| BackgroundRenal cancer is one of the most common malignant tumors in urogenital system,more than 400,000 patients are diagnosed every year in the world.The incidence rate of renal cell carcinoma has been increasing during recent years,and its pathogenesis is still elusive.Recently,it has been found that telomere and telomere activity might be important in the oncogenesis and development of tumor,including renal cancer.Telomerase is a ribonucleoprotein polymerase that could maintain telomere length by adding 5'-TTAGGG-3' repeats to the ends of human chromatin DNA.Its expression is normally repressed in postnatal somatic cells and its deregulation is associated with carcinogenicity.Telomerase activity is indeed detectable in up to 90%of human malignancies,whereas it is repressed in most normal human cells.Human telomerase reverse transcriptase(hTERT),the catalytic subunit of telomerase holoenzyme,is the rate-limiting factor in reconstituting telomerase activity.The expression and function of the human hTERT gene are known to be regulated through multiple avenues,including transcription factors,promoter methylation/mutations,chromatin remodeling,alternation splicing,protein modification,and subcellular localization.Among them,transcription regulation is the most critical and well established approach.DNA methylation,which always occurs in CpG sites,has got extensive interests since its discovery for the hypermethylation in the promoters of most of oncogenes on normal cells.hTERT promoter contains a dense CG-rich CpG island,which indicates a potential role of DNA methylation in hTERT expression regulation.Using bisulfate genomic sequencing,Theodora et al investigated the promoter methylation status of hTERT in 37 cell lines,including normal,immortalized,and cancer cells.They reported that no site-or region-specific methylation generalized pattern were found to be correlated with hTERT expression,while demethylation treatment in SUSM-1 cells induced hTERT expression.Some other studies also suggested that CpG island methylation of the hTERT promoter is associated with lower telomerase activity in vivo[9].However,more studies demonstrated that hyper-methylation in hTERT promoter region is positively associated with hTERT mRNA expression.Therefore,it is still elusive for the potential role of DNA methylation in hTERT expression and telomerase activity regulation.Renal cell carcinoma(RCC)represents the most common of kidney cancer with 25?30%metastasis rate at diagnosis.It has been reported that hTERT gene expression can be regulated by various trans-acting elements and cis-acting elements,including c-Myc,Spl,WT1,HIF-1?,P53,TGF-?,SMYD3,chromatin remodeling,and promoter mutation.However,few study has addressed DNA methylation status of hTERT promoter in RCC.For this purpose,we evaluated methylation levels of 5 CpG sites in hTERT promoter region using pyro-sequencing and explored the relationships between methylation level and hTERT expression and telomerase activity.To further explore the relationship between the percentage of hTERT promoter methylation and hTERT expression,telomerase activity,clinical stage and prognosis of patients,further study the potential role of hTERT promoter methylation in the occurrence and development of renal tumors,and screen biomarkers to predict the prognosis of renal tumorsObjective:A cohort of 103 RCC was investigated for hTERT promoter methylation percentage,hTERT mRNA abundance,telomerase activity,Fuhrman grade,clinical stage and clinical outcomes.The hTERT promoter methylation level in RCC and normal adjacent tissues was quantified by pyrosequencing following DNA extraction and bisulfate treatment.hTERT mRNA level was detected by realtime RT-PCR and telomerase activity was determined by a quantitative telomeric repeat amplification protocol(TRAP).To analyze the relationship between hTERT promoter methylation level,hTERT mRNA level,telomerase activity and clinical pathology of renal cell carcinoma patients,to analyze the prognosis of renal cell carcinoma by single factor analysis and Cox multivariate model,and to explore the mechanism of action of hRERT promoter methylation in renal cell types.Methods:Paired RCC tissue and normal tissue adjacent to tumor(NAT)were obtained by radical nephrectomy(n=99)or nephron sparing surgery(n=4)from 103 patients at Shandong University Qilu Hospital between 2009 and 2014.All diagnosis was confirmed by histopathological examination.Clinical staging was determined according to the 2010 UICC/AJCC TNM staging system for RCC(7th edition).Patients' clinical characteristics are summarized.Freeze the specimen at-80?.These patients were followed up from January 1,2009 to November 30,2016.The age,sex,left and right,pathological type,TNM stage,Fuhrman grade,survival time and other clinical data of each pathology were recorded.DNA was extracted from frozen tissue samples using a DNeasy tissue kit(Qiagen Ltd,Venlo,Netherlands,https://www.qiagen.com/us/)PCR was performed using the PyroMark PCR Kit(Qiagen).Primers were designed using PyroMark Assay Design Software 2.0.The sequences of the PCR primers and sequencing primers are shown in Supplementary Table.Total cellular RNA was extracted from tissue specimens using TRIzol reagent(Invitrogen)according to the manufacturer's instruction.A total of 1?g of RNA was used for reverse transcription with M-MLV reverse transcriptase(Thermo,Lithuania).The PCR primers are shown in Supplementary Table.QPCR was performed using SYBR Green PCR Master Mix(Applied Biosystems,Foster City,CA)in a 7000 Real-Time PCR System(Applied Biosystems).? 2-M expression was used as a reference.Relative levels of hTERT mRNA in given samples were calculated using the 2-??Ct method according to threshold cycle values.Telomerase activity was assayed using a commercial TeloTAGGG Telomerase PCR ELISA kit(Roche Diagnostics).For each assay,1.0 ?g of proteins from extraction was used,and 25 PCR cycles were performed,after the telomerase-primer elongation reaction.The PCR products were detected using ELISA color reaction.The level of telomerase activity was expressed as absorbance[optimal density(OD)in arbitrary units].T test was used for normal distribution.The Mann-Whitney U test was used to analyze the differences between two groups.The Wilcoxon rank-sum test for the paired groups was used to calculate two-sided P-values of hTERT promoter methylation level in RCC tissue and corresponding NAT.Mann-Whitney U test for the unpaired groups were used to compare the methylation level between lesion stages or differentiation levels.For Kaplan-Meier analysis,Cox multiple factor regression analysis for meaningful single factor analysis,P value is calculated using log-rank test.Results:The status of hTERT promoter methylation is not identical and may exhibit different methylation scenario in different types of cancer.For the selection of candidate CpG sites for the following analysis,we conducted pyrosequencing following bisulfite conversion of DNA in three regions at hTERT promoter on 12 RCC tissue samples and corresponding normal tissues adjacent to tumor(NAT)using 3 pairs of PCR primer and corresponding sequencing primer.We found in all the 14 examined CpG sites,the methylation percentage was significantly higher in RCC tissues compared with in NAT,which indicates that hTERT promoter is hyper-methylated in RCC tissues?To further evaluate methylation status in hTERT promoter in RCC,we examined the methylation percentage in Site 1 to Site 5 using pyrosequencing in samples of 103 RCC patients.The reason we choose this region is that Site 1 to 5 presented the most stable signal among the three PCR amplified regions in all samples.We found all the 5 CpG site were remarkably hypermethylated in RCC tissue compared with NAT.In all 103 RCC tissues and paired NAT,no difference in methylation density(means of methylation percentage of CpG site 1 to 5,MMP 1-5)was observed in relation to patient age.We next explored the relationship between hTERT promoter methylation level and hTERT full-length mRNA expression in RCC tissues.It has been reported that hTERT expression and telomerase activity was achieved via induction of full-length hTERT mRNA rather than alternative splicing in RCC.As shown significant positive correlation was observed between MMP 1-5 and hTERT full-length mRNA expression(R2=0.450)in RCC tissues.Consistently,positive correlation was also found between MMP 1-5 and telomerase activity(R2=0.446).Taken together,our data suggest that hTERT promoter is hypermethylated in RCC tissues and the methylation level is closely associated with hTERT mRNA expression and telomerase activity.We then sought to determine the association between hTERT promoter methylation level and clinic-pathological characters.The level of methylation has nothing to do with age and sex in all the patients.We found that higher levels of the hTERT promoter methylation level was significantly correlated with disease stages.However,our data showed no statistic significant difference of between hTERT methylation level and Fuhrman grades.Meanwhile,there is no significant difference in hTERT promoter methylation level between clear cell RCC(ccRCC)and non-ccRCC.Furthermore,follow-up data showed that RCC samples with higher hTERT methylation level were associated with a poor over survival,There was a linear correlation between hTERT expression and telomerase activity.By single factor analysis,TNM stage,Fuhrman grade,hTERT promoter methylation level,hTERT mRNA expression level,telomerase activity have influence on the survival rate of renal cancer patients after operation.By multiple factor Cox regression analysis,AJCC stage and hTERT promoter methylation level are independent factors affecting the prognosis of renal cancer patients.which suggested that hypermethylation in the hTERT promoter is a marker for unfavorable prognosis of RCCConclusion:In conclusion,we found that there was a positive correlation between hTERT promoter methylation and hTERT expression/telomerase activity,and a positive correlation between hTERT expression and telomerase activity.In addition,we found that hTERT promoter methylation,hTERT mRNA expression and telomerase activity are closely related to clinical results,which are risk factors for poor prognosis of renal cancer.Caavg(48.3%)stratification of hTERT promoter and AJCC stratification are independent factors influencing prognosis of renal cancer patients after operation.Methylation level of hTERT promoter affects hTERT mRNA expression and telomerase activity,and was closely correlate with prognosis of renal cancer.It is revealed that this epigenetic alteration may serve as a valuable biomarker for diagnosis and prognosis of RCC. |
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