人微小RNA-302s表达载体的构建及其对细胞周期的影响

Construction of Human miR-302s Expression Plasmid and Its Effect on Cell Cycle

PCR扩增人miR-302s的启动子区和编码区，亚克隆pEGFP—C1载体，构建miR-302s重组表达载体pEGFP—C1-miR-302s。经过测序鉴定该载体含有核心启动子区（TATA）和miR-302s（miR-302a、miR-302a#、miR-302b、miR-302b^#、miR-302c、miR-302c^#、miR-302d、miR-367和miR-367#簇）的编码子。荧光素酶报告基因实验结果显示pEGFP-C1-miR-302s转染HEK293细胞后抑制细胞周期相关基因cyclin D1和PCAF 3’UTR的活性，提示cyclin D1和PCAF是miR-302s的靶基因。pEGFP—C1-miR-302s转染HEK293细胞后，Real—time Q-PCR检测发现miR-302s高表达，west—ernblot分析发现cyclin D1和PCAF的表达降低，流式细胞术分析进一步表明细胞转染后细胞周期G1期缩短，S期延长，表明miR-302s对细胞周期Grs期转换具有重要调节作用。

Promoter region and coding region of Human miR-302s were amplified from human genomic DNA and inserted to pEGFP-C1 vector to construct human pEGFP- C1-miR-302s expression plasmid. The plasmid contained the core promoter （TATA） and miR-302s cluster （miR-302a, miR-302a^#, miR-302b, miR-302b^#, miR- 302c, miR-302c^#, miR-302d, miR-367 and miR-367^#） encoding sequence. Decreased levels of relative luciferase activity of PCAF and cyclin D1 were detected in HEK 293 cells transfected with pEGFP-CI-miR-302s, which indicated that cyclin D 1 and PCAF were targets of miR-302s. Real-time Q-PCR analysis demonstrated that miR- 302s highly expressed, and Western blot analysis showed that the protein levels of cyclin D1 and PCAF significantly decreased. Furthermore, the flow cytometry analysis showed the S phase extended and G1 phase shortened in HEK293 ceils after the transfection of pEGFP-CI-miR-302s. These results confirmed the important regulatory role of human miR-302s in the G1-S transition of cell cycle.