沉默ELAVL1基因表达体外和体内抑制前列腺癌PC-3细胞的增殖

Silencing ELAVL 1 gene inhibits proliferation of prostate cancer PC-3 cells in vivo and in vitro

目的:观察沉默胚胎致死性异常视觉1(embryonic lethal abnormal vision-like 1,ELAVL1)基因表达对前列腺癌PC-3细胞生长的影响,并探讨可能的分子作用机制。方法:分别采用实时荧光定量PCR法、蛋白质印迹法、免疫组织化学法及免疫荧光法检测前列腺癌PC-3和正常前列腺上皮RWPE-1细胞中ELAVL1 mRNA和蛋白的表达水平并定位。将携带有特异性针对ELAVL1基因-shRNA(shELAVL1)的重组腺病毒Ad5-shELAVL1-GFP感染PC-3细胞以沉默ELAVL1基因的表达,同时以腺病毒空载体Ad5-GFP感染PC-3细胞作为对照组。通过CCK-8法检测细胞的增殖能力,FCM检测细胞周期,蛋白质印迹法检测PC-3细胞中环氧合酶2(cyclooxygenase-2,Cox-2)、细胞周期蛋白D1(cyclin D1)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)、蛋白激酶B(protein kinase B,PKB,又称AKT)和磷酸化-AKT(phospho-Akt,p-AKT)蛋白的表达水平。建立PC-3细胞裸鼠皮下移植瘤模型,分别给予瘤内注射Ad5-shELAVL1-GFP、Ad5-GFP(对照组)及0.9%氯化钠溶液(空白对照组),观察移植瘤的生长情况。结果:PC-3细胞中ELAVL1 mRNA和蛋白的表达水平均高于RWPE-1细胞(P值均<0.001);ELAVL1蛋白在PC-3细胞中主要表达于细胞质中,在RWPE-1细胞中则主要表达于细胞核中。沉默ELAVL1基因表达后,与对照组相比,PC-3细胞的增殖能力明显下降,G1期细胞所占百分比明显提高,而G2期细胞所占百分比明显减少(P值均<0.05);同时,与细胞增殖有关的分子cyclin D1和Cox-2蛋白的表达水平明显降低,PI3K/AKT信号通路中PI3K、AKT及p-AKT的蛋白表达也明显下调(P值均<0.05)。与对照组相比,裸鼠移植瘤在经过Ad5-shELAVL1-GFP处理后移植瘤的生长减缓,移植瘤的体积和质量明显减小(P值均<0.05)。结论:ELAVL1蛋白在前列腺癌PC-3细胞中高表达且主要表达于细胞质中;ELAVL1蛋白可能通过PI3K/AKT经典肿瘤信号通路对前列腺癌细胞的生长起着促进作用。

Objective:To investigate the effect of silencing embryonic lethal abnormal vision-like 1(ELAVL1)gene expression on the growth of prostate cancer PC-3 cells,and to explore its molecular mechanism.Methods:Real-time fluorescent quantitative PCR,Western blotting,immunohistochemistry and immunofluorescence were used to detect the mRNA and protein expression levels and localization of ELAVL1 mRNA and protein in PC-3 cells and normal prostate epithelial RWPE-1 cells.PC-3 cells were infected with the recombinant adenoviral vector Ad5-shELAVL1-GFP specifically targeting ELAVL1 gene-shRNA(shELAVL1)to silence the ELAVL1 gene,and PC-3 cells were infected with the adenoviral empty vector Ad5-GFP as a control group.CCK-8 assay was employed to evaluate the proliferation ability,FCM was used to detect the cell cycle and Western blotting was applied to measure the protein expression of cyclooxygenase-2(Cox-2),cyclin D1,phosphatidylinositol 3-kinase(PI3K),protein kinase B(PKB,also known as AKT)and phospho-AKT(p-AKT).The xenograft tumor models of PC-3 cells in nude mice were established,and were injected with Ad5-shELAVL1-GFP,Ad5-GFP(control)or 0.9%sodium chloride solution(blank control)respectively to observe the growth of xenograft tumor.Results:The expression levels of ELAVL1 mRNA and protein in PC-3 cells were higher than those in RWPE-1 cells(both P<0.001)and the ELAVL1 protein in PC-3 cells was mainly expressed in the cytoplasm,while in RWPE-1 cells it was mainly expressed in nucleus.After silencing ELAVL1 gene,compared with the control group,the proliferation ability of PC-3 cells was significantly decreased,the cells in the G1 phase were significantly increased,and the cells in the G2 phase were significantly decreased(all P<0.05);at the same time,the expression levels of cell proliferation-related molecules including cyclin D1 and Cox-2 were significantly decreased(both P<0.05).In addition,the protein expressions of PI3K,AKT and p-AKT in the PI3K/AKT signaling pathway were also significantly decreased(all P<0.05).Finally,compared with the control group,the growth of xenograft tumors of PC-3 cells in nude mice was slowed down after treatment with Ad5-shELAVL1-GFP,and the volume and weight were significantly reduced(both P<0.05).Conclusion:ELAVL1 protein is highly expressed in prostate cancer PC-3 cells and mainly expressed in the cytoplasm;ELAVL1 protein can promote the growth of prostate cancer PC-3 cells through the PI3K/AKT signaling pathway.