Study On The Role Of HMLH1 Promoter Hypermethylation In The Cisplatin-resistance And Prognosis Of NSCLC

Background Lung cancer is one kind of malignant tumors with the highest incidence and mortality,while non-small cell lung cancer(NSCLC) is the most common type which accounts for 80%-85%of the lung cancer. Platinum-based chemotherapy is the standard chemotherapy for both early and advanced NSCLC,so platinum-resistance is the main reason for chemotherapy failure which causes tumor recurrence and metastasis,and has been a problem in the clinical practice.With in-depth study of epigenetics,the latest research showed that hMLH1 gene promoter methylation probably mediated cisplatin-resistance in ovarian cancer and colorectal cancer,demethylation intervention could well reverse the chemoresistance.However,there is no relevant research on hMLH1 methylation in NSCLC,and whether it could be used as a target for resistance reversal.This study first reported the hMLH1 methylation status in NSCLC cell line A549 and cisplatin -resistant cell line A549/DDP,in order to clear whether or not it correlate with platinum-resistance of NSCLC,then we observed whether demethylation intervention(5-Aza-dc) could remove the hMLH1 gene methylation and reverse platinum-resistance in A549/DDP cell line.In the next clinical study,we reported the hMLH1 methylation status of 83 NSCLC patients,to explore whether hMLH1 methylation was associated with the clinicopathological features of NSCLC,and if it could be used as an effective biomarker for the prediction of survival,recurrence and metastasis of NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy.Chapterâ… Study on the role of hMLH1 gene promoter methylation in the platinum-resistance of NSCLC cell lineObjective To explore whether the hMLH1 promoter methylation correlate with the cisplatin-resistance in NSCLC A549/DDP cell line;the impact of demethylation drug 5-Aza-dc on hMLH1 gene methylation,gene expression and cisplatin-resistance of A549/DDP cell line.Methods MSP for detection of hMLH1 methylation status in cisplatin-resistant NSCLC cell line A549/DDP and its parental cell line A549.MTT method and flow cytometry(FCM) for test of cisplatin resistance index in A549/DDP cell,the cytotoxicity of 5-Aza-dc on A549/DDP,and the IC₅₀ and cisplatin resistance index of A540/DDP cells 48 hours after different concentration of 5-Aza-dc(0μM,20μM,40μM) treatment.MSP,Fluorescence quantitative PCR(Real-time RT-PCR) and western blot for detection of hMLH1 methylation status,mRNA and protein expression.Results The IC₅₀ values of cisplatin in A549 and A549/DDP cells were 5.20±0.14μM and 30.15±0.76μM,the resistance index of A549/DDP was 5.80,A549/DDP cells was identified as cisplatin-resistant NSCLC cell line.MSP showed that A549 was hMLH1 unmethylated cell,A549/DDP was hMLH1 partly methylated cell.MTT and FCM results demonstrated that during 48 hours' 5-Aza-dc treatment,20μM was non-toxic dose,40μM was low-toxic dose,non-toxic dose and low-toxic dose of 5-Aza-dc(20μM, 40μM) reduced IC₅₀ values of cisplatin to 16.54±0.35μM(RI=1.82) and 6.82±0.16μM(RI=4.42).MSP,Fluorescence quantitative PCR(Real-time RT-PCR) and western blot showed that non-toxic and low-toxic 5-Aza-dc could well remove the hMLH1 methylation,hMLH1 mRNA and protein expression increased.Conclusion hMLH1 promoter methylation may be involved in the cisplatin-resistance of NSCLC.Low dose 5-Aza-dc cause no significant growth inhibition and apoptosis to cisplatin-resistant NSCLC cells,but it can partly reverse the chemoresistance through removal of hMLH1 methylation and increased expression of hMLH1 gene. Chapterâ…¡The clinical significance of hMLHl gene promoter methylation in NSCLCObjective To detect the hMLHl methylation status in the surgical resection specimens of NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy,and value its clinical significance, such as its relation to clinicopathological features and prognosis.Methods we used modified saturated phenol/chloroform method to extract FFPE(Formalin-fixed and Parrffin-Embedded) tumor tissue DNA from 83 NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy,nested MSP(nMSP) to detect the hMLH1 methylation status.x² test to analysis the relationship between hMLH1 methylation and clinicopathological features,univariate Kaplan-Meier (Log-rank test) and multivariate Cox regression for the hMLH1 methylation and overall survival(OS) analysis,Logistic regression for the hMLHl methylation and disease-free survival(DFS) analysis.Results In 80 patients with successful amplification of nested MSP, 33 were hMLH1 methylation cases(41.25%).hMLH1 methylation was not associated with age,sex,smoking status,T stage,phathologic type, differentiation and 3-year survival,but correlated with N status and 3-year disease-free survival.Univariate Kaplan-Meier and multivariate Cox analysis of survival revealed that N status significantly affect the patients' survival,but hMLH1 methylation was not associated with the overall survival.Logistic regression analysis showed that hMLH1 methylation and N staging were both risk factors of tumor recurrence and metastasis.Conclusion hMLH1 gene promoter methylation is a common event in NSCLC,it correlats with N status and disease-free survival of NSCLC, but is not associated with overall survival.hMLH1 hypermethylation may become a recurrence and metastasis prediction of NSCLC patients who accepted postoperative adjuvant cisplatin-based chemotherapy,and is expected to become a biomarker for NSCLC patients.