Objective The objective of this study was to investigate the proliferation,autophagy and the potential mechanism of Ubiquitin-like with PHD and ring finger domains 1(UHRF1)in lung adenocarcinoma cells. Methods The expression of UHRF1 in lung adenocarcinoma tissues was determined by the bioinformatics website(TCGA). The expression of UHRF1 in lung adenocarci-noma cell lines(PC-9,A549 and H1299)and human bronchial epithelial cells(16HBE)was detected by qRT-PCR and Western blot. After transfection of UHRF1-shRNA,CCK-8,clone formation and ki67 were performed to detect the changes in the prolifera-tive capacity of lung adenocarcinoma A549 cells. Western blot was used to detect the changes of autophagy-associated proteins(LC3-I/II and Beclin-1)and proliferation-related proteins(CDK6,Rb and PCNA). Transmission electron microscopy was used to ob-serve the effect of UHRF1 on autophagosomes in A549 cells. Results The expression of UHRF1 in lung adenocarcinoma tissues was significantly higher than that in adjacent tissues. Compared with normal bronchial epithelial 16HBE cells,the mRNA and protein levels of UHRF1 in lung adenocarcinoma A549 and H1299 cells were significantly increased. In addition,CCK-8 assay and colony forma-tion experiments showed that silencing UHRF1 reduced the growth of A549 cells. Ki-67 immunofluorescence staining showed that the proliferation ability of A549 cells after knocking out UHRF1 was significantly lower than that in the normal control group. Further-more,knockdown of UHRF1 resulted in an increased expression of CKD6 and PCNA proteins in comparison with the control-siRNA group. The expression of Rb protein was down-regulated in the UHRF1-siRNA group. Silencing UHRF1 increased the ratio of LC3-II/LC3-I, induced up -regulation of Beclin -1 expression and promoted the formation of autophagic bodies in A549 cells. Conclusion UHRF1 is highly expressed in lung adenocarcinoma,and silencing UHRF1 can inhibit proliferation. This effect may be regulated by promoting autophagy.%目的 探讨泛素样含PHD和环指结构域1(Ubiquitin like with PHD and ring finger domains 1,UHRF1)在肺腺癌(Lung adenocarcinoma)细胞的增殖、自噬中的作用及其潜在机制.方法 通过生物信息学网站( TCGA)检测UHRF1在肺腺癌组织中的表达. qRT-PCR和Western blot检测肺腺癌细胞系(PC-9、A549和H1299)以及人支气管上皮细胞(16HBE)中UHRF1的表达.转染UHRF1-shRNA后,采用CCK-8、克隆形成以及Ki-67检测肺腺癌A549细胞活性和增殖能力的改变;蛋白免疫印记检测自噬相关LC3-I/II、Beclin-1蛋白以及增殖相关蛋白CDK6、Rb和PCNA蛋白的变化;透射电镜观察敲除UHRF1后A549细胞中对于自噬小体的影响.结果 在肺腺癌患者组织中UHRF1表达明显高于癌旁组织,而相对于正常的支气管上皮细胞16HBE,肺腺癌细胞系A549与H1299中UHRF1的mRNA和蛋白水平均明显升高.此外,CCK-8法和克隆形成实验表明沉默 UHRF1 能够降低肺腺癌细胞系 A549 的生长效率. Ki-67 免疫荧光法检测结果显示敲除UHRF1后A549细胞增殖能力相对于正常对照组明显降低.此外我们发现,敲除UHRF1导致CKD6和PCNA蛋白水平相对于Control-siRNA组表达升高,而Rb蛋白表达下调.我们同时还发现,沉默UHRF1后能够提高LC3-II/LC3-I的比例,诱导Beclin-1的表达上调.沉默UHRF1促进A549细胞中自噬小体的形成.结论 UHRF1在肺腺癌中高表达,沉默UHRF1能够发挥抑制增殖的作用.而这种作用可能是通过促进细胞自噬产生的.
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