Preliminary Application In Detection For Promoter Methylation Of Non-small Cell Lung Cancer Related Genes Based On MS-HRM

In recent years,incidence and fatality rate of lung cancer continue to increase in China,causing more and more dangerous social impact.However,the present clinical treatment methods work poorly.Thus,precise diagnosis and timely control efforts are considered to be crucial approaches for improving the prospects of survival.Among numerous diagnostic methods,methylation status of sensitive target genes has been demonstrated to be closely related to the development of lung cancer,which provides valued research orientation for clinical diagnosis.Herein,we have evaluated methylation levels of potential related genes of non-small cell lung cancer(NSCLC)i.e.PCDHGB6,HOXA9,MGMT,SOCS3,NORE1A and miR-126 based on Methylation-sensitive high-resolution melting(MS-HRM),and explored clinical application value of these target genes in diagnosis of NSCLC.Study contents and results are as follows:(1)Establishment of MS-HRM system:optimized standard MS-HRM curves with high fluorescent efficiency and distinguishable gradient were obtained through adjustment of reaction systems and amplification conditions.Based on melting curves,regressive relation between temperature and relative signal difference of each gene was calculated,and all of them exhibited good linear relations.(2)Based on the above established MS-HRM systems,we detected methylation levels of six genes in tissue samples of NSCLC patients that have been reported as oncology related genes.Results showed more remarkable methylation frequency in PCDHGB6 and HOXA9 as 64.8%and 68.5%,respectively.HOXA9 took the second place with methylation frequency of 42.6%.The frequency of MGMT in NSCLC was 25.9%.In addition,neither SOCS3 nor NORE1A was found methylated in all of the tissue samples.(3)Relationships between methylation status and clinicopathological and physiological features of NSCLC patients.We have connected detection results of each target gene with patients' clinical data.The study found that methylation levels of PCDHGB6 and HOXA9 rising along with deterioration of disease,supplying potential monitoring factors in development of NSCLC.As for miR-126,it presented obviously higher methylation frequencies in stage ? and stage ?(73.1%and 72.7%,respectively)than in advanced stages(33.3%).Thus,miR-126 could be taken as important reference for early diagnosis of NSCLC.All of these genes performed more sensitive in detection of lung squamous cell carcinomas than in lung adenocarcinoma.In addition,there was no significant correlation between age and methylation status,while NSCLC was more prevalent in males than in females,concluded from this study.(4)Receiver operating characteristic curve(ROC curve)and pyrosequencing were performed to assess the results of methylation detection.PCDHGB6 led the highest sensitivity of 66.7%,while area under the curve(AUC)of PCDHGB6 was also the maximum among these genes,reaching 0.796.In t-test,PCDHGB6,HOXA9 and miR-126 were all statistically significant(P<0.01).Joint detection of PCDHGB6,HOXA9,MGMT and miR-126 has achieved sensitivity of 85.2%,specificity of 81.9%and AUC value of 0.891 that were superior to any of the single gene.Moreover,methylation levels of PCDHGB6,HOXA9 and miR-126 were tested by pyrosequencing,results have confirmed high coincidence between these two techniques(P<0.05).In conclusion,this research has optimized MS-HRM conditions for each selected gene,and provided a panel of effective genes for NSCLC diagnosis.Moreover,clinical application of several specific genes has potential to provide reliable datum for exploration of cause and development of NSCLC.