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Construction of Human miR-302s Expression Plasmid and Its Effect on Cell Cycle
Qiu-Yan Ge1,2, Li-Jun Ding2, Gui-Jun Yan2, Zhen-Yu Diao2, Hai-Xiang Sun1,2, Ya-Li Hu1,2*
1Drum Tower School of Clinical Medicine, Nanjing Medical University, Nanjing 210029, China; 2Center of Reproductive Medicine,Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, China
Abstract: 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-C1-miR-302s， which indicated that cyclin D1 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 cells after the transfection of pEGFP-C1-miR-302s. These results confirmed the important regulatory role of human miR-302s in the G1-S transition of cell cycle.
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