Rab25沉默对转化生长因子-β诱导的前列腺癌LNCaP细胞上皮-间充质转化的影响

Effect of Rab25 silencing on epithelial-mesenchymal transition induced by transforming growth factor-βin prostate cancer LNCaP cells

目的探讨Rab25沉默对转化生长因子-β(TGF-β)诱导的前列腺癌LNCaP细胞上皮-间充质转化(EMT)的影响。方法设计针对Rab25基因的3个干扰靶点(shRab25-1、shRab25-2、shRab25-3),将目的基因片段克隆到慢病毒载体中,连接产物转化到前列腺癌LNCaP细胞后提取重组质粒,分别作为shRab25-1组、shRab25-2组、shRab25-3组,并设置空白对照NC组。采用定量逆转录聚合酶链反应(qRT-PCR)及蛋白质印迹法(Western blot)检测Rab25 mRNA和蛋白相对表达量,筛选高干扰效率靶序列。培养NC组和shRab25-1组细胞,加入TGF-β(5 ng/ml)或不加入TGF-β,分别作为NC组、NC+TGF-β组、shRab25-1组、shRab25-1+TGF-β组,使用相差显微镜观察各组LNCaP细胞的形态学变化,采用Western blot检测各组LNCaP细胞中E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)表达情况。结果测序结果显示成功构建了Rab25的短发夹RNA(shRNA)慢病毒载体。qRT-PCR和Western blot检测结果显示,Rab25沉默后shRab25-1组细胞中Rab25表达量最低。相差显微镜显示Rab25沉默后细胞密度降低,加入TGF-β会部分逆转这种抑制作用,同时各组细胞形态结构未见明显破坏。shRab25-1组细胞中E-cadherin蛋白相对表达量高于NC组,N-cadherin蛋白相对表达量低于NC组,差异均有统计学意义(P﹤0.05);NC+TGF-β组细胞中E-cadherin蛋白相对表达量低于NC组,N-cadherin蛋白相对表达量高于NC组,差异均有统计学意义(P﹤0.05);shRab25-1+TGF-β组细胞中E-cadherin蛋白相对表达量低于shRab25-1组,N-cadherin蛋白相对表达量低于NC+TGF-β组,差异均有统计学意义(P﹤0.05)。结论Rab25沉默可逆转TGF-β介导的前列腺癌LNCaP细胞的EMT。

Objective To investigate the effect of Rab25 silencing on epithelial-mesenchymal transition(EMT)induced by transforming growth factor-β(TGF-β)in prostate cancer LNCaP cells.Method Three interference targets(shRab25-1,shRab25-2,and shRab25-3)targeting the Rab25 gene were designed,and then the target gene fragment were cloned into a lentiviral vector,and the ligated product was transformed into prostate cancer LNCaP cells to extract the recombinant plasmid,and divided into shRab25-1 group,shRab25-2 group,and shRab25-3 group,respectively,and a blank control NC group was also set.The relative expression levels of Rab25 mRNA and protein were detected using quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blot,and target sequence with high interference efficiency was screened.Cells in NC group and shRab25-1 group were cultured,adding TGF-β(5 ng/ml)or not adding TGF-β as NC group,NC+TGF-β group,shRab25-1 group,shRab25-1+TGF-β group,respectively.The morphological changes of LNCaP cells in each group were observed using a phase contrast microscope.The expression of E-cadherin and N-cadherin in LNCaP cells in each group were detected by Western blot.Result The sequencing results showed that the Rab25 short hairpin RNA(shRNA)lentiviral vector was successfully constructed.The results of qRT-PCR and Western blot showed that the expression of Rab25 in cells of the shRab25-1 group was the lowest after Rab25 was silenced.Phase contrast microscopy showed that the cell density decreased after Rab25 was silenced,and the addition of TGF-β partially reversed this inhibitory effect,at the same time,there were no obvious damage to the cell morphology and structure of each group.The relative expression of E-cadherin protein in the shRab25-1 group was higher than that in the NC group,and the relative expression of N-cadherin protein was lower than that in the NC group,and the differences were statistically significant(P<0.05).The relative expression of E-cadherin protein in the NC+TGF-β group was lower than that in the NC group,and the relative expression of N-cadherin protein was higher than that in the NC group,and the differences were statistically significant(P<0.05).The relative expression of E-cadherin protein in the shRab25-1+TGF-β group was lower than that in the shRab25-1 group,the relative expression of N-cadherin protein was lower than that in the NC+TGF-βgroup,and the differences were statistically significant(P<0.05).Conclusion Rab25 silencing could reverse TGF-β-mediated EMT in prostate cancer LNCaP cells.