沉默TMEM45A基因表达可促进肾癌CAKI-1细胞的体外增殖和迁移

发布时间：2018-06-04 01:30

  本文选题：肾肿瘤 + 慢病毒感染　； 参考：《肿瘤》2017年03期

【摘要】：目的:探讨跨膜蛋白45A(transmembrane protein 45A,TMEM45A)基因表达对肾癌细胞CAKI-1体外增殖和迁移能力的影响,并探讨其可能的作用机制。方法:构建特异性针对TMEM 45A基因的TMEM45A-shRNA慢病毒载体,并制备为慢病毒后感染CAKI-1细胞,沉默CAKI-1细胞中TMEM 45A基因的表达。分别采用实时荧光定量PCR及蛋白质印迹法检测TMEM45A mRNA及蛋白在CAKI-1细胞中表达水平的改变。通过CCK-8法和平板克隆法检测TMEM 45A基因沉默对CAKI-1细胞增殖和克隆形成能力的影响。通过Transwell小室迁移实验检测TMEM 45A基因沉默对CAKI-1细胞迁移能力的影响。采用蛋白质印迹法检测TMEM 45A基因沉默对CAKI-1细胞中蛋白激酶B(protein kinase B,PKB,又称为Akt)及磷酸化Akt(phospho-Akt,p-Akt)蛋白表达的影响。结果:成功构建了LV-TMEM45A-shRNA慢病毒载体,并感染CAKI-1细胞株。携带有TMEM45A-shRNA的慢病毒载体转入CAKI-1细胞后,TMEM45A mRNA及蛋白的表达水平均被明显下调(P值均0.01);CAKI-1细胞的增殖能力明显增强(P值均0.01);CAKI-1细胞的迁移能力明显增强(P0.01)。沉默TMEM 45A基因表达后,CAKI-1细胞中Akt蛋白的表达水平无明显变化(P0.05),而p-Akt蛋白的表达水平明显升高(P0.01)。结论:沉默TMEM 45A基因的表达可增强人肾癌细胞CAKI-1的增殖和迁移能力,其机制可能与磷脂酰肌醇3-激酶(phosphatidylinositol-3-kinase,PI3K)/Akt信号转导通路的活化有关。
[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.
【作者单位】： 上海交通大学医学院附属仁济医院临床干细胞研究中心;
【基金】：国家自然科学基金资助项目(编号:81630073,31571399) 上海市科委重点资助项目(编号:16JC1405700)~~
【分类号】：R737.11
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本文编号：1975213