【摘要】：目的:明確蛋白質(zhì)精氨酸甲基轉(zhuǎn)移酶2(protein arginine methyltransferase 2,PRMT2)穩(wěn)定過表達(dá)后對(duì)人乳腺癌MCF-7細(xì)胞自噬的影響,并初步探索其引起自噬現(xiàn)象改變可能的分子機(jī)制。方法:1.電子透射電鏡觀察PRMT2穩(wěn)定過表達(dá)后細(xì)胞內(nèi)自噬現(xiàn)象(自噬性囊泡)的改變;2.細(xì)胞免疫熒光技術(shù)觀察PRMT2穩(wěn)定過表達(dá)后對(duì)人乳腺癌MCF-7細(xì)胞胞內(nèi)自噬相關(guān)蛋白表達(dá)是否改變;3.蛋白質(zhì)免疫印跡(Western Blot,WB)以檢測(cè)自噬相關(guān)蛋白表達(dá)量的改變,進(jìn)一步分析PRMT2對(duì)人乳腺癌MCF-7細(xì)胞自噬影響;4.采用高通量抗體芯片技術(shù)檢測(cè)PRMT2影響細(xì)胞的相關(guān)信號(hào)通路關(guān)鍵位點(diǎn)蛋白,WB技術(shù)驗(yàn)證通路相關(guān)蛋白的表達(dá),以初步探索PRMT2穩(wěn)定過表達(dá)影響乳腺癌..MCF-7細(xì)胞自噬的相關(guān)分子機(jī)制。結(jié)果:1.透射電鏡下觀察自噬現(xiàn)象(自噬性囊泡面積)的改變:在PRMT2穩(wěn)定過表達(dá)的人乳腺癌細(xì)胞MCF-7中,自噬小體數(shù)量明顯減少,且隨時(shí)間呈遞減趨勢(shì);2.細(xì)胞免疫熒光結(jié)果顯示:PRMT2穩(wěn)定過表達(dá)后微管相關(guān)蛋白輕鏈3(Microtubuleassociated protein light chain 3,LC3A/B)表達(dá)減少;3.WB檢測(cè)結(jié)果表示:PRMT2穩(wěn)定過表達(dá)后LC3A/B、酵母自噬相關(guān)基因6同源物(Beclin1,BECN1)的表達(dá)減少(p0.05),且隨時(shí)間呈遞減趨勢(shì);4.高通量抗體芯片檢測(cè)、分析結(jié)果提示:PRMT2下調(diào)哺乳動(dòng)物雷帕霉素靶蛋白復(fù)合物(Mammalian target of rapamycin complex,mTOR)水平,WB檢測(cè)結(jié)果提示磷酸化的腺苷活化蛋白激酶(AMP-activated protein kinase,AMPK)、自噬相關(guān)蛋白1(Unc-51 like autophagy activating kinase 1,ULK1)水平下調(diào)(p0.05)。結(jié)論:1.PRMT2能抑制MCF-7細(xì)胞自噬,且與時(shí)間呈遞減關(guān)系;2.PRMT2抑制MCF-7細(xì)胞自噬現(xiàn)象可能是通過下調(diào)AMPK-ULK1信號(hào)通路所實(shí)現(xiàn)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.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9

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