miR-34a阻断Wnt/β-catenin信号通路下调PD-L1表达抑制肺腺癌细胞的生长和转移

miR-34a inhibits the growth and metastasis of lung adenocarcinoma cells by blocking Wnt/β-catenin signaling pathway to downregulate PD-L1 expression

目的:研究miR-34a对肺腺癌细胞增殖、转移及凋亡的影响及其作用机制。方法:选取105例肺腺癌患者,根据患者PD-L1表达分为高、中、低三组,收集患者腺癌组织及癌旁组织,运用实时荧光定量PCR法(qRT-PCR)和Western blot法(WB)检测miR-34a和Wnt信号相关基因mRNA和蛋白表达量。构建miR-34a mimcs-pCMV、miR-34a inhibitor-pCMV载体和空载pCMV,利用脂质体转染肺腺癌A549细胞,采用MTT法检测miR-34a高表达、低表达和阴性对照NC组细胞增殖活性,采用Transwell小室实验、细胞划痕实验、EdU实验检测细胞侵袭、迁移和增殖能力;采用qPCR和WB法检测各组细胞中PD-L1、Wnt信号相关基因以及肺腺癌相关基因及蛋白表达水平;采用双荧光素酶系统检测miR-34a和Wnt1、PD-L1靶向结合作用。结果:在三组患者中miR-34a的表达与PD-L1表达水平呈负相关(P<0.05),而Wnt信号通路相关蛋白的表达水平与PD-L1表达呈正相关(P<0.05)。与对照NC组相比,miR-34a mimcs组细胞增殖活性、迁移能力和侵袭能力均显著降低,而miR-34a inhibitor组细胞的增殖及迁移、侵袭能力显著提高(均P<0.05);qPCR和WB结果显示,PD-L1、Wnt通路相关蛋白Wnt1、β-catenin、GSK-3β、PTEN,凋亡相关蛋白Bcl-2,肺腺癌相关驱动基因EGFR、ROS1、ALK,转移相关基因MMP2、Vimentin的表达量在miR-34a mimcs组中显著低于它们在miR-34a inhibitor组中的表达水平;而细胞凋亡相关的Cleaved caspase-3、p53蛋白表达量则在miR-34a mimcs组显著高于miR-34a inhibitor组。双荧光素酶报告基因实验验证miR-34a与Wnt1和PD-L1均能特异性结合,Wnt1和PD-L1是miR-34a调控的靶基因。结论:miR-34a能够抑制PD-L1蛋白和肺腺癌相关驱动基因的表达,并通过调控Wnt1蛋白抑制Wnt信号通路从而抑制肺腺癌细胞的增殖、迁移和侵袭过程;同时诱导细胞凋亡相关基因表达,促进肺腺癌细胞的凋亡。

Objective:To study the effect and mechanism of miR-34a on proliferation,metastasis,and apoptosis of lung adenocarcinoma cells.Methods:105 lung adenocarcinoma patients were selected and divided into three groups based on their PD-L1 expression level(high,medium,low).Adenocarcinoma and adjacent tissues were collected,and miR-34a and Wnt signaling related gene mRNA and protein expression levels were detected using real-time fluorescence quantitative PCR(qRT-PCR)and Western blot(WB)methods.Construct miR-34a mimcs-pCMV,miR-34a inhibitor-pCMV vectors,and pCMV.Use liposomes to transfect lung adenocarcinoma A549 cells.CKK-8 assay was used to detect the proliferation activity of high expression,low expression,and negative control NC cells in miR-34a.Transwell cell assay,cell scratch assay,EdU assay were used to detect the invasion,migration and proliferation ability of each group of cells.The expression levels of PD-L1,Wnt signaling related genes,and lung adenocarcinoma related genes and proteins in each group of cells were detected by qPCR and WB methods.The targeted binding of miR-34a to Wnt1 and PD-L1 was detected by a dual luciferase system.Results:The expression of miR-34a was negatively correlated with the expression level of PD-L1 in the three groups of patients(P<0.05),while the expression level of Wnt signaling pathway related proteins was positively correlated with PD-L1 expression(P<0.05).Compared with the control NC group,the proliferation activity,migration ability,and invasion ability of cells in the miR-34a mimcs group were significantly reduced,while the proliferation,migration,and invasion ability of cells in the miR-34a inhibitor group were significantly increased(P<0.05).qPCR and WB results showed that the expression levels of PD-L1,Wnt pathway related proteins Wnt1,β-catenin,GSK-3β,PTEN,the expression of apoptosis-related protein Bcl-2,lung adenocarcinoma related driving genes EGFR,ROS1,ALK,and metastasis related genes MMP2,and Vimentin were significantly lower in the miR-34a mimcs group than in the miR-34a inhibitor group.The expression levels of Cleaved caspase-3 and p53 proteins related to cell apoptosis were significantly higher in the miR-34a mimcs group than in the miR-34a inhibitor group.The double luciferase reporter gene experiment verified that miR-34a can specifically bind to Wnt1 and PD-L1,and Wnt1 and PD-L1 were target genes regulated by miR-34a.Conclusion:miR-34a can inhibit the expression of PD-L1 protein and lung adenocarcinoma related driving genes,and inhibit the proliferation,migration,and invasion process of lung adenocarcinoma by regulating the Wnt1 protein to inhibit the Wnt/β-catenin signaling pathway,simultaneously induce the expression of apoptosis related genes and promote apoptosis in lung adenocarcinoma cells.