Methylation And Functional Study Of Peroxidase-activated Receptor D Gene In The Development Of Lung Adenocarcinoma

OBJECTIVE Lung cancer has become the most common disease with highest incidence of malignancy in the world at present.In China,lung cancer has been more spread out than any other male cancersand only after breast cancer in female.Non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC)are two major types of lung cancer which consist80% of all cases.The lung adenocarcinoma is the most common subtype of NSCLC.This study aims to select the early epigenetic bio-markers of lung adenocarcinoma by exploring the key methylations in specific genes and related mechanisms.Our findings provide scientific clues and theoretical basis for the future diagnosis and treatment of lung adenocarcinoma.METHODS1.The whole genomic methylation status of normal lung epithelial and the lung adenocarcinoma cells were analyzed by the,Illumina 850 k DNA methylation chip.The key genes with abnormal methylation alterations were then submitted to David Bioinformatics Resources 6.8(https://DAVID.ncifcrf.gov/)to evaluate the biological function.We further identified PPARD as the potential prognostic marker of lung adenocarcinoma using gene expression data and clinicopathological information from the TCGA database.2.Methylation specific PCR(MSP),real-time fluorescence quantitative PCR(qPCR)and Western blot analysis were use to verify the variation of DNA methylation in PPARD promoter regions and its expressions in lung adenocarcinoma tissues(n=12)respectively.3.PPARD-knockdown lung adenocarcinoma cells(NCI-H1975 and NCI-H1395)were established using lentivirus mediated transfection assay.We further evaluated the role of PPARD gene in proliferation,invasion and metastasis of lung adenocarcinoma cells by CCK8 assay,Transwell assay and cell scratching assay.RESULTS1.A total of 2,414 different methylation sites in 1676 genes were identified by the Illumina 850 k DNA methylation chip.Further analysis revealed that the key sites of PPARD gene promoter in lung adenocarcinoma cells were abnormally methylated in lung adenocarcinoma.Moreover,PPARD was found elevated and correlated with prognosis in lung adenocarcinoma by the evaluation of gene expression data from TCGA database..2.PPARD methylation rate was abnormally decreased in the lung adenocarcinoma tissues compared with the cancer-adjacent tissues(P<0.05).qPCR and Western blot analysis suggested that PPARD was elevated in lung adenocarcinoma tissues compared with cancer-adjacent tissues(1.44±0.46,P<0.05;2.04±1.61,P<0.05).3.Knockdown of PPARD significantly suppressed the migration and cell viability of lung adenocarcinoma cells however no impairment on the invasive ability was observed.CONCLUSION The key methylation sites of PPARD promoter region in lung adenocarcinoma may play an important role in its epigenetic regulation.Functional studies indicated that PPARD promotes the proliferation and metastasis of lung adenocarcinoma cells.In summary,PPARD is a potential diagnostic and therapeutic target for lung cancer.