Research For Epigenetic Mechanism Of Lung Cancer

Lung cancer remains the most common primary neoplasm of the respiratry system.NSCLC accounts for approximately80～85%of all lung cancer. Unfortunately, currenttherapeutic modalities including surgical resections, chemotherapy, radiotherapy, targettherapy or combinations can not ensure a cure. The precise mechanisms underlying theinitiation, maintenance and progression of NSCLC still remain largely unknown. Lungcancer originated in a series of genetic and epigenetic changes. The major epigeneticmechanisms of NSCLC include DNA promoter methylation and microRNAs. Hence,identification and characterization of the epigenetic alterations that involved in theNSCLC may offer biomarkers for cancer detection.The experiment is divided into two parts:Part I: We evaluated the promoter hypermethylation differences of hOGG1andHYAL1in Paracancerous tissue, cancer tissue, normal tissue, and evaluated hOGG1andHYAL1gene promoter methylation differences in the peripheral blood of NSCLCpatients and benign control patients. We sough to determine whether their promotermethylation were associated with clinicopathological features of NSCLC.Part II: We investigate whether plasma miRNAs can be used as biomarkers for theearly detection of NSCLC，and whether the differential expression of peripheral bloodmiRNAs can be used to predict recurrence of early stage lung cancer.Part ⅠHYAL1and hOGG1promoter methylation in NSCLCObjective: The aim of this study was evaluate the promoter hypermethylation differences of hOGG1and HYAL1in Paracancerous tissue, cancer tissue, normal tissue,and to assess hOGG1and HYAL1gene promoter methylation differences in theperipheral blood of NSCLC patients and benign control patients. The aim of this studywas also to determine whether their promoter methylation were associated withclinicopathological features of NSCLC.Methods: We evaluate80cases with NSCLC lung cancer,80cases ofparaneoplastic organizations, and20patients with benign lung lesions in hOGG1andHYAL1gene promoter methylation differences, and analyze the relationship betweenlung cancer group methylation status with clinicopathological data. We evaluate80casesNSCLC patients and20patients with benign lung lesions the peripheral blood thehOGG1and HYAL1gene promoter methylation status. And we evaluate10cases of lungcancer and the mediastinal lymph nodes for promoter methylation status of hOGG1andHYAL1.Results: HYAL1promoter methylation positive rate of NSCLC was significantlyhigher than the benign group (48.8%VS10%, p=0.002); HYAL1promoter methylationof paraneoplastic tissues was higher than the benign control group(32.5%VS10%, p=0.045); NSCLC plasma HYAL1promoter methylation positive rate was significantlyhigher than that in the benign control group (40%VS5%, p=0.003).hOGG1promoter methylation positive rate of NSCLC was significantly higherthan the benign group(52.5%VS15%, p=0.003); hOGG1promoter methylation ofparaneoplastic tissues was higher than the benign control group,but the difference wasnot significant(23.8%VS15%, p=0.587); NSCLC plasma hOGG1promoter methylationpositive rate was significantly higher than that in the benign control group(42.5%VS10%, p=0.007).In lung cancer tissue, there were no significant correlation between promotermethylation rates of HYAL1and hOGG1and the patient’s gender, age group, tumor size, histological type, histological grade, T staging(P>0.05).In lung cancer tissue,HYAL1promoter methylation positive rate of the stage III group was significantly higherthan the stage I-II group (66.7%VS39.6%, p=0.022). In lung cancer tissue, HYAL1promoter methylation positive rate of the Lymph node-positive group was significantlyhigher than the node-negative group (63.6%VS38.3%, p=0.026). In lung cancer tissue,hOGG1promoter methylation positive rate of the stage III group was significantly higherthan the stage I-II group (74.1%VS s41.5%, p=0.006). In lung cancer tissue, hOGG1promoter methylation positive rate of the Lymph node-positive group was significantlyhigher than the node-negative group (69.7%VS40.4%, p=0.001).In lung cancer plasma, there were no significant correlation between promotermethylation rates of HYAL1and hOGG1and the patient’s gender, age group, tumorsize, histological type, histological grade, T staging(P>0.05). In lung cancer plasma,HYAL1promoter methylation positive rate of the stage III group was significantly higherthan the stage I-II group (66.7%VS26.4%, p=0.001). In lung cancer plasma, HYAL1promoter methylation positive rate of the Lymph node-positive group was significantlyhigher than the node-negative group (60.6%VS25.5%, p=0.002). In lung cancer plasma,hOGG1promoter methylation positive rate of the stage III group was significantly higherthan the stage I-II group(59.3%VS34.0%, p=0.030). In lung cancer plasma, hOGG1promoter methylation positive rate of the Lymph node-positive group was higher than thenode-negative group, but the difference was not significant(54.5%VS34.0%, p=0.068)HYAL1promoter methylation in lung cancer plasma yielded an AUC of0.675(95%CI:56%-79%) in discriminating early-stage NSCLC from controls. hOGG1promoter methylation in lung cancer plasma yielded an AUC of0.663(95%CI:54.3%-78.2%) in discriminating early-stage NSCLC from controls. Combined ROCanalyses revealed an elevated AUC of0.728(95%CI：61.9%-83.8%) in discriminating early-stage NSCLC from controls.There were four cases with HYAL1promoter methylation status differencesbetween the NSCLC tissue and lymph nodes. And there were four cases with hOGG1promoter methylation status differences between the NSCLC tissue and lymph nodes.Conclusion: hOGG1and HYAL1in NSCLC tissues and plasma exists promoterhypermethylation. The promoter hypermethylation of hOGG1and HYAL1wereassociated with lymph nodal metastases and advanced pathological stages.The hOGG1and HYAL1promoter hypermethylation may be involved in the occurrence, developmentand metastasis of NSCLC. The plasma hOGG1and HYAL1detection can be used to aidin the diagnosis of NSCLC. The tumor may exist within the NSCLC tumorheterogeneity. Part IIThe study on plasma miRNAs as non-invasive biomarkersfor early-stage non-small cell lung cancerObjective: The aim of this study was to investigate whether plasma miRNAs can beused as biomarkers for the early detection of NSCLC，and whether the differentialexpression of peripheral blood miRNAs can be used to predict recurrence of early stagelung cancer.Methods: We measured the levels of9miRNAs(miR-30d，miR-383， miR-223，miR-25，miR-21、miR-126、miR-210、miR-155、miR-182) in plasma samples fromNSCLC patients and benign pulmonary lesions using real-time RT-PCR. We screened miRNAs which may have diagnostic value in20NSCLC patients and20patients withbenign control group. In a total of135patients with early NSCLC and85cases ofbenign control group, receiver operating characteristic (ROC) curves were generated forthree of them(miR-21, miR-155and miR-182) and the combination of the miRNAs；Follow-up of135patients for3years, assess the recurrence group and non-recurrencegroup miRNAs differentially expressed.Results: Our observations indicated that three of them, miR-21, miR-155andmiR-182, were significantly upregulated in serum of NSCLC compared with incorresponding controls. MiR-21yielded an AUC(the areas under the ROC curve) of0.805(95%CI:0.749-0.861)with68.1%sensitivity and78.8%specificity，miR-155yielded an AUC of0.821(95%CI：0.767-0.875)with81.5%sensitivity and69.4%specificity，and miR-182yielded an AUC of0.745(95%CI：0.681-0.809)with55.6%sensitivity and82.4%specificity，in discriminating early-stage NSCLC from controls.Combined ROC analyses using these3miRNAs revealed an elevated AUC of0.894(95%CI：0.862-0.936) with73.3%sensitivity and95.3%specificity indiscriminating early-stage NSCLC from controls.Conclusion: Plasma of miR-21, miR-155and miR-182can be used for differentialdiagnosis of small pulmonary lesions，and circulating miRNAs can accurately detectNSCLC at an early stage. Plasma of miR-21, miR-155and miR-182was found to beassociated with a high risk of recurrence in early stage NSCLC.