Association Study On MicroRNA Binding Site SNPs And Survival Of Non-small Cell Lung Cancer

Lung cancer is the most common cancer worldwide.In China,the morbidity and mortality of lung carcinoma have been increasing rapidly in the last two decades,mainly due to continuous increases in tobacco consumption and environmental pollution.Non-small cell lung cancer(NSCLC)accounts for?85%of lung cancers.Surgical operation is the major treatment for early stage NSCLC cases.However,approximately two thirds of NSCLC patients are presented and diagnosed at an advanced stage and thus chemotherapy is the standard initial treatment.Although the major prognostic determinant for NSCLC are the TNM staging system and histology,there are differences in survival among patients with similar disease.Until now,researchers have revealed that genetic variants,such as single nucleotide polymorphisms(SNPs)play an important role in the prognosis of NSCLC and thus inter-individual survival variability between patients.Therefore,we believe that in the future,better understanding of prognostic factors would be helpful for individualizing treatment for NSCLC patients.In our study,we mainly focused on functional SNPs in microRNA(miRNA)binding sites and survival of NSCLC patients.Part I:Evaluation of genetic variants in miRNA-200b/c bindingsites and survival of advanced Non-small cell lung cancer in aChinese populationMiR-200 is a miRNA family with tumor suppressive functions in a wide range of cancer.MiR-200b and miR-200c are the most widely and in-depth studied ones.Single nucleotide polymorphisms(SNPs)in may infulence miRNA-mRNA interaction through modulate binding affinity,destroying or creating miRNA-binding sites.In this study,we systematically selected 10 functional SNPs located in both miR-200b and miR-200c binding sites and investigated the association between these SNPs and survival of advanced non-small cell lung cancer(NSCLC)in a Chinese population.Our study was restricted to stage ? or ? NSCLC patients treated with first-line platinum-based chemotherapy without surgery.Stage ? included 303 patients recruited at the First Affiliated Hospital of Nanjing Medical University and the Affiliated Cancer Hospital.Stage ? included 340 NSCLC patients recruited from Shanghai Chest Hospital and Nanjing Thoracic Hospital.All patients were histopathologically or cytologically confirmed NSCLC,which was reviewed by at least two local pathologists.10 putative SNPs predicted to simultaneously influence miR-200b/c and target mRNA binding affinity were selected for genotyping.The sequenom MassARRAY iPLEX platform was used to genotype SNPs in stage ? and stage ? samples.Functions of the promising SNP were investigated using luciferase activity assay.All 10 SNPs were firstly genotyped in stage 1(303 cases)and no SNPs showed significant departure from HWE(P<0.5).One promising SNP(rsl7224177)was selected for further validation in stage 2(340 cases).We found that rs17224177 located in the 3'UTR of GLT8D3 was consistently associated with an increased risk of death in two stages with a combined hazard ratio(HR)of 1.70[95%confidence interval(CI):1.24-2.34].Based on the bioinformatics prediction,rs17224177 A>G in the 3'UTR of GLT8D3 destroys the binding sites of miR-200b and miR-200c.To evaluate whether this SNP affects the binding capacity of miR-200b/c,we cotransfected luciferase reporter plasmids of GLT8D3 3'UTR and miR-200b/c in three cell lines.Luciferase activity assay showed a higher expression level with rs17224177 G allele compared with A allele in A549 cell lines(P<0.0001 for miR-200b and P=0.0053 for miR-200c).Our findings suggested that GLT8D3 rs 17224177 A>G might affect advanced NSCLC survival through the interruption of miR-200b/c binding.Part II:Genetic variation in a microRNA-502 binding site in SET8 gene confers clinical outcome of non-small cell lung cancer in a Chinese populationGenetic variants may influence microRNA-target interaction through modulate their binding affinity,creating or destroying miRNA-binding sites.SET8,a member of the SET domain-containing methyltransferase,has been implicated in a variety array of biological processes.All subjects were recruited from the First Affiliated Hospital of Nanjing Medical University.Using Taqman assay,we genotyped a polymorphism rs 16917496 T>C within the miR-502 binding site in the 3'-untranslated region of the SET8 gene in 576 NSCLC patients.Functions of rs16917496 were investigated using luciferase activity assay and validated by immunostaining.Log-rank test and cox regression indicated that the CC genotype was associated with a longer survival and a reduced risk of death for NSCLC[58.0 vs.41.0 months,P=0.031;HR=0.44,95%CI:0.26-0.74].Further stepwise regression analysis suggested rs16917496 was an independently favorable factor for prognosis and the protective effect more prominent in never smokers,patients without diabetes and patients who received chemotherapy.A significant interaction was observed between rs 16917496 and smoking status in relation to NSCLC survival(P<0.001).Luciferase activity assay showed a lower expression level for C allele as compared with T allele,and the miR-502 had an effect on modulation of SET8 gene in vitro.The CC genotype was associated with reduced SET8 protein expression based on immunostaining of 192 NSCLC tissue sample(P=0.007).Lower levels of SET8 were associated with a non-significantly longer survival(55.0 vs.43.1 months).Our data suggested that the rs 16917496 T>C located at miR-502 binding site contributes to NSCLC survival by altering SET8 expression through modulating miRNA-target interaction.