Methylatlon Level Of ERRα And Its Mechanism Of Action In The Development Of Lung Adenocarcinoma

Purpose:Lung cancer is the most life threatening cancer worldwide with high morbidity and mortality worldwide.The pathogenesis of lung cancer is not yet clear.Lack of early diagnosis and better therapy,it is necessary to find new important genes in lung cancer.The estrogen-related receptor alpha(ERRα)is a multifunctional protein not limited to bind ligands and has been reported to be associated with numerous cancers.Studies have reported that it can play its role as an oncogene by promoting the proliferation,migration and invasion of tumor cells.There are also a few reports in lung cancer,but the expression regulation,function and prognosis of ERRα in lung adenocarcinoma are unknown.This study aimed to investigate the role of ERRα in the development of lung adenocarcinoma and provide a new perspective for early diagnosis,targeted therapy and prognosis evaluation of lung cancer.Methods:1.Detection of mRNA expression of ERRα in lung adenocarcinoma cells(A549,NCI-H1395,NCI-H1975),lung squamous carcinoma cells(SW900,NCI-H520)and normal lung bronchial epithelial cells(BEAS-2B)by qPCR.Protein expression levels of ERRα in lung adenocarcinoma cells(A549,NCI-H1395,NCI-H1975)and normal lung bronchial epithelial cells(BEAS-2B)were detected by Western blot.ERRα protein levels were detected by immunohistochemical method in 88 matched lung adenocarcinoma clinical tissues and adjacent normal tissues(and 4 separate lung adenocarcinoma tissues).2.Differential methylation sites were screened for normal lung bronchial epithelial cells(BEAS-2B),lung adenocarcinoma cells(A549),and lung squamous carcinoma cells(SW900,NCI-H520)using an Illumina 850 k methylation chip.Methylation-specific PCR(MSP)was used to verify the methylation level of methylation differential sites.3.The correlation between ERRα mRNA expression and survival time of the online clinical data(TCGA、EGA)about lung cancer was analysed by using Kaplan Meier plotter.Immunohistochemistry was performed to detect the expression level of ERRα in tissue microarrays containing 88 lung adenocarcinoma tissues and 92 paracancerous tissues.The ERRα level was scored using the IRS scoring criteria and then combined with the patient’s clinical information for prognostic analysis.4.To construct a BALB/c mouse model of lung adenocarcinoma in situ cancer.Immunohistochemistry,Western blot and enzyme-linked immunosorbent assay(ELISA)were used to qualitatively and quantitatively detect the expression level of key gene ERRα in tumor tissues of lung adenocarcinoma mice.5.To construct ERRα knockdown lung adenocarcinoma cell lines(A549,NCI-H1395,NCI-H1975).CCK-8 assay,scratch assay,tranwell assay,and cell cycle assay were used to analyze the effects of ERRα on cell proliferation,migration,invasion and cell cycle,respectively.Detection of mitochondrial DNA copy number and ATP was performed on the construction of ERRα knockdown lung adenocarcinoma cell lines.Results:1.The results of qPCR and Western blot showed that ERRα was highly expressed in lung adenocarcinoma cell lines(A549,H1395,H1975)(P<0.01).Immunohistochemistry results showed that ERRα was highly expressed in lung adenocarcinoma tissues.The Illumina 850 k DNA methylation chip screened 2,413 differential methylation sites,corresponding to 1675 genes.29 ERRα methylation sites were screened out,in which there was a hypomethylation change in the ERRα key methylation site in lung adenocarcinoma cells(with normal lung epithelial cells as controls).MSP verification results indicated that the key sites were hypomethylated(P<0.001).2.The KM plotter analysis suggested that ERRα is correlated with poor prognosis in LUAD(P<0.001,HR=1.68)rather than in LSCC.The immunohistochemical scoring of tissue microarray combined with clinical data showed that the high expression of ERRα in lung adenocarcinoma was positively correlated with low survival rate(P<0.001,HR=1.597).3.Successfully constructed lung adenocarcinoma in situ cancer model BALB/c mice.Qualitative and quantitative detection by immunohistochemistry,Western blot and enzyme-linked immunosorbent assay(ELISA)showed that ERRα was highly expressed in tumor tissues of lung adenocarcinoma mice(P<0.01).4.Successfully constructed ERRα knockdown lung adenocarcinoma cell line(A549,H1395,H1975).Functional experiments such as CCK-8 assay,scratch assay,tranwell assay and cell cycle assay showed that the down-regulation of ERRα inhibited the proliferation,invasion and migration of lung adenocarcinoma cells and arrested cells in G2/M phase(P< 0.01).The mitochondrial DNA copy number and ATP content of ERRα knockdown lung adenocarcinoma cells decreased(P<0.05).Conclusions:1.ERRα is highly expressed in lung adenocarcinoma cell lines(A549,H1395,H1975),lung adenocarcinoma BALB/c mouse model,and clinical tissues.Up-regulation of ERRα may be associated with hypomethylation of methylation sites in lung adenocarcinoma cells.2.ERRα may participate in the development of lung adenocarcinoma by promoting the proliferation,migration,invasion and affecting the cell cycle of lung adenocarcinoma cells.It was also found that it can cause changes in mitochondrial DNA copy number and ATP content,suggesting that it may promote the proliferation,migration and invasion of tumor cells by affecting the biosynthesis and function of mitochondria.3.ERRα may be used as an indicator of poor prognosis in lung adenocarcinoma.