Predictive Value Of LDHC And HK3 Promoter Methylation On The Diagnosis And Prognosis Of Non-small Cell Lung Cancer

Objective: To analyze the differences in promoter methylation levels of glycolysis related genes PFKFB3,PKLR,HK3,LDHB and LDHC in non-small cell lung cancer(NSCLC)tissues,paracancerous tissues and distal tissues,and to explore the possibility of such differences as diagnostic and prognostic markers of NSCLC.Methods: A total of 105 NSCLC patients with cancer tissues and their matched non-cancer tissues were collected by QMSP(Quantitative Methylation Specific Polymerase Chain Reaction).The differences of methylation levels of glucose metabolism-related genes PFKFB3,PKLR,HK3,LDHB and LDHC promoters in NSCLC paired cancer tissues,paracancerous tissues and distal tissues were analyzed to screen positive genes.At the cellular level,the functions of these promoters were further confirmed by 5 ’-Aza-deoxycytidine(5’-Aza)treatment and double luciferase assay.At the same time,combined with online data and experimental data,the clinical diagnostic value of related gene promoter methylation sites was comprehensively analyzed.Results: 1.After methylation primer detection of several glycolysis related gen es,five target genes were identified for lung cancer tissue screening: pFKFB3,pKLR,HK3,LDHB,LDHC.2.The promoter methylation levels of 5 genes were measured by q MSP,and it was found that the promoter methylation levels of 2 genes(LDHC,HK3)were statistically different among the matched cancer tissues,paracancerous tissues and distal tissues.Further analysis showed that the methylation level of LDHC had diagnostic significance for lung cancer.The methylation level of LDHC in tumor tissues was lower than that in adjacent paracancerous tissues(median PMR :40.84% vs.49.89%,P = 0.005)and was significantly lower than that in distal paracancerous tissues(median PMR :40.84% vs.54.51%,P = 0.0004).Further analysis of clinical data showed that,except for the younger group(Age≦60),where the lesion was located on the right side,and the non-smoking group,the methylation levels of LDHC in all subgroups were different in the cancer tissues and distal paracancerous tissues(all P <0.05).There was no significant difference in the level of HK3 methylation between lung adenocarcinoma and adjacent paracancerous tissues(median PMR :29.34% vs.31.89%,P = 0.131),but it was lower than that in distal paracancerous tissues(median PMR :29.34% vs.40.79%,P = 0.016).In other clinical groups,No significant significance.3.At the cellular level,DNA demethylation agent 5 ’-Aza was used to treat A549 and H1299 lung cancer cells and HB2 B human bronchial epithelial cells,and the results showed that m RNA expression of the two genes was up-regulated to varying degrees after demethylation.LDHC(A549: Fold change(FC)=2.553,P = 0.0001;H1299:Fold change(FC)=1.290,P = 0.0068;HB2b: Fold change(FC)=1.737,P=0.0019);HK3(A549:Fold change(FC)=7.934,P = 0.0031;H1299:FC=2.71,P = 0.0015;HB2b: Fold change(FC)=2.768,P=0.0005),indicating that the expression of LDHC and HK3 genes is regulated by promoter methylation.Dual luciferase assay was used to verify the transcriptional activity of the promoter fragments of the two genes we detected,and the results showed that both promoter fragments could significantly improve the transcription level of the reporter genes,LDHC(FC=7.19,P <0.0001)and HK3(FC= 1.570,P = 0.011).We then modified the promoter reporter plasmid with methylation,and found that methylation significantly reduced the ability of LDHC(FC=8.89,P <0.0001)and HK3(FC= 1.570,P = 0.011)to activate the reporter gene expression.4、Online analysis of the correlation between the expression of LDHC and HK3 and promoter methylation showed that compared with normal lung tissue,the degree of methylation of LDHC and HK3 was lower in NSCLC tissue(P<0.01),and the expression of LDHC and HK3 was negatively correlated with the methylation of Cp G island and promoter region.5、The area under the curve(AUC)was used to evaluate the diagnostic ability of LDHC methylation to NCSLC.The results showed that LDHC hypopmethylation was effective for specificity and specificity of lung cancer(AUC=0.768,P = 0.041,sensitivity=87.5%,specificity=71.4%)and for specificity and specificity of lung cancer(AUC=0.707,P = 0.017).Sensitivity =84.6%,specificity=51.6%)was of diagnostic significance,and HK3 methylation had no diagnostic significance in patients with non-small cell lung cancer.TCGA database was used to collect Cp G loci related to LDHC promoter region(CHR11: 18412808,Human GRCH37 / HG19)and a large-scale analysis of the clinical data.Data analysis showed that methylation of Cp G sites in LDHC had no prognostic significance for NSCLC patients.The prognostic significance of Cp G sites related to the HK3 promoter region for NSCLC was analyzed.Results showed that CG17393572 methylation had prognostic significance in lung adenocarcinoma(HR=1.434,P=0.027).Patients with high levels of CG29312072(HR=0.646,P=0.034)and CG06783121(HR=0.618,P=0.013)had longer survival.6 、 The TCGA databa was used to determine the potential biological function of LDHC and HK3.According to the median expression of LDHC and HK3,566 cases of LUAD and 551 cases of LUSC were divided into high expression group and low expression group.GSEA software was used for KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway and GO(Gene Ontology)enrichment analysis,and the analysis results showed that The results showed that LDHC and its related genes were mainly concentrated in ATP generation,extracellular matrix formation,cell cycle regulation,regulation of fat metabolism and other processes,while HK3 and its related genes were involved in inflammatory response and immune response.According to the median expression of LDHC,566 cases of LUAD and 551 cases of LUSC were divided into high expression group and low expression group of LDHC.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway and GO(Gene ontology)enrichment analysis were performed by GSEA software.The results showed that in LDHC high expression group,the main enrichment pathways included amino acid metabolism,glycolysis,TGF signal pathway,cell cycle regulation and so on.GO analysis showed that LDHC and its related genes were mainly enriched in the processes of ATP production,extracellular matrix formation,cell cycle regulation,biological behavior regulation,transmembrane transport,regulation of fat metabolism and so on.Conclusion: The promoter methylation levels of LDHC and HK3 are different in lung cancer,paracancerous and distal paracancerous tissue samples,and their promoter methylation can regulate gene expression and may serve as a biomarker for the diagnosis of non-small cell lung cancer.