| Purpose:Lung cancer is one of the most common malignancy and the leading cause of cancer related deaths worldwide. Most patients die of cancer recurrence and metastasis. Approximately 30% to 40% of patients with stage I non-small cell lung cancer (NSCLC) that undergo curative resection die of recurrent disease. Therefore, it is much more important to identify diagnostic and prognostic biomarkers of NSCLC. MicroRNA profiles have been reported to have diagnostic and prognostic values for lung cancer. Recently, miR-34 family has been shown to be part of the p53 pathway which is frequently involved in lung cancer, and the expression of miR-34 has been reported to be regulated by DNA methylation. In this study, we analyzed the association between DNA methylation of miR-34 family and recurrence of stage I NSCLC.Materials and Methods:From Jan 1999-Dec 2005, a total of 4,702 patients with lung cancer were diagnosed at our hospital, in which 833 patients received a pathological diagnosis of stageâ… (Tla-b N0M0 and T2aN0M0) NSCLC, account for 17.72% of all lung cancer. All patients were not treated with adjuvant chemotherapy, irradiation therapy and biological therapy. Formalin-fixed paraffin embedded (FFPE) tumor tissue samples from 161 patients could be obtained from department of Pathology. All patients were followed up for at least 5 years. All recurrent cases had radiographic imaging or histologic verification of recurrence and all death was caused by cancer or cancer related complications. miR-34a and miR-34b/c promoter methylation status were determined by nested methylation-specific PCR (MSP) in FFPE tumor tissues from 161 patients of stageâ… NSCLC. Furthermore, mature miR-34b and miR-34c expression were analyzed by qRT-PCR in the same panel tissues.All statistical analyses were performed with SPSS 13.0 software. The Chi-square tests and was used to assess the relationship between recurrence and the clinical characteristics including gender, stage, histological characteristics, surgical procedure, history of smoking and family history of cancer. The same method was used to estimate the association of DNA methylation status of miR-34 family and recurrence. Multivariable logistic regression modeling was used to estimate the association between miR-34 promoter methylation and patients characteristics. Overall survival and recurrence-free survival were calculated with the Kaplan-Meier method and compared using the log-rank test. Overall survival and recurrence-free survival of patients in relation to clinical characteristics and miR-34a, miR-34b/c methylation status were analyzed using multivariate Cox regression model. miR-34b and miR-34c relative expression of methylated and unmethylated groups was compared with nonparametric Wilcoxon rank sum (Mann-Whitney) test. All p-values were 2-sided and represent raw values. Statistical significance was set at p<0.05.Results:DNA was isolated from tumor tissues of 161 stage I NSCLC patients, and treated with sodium bisulfite. Bisulfite-modified DNA was amplified by nested MSP.98.1%(158/161) of cases could be amplified by both miR-34a unmethylated and methylated primers,96.9%(156/161) cases could not be amplified by both miR-34b/c unmethylated and methylated primers. Methylation for miR-34a were observed in 39.2%(20/51) of recurrence patients and in 35.5%(38/107) of recurrence-free cases; for miR-34b/c, methylation was found in 54.9%(28/51) of recurrence patients and in 36.2%(38/105) of recurrence-free cases. Aberrant promoter methylation of miR-34b/c but not miR-34a was found to have significant association with tumor relapse (p=0.026).We then analyzed the overall survival and recurrence-free survival after surgery in 156 patients according to the methylation status of miR-34b/c. DNA hypermethylation of miR-34b/c was significantly associated with poor overall survival (p=0.010) and disease-free survival (p=0.017). The median overall survival after surgery of unmethylated and methylated cases were 56 months and 51 months respectively; and the median recurrence-free survival of unmethylated and methylated cases were 52 months and 48 months respectively. Multivariate Cox regression analysis showed that promoter hypermethylation of miR-34b/c was an independent prognostic factor of stageâ… NSCLC.We detected the mature miR-34b and miR-34c expression by qRT-PCR in 156 FFEP tumor tissues of stageâ… NSCLC which miR-34b/c unmethylation band could be amplified by nested MSP. The median value of relative expression of miR-34b was 1.85 and 1.59 in unmethylated and methylated groups respectively,1.78 and 1.64 for miR-34c. No significant differences of relative expression of miR-34b and miR-34c were found between the unmethylated and methylated groups.We then analyzed the overall survival and recurrence-free survival of patients according to the expression of miR-34b and miR-34c. The median relative expression of miR-34b was 1.72, and we divided 156 patients as two groups:patients with relative expression of miR-34b higher than 1.72 as miR-34b high expression group, patients with relative expression of miR-34b lower than 1.72 as miR-34b low expression group. Likewise, the median relative expression of miR-34c was 1.76, and we divided 156 patients as two groups:patients with relative expression of miR-34c higher than 1.76 as miR-34c high expression group, patients with relative expression of miR-34c lower than 1.76 as miR-34c low expression group. No significant association was found for mature miR-34b and miR-34c expression and overall survival and recurrence-free survival of patients by using Kaplan-Meier analysis and log-rank test.Conclusion:DNA methylation of miR-34b/c was found to have significant association with survival of stage I NSCLC after surgery. Promoter hypermethylation of miR-34b/c was an independent prognostic factor of stageâ… NSCLC and could be a potential prognostic biomarker for stageâ… NSCLC. DNA methylation of miR-34a was not found to have significant association with tumor recurrence. No relationship between relative expression of mature miR-34b or miR-34c and miR-34b/c DNA methylation status was found.
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