摘要
目的:研究微小RNA-187*(miR-187*)在人结肠癌细胞株及正常结肠组织中的表达,同时分析miRNA-187*上调对人结肠癌细胞增殖和细胞周期的影响。方法:选取3例结直肠癌组织及配对正常黏膜组织进行miRNA芯片检测,筛选出大肠癌组织中异常表达的miRNA;提取8株结肠癌细胞株和10例正常结肠组织中总RNA,采用Taqman实时定量PCR方法检测miR-187*的表达;预测miR-187*的靶基因,采用实时定量PCR方法检测靶基因的表达;采用脂质体介导的转染方法将miR-187*模拟物转染人结肠癌细胞株HCT116;检测转染后miR-187*和靶基因的表达;通过MTS试剂盒检测细胞增殖能力,流式细胞术检测miR-187*对细胞周期的影响。结果:miRNA芯片检测结直肠癌组织中miR-187*的表达较正常黏膜明显降低;miR-187*在结肠癌细胞株中的表达较正常结直肠黏膜组织明显下调;而B细胞特异性莫洛尼小鼠白血病病毒整合位点1(BMI-1)mRNA在结肠癌细胞株中表达较正常黏膜组织中明显增高;转染miR-187*模拟物后,miR-187*表达明显增加;同时miR-187*的高表达可以显著抑制BMI-1 mRNA的水平;miR-187*模拟物转染组和阴性对照组相比,细胞增殖活力明显受抑制(P<0.05),同时增殖细胞核抗原表达亦明显降低;细胞周期检测结果显示,miR-187*模拟物转染组G2/M期细胞增多,和阴性对照组间差异有统计学意义。结论:miR-187*在人结肠癌细胞株中表达下调;上调miR-187*表达可抑制结肠癌细胞增殖活性,影响结肠癌细胞周期;miR-187*可能通过抑制BMI-1在结肠癌中发挥抑癌作用。
AIM: To investigate the expression of microRNA-187 * (miR-187 * ) in human colon cancer cell lines and normal colon tissues, and to determine the effects of miR-187 * up-regulation on the proliferation and cell cycle of human colon cancer cell line HCT116. METHODS: The expression profiling of miRNAs in 3 colorectal adenocarcinoma samples and their matched normal tissue samples was performed using miRNA microarray chip. Total RNA was isolated from 8 colon cancer cell lines and 10 normal colon tissues. The miR-187 * level was detected by Taqman real-time RT-PCR. B-cell-specific Moloney murine leukemia virus integration site 1 ( BMI-1 ), the possible target of miR-187 * , was also detected. Synthetic miR-187 * mimics were transfected into HCT116 cell line by LipofectamineTM 2000. The mRNA expression of miR-187 * and BMI-1 in HCT116 cell line was measured by real-time RT-PCR. Cell growth and cell cycle were assayed by MTS method and flow cytometry. RESULTS: miR-187 * was found to be differentially expressed between colorectal adenocarcinoma and normal tissues. The expression of miR-187 * in 8 colon cancer cell lines was down-regulated, while BMI-1 mRNA was up-regulated. Compared with blank control group, miR-187 * expression was remarkably increased after transfection with miR-187 * mimics, and ectopic expression of miR-187 * significantly inhibited the mRNA expression of BMI-1. The cell growth was inhibited in miR-187 * mimics group, and proliferating cell nuclear antigen(PCNA) mRNA expression was decreased. The cells at G2/M phase in miR-187 * mimics group were significantly increased. CONCLUSION: miR-187 * is down-regulated in human colon cancer cell lines. Up-regulation of miR-187 * not only inhibits the proliferation but also influence the cell cycle of HCT116 cells, which might act as a tumor suppressor in colorectal cancer by inhibiting the expression of BMI-1.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2013年第11期1946-1951,共6页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81072034)
河北省自然科学研究计划(No.C2011206103)
河北省国际合作项目(No.12396105D)