miR-210靶基因HBVSP2的鉴定

目的：构建miR-210的过表达载体，利用双荧光蛋白报告基因分析系统验证miR-210的靶基因HBVSP2。方法：构建含有miR-210前体序列的miR-210的过表达质粒pcDNA3.1(+)/pri-miR-210；选取表达绿色荧光蛋白(EGFP)的质粒 pcDNA3/EGFP，将miR-210在HBVSP2上靶位点的一段特异性序列插入该质粒中，构建EGFP报告载体pcDNA3/EGFP-HBVSP2，并与miR-210ASO或者pcDNA3.1(+)/pri-miR-210及表达红色荧光蛋白质(RFP)的pDsRed2 -N1共同转染HEK293以及HepG2 2215细胞，用荧光分光光度计定量检测转染后提取的蛋白样品的荧光值。结果：共同转染pcDNA3/pri-miR-210 和pcDNA3/ EGFP-HBVSP2质粒后，EGFP/RFP的表达量明显低于pcDNA3和pcDNA3/ EGFP-HBVSP2共转染组，差异具有统计学意义(P<0.05)。pcDNA3/pri-miR-210和pcDNA3/EGFP-HBVSP2组EGFP/RFP低于pcDNA3和pcDNA3/EGFP-HBVSP2共转染组(P<0.05)。共转染miR-210ASO和pcDNA3/EGFP-HBVSP2组EGFP/RFP的表达量高于共转染LacZ和pcDNA3/EGFP-HBVSP2组，差异具有统计学意义(P<0.05)。LacZ和pcDNA3/EGFP-HBVSP2组EGFP/RFP低于LacZ和pcDNA3/EGFP组(P<0.05)。结论：成功构建miR-210的过表达质粒pcDNA3.1(+)/pri-miR-210和含有miR-210靶位点的pcDNA3/ EGFP-HBVSP2，HBVSP2 可能是miR-210的直接靶基因。

Identifcation of miR-210 target gene HBVSP2

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.