本文選題：液泡型ATP酶 + ATPVC��； 參考：《北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年06期

【摘要】：目的:采用RNA干涉技術(shù)沉默液泡型ATP酶c亞基ATP6V0C的表達(dá),探索ATP6V0C在前列腺癌侵襲中的分子作用機(jī)制,并研究其與LASS2/TMSG1的關(guān)系。方法與結(jié)果:ATP6V0C在高轉(zhuǎn)移潛能人前列腺癌細(xì)胞系(PC-3M-1E8和PC-3M)中的表達(dá)明顯高于低轉(zhuǎn)移潛能人前列腺癌細(xì)胞系(PC-3M-2B4和PC-3),選擇ATP6V0C表達(dá)量最高的PC-3M-1E8細(xì)胞進(jìn)行基因沉默實(shí)驗(yàn),發(fā)現(xiàn)ATP6V0C的siRNA轉(zhuǎn)染PC-3M-1E8后,其基質(zhì)金屬蛋白酶2(matrix metalloprotein-2,MMP-2)和基質(zhì)金屬蛋白酶9(MMP-9)蛋白的表達(dá)量及MMP-2的分泌量均無明顯變化,但上清液中MMP-9的活性明顯減弱,與空白對(duì)照組及陰性對(duì)照組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.05);干擾組細(xì)胞外氫離子濃度及液泡型ATPase的活性明顯降低(P0.05);細(xì)胞劃痕修復(fù)實(shí)驗(yàn)及transwell體外侵襲實(shí)驗(yàn)結(jié)果顯示ATP6V0C siRNA干擾組細(xì)胞的體外遷移及侵襲能力明顯減弱(P0.05)。PC-3M-1E8細(xì)胞轉(zhuǎn)染ATP6V0C siRNA后,LASS2/TMSG1的表達(dá)明顯減弱(P0.05)。免疫熒光雙重染色結(jié)果顯示ATP6V0C蛋白與LASS2/TMSG1共定位于胞漿和胞膜,干擾組共定位信號(hào)與對(duì)照組相比明顯減弱。結(jié)論:特異性siRNA沉默ATP6V0C能抑制人前列腺癌細(xì)胞的遷移和侵襲能力,其機(jī)制與ATP6V0C沉默后抑制V-ATPase的活性,使細(xì)胞外氫離子濃度降低,抑制MMP-9的激活,從而影響細(xì)胞外基質(zhì)的降解和重塑有關(guān)。靶向siRNA干擾ATP6V0C的表達(dá)后,可能通過反饋調(diào)節(jié)機(jī)制抑制LASS2/TMSG1的表達(dá),但其具體分子機(jī)制尚待進(jìn)一步研究。
[Abstract]:Aim: to silence the expression of the vacuolar ATPase c subunit ATP-6V0C by RNA interference technique, and to explore the molecular mechanism of ATP-6V0C in the invasion of prostate cancer, and to investigate its relationship with LASS2 / TMS320G1. Methods and results the expression of ATP 6V0C in high metastatic potential human prostate cancer cell lines (PC-3M-1E8 and PC-3M) was significantly higher than that in low-metastatic human prostate cancer cell lines (PC-3M-2B4 and PC-3). It was found that the expression of matrix metalloprotein-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) and the secretion of MMP-2 were not significantly changed after transfection of ATP 6V0C siRNA into PC-3M-1E8, but the activity of MMP-9 in the supernatant was significantly decreased, compared with the control group and the negative control group. The difference was statistically significant (P0.05), the extracellular hydrogen ion concentration and the activity of vacuolar ATPase were significantly decreased in the interference group (P0.05). The results of cell scratch repair and transwell invasion in vitro showed that the extracellular migration and invasion ability of the cells in vitro were observed in the ATT6V0C siRNA interference group (P0.05). PC-3M-1E8 cells were transfected with ATP-6V0C siRNA and the expression of LASS2 / TMS320G1 was significantly decreased (P0.05). The results of immunofluorescence double staining showed that ATP 6V0C protein and LASS2 / MS G1 co-located in the cytoplasm and membrane, and the co-localization signal in the interference group was significantly lower than that in the control group. Conclusion: the specific siRNA silencing ATP-6V0C can inhibit the migration and invasion of human prostate cancer cells. The mechanism of inhibition of V-ATPase activity, the decrease of extracellular hydrogen ion concentration, and the activation of MMP-9 after the silencing of ATP 6V0C. Thus affecting the degradation and remodeling of extracellular matrix. After targeted siRNA interference, the expression of ATP 6V0C may be inhibited by feedback regulation mechanism, but its molecular mechanism needs to be further studied.
【作者單位】： 北京大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系;青島中心醫(yī)院病理科;內(nèi)蒙古醫(yī)學(xué)院基礎(chǔ)醫(yī)學(xué)院病理學(xué)系;內(nèi)蒙古醫(yī)學(xué)院附屬醫(yī)院病理科;北京科技大學(xué)機(jī)械學(xué)院;首都師范大學(xué)附屬育新學(xué)校;
【基金】：國(guó)家自然科學(xué)基金(81572533)資助~~
【分類號(hào)】：R737.25

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