本文選題：腎腫瘤 + 慢病毒感染　； 參考：《腫瘤》2017年03期

【摘要】：目的:探討跨膜蛋白45A(transmembrane protein 45A,TMEM45A)基因表達(dá)對腎癌細(xì)胞CAKI-1體外增殖和遷移能力的影響,并探討其可能的作用機(jī)制。方法:構(gòu)建特異性針對TMEM 45A基因的TMEM45A-shRNA慢病毒載體,并制備為慢病毒后感染CAKI-1細(xì)胞,沉默CAKI-1細(xì)胞中TMEM 45A基因的表達(dá)。分別采用實(shí)時熒光定量PCR及蛋白質(zhì)印跡法檢測TMEM45A mRNA及蛋白在CAKI-1細(xì)胞中表達(dá)水平的改變。通過CCK-8法和平板克隆法檢測TMEM 45A基因沉默對CAKI-1細(xì)胞增殖和克隆形成能力的影響。通過Transwell小室遷移實(shí)驗(yàn)檢測TMEM 45A基因沉默對CAKI-1細(xì)胞遷移能力的影響。采用蛋白質(zhì)印跡法檢測TMEM 45A基因沉默對CAKI-1細(xì)胞中蛋白激酶B(protein kinase B,PKB,又稱為Akt)及磷酸化Akt(phospho-Akt,p-Akt)蛋白表達(dá)的影響。結(jié)果:成功構(gòu)建了LV-TMEM45A-shRNA慢病毒載體,并感染CAKI-1細(xì)胞株。攜帶有TMEM45A-shRNA的慢病毒載體轉(zhuǎn)入CAKI-1細(xì)胞后,TMEM45A mRNA及蛋白的表達(dá)水平均被明顯下調(diào)(P值均0.01);CAKI-1細(xì)胞的增殖能力明顯增強(qiáng)(P值均0.01);CAKI-1細(xì)胞的遷移能力明顯增強(qiáng)(P0.01)。沉默TMEM 45A基因表達(dá)后,CAKI-1細(xì)胞中Akt蛋白的表達(dá)水平無明顯變化(P0.05),而p-Akt蛋白的表達(dá)水平明顯升高(P0.01)。結(jié)論:沉默TMEM 45A基因的表達(dá)可增強(qiáng)人腎癌細(xì)胞CAKI-1的增殖和遷移能力,其機(jī)制可能與磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)/Akt信號轉(zhuǎn)導(dǎo)通路的活化有關(guān)。
[Abstract]:Aim: to investigate the effect of transmembrane protein 45A(transmembrane protein 45A (TMEM45A) gene expression on the proliferation and migration of renal cancer cell line CAKI-1 in vitro and its possible mechanism. Methods: the TMEM45A-shRNA lentivirus vector targeting TMEM 45A gene was constructed and was prepared into CAKI-1 cells infected with lentivirus and silenced the expression of TMEM 45A gene in CAKI-1 cells. The expression of TMEM45A mRNA and protein in CAKI-1 cells was detected by real-time fluorescence quantitative PCR and Western blot respectively. The effects of TMEM 45A gene silencing on the proliferation and clone formation of CAKI-1 cells were detected by CCK-8 and plate cloning. The effect of TMEM 45A gene silencing on the migration ability of CAKI-1 cells was detected by Transwell chamber migration assay. The effects of TMEM 45A gene silencing on the expression of protein kinase (B(protein kinase) and phosphorylated Aktophospho-Aktp-Aktb in CAKI-1 cells were detected by Western blot. Results: LV-TMEM45A-shRNA lentivirus vector was successfully constructed and infected with CAKI-1 cell line. The expression level of TMEM45A mRNA and protein were significantly down-regulated by lentivirus vector carrying TMEM45A-shRNA into CAKI-1 cells. The proliferative ability of CAKI-1 cells was significantly enhanced by 0.01P value, and the migration ability of CAKI-1 cells was significantly enhanced by P0.01. After silencing the expression of TMEM 45A gene, the expression level of Akt protein in CAKI-1 cells did not change significantly, but the expression level of p-Akt protein increased significantly. Conclusion: silencing the expression of TMEM 45A gene can enhance the proliferation and migration of CAKI-1 in human renal cancer cells, and its mechanism may be related to the activation of phosphatidylinositol-3-kinase- PI3K / Akt signal transduction pathway.
【作者單位】： 上海交通大學(xué)醫(yī)學(xué)院附屬仁濟(jì)醫(yī)院臨床干細(xì)胞研究中心;
【基金】：國家自然科學(xué)基金資助項(xiàng)目(編號:81630073,31571399) 上海市科委重點(diǎn)資助項(xiàng)目(編號:16JC1405700)~~
【分類號】：R737.11
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本文編號：1975213