摘要
目的探讨微小RNA92a（miR92a）靶向调控Krüppel样因子4（KLF4）对结肠癌细胞增殖的影响。方法采用实时荧光定量PCR（qRTPCR）方法检测21例结肠癌和对应癌旁正常组织及4种结肠癌细胞（HT29、SW480、SW620、HCT116）中miR92a的表达水平。选取结肠癌细胞株HCT116、SW620分别瞬时转染miR92a mimic、inhibitor，在构建miR92a高表达和抑制表达的细胞模型后，采用CCK8比色法检测细胞增殖活性，运用流式细胞仪检测细胞周期，采用Western blotting方法检测KLF4的蛋白表达水平，采用双荧光素酶报告基因检测细胞荧光素酶活性。结果结肠癌组织中miR92a的表达水平为（0.648±0.489）fmol/μg总RNA，显著高于对应癌旁正常组织的（0.064±0.062）fmol/μg总RNA，差异具有统计学意义（t=-5.420，P＜0.001）。4种人结肠癌细胞株中HCT116的miR92a表达水平相对最低，SW620的表达水平相对最高。上调miR92a表达，HCT116细胞72 h增殖活性高于阴性对照组（0.919±0.014∶0.765±0.025），差异具有统计学意义（t=-9.309，P=0.001），S期细胞增多［（41.670±0.461）%∶（38.703±0.554）%，t=-7.127，P=0.002］，KLF4蛋白表达水平下调（0.460±0.048∶0.758±0.109，t=22.865，P=0.028）；抑制miR92a表达，SW620细胞72 h增殖活性低于阴性对照组（0.608±0.011∶0.713±0.005），差异具有统计学意义（t=15.920，P＜0.001），S期细胞减少［（31.935±0.365）%∶（34.955±0.465）%，t=8.849，P=0.001］，KLF4蛋白表达水平上调（0.694±0.121∶0.479±0.044，t=-5.246，P=0.034）。miR92a3p mimic与KLF4 3′UTR野生型质粒共转染HCT116细胞后，与阴性对照组比较，上调细胞miR92a表达能使Firefly荧光素酶活性略有降低，但差异无统计学意义（t=0.878，P=0.429）。结肠癌组织中miR92a表达与KLF4蛋白表达之间有一定的负相关趋势，但无统计学意义（r=-0.163，P=0.699）。结论miR�
ObjectiveTo evaluate the effect of microRNA92a （miR92a） on regulating cell proliferation by targeting Krüppellike factor 4 （KLF4） in colon cancer. MethodsThe miR92a expressions in 21 colon cancer tissues and matched normal tumoradjacent tissues and 4 colon cancer cells （HT29, SW480, SW620, HCT116） were detected using quantitative realtime polymerase chain reaction （qRTPCR）. Models of overexpression and suppression of miR92a were established by transient transfection of miR92a3p mimic to HCT116 and transient transfection of miR92a3p inhibitor to SW620, respectively. Cell proliferation activity was detected by the CCK8 colorimetry method, cell cycles were detected by flow cytometry, KLF4 protein expression was detected by Western blotting, and cell luciferase activity was detected by the dual luciferase reporter gene experiment. ResultsThe expression level of miR92a in colon cancer tissues was （0.648±0.489） fmol/μg total RNA, significantly higher than that in matched normal tumoradjacent tissues [（0.064±0.062） fmol/μg total RNA], with statistically significant difference （t=-5.420, P〈0.001）. In 4 colon cancer cell lines, the miR92a expression level in HCT116 cells was the lowest, and highest in SW620 cell. When the expression of miR92a was upregulated, the cell proliferation activity of 72 h in HCT116 cells was higher than that in the negative control group （0.919±0.014 vs. 0.765±0.025）, with statistically significant difference （t=-9.309, P=0.001）, the proportion of S phase cells was also significantly increased [（41.670±0.461）% vs. （38.703±0.554）%, t=-7.127, P=0.002）, and KLF4 protein expression was decreased （0.460±0.048 vs. 0.758±0.109, t=22.865, P=0.028）. When the expression of miR92a was downregulated, the cell proliferation activity of 72 h in SW620 cells was lower than that in the negative control group （0.608±0.011 vs. 0.713±0.005）, with statistically significant difference �
出处
《国际肿瘤学杂志》
CAS
2017年第11期812-818,共7页
Journal of International Oncology
基金
深圳市科技计划（JCYJ20140411092959841）
