Let-7a过表达对结肠黏液腺癌细胞LS-174T增殖及侵袭性的影响

The effect of Let-7a over-expression on proliferation and invasiveness of colonic mucinous adenocarcinoma cell line LS-174T

目的:研究let-7a过表达对结肠黏液腺癌LS-174T细胞增殖及侵袭性的影响。方法:构建let-7a过表达慢病毒载体并感染结肠黏液腺癌LS-174T细胞,设立空白对照组(A组,不进行病毒转染);转染空病毒组(B组,只含有EGFP,浓度1×10^7 TU/L);C1转染组(C1组,浓度1×10^6 TU/L):转染hsa-Let-7a(含let-7a病毒和EGFP);C2转染组(C2组,浓度1×10^7 TU/L);C3转染组(C3组,浓度1×10^8 TU/L)。通过倒置荧光显微镜观察各组感染效率;RT-qPCR检测各组细胞中let-7a基因表达水平;MTT法检测各组细胞增殖活力;Transwell侵袭和划痕实验测评估细胞的侵袭和迁移能力;RT-qPCR和Western blot分析TGF-β1 mRNA和蛋白水平的表达。结果:倒置荧光显微镜观察各组细胞48 h绿色荧光表达情况,C3组荧光表达最强,感染效率可达90%以上,与初始病毒浓度成正相关(P<0.05);MTT检测结果显示各组OD值均随时间延长而增高,在第0、第1天,5组OD值差异无统计学意义(P>0.05),第2、第3、第4天,与A组比,C组的OD值增幅明显降低(P<0.05);各组细胞let-7a基因表达水平行RT-q PCR检测结果显示,与A组比,C组let-7a的基因表达水平显著升高,且转染细胞let-7a的表达水平与含let-7a的慢病毒浓度梯度呈正相关(P<0.05),而RT-qPCR及Western blot检测显示let-7a过表达后TGF-β1 mRNA和蛋白水平逐渐降低(P<0.05)。侵袭和划痕实验结果表明,let-7a的过表达降低了LS-174T细胞的侵袭和迁移能力,且与初始病毒浓度成负相关(P<0.05)。结论:Let-7a过表达对LS-174T细胞增殖活力有明显抑制作用,可通过下调TGF-β1含量而抑制细胞的侵袭和转移。

Objective: To study the effect of let-7 a overexpression on the proliferation and invasiveness of colonic mucinous adenocarcinoma cell line LS-174 T. Methods: The let-7 a overexpression lentiviral vector was constructed and infected with colonic mucinous adenocarcinoma LS-174 T cells. A blank control group was established: no virus transfection;B transfected empty virus group;C1 transfection group(concentration 1× 10^6 TU/L): transfection of hsa-let-7 a [containing let-7 a virus and enhanced green fluorescent protein(EGFP)];C2 transfection group(concentration 1×10^7 TU/L);C3 transfection group(concentration 1×10^8 TU/L). The infection efficiency of each group was observed by fluorescence microscope. The expression of let-7 a was detected by RT-qPCR. The proliferation of each groupcells was detected by MTT assay. The invasion and migration ability of cells were evaluated by Transwell invasion and scratch test. RT-qPCR and Western blot analysis of TGF-β1 mRNA and protein levels were expressed. Results: The expression of green fluorescence protein in each group was observed by inverted fluorescence microscope. The fluorescence expression of C3 transfection group was the strongest, with transfection efficiency over 90%, which was positively correlated with the initial virus concentration(P<0.05). The expression levels of let-7 a in each group were detected by RT-qPCR. The results showed that the expression level of let-7 a in the transfected group was significantly higher than that in the negative control group, and the expression level of let-7 a in the transfected cells was positively correlated with the gradient of lentivirus concentration(P<0.05). The results of Western blot and RT-qPCR showed that the expression of TGF-β1 mRNA and protein decreased gradually after let-7 a overexpression(P<0.05). The results of invasion and scratching showed that the overexpression of let-7 a reduced the invasion and migration ability of LS-174 T cells, and was negatively correlated with the initial virus concentration(P<0.05). Conclusion: Overexpression of let-7 a can inhibit the invasion and metastasis of LS-174 T cells by down-regulating the content of TGF-β1.