Construction And Activity Identification Of MiRNA Eukaryotic Expressing Vector Of PRMT2

Objective:To construct PRMT2gene expressing eukaryotic miRNA recombiants，and to identifybiological activity of recombinat in breast cancer cell line MCF7cells after transfection.Methods:According to sequence of PRMT2gene, the pre-miRNA was designed and synthesized,then cloned into the GFP reporter pcDNATM6.2-GW/EmGFP-miR, and transfected intoMCF7cell line. The integrity of inserted fragment sequence was tested through colony PCRand sequence analysis, and the biological activity of recombinants was identified by detectingthe interference efficiency of PMRT2employing Western blotting, and the system offirefly luciferase reporter gene was used to detect the influence of the PRMT2andrecombinants on the transcription of ER α.Results:1. Sequences of insert fragment in four miRNA expressing recombinants was proved tobe correct, and the expression of PRMT2was inhibited after the recombinants transienttransfection in MCF7cells.2. During the four miRNA expressing recombinants group such as miPRMT2-1miPRMT2-2miPRMT2-3and miPRMT2-4, the expression of protein reduced obvious themost in the group of miPRMT2-3.3. Compare with the untransfected group and the control-miRNA group, the group inwhich the miPRMT2was stably transfected found that the sensitivity of E2decreasedobviously in MCF7cell.4. The PRMT2and recombinants can inhibit the transcription activity of ERE-LUC, the transcription activity is hormone-dependen. Without estrogen, the transcription activityhas no significant effect on ERE-LUC.Conclusion:1. The expressing eukaryotic miRNA recombinants were constructed successfully, andthe recombinants group can inhibit the protein expression.2. The recombinant of PRMT2can inhibit the transcription activity of ERE-LUC.