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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(P<0.01)。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(P>0.05),而p-Akt蛋白的表达水平明显升高(P<0.01)。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。
Objective: To investigate the effect of transmembrane protein 45A (TMEM45A) gene expression on the proliferation and migration of human renal cell carcinoma cell line CAKI-1 in vitro and to explore its possible mechanism. Methods: TMEM45A-shRNA lentiviral vector targeting TMEM 45A gene was constructed. The lentiviral vector was used to infect CAKI-1 cells after lentivirus infection. The expression of TMEM45A gene was silenced 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 clonality of CAKI-1 cells were examined by CCK-8 assay and plate cloning assay. The effect of TMEM 45A gene silencing on the migration of CAKI-1 cells was examined by Transwell chamber migration assay. The effect of TMEM 45A gene silencing on the expression of protein kinase B (PKB), Akt and phospho-Akt (p-Akt) in CAKI-1 cells was detected by Western blotting. Results: LV-TMEM45A-shRNA lentivirus vector was successfully constructed and infected with CAKI-1 cell line. The expression level of TMEM45A mRNA and protein in CAKI-1 cells transfected with TMEM45A-shRNA lentiviral vector was significantly down-regulated (P <0.01), and the proliferation of CAKI-1 cells was significantly enhanced (P < 0.01). The migration ability of CAKI-1 cells was significantly enhanced (P <0.01). The expression of Akt protein in CAKI-1 cells was not significantly changed (P> 0.05), but the expression of p-Akt protein was significantly increased after silencing TMEM 45A gene expression (P <0.01). CONCLUSION: The silencing of TMEM 45A gene expression enhances the proliferation and migration of human renal cell carcinoma cell line CAKI-1, and its mechanism may be related to the phosphorylation of phosphatidylinositol 3-kinase (PI3K) / Akt signaling pathway Activation related.