Construction of dsRNA expression vector and research of RNA activation

Objective To construct the small-molecule double-stranded RNA (dsRNA) expression vector plasmid,and to investigate its activation ability of the expression of cell cycle repressor protein p21WAF1/CIP1(p21) in human prostate cancer cell line PC-3 in vitro.Methods Synthetic the sequence-specific corresponding DNA fragments of the promoter with the p21 gene,as well as the same length of random DNA fragments,by means of the recombinant DNA technology to construct these fragments into the eukaryotic expression plasmids pGenesil-1,constructed with the purpose of the expression of dsRNA fragments plasmid vectors (dsRNAp21-pGenesil-1) and the random fragments of the control dsRNA expression vectors (dsRNACon-pGenesil-1).The dsRNAp21-pGenesil-1 (experimental group),dsRNACon-pGenesil-1 (negative control group) and a blank plasmid pGenesil-1 (control group) were transfected into human prostate cancer cell line PC-3 in vitro respectively with lipofectamine reagent,and transfection efficency was detected by laser scanning confocal microscope.Immunohistochemistry and reverse transcription-PCR were performed to examine the difference expression level of p21 in each group.Results These dsRNAp21-pGenesil-1 and the dsRNACon-pGenesil-1 eukaryotic cell expression vectors were successfully constructed.For each of the groups of plasmids transfected into PC-3 cells,p21 gene expression in the experimental group significantly increased,while there was no significant difference between the negative control group and the blank control group.Conclusions The dsRNAp21-pGenesil-1 express dsRNA molecule successfully,and active the level of expression of p21 mRNA and protein,and provide an effective tool for studying the activation effect of dsRNA.