结肠癌中相关miRNA对NRP2调控的预测及鉴定

Prediction and identification of microRNAs involved in regulating neuropilin-2 in colorectal carcinoma

目的:预测可能调控神经纤毛蛋白2(neuropilin-2,NRP2)的微小RNA(microRNA,miRNA),鉴定其对NRP2的调控作用及可能的调控机制.方法:使用生物信息学软件预测可能调控NRP2的miRNA,选择可能性最大的miRNA-486-5p进一步研究,构建miRNA-486-5p过表达质粒,采用脂质体法将质粒转染入人结肠癌细胞株SW620,实时定量PCR(qRT-PCR)验证miRNA-486-5p的表达丰度,Western blot检测NRP2的表达,双荧光素酶实验验证miRNA-486-5p对NRP2的调控机制.结果:生物信息学技术预测共有25条miRNA可能调控NRP2的表达,结合文献选择miR-486-5p进一步研究;miR-486-5p过表达质粒转染SW620细胞后,表达丰度升高,NRP2蛋白表达明显下调,双荧光素酶实验证实miRNA-486-5p可与NRP2 mRNA 3'非转录区(3'untranslated region,3'UTR)直接结合,从而发挥对NRP2转录后翻译的抑制作用.结论:miRNA-486-5p可调控的NRP2的表达,其作用机制是通过结合NRP2 3'UTR而抑制NRP2 mRNA的正常翻译.

AIM： To predict and identify the possible microRNAs （miRNAs） involved in regulating neuropilin-2 （NRP2） in colorectal carcinoma. METHODS： Bioinformatic technique was used to predict the possible miRNAs which might participate in regulating NRP2. miR-486-5p, which is most possibly involved in regulating NRP2, was selected for further study. A miR- 486-5p over-expressing plasmid was constructed and transfected into the human colorectal carcinoma cell line SW620 using Lipofectamine 2000.The expression of miR-486-5p in transfected cells was measured by quantitative real-time PCR （qRT-PCR）. Western blot was conducted to detect the expression of NRP2 protein, and miR- 486-5p targeting NRP2 3＇UTR was verified by dual luciferase reporter assay. RESULTS： Twenty-five possible NRP2-regulating miRNAs were predicted using bioinformatic software. By searching the literature, we chose rniR-486-5p for further study. The expression of rniR486-5p in SW620 cells transfected with miR- 486-5p over-expressing plasmid was obviously up-regulated. Western blot analysis revealed that miR-486-5p reduced the expression level of NRP2. Dual luciferase reporter assay demonstrated that miR-486-5p could directly target the NRP2 3＇UTR. CONCLUSION： miR-486-5p is involved in the post-transcriptional regulation of NRP2 by binding to the NRP2 3＇UTR and interfering the normal translation of NRP2.