中文摘要：

目的：构建靶向人蛋白质精氨酸甲基转移酶 2 （protein arginine methyltransferase2,PRMT2）基因的microRNA真核表达载体,鉴定其稳定转染乳腺癌细胞株MCF7后对细胞增殖的影响。方法：根据PRMT2mRNA序列设计合成pre-microRNA片段,定向克隆至pcDNATM6.2-GW/EmGFP-miR真核表达载体,并稳定转染入MCF7细胞。采用间接免疫荧光法和蛋白质印迹法检测重组体对PRMT2表达的干扰效果以确定其生物活性。采用结晶紫实验和平板克隆形成实验检测重组体转染对MCF7细胞体外增殖和细胞克隆形成能力的影响。FCM法检测重组体转染对MCF7细胞周期的影响。结果：构建的重组体插入片段的碱基序列完全正确,并且重组体稳定转染MCF7细胞后可成功干扰PRMT2基因的表达。在无处理因子的情况下,重组体转染对细胞的生长速度无明显影响;与转染空载体的MCF7细胞相比,稳定转染重组体的MCF7细胞对雌激素的敏感性增强,但其对雌激素拮抗剂4-OHT处理的敏感性无明显差异。平板克隆形成实验表明,重组体能够增强雌激素介导的MCF7细胞的克隆形成能力。FCM法检测表明,转染重组体的MCF7细胞中G2期细胞比例明显升高（P〈0.05）。结论：成功构建靶向PRMT2基因的pcDNATM6.2-GW/EmGFP-miR真核表达载体,且在稳定转染MCF7细胞后具有生物学活性。抑制内源性PRMT2基因的表达能够提高雌激素介导的MCF7细胞的增殖和克隆形成能力,上调雌激素受体α目标基因cyclinD1和c-myc的表达,促进细胞周期进程。

英文摘要：

Objective：To construct human protein arginine methyltransferase 2（PRMT2） gene-targeted microRNA expressing eukaryotic recombinant,and to investigate its effect on cell proliferation of breast cancer MCF7 cells transfected with PRMT2 gene-targeted microRNA.Methods：According to the sequence of PRMT2 mRNA,the PRMT2 pre-microRNA was designed and synthesized,and then it was cloned into the GFP tagged pcDNATM6.2-GW/EmGFP-miR vector and transfected into breast cancer MCF7 cells.The integrity of inset fragment was detected by sequencing analysis.The biological activity of recombinant was identified through detecting interference efficiency of PRMT2 microRNA recombinant by indirect immunofluorescence and Western blotting.The proliferation and colony formation of MCF7 cells were measured by crystal violet assay and colony formation assay,and the cell cycle was determined by flow cytometry（FCM）.Results：The sequence of inset fragment in microRNA expressing recombinat was correct,and the expression of PRMT2 protein was decreased after transfection.The knockdown of PRMT2 expression induced by microRNA did not change the growth property of MCF7 cells with no treatment.The microRNA recombinant-transfected MCF7 cells treated with 4-OHT proliferated at the same rate as the control cells,whereas a stronger promotion of the proliferation of the microRNA recombinant-transfected MCF7 cells cultivated with estrogen was observed.The colony formation assay showed that PRMT2 knockdown induced by microRNA enhanced the estrogen-mediated colony formation ability of MCF7 cells.FCM assay indicated that the percentages of MCF7 cells at G2 phase were increased after PRMT2 knockdown induced by microRNA（P0.05）.Conclusion：The PRMT2 gene-targeted pcDNATM6.2-GW/EmGFP-miR expressing eukaryotic recombinant plasmid can be successfully constructed,and has biological activity in MCF7 cells after stable transfection.PRMT2 knockdown induced by microRNA can enhance the estrogen-mediated proliferation and colony formation of MCF7 cells,up-