Association Of CpG Island Methylator Phenotype Involving Tumor Suppressor Genes Located On Chromosome 3p And Hogg1 Variation With Non-Small Cell Lung Cancer

Objective: To explore the association of CpG island methylator phenotype (CIMP) involving tumor suppressor genes (TSGs) on short arm of chromosome 3 (3p) and hOGG1 variation with risk of non-small cell lung cancer (NSCLC).Methods: In the present study,1:Four NSCLC cell lines ( SPC-A-1,A-549,95-D,and LTEP-α-2) were cultured, and peripheral blood mononuclear cell (PBMC) specimens from 80 patients with NSCLC and 80 matched controls were collected for 3p-involved CIMP (3pCIMP) analysis. 3pCIMP was referred to as having at least three synchronously methylated genes of 3p per sample. Methylation-specific PCR (MSP) was performed to examine methylation status of each gene. DNA demethylation of NSCLC cell lines was achieved through the treatment with 5-aza-deoxycytidine (5-aza-CdR). Proliferation of NSCLC cells before and after treatment of demethylation agent 5-aza-CdR was assessed with the MTT method.2:Five NSCLC cell lines (SPC-A-1,A-549,95-D, NCI-H460 and LTEP-α-2 ) were cultured, and NSCLC tissue and matched non cancerous tissue from 117 NSCLC patients were collcted for hOGG1 methyaltion status analysis. Logistic regression was used to assess odds ratios (OR) and 95% confidence intervals (CI) of NSCLC, which were adjusted for gender, age and smoking status. Moreover, 117 NSCLC patients and 121 cancer-free controls were enrolled to assess the association of genetic variants in hOGG1 gene with risk of NSCLC. Four haplotype-tagging single nucleotide polymorphisms (tSNPs) were selected using Haploview software V4.1. The tSNPs were genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP) and single-strand conformation polymorphism (SSCP) assay. Haplotypes were reconstructed according to the genotyping data and linkage disequilibrium status of these 4 tSNPs. Results: Demethylation experiment showed that 3pCIMP status could play an important role in NSCLC cell proliferation. 97.5% of PBMC specimens from NSCLC patients presented promoter methylation of any one of six genes (hOGG1, RAR-B, SEMA3B, RASSF1A, BLU, or FHIT) on 3p. Interestingly, 3pCIMP+ was found in 43.8% of NSCLC PBMC specimens but only in 6.3% of normal PBMC samples. The data suggest that 3pCIMP status is significantly associated with NSCLC and normal PBMC samples (P<0.001). More importantly, the results show that 3pCIMP positive carriers have a 12.8-fold increased risk of NSCLC (adjusted OR, 12.8; 95% CI, 4.38 to 37.4, P<0.001) in Chinese population.The distribution of genotype and allelic frequencies of the 4 htSNPs in hOGG1 gene is not significantly different among NSCLC cases and controls. Interestingly, demethylation experiment showed that hOGG1 gene methylation status plays an important role in mRNA expression of hOGG1. Significant difference in mRNA expression was observed in three NSCLC cell lines. Futhermore, our data showed that the promoter region of hOGG1 gene is more frequently methylated in NSCLC cancerous tissue than in matched non-cancerous tissue (68.38% vs. 40.17%, P < 0.001). The hOGG1 gene methylation appears to be more susceptible to squamous cell carcinoma (P=0.022).Conclusion: This is the first evidence of an association between PBMC 3pCIMP and risk for NSCLC. The methylation status of hOGG1 might be an important factor in mRNA down-regulation of hOGG1.