MicroRNA-101真核表达载体的构建及其在人胎盘绒毛癌中的表达

目的：研究外源性microRNA-101前体的真核表达载体构建方法和其在人胎盘绒毛癌细胞JAR中的表达,及其对靶基因EZH2的沉默作用。方法：利用化学合成法合成microRNA-101前体分子（pre-microRNA-101）并克隆至真核表达载体pR-NAT-CMV3.2/Neo。通过酶切及测序法验证该阳性重组质粒pRNAT-CMV3.2-mir101的正确性。体外培养人胎盘绒毛癌细胞JAR;利用脂质体转染法将空白质粒及重组子pRNAT-CMV3.2-mir101转染至JAR细胞。应用Northern blotting检测各组细胞之间成熟microRNA-101（mature microRNA-101）的表达情况;应用Western blotting检测各组细胞中JAR的表达情况。结果：质粒酶切及测序结果显示pRNAT-CMV3.2-mir101上的microRNA-101与合成的pre-microRNA-101寡核苷酸序列完全一致,无碱基的突变和缺失。Northern blotting结果显示,pRNAT-CMV3.2-mir101转染组细胞的总RNA中含有mi-croRNA-101阳性条带,说明该细胞中mature microRNA-101呈高水平表达。Western blotting结果表明,pRNAT-CMV3.2-mir101转染组JAR细胞中的EZH2蛋白表达量（52.76±3.12）%低于对照组（81.18±2.96）%,两者具有显著差异（P〈0.05）。结论：通过构建真核细胞表达质粒可以高效表达外源性microRNA分子;microRNA-101可以有效地沉默宿主细胞JAR中靶基因EZH2的表达。

The Built Eukaryotic Expression Vector of microRNA-101 and the Expression in the Human Placenta Carcinoma Cells

Objective： To study the eukaryotic expression vector of microRNA-101 built,its expression in the human placenta carcinoma cells JAR,and its silent function of target gene EZH2.Methods：The molecular pre-microRNA-101 was synthesized by a chemical method,which was cloned on the eukaryotic expression vector pRNAT-CMV3.2/Neo.Then the restriction endonuclease cleaved and DNA sequenced methods were used to assay the positive clone pRNAT-CMV3.2-mir101 accurately.Human placenta carcinoma cell JAR was cultured in vitro,both blank plasmid and positive clone pRNAT-CMV3.2-mir101 were transfected by lipofectamine 2000 kit.The mature microRNA-101 expression in each groups were tested by Northern Blotting.The total proteins of different groups were extracted and the expression of EZH2 each groups were tested by Western blotting.Results： The results of restriction endonuclease cleaved and DNA sequenced indicated that the DNA sequences of positive recombinant clone were in conformity with the pre-microRNA-101 fragment.The results of Northern blotting indicated there was an obvious hybridization band from the pRNAT-CMV3.2-mir101 transfected group.And the microRNA-101 expressed in JAR cells could decrease the expression of EZH2（52.76±3.12） % compared with the blank plasmid.There were significant difference in two groups（P0.05）.Conclusions： The mature microRNA-101 expression vector pRNAT-CMV3.2-mir101 is built succeed.In addition the microRNA-101 could interfere the target gene EZH2 expression in JAR growth in vitro.