PRMT2β对MCF-7细胞增殖的影响及其相关机制研究

发布时间：2018-06-28 11:04

本文选题：乳腺癌细胞 + PRMT2β　；参考：《南华大学》2015年硕士论文

【摘要】：目的:初步探讨PRMT2β对乳腺癌MCF-7细胞增殖的影响及其相关机制,为乳腺癌的靶向治疗提供新思路。方法:通过显微镜观察及蛋白免疫印迹(Western blot)技术鉴定稳转PRMT2β的MCF-7细胞株构建成功。设立实验组即加四环素处理48h后可诱导PRMT2β高表达组(over expression,OE),阴性对照组即未加四环素处理组(negative control,NC)。以上述两组细胞为实验对象,分别采用MTT检测法、软琼脂克隆形成实验检测两组细胞的增殖及克隆形成能力;通过流式细胞术检测两组细胞的细胞周期及凋亡情况;利用蛋白质免疫印迹法,从蛋白水平分析MCF-7细胞中,PRMT2β对CCND1及Akt磷酸化水平的影响。结果:1.显微镜下观察,与NC组细胞相比,OE组细胞呈梭形,且细胞生长密度相对减少;Western blot结果表明,四环素能成功诱导MCF-7细胞表达PRMT2β-3Flag融合蛋白,提示PRMT2β-MCF7稳定细胞株构建成功。2.MTT检测法表明,与NC组相比,OE组细胞的增殖率明显减少,且具有统计学意义(P0.05),提示PRMT2β可抑制MCF-7细胞的增殖。3.克隆形成实验表明,相对于NC组,OE组克隆形成能力减弱,结果说明PRMT2β可以抑制MCF-7细胞体外增殖的能力。4.流式细胞术结果显示:与NC组相比,OE组细胞凋亡率相对增多,有统计学意义(P0.05);细胞周期检测结果显示OE组细胞增殖相对受抑制,有统计学意义(P0.05);结果说明PRMT2β能够促进MCF-7细胞的凋亡,延长细胞周期,抑制细胞增殖。5.Western Blot结果显示:相对于NC组,OE组细胞中CCND1的表达水平下调,且Akt磷酸化水平下调,有统计学意义(P0.05);在加入两种PI3K/Akt通路抑制剂(LY294002、Wortmannin)后,NC组与OE组中,与各自空白对照组相比,两种抑制剂处理组的CCND1及Akt磷酸化表达水平都下调,有统计学意义(P0.05)。结论:PRMT2β能够抑制乳腺癌MCF-7细胞的增殖,其机制可能与PRMT2β下调CCND1表达及Akt磷酸化水平有关。
[Abstract]:Objective: to explore the effect of PRMT2 尾 on the proliferation of breast cancer MCF-7 cells and its related mechanisms, and to provide a new idea for the targeted treatment of breast cancer. Methods: the MCF-7 cell line was successfully constructed by microscopical observation and Western blot analysis. The experimental group was treated with tetracycline for 48h, and the over expression group (over expression OE) was induced, while the negative control group was not treated with tetracycline (negative control group). MTT assay and soft Agar clone formation assay were used to detect the proliferation and clone forming ability of the two groups of cells, and the cell cycle and apoptosis of the two groups were detected by flow cytometry. The effects of PRMT2 尾 on the phosphorylation of CCND1 and Akt in MCF-7 cells were analyzed by Western blot. The result is 1: 1. Under microscope, compared with NC group, the cells in OE group were fusiform, and the cell growth density was decreased. The results showed that tetracycline could induce the expression of PRMT2 尾 -3Flag fusion protein in MCF-7 cells successfully, and the expression of PRMT2 尾 -3Flag fusion protein in MCF-7 cells could be induced by tetracycline. The results indicated that PRMT2 尾 -MCF7 stable cell line was successfully constructed. 2. MTT assay showed that the proliferation rate of OE group was significantly lower than that of NC group (P0.05), suggesting that PRMT2 尾 could inhibit the proliferation of MCF-7 cells. Clone formation assay showed that compared with NC group, the clone forming ability of OE group was weakened. The results showed that PRMT2 尾 could inhibit the proliferation of MCF-7 cells in vitro. 4. The results showed that PRMT2 尾 could inhibit the proliferation of MCF-7 cells in vitro. The results of flow cytometry showed that the apoptosis rate of OE group was higher than that of NC group (P0.05), and the cell cycle test showed that the proliferation of OE group was inhibited. The results showed that PRMT2 尾 could promote the apoptosis of MCF-7 cells, prolong cell cycle and inhibit cell proliferation. 5. Western blot showed that the expression level of CCND1 was down-regulated and the phosphorylation of Akt was down-regulated compared with NC group. After adding two kinds of PI3K / Akt pathway inhibitor (LY294002Wortmannin), the expression of CCND1 and Akt phosphorylation was significantly decreased in NC group and OE group compared with control group (P0.05). Conclusion the proliferation of breast cancer MCF-7 cells can be inhibited by 1: PRMT2 尾, which may be related to the down-regulation of CCND1 expression and Akt phosphorylation by PRMT2 尾.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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