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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HepG22215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。
OBJECTIVE: To construct an overexpression vector of miR-210 and verify the target gene HBVSP2 of miR-210 by using double fluorescent protein reporter gene analysis system. Methods: The overexpression plasmid pcDNA3.1 (+) / pri-miR-210 containing miR-210 precursor sequence was constructed. The plasmid pcDNA3 / EGFP expressing green fluorescent protein (EGFP) A specific sequence of HBVSP2 was inserted into the plasmid to construct EGFP reporter vector pcDNA3 / EGFP-HBVSP2, which was used in combination with miR-210ASO or pcDNA3.1 (+) / pri-miR-210 and the expression of red fluorescent protein ) PDsRed2-N1 was co-transfected into HEK293 and HepG22215 cells, and the fluorescence value of protein samples extracted after transfection was quantitatively determined by fluorescence spectrophotometer. Results: The co-transfected pcDNA3 / pri-miR-210 and pcDNA3 / EGFP-HBVSP2 plasmids, EGFP / RFP expression was significantly lower than the pcDNA3 and pcDNA3 / EGFP-HBVSP2 co-transfected group, the difference was statistically significant (P < 0.05). EGFP / RFP in pcDNA3 / pri-miR-210 and pcDNA3 / EGFP-HBVSP2 groups was lower than that in pcDNA3 and pcDNA3 / EGFP-HBVSP2 groups (P <0.05). The expression of EGFP / RFP in co-transfected miR-210ASO and pcDNA3 / EGFP-HBVSP2 groups was significantly higher than that in co-transfected LacZ and pcDNA3 / EGFP-HBVSP2 groups (P <0.05). EGFP / RFP in LacZ and pcDNA3 / EGFP-HBVSP2 groups was lower than that in LacZ and pcDNA3 / EGFP groups (P <0.05). Conclusion: The overexpression plasmid pcDNA3.1 (+) / pri-miR-210 and miR-210 containing pcDNA3 / EGFP-HBVSP2 were successfully constructed. HBVSP2 may be the direct target of miR-210.