低表达UHRF1通过Wnt/β-catenin信号通路抑制非小细胞肺癌细胞增殖能力的研究

Silence of UHRF1 inhibit lung adenocarcinoma proliferation via Wnt/β-catenin signaling pathway

目的探讨泛素样含PHD和环指域蛋白1(Ubiquitin like PHD and ring finger domain 1,UHRF1)与肺腺癌的作用及其潜在的作用机制。方法通过生物信息方法检测UHRF1在肺腺癌中的表达。实时定量PCR及免疫蛋白印记检测UHRF1在肺腺癌细胞中的表达。MTT实验,Ki67染色和克隆形成方法检测正常组,阴性对照组低表达UHRF1组肺腺癌细胞系A549活性的变化。通过免疫蛋白印记的方法检测增殖相关蛋白PCNA,Rb,CDK4/6的变化,以及Wnt/β-catenin信号通路中经典蛋白β-catenin以及GSK-3β的变化。结果UHRF1在肺腺癌中相对于癌旁组织表达升高(P<0.05)。在肺腺癌细胞系(A549和H1299)相对于16HBE表达上调(P<0.05)。MTT法和克隆形成实验表明敲除UHRF1能够降低A549细胞生长效率(P<0.05)。Ki-67免疫荧光染色显示沉默UHRF1后A549细胞增殖能力下降(P<0.05)。此外,沉默UHRF1导致PCNA CDK4/6表达相对于对照组明显降低,而Rb蛋白相对于对照组表达升高(P<0.05)。沉默UHRF1抑制能够使β-catenin的蛋白相对于对照组表达下调,GSK-3β的蛋白相对于对照组表达上调(均P值<0.05)。结论UHRF1基因在非小细胞肺癌尤其是肺腺癌中具有重要作用,UHRF1可能是一个潜在预防、治疗的肺腺癌的一个重要因子。

Objective To investigate the role and potential mechanism of Ubiquitin like PHD and ring finger domain 1(UHRF1)in Lung adenocarcinoma.Methods The expression of UHRF1 in lung adenocarcinoma was detected by bioinformatics(TCGA).Real-time quantitative PCR and western blot were used to detect the level of UHRF1 in lung adenocarcinoma cells.The cells in control NC.UHRF1-SiRNA group viability were detected by MTT and colony formation.Ki67 immunofluorescence staining was used to test the proliferation of A549 cells transfected with UHRF1-siRNA or control-siRNA.The proliferation related protein PCNA,Rb,CDK4/6,β-catenin,and GSK-3βin Wnt/β-catenin signaling pathway were determined by Western blotting.Results The expression of UHRF1 in lung adenocarcinoma was higher than that in adjacent tissues(P<0.05).The expression of UHRF1 in 16HBE was lower than that in lung adenocarcinoma cell lines(A549 and H1299)(P<0.05).In addition,MTT and clonogenic assay showed that knocking down UHRF1 could inhibit A549 cell viability(P<0.05).Ki-67 immunofluorescence staining showed that the proliferation of A549 cells decreased after UHRF1 knockdown(P<0.05).In addition,knockdown of UHRF1 decreased the level of PCNA CDK4/6 expression compared with NC group,while the expression of Rb was higer than that of the control group(P<0.05).Knockdown of UHRF1 reduced the expression ofβ-catenin,while the level of GSK-3βwas higher than that in control-siRNA(both P<0.05).Conclusion The UHRF1 may play an important role in the progress of lung adenocarcinoma.