| 摘要: |
| 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。 |
| 关键词: 微RNAs S100A6蛋白；结直肠肿瘤；HCT116细胞；细胞增殖；细胞凋亡 |
| DOI：10.3724/SP.J.1008.2012.00940 |
| 投稿时间：2012-05-08修订日期：2012-08-09 |
| 基金项目: |
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| Effects of miRNA-136 overexpression on S100 calcium binding protein A6 contents, proliferation and apoptosis in HCT116 cells |
| HUO Zhong-hua*,HU Jun,CHU Zhu-ling,SONG Bo,Lü Sheng,YIN Peng |
(Department of General Surgery, No.454 Hospital of PLA, Nanjing 210002, Jiangsu, China
*Corresponding author.) |
| Abstract: |
| ObjectiveTo investigate the effect of exogenous microRNA 136(mir136) on contents of S100 calcium binding protein A6 (S100A6) and proliferation in human colon cancer cell line HCT116 through constructing a eukaryotic expression vector of mir136 (pcDNA-mir136) and cationic liposome-mediated transfection.MethodsHuman genomic DNA was extracted from HEK293 cells and the DNA fragment containing mir136 precursor sequence was amplified by PCR and cloned into pcDNA to construct pcDNA-mir136. The plasmid was transfected into HCT116 cells via cationic liposome. The content of mir136 was determined by Real-time PCR, S100A6 protein was examined by Western blotting analysis, and apoptosis was detected by Flow Cytometry at 48 h after transfection. The proliferation ability of the tumor cells was examined by CCK-8 at 24 and 48 hours after transfection. ResultsThe eukaryotic expression vector of mir136 was successfully constructed and transfected into HCT116 cells. Expression of mir136 was significantly higher in the transfected group than that in the untransfected group (P<0.01) and the content of S100A6 protein was significantly lower than that of the untransfected group (P<0.05) 48 h after transfection. Meanwhile, cell proliferation activity of the transfected group was significantly lower than that of the untransfected group (P＜0.05) and cell apoptosis was significantly increased compared with the untransfected group (P<0.01).ConclusionMir136 may inhibit proliferation and induce apoptosis of HCT116 cells by inhibiting the expression of S100A6 gene. |
| Key words: microRNAs S100A6 protein colorectal neoplasms HCT116 cells cell proliferation apoptosis |
