沉默PHLDA2通过PI3K/AKT/mTOR信号通路抑制胰腺癌细胞EMT和自噬

Silencing PHLDA2 to inhibit EMT and autophagy in pancreatic cancer through PI3K/AKT/mTOR pathway

目的:探讨沉默人普列克底物蛋白同源域家族A成员2(PHLDA2)基因通过磷脂酰肌醇激酶(PI3K)/丝-苏氨酸蛋白激酶(AKT)/雷帕霉素靶蛋白(mTOR)信号通路,进而抑制胰腺癌细胞上皮间质转化(EMT)和自噬的相关分子机制。方法:首先采用qRT-PCR检测人胰腺癌细胞PANC-1、SW1990和正常胰腺导管上皮细胞HPDE6-C7中PHLDA2 mRNA表达水平。然后选择PANC-1细胞分组为对照组、PHLDA2沉默组和阴性组,培养48 h后采用MTT法检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测EMT标志物包括波形蛋白(Vimentin)、N-钙黏蛋白(N-cadherin)和E-钙黏蛋白(E-cadherin),自噬标志物包括Beclin1和微管相关蛋白轻链3(LC3)以及p-PI3K、p-AKT和p-mTOR蛋白。结果:与HPDE6-C7相比,PANC-1和SW1990中PHLDA2 mRNA表达水平显著升高,且PANC-1高于SW1990(P<0.05)。与对照组相比,PHLDA2沉默组PHLDA2 mRNA表达水平显著降低(P<0.05)。与对照组相比,沉默组细胞增殖率明显下降,凋亡率升高,E-cadherin增加,Vimentin、N-cadherin、Beclin1、LC3、p-PI3K、p-AKT和p-mTOR降低(P<0.05)。结论:PHLDA2基因上调可能参与了胰腺癌的发生,通过激活PI3K/AKT/mTOR信号通路参与肿瘤EMT和自噬的发生,PHLDA2可作为胰腺癌靶向干预的新位点。

Objective:To investigate the molecular mechanism of silencing human plec substrate protein homeodomain family A member 2(PHLDA2)gene through phosphatidylinositol kinase(PI3K)/silk threonine protein kinase(AKT)/rapamycin target protein(mTOR)signaling pathway to inhibit epithelial mesenchymal transition(EMT)and autophagy in pancreatic cancer cells.Methods:Firstly,the expression of PHLDA2 mRNA in human pancreatic cancer cells PANC-1,SW1990 and normal pancreatic duct epithelial cells HPDE6-C7 was detected by qRT-PCR.Then,PANC-1 cells were divided into control group,PHLDA2 silencing group,and negative group.After 48 hours of cultivation,cell proliferation was detected with MTT method,cell apoptosis was detected with flow cytometry,and Western blot was used to detect EMT markers including Vimentin,N-cadherin,and E-cadherin,autophagy markers including Beclin1 and microtubule associated protein light chain 3(LC3),as well as p-PI3K,p-AKT,and p-mTOR proteins.Results:Compared with HPDE6-C7,the expression level of PHLDA2 mRNA in PANC-1 and SW1990 was significantly higher,and PANC-1 was higher than SW1990(P<0.05).Compared with the control group,PHLDA2 mRNA expression level in PHLDA2 silencing group was significantly less(P<0.05).Compared with the control group,cell proliferation rate in silencing group was significantly lower,apoptosis rate was higher,E-cadherin was more,the expressions of Vimentin,N-cadherin,Beclin1,LC3,p-PI3K,p-AKT,and p-mTOR were less,too(P<0.05).Conclusion:Up-regulation of PHLDA2 gene may be involved in the occurrence of pancreatic cancer,which may be involved in the occurrence of tumor EMT and autophagy by activating PI3K/AKT/mTOR signaling pathway.PHLDA2 may be a new site for targeted intervention in pancreatic cancer.