MiR-27靶基因的分析及鉴定

MicroRNAs(miRNAs)是一类约20～25nt的小分子核苷酸,在细胞内的多种生物学过程,如细胞增殖、凋亡、生长、分化和代谢等过程中具有重要的功能。已知miR-27在脂肪细胞和肌肉细胞的发育过程中起了重要作用,其在神经细胞中的表达调节至今仍不清楚。在本研究中,通过miRBase和TargetScan数据库分析了miR-27的靶基因,构建了miR-27的真核表达载体,改造了萤火虫荧光素酶和海肾荧光素酶报告载体,将miR-27的靶基因Bmi1的3′-UTR融合到报告载体中,转染神经胶质瘤细胞,利用双荧光素酶检测系统分析荧光素酶的活性。研究发现miR-27a和miR-27b共同的靶基因主要调节发育过程。MiR-27真核表达载体能产生成熟态的miR-27。MiR-27a、miR-27b或miR-27a和miR-27b联合与Bmi1的3′-UTR的正义序列共转染U343细胞能明显降低萤火虫荧光素酶的活性(分别P0.05,P0.05,P0.01),这提示了Bmi1可能为miR-27的靶基因。

The Analysis and Identification of MiR-27 Target Gene

MicroRNAs (miRNAs) is singlestranded oligoribonucleotides about 20 -25 nucleotides (nt) in length and function at many biological process, for example, cell proliferation, apoptosis, growth, differentiation and metabolism. MiR-27 play an important role in adipogenesis and muscle stem cell behavior,but it is unclear about its role in the development of neural cell. In the present study,the target genes of miR-27 was analyzed by miRBase and TargetScan databases. A recombinant miR-27 eukaryotic expression vector was constructed and expressed it in human glioraa cells U343. We reconstructed the firefly luciferase and renilla luciferase vector and fused the 3'-UTR of miR-27 target gene Bmil with report gene that was transfected in U343 cells, luciferase activity was detected by Dual-Luciferase Reporter (DLR) Assay. Results showed the common target genes of miR-27a and miR-27b regulated mainly the process of development. MiR-27 eukaryotic expression vector can produce mature miR-27a and miR-27b. Firefly luciferase activity was significantly reduced by cotransfection of miR-27a (P <0.05) ,miR-27b (P < 0. 05), combination of both (P<0.01) and sense 3 '-UTR of Bmil. These results implied that Bmil may be the one of miR-27 target genes.