Department of General Surgery, Nanfang Hospital of Southern Medical University, Guangzhou, China.
Acta Pharmacol Sin. 2011 Mar;32(3):375-84. doi: 10.1038/aps.2010.206. Epub 2011 Jan 31.
To investigate the regulatory effect of microRNA-221 (miR-221) on CDKN1C/p57 expression in colorectal carcinoma (CRC).
Thirty four CRC and adjacent non-tumorous tissue samples were collected individually. Total RNA and protein were isolatedand from these samples and four human CRC-derived cell lines (including HT-29, Lovo, SW-480 and Caco2). MiR-221 expression was examined using real-time RT-PCR. CRC cells were treated with or without anti-p57-siRNA prior to the addition of pre-miR-221 or anti-miR-221. The mRNA and protein levels of CDKN1C/p57 were examined using semi-quantitative RT-PCR and Western blot, respectively. CRC cell proliferation and apoptosis were assessed using MTT assay and flow cytometry, respectively. The CDKN1C/p57 3'-UTR fragment was amplified using PCR from the genomic DNA of human colon cells and inserted into a luciferase reporter construct. The reporter construct was then transfected into CRC cells together with pre-miR-221 or anti-miR-221, and the luciferase activity in the transfected cells was examined.
MiR-221 expression was significantly up-regulated in 90% of CRC samples compared to that in the adjacent non-tumorous tissue, and the expression level was positively correlated to an advanced TNM stage and local invasion. There was no significant difference in CDKN1C/p57 mRNA expression between CRC and corresponding non-tumorous tissues, whereas CDKN1C/p57 protein expression was markedly decreased in the CRC samples. A significant inverse correlation between miR-221 and CDKN1C/p57 expression was found in CRC cells. Moreover, a miR-221-specific inhibitor significantly increased CDKN1C/p57 protein expression in CRC cells. Anti-miR-221 markedly inhibited CRC cell proliferation and induced apoptosis. This inhibitory effect was abolished by pretreatment with anti-p57-siRNA, suggesting that the inhibition was mediated by CDKN1C/p57. A significant increase of the luciferase activity was observed in CRC cells co-transfected with the luciferase reporter construct and anti-miR-221.
MiR-221 binds to the target site in the 3'-UTR of the CDKN1C/p57 mRNA to inhibit CDKN1C/p57 expression by post-transcriptional gene silencing to promote CRC occurrence and progress, therefore serving as a potential therapeutic target for the prevention and treatment of CRC.
研究微小 RNA-221（miR-221）对结直肠癌（CRC）中 CDKN1C/p57 表达的调控作用。
分别收集 34 例 CRC 及相邻非肿瘤组织标本和 4 个人结直肠癌细胞系（包括 HT-29、Lovo、SW-480 和 Caco2）。采用实时 RT-PCR 检测 miR-221 的表达。用抗 p57-siRNA 预处理 CRC 细胞后，加入 pre-miR-221 或 anti-miR-221。分别采用半定量 RT-PCR 和 Western blot 检测 CDKN1C/p57 的 mRNA 和蛋白水平。采用 MTT 法和流式细胞术分别检测 CRC 细胞增殖和凋亡。采用 PCR 从人结肠细胞基因组 DNA 扩增 CDKN1C/p57 3'-UTR 片段，并插入荧光素酶报告载体。将报告载体与 pre-miR-221 或 anti-miR-221 共转染 CRC 细胞，检测转染细胞中的荧光素酶活性。
与相邻非肿瘤组织相比，90%的 CRC 样本中 miR-221 表达显著上调，且表达水平与 TNM 分期较晚和局部浸润呈正相关。CRC 与相应非肿瘤组织之间 CDKN1C/p57 mRNA 表达无明显差异，而 CRC 样本中 CDKN1C/p57 蛋白表达明显降低。在 CRC 细胞中发现 miR-221 与 CDKN1C/p57 表达呈显著负相关。此外，miR-221 特异性抑制剂可显著增加 CRC 细胞中 CDKN1C/p57 蛋白表达。抗 miR-221 显著抑制 CRC 细胞增殖并诱导细胞凋亡。用抗 p57-siRNA 预处理可消除这种抑制作用，表明抑制作用是通过 CDKN1C/p57 介导的。与共转染荧光素酶报告载体和 anti-miR-221 的 CRC 细胞相比，观察到荧光素酶活性显著增加。
miR-221 通过靶向 CDKN1C/p57 mRNA 的 3'-UTR 结合位点，通过转录后基因沉默抑制 CDKN1C/p57 表达，促进 CRC 的发生和进展，因此可作为预防和治疗 CRC 的潜在治疗靶点。
