CHD5对肺腺癌细胞增殖、凋亡及p16^(INK4a)/RB通路的影响

Effects of CHD5 on proliferation,apoptosis and p16^(INK4a)/RB pathway of lung adenocarcinoma cells

目的:探讨染色质解旋酶DNA结合蛋白5(CHD5)在肺腺癌(LUAD)中的临床意义及对LUAD细胞增殖、凋亡和p16^(INK4a)/RB通路的影响。方法:通过UALCAN、K-M数据库分析CHD5在LUAD中表达情况及其与预后的关系;通过实时荧光定量聚合酶链反应(qRT-PCR)和蛋白质印迹法(Western blotting)检测LUAD组织、癌旁组织和各细胞系(H1299、H1650、H1975、A549)及正常肺上皮细胞系(BEAS-2B)中CHD5的表达情况;免疫组化染色检测LUAD组织中CHD5的表达情况,并分析其与患者临床病理特征及预后的关系。CHD5基因的sgRNA载体感染H1975和A549细胞作为过表达组(CHD5-OE组),同时设置空载体感染细胞为阴性对照组(Ctrl-OE组)、未感染病毒细胞为空白组(NC组),并通过CCK-8实验、克隆形成实验、TUNEL凋亡实验、细胞划痕和Transwell实验,评估CHD5表达上调对LUAD细胞增殖、凋亡、迁移及侵袭能力的影响,Western blotting实验检测CHD5表达上调对p16^(INK4a)/RB介导的信号通路和细胞周期蛋白表达情况的影响。结果:生物信息数据库分析显示CHD5在LUAD中显著低表达,且CHD5低表达与更差的总生存时间(OS)显著相关;qRT-PCR和Western blotting检测发现,CHD5在LUAD组织和癌细胞(H1299、H1650、H1975、A549)中表达水平均显著低于癌旁组织和正常肺上皮细胞系BEAS-2B,差异具有统计学意义(P<0.05);免疫组化结果显示,65.7%的LUAD组织样本中CHD5呈低表达或不表达,与临床分期、肿瘤直径及淋巴结转移显著相关,与年龄、性别、吸烟及远处转移无关;CHD5低表达的患者无进展生存期(PFS)更短;Cox多因素分析显示CHD5表达可作为LUAD患者预后影响因素(P<0.001)。CHD5过表达明显抑制体外细胞增殖、迁移和侵袭;CyclinD1、CDK4、p-RB、MMP-9的表达水平降低,caspase-9、p16、RB的表达水平升高;结论:CHD5在LUAD中表达下调且与患者的不良预后相关,CHD5过表达可抑制LUAD细胞增殖、迁移及侵袭,其作用机制可能与抑制p16^(INK4a)/RB信号通路活化有关。

Objective:To investigate the clinical significance and molecular mechanism of chromatin helicase DNA binding protein 5(CHD5)in lung adenocarcinoma(LUAD)and its effect on LUAD cell proliferation,apoptosis and p16;/RB pathway.Methods:UALCAN and Kaplan-Meier Plotter(K-M)databases were used to analyze the expression of CHD5 in LUAD and its relationship with prognosis.Real-time quantitative polymerase chain reaction(qRT-PCR)and Western blotting were used to detect the expression of CHD5 in LUAD tissues,paracancer tissues and cell lines(H1299,H1650,H1975,A549,BEAS-2 B).The relationship between CHD5 and clinicopathological features and prognosis of patients were analyzed.The sgRNA vector of CHD5 gene infects H1975 and A549 cells as the overexpression group(CHD5-OE group),and empty vector infected cells as the negative control group(CTRL-OE group),and uninfected cells as the blank group(NC group).The effects of up-regulated of CHD5 expression on the proliferation,apoptosis,migration and invasion of LUAD cells were evaluated by CCK-8,clonogenesis assay,TUNEL apoptosis assay,cell scratching and Transwell assay.Western blotting detected the effect of up-regulation of CHD5 expression on p16;/RB-mediated signaling pathway and cyclin expression.Results:Bioinformatics database analysis showed that CHD5 expression was significantly low in lung adenocarcinoma.Low expression of CHD5 was significantly associated with worse overall survival time(OS).qRT-PCR and Western blotting analysis showed that the expression level of CHD5 in LUAD tissues and cancer cells(H1299,H1650,H1975,A549)was significantly decreased,the difference is statistically significant(P<0.05).Immunohistochemical results showed low or no CHD5 expression in 65.7%lung adenocarcinoma tissue samples.CHD5 expression was related to clinical staging,tumor diameter,lymph node metastasis,but was not related to age,gender,smoking and distant metastasis;Patients with low CHD5 expression had shorter progression-free-survival(PFS).Cox multivariate analysis showed that CHD5 expression was a prognostic factor in LUAD(P<0.001).Overexpression of CHD5 significantly inhibited cell proliferation,migration and invasion in vitro;The expression levels of CyclinD1,CDK4,P-Rb and MMP-9 decreased.The expression levels of Caspase-9,P16 and RB increased.Conclusion:The down-regulated expression of CHD5 in LUAD is related to the poor prognosis of patients.Overexpression of CHD5 can inhibit the proliferation,migration and invasion of LUAD cells,and its mechanism may be related to the inhibition of p16 INK4 a/RB signaling pathway activation.