目的 检测结肠癌细胞系中miR-181d的表达水平,研究miR-181d对结肠癌细胞增殖及凋亡的影响。方法 RT-qPCR检测结肠癌细胞HCT116,HT29,LoVo,SW480及SW620和永生化肠黏膜上皮细胞HIEC中miR-181d的表达。在LoVo细胞中转染miR-181d模拟物及对照,SW620细胞中转染miR-181d抑制物及对照,RT-qPCR检测转染效率,MTT法检测细胞增殖,流式细胞术检测细胞周期和细胞凋亡比例。结果 miR-181d表达水平在各结肠癌细胞系中均较正常肠黏膜上皮细胞HIEC显著降低（F=29.34,P〈0.01）。在LoVo细胞中过表达miR-181d能够显著减慢细胞体外增殖（F=5.403,P〈0.01）,细胞周期检测示S期细胞比例降低（t=4.71,P〈0.05）,凋亡细胞显著增多（t=3.47,P〈0.05）。在SW620细胞中抑制miR-181d的表达能够显著加快细胞体外增殖（F=20.82,P〈0.01）,细胞周期检测示细胞周期加速,S期细胞比例增高（t=2.92,P〈0.05）,细胞凋亡率显著减少（t=4.14,P〈0.05）。结论 miR-181d在结肠癌细胞系中表达普遍下调,有望成为结肠癌新的诊断标志物。miR-181d能够通过抑制结肠癌细胞增殖和促进凋亡,发挥抑癌基因的作用。
Objective To detect miR-181d expression levels in colon cancer cell lines,and to study the functions of miR-181d on colon cancer cell proliferation and apoptosis. Methods RT-qPCR was employed to study miR-181d expression levels in colon cancer cell line HCT116, HT29, LoVo,SW480 and SW620 cells, as well as in colon normal epithelial cell line HIEC. miR-181d mimic and control were transfected into LoVo cells while miR-181d inhibitor and control were transfected into SW620 cells, qRT-PCR was performed to validate the transfection efficiency. MTT assay was performed to measure cell pro- liferation while flow cytometry was performed to detect cell cycle and apoptosis rate. Results miR-181d was universally downregulated in all colon cancer cell lines compared to the colon normal epithelial cell line HIEC （F= 29.34, P〈0.01 ）. Overexpression of miR-181d in LoVo cells significantly decreased in vitro cell proliferation rate （F= 5. 403 ,P〈0.01）. Flow cytometry indicated that cells at S phase were greatly decreased （t= 4.71, P〈0.05） and apoptotic cells were greatly in- creased compared to the control cells （t = 3.47, P〈 0.05）. On the contrary, inhibition of miR-181d in SW620 cells signifi- cantly promoted cell proliferation （F= 20.82,P〈0.01）. Cell cycle was accelerated with significant increase in S phase com- pared to the control ceils （t=~ 2.92, P〈0.05）, whereas apoptosis ratio was significantly decreased （t= 4.14, P〈0.05）. Con- clusion miR-181d was universally downregulated in colon cancer cell lines compared to the normal epithelial cell line. miR- 181d inhibits cell proliferation and induces apoptosis,thus functions as an tumor suppressive miRNA.
Journal of Modern Laboratory Medicine