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目的构建携带微小RNA136(mir136)的真核表达载体并将其转染人结直肠癌细胞,观察外源性mir136表达对人结直肠癌细胞中钙结合蛋白S100A6表达及细胞增殖活性的影响。方法提取人肾上皮HEK293细胞基因组DNA,PCR扩增包含mir136前体序列的DNA片段并构建重组载体,采用阳离子脂质体法将重组载体转染人结直肠癌HCT116细胞。转染后48 h,采用实时定量PCR法检测细胞内mir136含量变化,以蛋白质印迹法检测细胞中S100A6蛋白的表达;转染后24和48 h,采用CCK-8试剂盒检测肿瘤细胞增殖活性;转染后48 h,以双染法检测细胞凋亡情况。结果成功构建重组mir136真核表达载体并转染HCT116细胞。转染后48 h,重组载体转染组HCT116细胞内mir136相对表达量高于未转染组(P<0.01),而S100A6蛋白的相对表达量则低于未转染组(P<0.01)。同时,重组载体转染组HCT116细胞增殖活性较未转染对照组降低(P<0.05),而细胞凋亡则较未转染对照组增加(P<0.01)。结论 Mir136可能通过抑制S100A6基因表达而抑制结直肠癌细胞增殖活性,同时引起细胞的凋亡。
Objective To construct eukaryotic expression vector carrying microRNA136 (mir136) and transfect it into human colorectal cancer cells to observe the effect of exogenous mir136 expression on the expression of calcium binding protein S100A6 and cell proliferation in human colorectal cancer cells. Methods The genomic DNA of human renal epithelial HEK293 cells was extracted. The DNA fragment containing mir136 precursor was amplified by PCR and the recombinant vector was constructed. The recombinant vector was transfected into human colorectal cancer HCT116 cells by cationic liposome method. Forty-eight hours after transfection, the expression of mir136 in cells was detected by real-time quantitative PCR, and the expression of S100A6 protein was detected by Western blotting. The proliferation of tumor cells was detected by CCK-8 kit 24 and 48 hours after transfection; 48 h after transfection, cell apoptosis was detected by double staining. Results The eukaryotic expression vector of recombinant mir136 was successfully constructed and transfected into HCT116 cells. 48 h after transfection, the relative expression level of mir136 in HCT116 cells transfected with recombinant vector was higher than that in untransfected cells (P <0.01), while the relative expression level of S100A6 protein in transfected HCT116 cells was lower than that in untransfected cells (P <0.01). At the same time, the proliferation of HCT116 cells in the recombinant vector transfected group was lower than that in the untransfected control group (P <0.05), while the apoptosis of HCT116 cells increased compared with the untransfected control group (P <0.01). Conclusion Mir136 may inhibit the proliferation of colorectal cancer cells by inhibiting the expression of S100A6 gene and cause cell apoptosis.