β1a和pshRNA-TGF-β1b组细胞TGF-β1基因mRNA的转录水平和蛋白的表达水平均明显减低(P<0.05);SW480细胞的侵袭能力也明显降低(P<0.05)。结论成功构建了TGF-β1基因shRNA重组表达质粒,其能有效抑制结肠癌细胞SW480中TGF-β1基因的表达,并可显著降低细胞的侵袭能力,为结肠癌的基因治疗提供了新的思路。

Objective To construct the short hairpin RNA （shRNA） expression vector for human transforming growth factor- β1 （TGF-β1） gene and investigate its effect on invasion ability of human colon cancer SW480 cells. Methods Two pairs of RNA interference sequences targeting TGF-β1 and one pair of negative control sequence were designed and synthesized, based on which recombinant plasmids pshRNA-TGF-β1a, pshRNA-TGF-β1b and pshRNA-TGF-β1c（negative control） were constructed and transfected into SW480 ceils in mediation of Lipofectamine 2000. The transfected cells were observed by fluorescent microscopy, in which the transcription level of TGF-β1 mRNA was determined by RT-PCR, and the expression level of TGF-β1 protein by Western blot. The effect of TGF-β1 gene expression on invasion ability of SW480 cells was evaluated by Transwell test. Results Restriction analysis and sequencing showed that all the recombinant plasmids were constructed correctly. As compared with those in blank control group, strong green fluorescence was observed in the cells transfected with the three recombinant plasmids; both mRNA transcription and protein expression levels of TGF-β1, as well as the invasion abilities of cells transfected with recombinant plasmids pshRNA-TGF-β1a and pshRNA-TGF-β1b decreased significantly （each P 〈 0. 05）. Conclusions The recombinant shRNA expression vectors for TGF- β1 gene was constructed successfully, which effectively inhibited the expression of TGF-β1 gene in SW480 ceils and decreased the invasion ability of the cells. It provided a novel idea for gene therapy of colon cancer.