Abstract:

Objective To construct  overexpression vector of miR-210 and to identify the miR-210 target gene  HBVSP2 by using a dual fluorescent protein repoter assay system.Methods The primary sequence of miR-210 was cloned to form a new plasmid named pcDNA3.1(+)/pri-miR-210.A sequence of  HBVSP2 was inserted into the plasmid which expressed green fluorescent protein (EGFP) pcDNA3/EGFP.The plasmid pcDNA3/EGFP-HBVSP2 and miR-210ASO or pcDNA3.1(+)/pri-miR-210 and the plasmid expressed red fluorescent protein (RFP) pDsRed2 -N1  were cotransfected into HEK 293 cells and  HepG2 2215 cells.The fluorescence value  of the extracted protein was detected by fluorescence spectrophotometer respectively.Results After pcDNA3/miR-210 and the plasmid of pcDNA3/EGFP-HBVSP2 being cotransfected,the indensity of EGFP/RFP   was significantly lower than that in   pcDNA3+pcDNA3/EGFP-HBVSP2 group(P<0.05).The EGFP/RFP was lower in PCDNA3/pri-miR-210+pcDNA3/EGFP-HBVSP2 group compared with  pcDNA3+pcDNA3/EGFP-HBVSP2 group(P<0.05).After miR-210 ASO and the plasmid  pcDNA3/EGFP-HBVSP2 being cotransfected,the indensity of EGFP/RFP was significantly higher than that in  pcDNA3/EGFP-HBVSP2+LacZ group(P<0.05).The EGFP/RFP was lower in LacZ+pcDNA3/EGFP-HBVSP2 group compared with  LacZ+pcDNA3/EGFP group(P<0.05).Conclusion The miR-210 overexpression plasmid pcDNA3.1(+)/pri-miR-210 and pcDNA3/EGFP-HBVSP2 contained miR-210 target condition are successfully constructed,and HBVSP2 may be a direct target gene of miR-210.

Key words: plasmid;MiR-210;gene;expression;transfection