摘要目的 观察结直肠癌组织和细胞中微小RNA-221(miR-221)和CDKN1C/P57表达情况,并探讨特异性miR-221抑制剂对癌细胞增殖、凋亡及CDKN1C/P57表达的影响.方法 应用实时荧光定量PCR检测34例结直肠癌组织和相应癌旁组织、以及4种人结直肠癌细胞系HT-29、Lovo、SW-480、Caco2中miR-221表达情况;采用半定量RT-PCR和Western-blot法分析CDKN1C/P57mRNA及蛋白表达情况;将miR-221和anti-miR-221寡核苷酸通过脂质体转染Caco2细胞系,通过MTT法及流式细胞仪观察细胞的增殖及凋亡情况.结果 miR-221在结直肠癌组织中的相对表达量为2.041±1.401,明显高于癌旁组织(0.806±0.341,P＜0.01);同样,miR-221在4种人结直肠癌细胞系中的表达亦高于正常对照细胞.CDKN1C/P57 mRNA水平在结直肠癌与癌旁组织中差异并不显著;但其蛋白相对表达量结直肠癌组织显著高于癌旁组织(3.019±1.708比0.972±0.316,P＜0.01).癌细胞转染anti-miR-221后,CDKN1C/P57蛋白表达上调,细胞增殖受抑,细胞凋亡率增高,G0/G1期细胞比例增加同时S期细胞比例下降,差异均有统计学意义(P＜0.01).结论 miR-221可能通过转录后基因沉默机制使CDKN1C/P57蛋白表达水平下降,从而促进结直肠癌发生发展;anti-miR-221可通过抑制细胞增殖同时诱导细胞凋亡,以抑制结直肠癌细胞生长;miR-221有可能成为结直肠癌生物治疗的新靶点.

abstractsObjective To investigate the expression of microRNA-221 (miR-221) and CDKN1C/ P57 in colorectal carcinoma (CRC) and adjacent non-cancerous tissues. The effect of miR-221-specific inhibitor on cell proliferation and apoptosis in CRC cells was also assessed. Methods The expression of miR-221 was detected by real-time RT-PCR. CDKN1C/P57 mRNA and corresponding protein expression pattern were detected by semi-quantitative RT-PCR and Western-blot. The specific 2'-methoxy-modified RNA oligonucleotide of miR-221 (miRNA inhibitor, anti-miR-221)was designed,synthesized and transfected into Caco2 cell by liposome. Finally, the status of CRC cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Results The expression of miR-221 was significantly up-regulated in CRC tissues as compared to the adjacent non-cancerous tissues (2.041 ±1.401 vs. 0.806±0.341, P＜0.01 ). There was no significant difference in CDKN1C/P57 mRNA expression between CRC and non-cancerous tissues, whereas CDKN1C/P57 protein markedly decreased in CRC (3.019± 1.708 vs. 0.972±0.316, P＜0.01 ). miR-221-specific inhibitor significantly enhanced CDKN 1C/P57 protein expression, inhibited proliferation of CRC cells and induced apoptosis of CRC cells (P＜0.01).Conclusions miR-221 inhibits CDKN1C/P57 expression by post-transcriptional gene silencing to promote CRC development and progression. miR-221-specific inhibitor potentially inhibits the growth of CRC cells. Therefore, it may be a new target for the biologic therapy for CRC.