PRMT2稳定过表达对MCF-7细胞自噬的影响及机制研究

发布时间：2018-09-13 10:13

【摘要】：目的:明确蛋白质精氨酸甲基转移酶2(protein arginine methyltransferase 2,PRMT2)稳定过表达后对人乳腺癌MCF-7细胞自噬的影响,并初步探索其引起自噬现象改变可能的分子机制。方法:1.电子透射电镜观察PRMT2稳定过表达后细胞内自噬现象(自噬性囊泡)的改变;2.细胞免疫荧光技术观察PRMT2稳定过表达后对人乳腺癌MCF-7细胞胞内自噬相关蛋白表达是否改变;3.蛋白质免疫印迹(Western Blot,WB)以检测自噬相关蛋白表达量的改变,进一步分析PRMT2对人乳腺癌MCF-7细胞自噬影响;4.采用高通量抗体芯片技术检测PRMT2影响细胞的相关信号通路关键位点蛋白,WB技术验证通路相关蛋白的表达,以初步探索PRMT2稳定过表达影响乳腺癌..MCF-7细胞自噬的相关分子机制。结果:1.透射电镜下观察自噬现象(自噬性囊泡面积)的改变:在PRMT2稳定过表达的人乳腺癌细胞MCF-7中,自噬小体数量明显减少,且随时间呈递减趋势;2.细胞免疫荧光结果显示:PRMT2稳定过表达后微管相关蛋白轻链3(Microtubuleassociated protein light chain 3,LC3A/B)表达减少;3.WB检测结果表示:PRMT2稳定过表达后LC3A/B、酵母自噬相关基因6同源物(Beclin1,BECN1)的表达减少(p0.05),且随时间呈递减趋势;4.高通量抗体芯片检测、分析结果提示:PRMT2下调哺乳动物雷帕霉素靶蛋白复合物(Mammalian target of rapamycin complex,mTOR)水平,WB检测结果提示磷酸化的腺苷活化蛋白激酶(AMP-activated protein kinase,AMPK)、自噬相关蛋白1(Unc-51 like autophagy activating kinase 1,ULK1)水平下调(p0.05)。结论:1.PRMT2能抑制MCF-7细胞自噬,且与时间呈递减关系;2.PRMT2抑制MCF-7细胞自噬现象可能是通过下调AMPK-ULK1信号通路所实现的。
[Abstract]:Aim: to investigate the effect of overexpression of arginine methyltransferase 2 (protein arginine methyltransferase 2 / PRMT2 on autophagy of human breast cancer MCF-7 cells and to explore the possible molecular mechanism of the change of autophagy. Method 1: 1. Electron transmission electron microscopy (TEM) was used to observe the changes of autophagy (autophagic vesicle) after PRMT2 overexpression. Cellular immunofluorescence technique was used to observe whether the expression of autophagy associated protein in human breast cancer MCF-7 cells changed after overexpression of PRMT2. Western blot (Western Blot,WB) was used to detect the changes of autophagy associated protein expression and to further analyze the effect of PRMT2 on autophagy of human breast cancer MCF-7 cells. High-throughput antibody chip technique was used to detect the expression of transduction pathway proteins at the key site of signal pathway related to PRMT2 and to explore the molecular mechanism of stable overexpression of PRMT2 affecting autophagy of breast cancer cell line ..MCF-7. The result is 1: 1. The changes of autophagy (area of autophagic vesicles) were observed under transmission electron microscope: in MCF-7 cells with stable expression of PRMT2, the number of autophagy bodies decreased significantly and decreased gradually with time. The results of cellular immunofluorescence showed that the expression of microtubule-associated protein light chain 3 (Microtubuleassociated protein light chain 3 + LC3A / B was decreased after the stable expression of microtubule related protein. The results showed that the expression of LC3A/B, yeast autophagy related gene 6 congener (p0.05) was decreased (p0.05) after the stable overexpression of the microtubule associated protein 3 (Microtubuleassociated protein light chain 3 + LC3A / B. The time shows a decreasing trend. High throughput antibody chip detection, The results suggested that the (Mammalian target of rapamycin complex,mTOR level of rapamycin target protein complex in mammals was down-regulated by: 1 / PRMT2. The results showed that phosphorylated adenosine activated protein kinase (AMP-activated protein kinase,AMPK) and autophagy associated protein 1 (Unc-51 like autophagy activating kinase 1 / ULK1) were down-regulated (p0.05). Conclusion: 1. PRMT2 can inhibit the autophagy of MCF-7 cells, and the relationship between PRMT2 and time is decreasing. 2. The inhibition of MCF-7 autophagy by PRMT2 may be achieved by down-regulating the AMPK-ULK1 signaling pathway.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R737.9

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