| Lung cancer is one of the most common cancers of human for several decades,with top incidence and mortality among all cancers.In recent years,due to the continued growth of tobacco consumption,environment contamination,lifestyle and diet changes,the incidence and mortality of lung cancer in our country trend to ascend straightly,and it has become one of the important serious problem of public health in our country.Lung cancer is divided into small cell lung cancer and non-small cell lung cancer(NSCLC),and the latter accounts for about 80-85 percent of lung cancer.Although the diagnosis and treatment of NSCLC technology has made great progress,the prognosis remains poor,the 5-year survival rate of lung cancer being only 15%in the United States and even lower in China.Clinically,the major prognostic determinant for NSCLC are the TNM staging system and pathologic histology,However,even with same pathological types,the same clinical stages,using the same treatment,there are still differences in prognosis among patients.Therefore,it has been a hot research topic that choosing stable molecular biomarkers such as single nucleotide polymorphisms(SNPs)to predict prognosis of patients and accordingly guiding individualized treatment to improve the patients'quality of life and prolong their survival time.In our study,we mainly focused on functional SNPs in microRNA(miRNA)-related genetic variants and the prognosis of lung cancer.Part I:Genetic variations in DROSHA and DICER and Survival of Advanced Non-Small Cell Lung Cancer:A Two-stage Study in ChineseMiRNAs undergo a complex processing procedure involving multiple proteins and RNAs to produce the mature functional unit.Primary miRNA is firstly synthesized in the nucleus by RNA polymerase and then processed into precursor miRNA by DROSHA.Subsequently,the precursor miRNA moves to the cytoplasm,where DICER,also a nuclear RNase III enzyme,cleaves them into mature double-stranded RNA fragments(miRNA).Accumulative evidence shows that impaired miRNA processing reduces stable miRNA levels and promotes tumorigenesis.Single nucleotide polymorphisms(SNPs)in microRNA genes,their processing machinery and target binding sites are associated with the development or progression of cancer by modulating expression levels of miRNAs and miRNA–mRNA interactions.In this study,we have systematically screened common polymorphisms(MAF?0.05)in DROSHA and DICER regions(including 10kb up-stream region of each gene)based on the HapMap SNP database(phase II+III Feb 09,on NCBI B36assembly,dbSNP b126)and the HaploView 4.2 software.Hardy-Weinberg equilibrium analysis and linkage disequilibrium(LD)analysis with an r~2 threshold of0.80,we finally included 36 potentially functional SNPs with call rates>95%,and used a two-stage study design to evaluate the association between these SNPs and NSCLC survival in Chinese Han population.Our study was restricted to stage III or IV NSCLC patients without surgery and with available follow-up survival data.We selected 303 patients from the Affiliated Cancer Hospital and the First Affiliated Hospital of Nanjing Medical University to form the discovery cohort and the remaining patients(340 patients)from Nanjing Thoracic Hospital and Shanghai Chest Hospital(both in Southeastern China)formed the replication cohort.The demographic information was collected by face to face questionnaire investigations,and the clinical information was gathered from patients'medical records.The iPLEXSequenom MassARRAY platform was used for the genotyping in the discovery cohort and the replication cohort.Thirty-six SNPs were detected in the discovery cohort and 12 promising SNPs were validated in the replication cohort.The results showed that DROSHA rs3805525was marginally associated with the survival of NSCLC patients in the replication cohort(dominant model:HR=0.69,95%CI=0.46-1.03,P=0.071),which was in the same direction as that in the discovery cohort.When combing all patients into one group,three SNPs(rs3805525,rs17410035 andrs7719497)in DROSHA showed significantly associations with NSCLC survival(additive model:HR=0.82,95%CI=0.68-0.99 for rs3805525;HR=0.79,95%CI=0.62-1.00 for rs17410035;HR=0.76,95%CI=0.62-0.93 for rs7719497).Additionally,the combined analysis of those 3SNPs showed a significant locus-dosage effect between number of favorable alleles and death risk of NSCLC(Trend P=0.002).Our findings suggested that genetic variations in DROSHA might be associated with the survival of advanced NSCLC patients in Chinese population.Part II:Genetic variations in MiR-125 family and the survival of Non-Small Cell Lung Cancer in Chinese PopulationMiR-125 family is composed of three homologs hsa-miR-125a,hsa-miR-125b-1and hsa-miR-125b-2.Members of the family play crucial roles in many different cellular processes like cell differentiation,proliferation and apoptosis by targeting many different transcription factors,matrix-metalloprotease,growth factors and so on.Moreover,the different members of miR-125 family have been reported controversial properties in different types of cancer,they may contribute to the initiation and progression of cancers including lung cancer by acting as either tumor suppressors or oncogenes.In this study,we investigate the SNPs in regulation regions of the miR-125 family(hsa-miR-125a,hsa-miR-125b-1 and hsa-miR-125b-2)to explore whether these genetic variants have an influence on clinical outcome in NSCLC patients.Finally,we selected 6 potentially functional SNPs in three pre-miRNAs and their surrounding regions and tested the association between these SNPs and NSCLC survival in Chinese Han population.All subjects were genetically unrelated ethnic Han Chinese from Nanjing City and surrounding regions in southeast China.All patients were newly diagnosed and histopathologically or cytologically confirmed NSCLC without prior history of other cancers.Since July 2003,a total of 1001 patients with NSCLC have been prospectively recruited from the Cancer Hospital of Jiangsu Province,and the First Affiliated Hospital of Nanjing Medical University,Nanjing,China.The genotyping was performed by Illumina Infinium?BeadChip(Illumina Inc.)We systematically selected 6 functional SNPs located in 3 miRNAs(miR-125a,miR-125b-1,miR-125b-2)and investigated the association between these SNPs and the prognosis of Non-Small Cell Lung Cancer in a clinical follow-up study.We found that rs2241490,rs512932 and rs8111742 were associated with the prognosis of NSCLC in a Chinese population(dominant model:P=0.014,adjusted HR=1.24,95%CI:1.05-1.48;P=0.013,adjusted HR=1.25,95%CI:1.05-1.48;P=0.047,adjusted HR=0.84,95%CI:0.71-1.00,respectively)and the combined analysis of the 3 SNPs showed a significant locus-dosage effect between number of favorable alleles(rs2241490-A,rs512932-G and rs8111742-G)and death risk of NSCLC(P for trend<0.001).Furthermore,luciferase reporter gene assay showed that the significantly higher levels of luciferase expression for the reporter gene with rs512932 variant G allele than with A allele in 293T,SPCA1 and A549 cells.Our findings indicated that genetic variations in miR-125 family might be associated with the survival of NSCLC patients.Part III:Genetic variations in miR-9 and the survival of Non-Small Cell Lung Cancer in Chinese PopulationMiR-9 is a regulator of neuronal progenitor cell fate during neurogenesis,which has recently been implicated in cancer.Studies indicate that miR-9 acts either tumor suppressors or oncogenes in different cancer cells.In our study,we hypothedized the function SNPs in regulation regions of miR-9(miR-9-1,miR-9-2,miR-9-3)were important in the survival of NSCLC in Chinese Han population.In order to verify the hypothesis,4 funtional SNPs were selected,we then genotyed these variants using Illumina Infinium?BeadChip.We observed a borderline significant association between miR-9-2 rs1501672and survival of NSCLC(additive model:P=0.056,adjusted HR:0.89,95%CI:0.79-1.00).Luciferase activity assay showed a lower expression level for G allele as compared with A allele.Our data suggested that the miR-9-2 rs1501672 A>G contributes to NSCLC survival. |
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