The Biological Effects Of Variation In TERT Promoter Region And ETV4 On Non-small Cell Lung Cancer

Introduction:Non-small cell lung cancer(NSCLC)accounts for 85%of lung carcinoma,which is the leading cause of cancer-related death in both male and female worldwide.Telomerase activity has been considered to be of importance in NSCLC development,it may be useful both as a diagnostic marker and as a target for therapeutic intervention.TERT(Telomerase reverse transcriptis)is a key determinant of the enzymatic activity of human telomerase and its transcriptional control is a major contributor to the regulation of telomerase activity in many types of human cells.Accordingly,ectopic TERT expression can lead to unlimited yet not tumorigenic growth.The regulatory mechanisms that impel telomerase reactivation in carcinogenesis are multifaceted and have only recently started to be revealed.The promoter region of TERT is believed to be responsible for transcriptional activity of the TERT mRNA by variety of transcription factors,including E-twenty-six/ternary complex transcription factors(Ets/TCF).Mutations affecting the promoter region of the TERT gene have been widely analyzed in numerous cancers.C228T and C250T(-124 and-146 base pairs upstream from the starting codon)are most reported TERT promoter mutation,leading to increased TERT transcriptional activities by generating a consensus binding site for Ets/TCF.Interestingly,another TERT promoter polymorphic variant(rs2853669)within an endogenous Ets2 transcription factor binding site was also revealed to be associated with escalating telomerase activity from T/T allele towards C/C allele,with T/C allele in the middle,notwithstanding the difference did not reach to a significant level.In this study,we will detect TERT promoter mutation and rs2853669 SNP in the patietns with NSCLC,we will also detect the expression of ETV4(one of memebers from Ets transcription factors),and we analyzed the relationship between TERT promoter diversity/ETV4 and clinicopathological parameters including prognosis of the NSCLC patients.Through the experiments in NSCLC cell line,the biological function and mechanism of ETV4 in NSCLC was explored.Part I Association of variation in TERT promoter region with clinical and telomere parameters in NSCLC patientsPurposeTo identify the mutation of TERT promoter region and rs2853669 SNP in NSCLC,and to analysis their potential association with clinical and telomere parameters in NSCLC patientsMethods1.Sanger sequencing was conducted to genotyping 250 NSCLC patients in the TERT promoter region.2.The TERT mRNA transcription level was detected by Real-time quantitive PCR,3.The relative telomere length was detected by monochrome multiplex quantitative PCR in all patients.4.Association between TERT promoter mutation and rs2853669 and clinicopathological parameters were analyzed by chi-square test or students' t-test.5.The comparison of RTL was performed using multivariable linear regression models that adjusted for binary variable which showed relative significant difference.6.Survival outcomes were estimated using the Kaplan-Meier method.The differences between the curves were analyzed with log-rank test.univariate Cox proportional hazard regression models were used to determine the effects of covariates on patient survival.7.All analyses were performed using IBM statistics SPSS version 24.0(SPSS Inc,Chicago,IL).Results1.We analyzed 250 NSCLC patients and identified point mutation in TERT promoter region in 11 patients(11/250).The TERT promoter mutation is associated with higher TERT mRNA transcription level and longer relative telomere length.And are associated inferior survival outcomes in NSCLC patients.2.rs2853669 SNP was analyzed in 250 NSCLC patients,rs2853669 T/T allele,T/C allele and CC allele were identified in 108 patients,106 patients and 36 patients,separately.T/C allele was associated with higher TERT mRNA transcript level and shortest telomere length.In additation,patients with T/C allele has shorter overall survival length and relapse free survival.3.Using multivariate cox regression model,we found an interaction effect of EGFR mutation and rs2853669 SNP on patient survival,and telomere parameter.The association between rs2853669 SNP and telomere parameter is only showed in patients without EGFR mutation.Similarly,the prognostic role of the SNP is confined to patients without EGFR mutation.Conclusions:TERT promoter mutation is associated with higher TERT mRNA transcription level and inferior clinical outcomes in NSCLC patients.Rs2853669 SNP has EGFR-depended effects on telomere parameters and survival outcomes in NSCLC patients.Part ? The biological effect of ETV4 on Non-small cell lung cancer PurposeTo investigate the effect and mechanism of ETV4 in non-small cell lung cancerMethods1.The ETV4 expression level in 76 NSCLC patients were analyzed by real-time quantitive PCR.2.The ETV4 expression were down-regulated by transfecting ETV4 siRNA to A549,HCC827 and H661 NSCLC cell line.3.Cell growth and proliferation capacity were measured by MTT assay.4.The TERT mRNA expression level were analyzed by real-time quantitive PCR.5.Cell cycle was analyzed by Flow cytometry.6.The protein levels were analyzed by western blot.Results:1.The expression level of ETV4 is higher in tumor tissue compared to pericarcinomatous tissue.Patients harboring TERT promoter mutation has higher ETV4 expression compared to patients without TERT promoter mutation.In addiation,patients with rs2853669 T/C allele harboring the highest ETV4 mRNA transcription level.2.Down regulation of ETV4 surpressed proliferation of A549,HCC827 and H661 NSCLC cell line by inducing G1-phase arrest.The arrest was induced by activating erk pathway.3.Down regulation of ETV4also surpressed TERT mRNA expression by synergetic effect with other transcript factors from AP-1 family in A549 and HCC827 cell lines.Conclusions:ETV4 down-regulation represes the proliferation of non-small cell lung cancer via inducing G1/G0 phase arrest by surpressing erk pathway,the ETV4 down-regulation can also regulate TERT mRNA transcription level by synergetic effect with other transcript factors.Our data indicate that ETV4 could be a therapeutic target in patients and may have a potential to be a diagnostics or predictive biomarker in non-small cell lung cancer.