论文部分内容阅读
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。
OBJECTIVE: To construct a eukaryotic expression vector of microRNA targeting human protein arginine methyltransferase 2 (PRMT2) gene and identify its effect on the proliferation of breast cancer cell line MCF7. Methods: The pre-microRNA fragment was designed and synthesized according to PRMT2mRNA sequence and cloned into pcDNATM6.2-GW / EmGFP-miR eukaryotic expression vector and stably transfected into MCF7 cells. Indirect immunofluorescence and Western blotting were used to detect the effect of recombinants on the expression of PRMT2 to determine its biological activity. The effects of recombinant transfection on the proliferation and cell cloning ability of MCF7 cells were examined by crystal violet assay and plate clone formation assay. Effect of Recombinant Transfection on Cell Cycle of MCF7 Cells Detected by. Results: The nucleotide sequence of the inserted fragment was completely correct, and the recombinant plasmid transfected into MCF7 cells successfully interfered with the expression of PRMT2 gene. In the absence of treatment factors, transfection of recombinant cells had no significant effect on cell growth rate; compared with MCF7 cells transfected with empty vector, MCF7 cells stably transfected with recombinant plasmid showed enhanced sensitivity to estrogen, There was no significant difference in the sensitivity to 4-OHT, an estrogen antagonist. Plate clone formation experiments showed that the recombinant can enhance the estrogen-mediated MCF7 cell clonality. FCM assay showed that the proportion of G2 phase cells in transfected MCF7 cells was significantly increased (P <0.05). CONCLUSION: The pcDNATM6.2-GW / EmGFP-miR eukaryotic expression vector targeting PRMT2 gene is successfully constructed and has biological activity after stable transfection of MCF7 cells. Inhibition of endogenous PRMT2 gene expression can increase estrogen-mediated proliferation and colony formation of MCF7 cells, up-regulate the expression of estrin receptor α target genes cyclinD1 and c-myc, and promote cell cycle progression.