The Role Of Retinoic Acid Recrptor Beta Methylaton In The Progression Of Non-small-cell Lung Cancer

Objective: Lung cancer is one of the most deadly cancers which pathogenesis is still unclear.This study aimed to investigate the role of retinoic acid receptor beta methylation in the progression of non-small cell lung cancer.To provide a theoretical basis and experimental basis for further clarifying the mechanism of action of retinoic acid beta receptor in non-small cell lung cancer.Methods:1.Detection of RAR-? gene expression in paracancerous tissues and cancer tissues by qPCR.Detection of RAR-? protein expression in tissues by immunohistochemistry.2.Construction of mouse lung cancer model.Fixation,embedding,dehydration and staining of mouse lung tissue.Hematoxylin-eosin staining stained mouse lung tissue and observed the tumor under microscope.Detection of RAR-? protein expression in tumor tissues by Western blot and Enzyme linked immunosorbent assay.Detection of DNMT3 B protein expression in mouse tumor tissues by ELISA.3.Screening of BEAM-2B,A549,SW-900 and NCI-H520 differential methylation sites with Illumina Infinium Methylation EPIC(850K)BeadChip.Identify key methylation sites and map them on DAVID 6.7 web.Detection of RAR-? expression in normal lung cells and lung cancer cells by Quantitative real-time PCR.To verify the expression of RAR-? protein and DNMT3 B protein in cells by Western blot.And detection of methylation status of RAR-? in cells by methylation-specific PCR.Results:1.The lung cancer and paracancerous tissues of 55 patients with lung cancer were collected.The real-time PCR results of total RNA showed that the expression of RAR-? gene in lung cancer tissues was significantly lower than that in adjacent tissues(P<0.01).A total of 100 paraffin sections of lung cancer were produced.The results of immunohistochemistry showed that the number of dark brown cells in the paracancerous tissues was more than that in lung cancer tissues,indicating that RAR-? was located in the nucleus,and the expression of RAR-? protein in lung cancer tissues was significantly lower than that in adjacent tissues.2.The formation of cancer cells was found in paraffin sections of lung tissue of mouse lung cancer model,indicating successful modeling.Extraction of 6 pairs of mouse lung cancer tissues and paracancerous tissues for Western blot,The results showed that RAR-? protein was lowly expressed in lung cancer tissues compared with adjacent tissues(P<0.001).The ELISA result of RAR-? were consistent with those of Western blot(P<0.05).The results of mouse lung cancer model showed that the expression of DNMT3 B protein in mouse tumor tissues was higher than that in adjacent tissues(P<0.001).3.The total number of differential methylation sites was 199 492 and the related genes were 1 675 by Illumina 850 K chip.Analysis of gene pathway maps revealed that RAR-? hypermethylation may be involved in tumor proliferation.Real-time quantitative PCR and Western blotting results showed that RAR-? was lowly expressed in non-small cell lung cancer.In vitro experiments with lung cancer cells showed that DNMT3 B protein was highly expressed in A549,SW-900 and NCI-H520 compared with BEAS-2B(P<0.01).Combined with methylation microarray analysis,71 different sites of RAR-? promoter region were screened,and some important methylation sites cg23302436 and cg06905304 were hypermethylated.At the same time,methylation-specific PCR results showed that some important methylation sites of RAR-? were hypermethylated in non-small cell lung cancer cells..Conclusions:1.In non-small cell lung cancer,the retinoic acid beta receptor is hypermethylated and its expression level is reduced.2.Increased expression of DNMT3 B in non-small cell lung cancer may induce hypermethylation of RAR-? methylation site and down-regulate RAR-? expression,which is closely related to the progression of non-small cell lung cancer.3.The important sites of RAR-? promoter region cg23302436 and cg06905304 may play an important role in the epigenetic regulation of non-small cell lung cancer.The specific methylation site of RAR-? promoter region can be used as a potential target of lung cancer drugs.