| Background and ObjectivePrimary liver cancer, one of the most common malignant tumors, is a serious threat to human health. More than 90% of primary liver cancer is hepatocellular carcinoma (HCC). HCC is the second most common cancer in China and causes the greatest mortality in world. HCC has seriousprognosis. Even the patients with early or tumor<3cm receiveing surgical resection, their 5-year survival rate is only 47% to 53% and 2-yearrecurrence rate up to 55%. As the tumor genomics and proteomics research and application progressing, more and more researchers focus on the molecular level of risk factors which regulate cancer metastasis, recurrence and survival time, in order to develop earlier interventions and improve liver cancer survival.Telomerase is a reverse transcriptase.It can use its own de novo synthesis of RNA as a template DNA sequence to make up for the loss of telomeric DNA in replication, maintaining stable telomere length, then giving the cells the ability to proliferate indefinitely. The loss of telomere function or telomerase activation is a significant pathological mechanism of oncogenesis, which maybe involved telomerase gene mutation thereby promoting oncogenesis. Therefore we hypothesized that:telomerase gene mutation may result in abnormal expression, dysregulation and disfunction of telomerase, causing change of the telomere length and activation, which influence tumor development and prognosis.The Objective of this study is that:Through the analysis the polymorphism of single nucleotide (SNP) of telomerase gene in hepatocellular carcinoma tissue form HCC patients to explore the influence of gene loci mutation on the metastasis, recurrence and mortality of HCC patients receiving operation.Methods(1) Patients:Eighty-four patients with pathological diagnosis HCC and receiving treatment in Ganmei Affiliated Hospital of Kunming Medical University were included in the study. The basic information of the patients, including sex, age, family history of cancer, serum markers of hepatitis B virus and antibody of hepatitis C virus, Barcelona staging, type of treatment were collected. Patients were followed for outcome of transfer, recurrence or mortality by phone from July 1,2006 to January 30, 2015.(2) AS-PCR:AS-PCR methods were used to detect the loci polymorphism of five SNPs in telomerase gene, using the HCC tissue as the samples. The primers of AS-PCR were designed by primer 5.0. The reaction system and the conditions of 10 AS-PCR methods were optimized. The results of polymorphism detected with these methods were verified by sequenceing of products of PCR for the five SNPs.(3) Genetyping:The polymorphisms of five SNPs locus of telomerase gene were detected with 10 AS-PCR and the frequencies of the allele and genotype were analyzed.(4) Analysis of correlation:The correlation between the polymorphisms of five SNPs and the prognosis of HCC patients were explored by COX regression analysis.Result(1) 84 patients were collected, including 69 males and 15 females. The mean age is 56 old-years, the maximum is 77 and the youngest is 31 years old. Among them there were 53 Patients with completed clinical information,59 Patients with DNA sample extracted from HCC tissueand 41 cases with both of the clinical information and DNA sample. Among 41 cases there were 13 mortality patients,5 patients with tumor relapsing and 6 cases with metastasis. The censored value was 17.(2) The optimal annealing temperatures were 53℃ for rs2735940,61℃ for rs2736100,63℃ for rs2736098,62.8℃ for rs35719940,66.3℃ for rs2293607; The concentration optimum primer was 20μm. The optimal concentration of TaqDNA polymerase was 0.25μl (1.25U). The results of polymorphism detected with the optimal methods were verified by sequencing of products of PCR for the five SNPs in five patients.(3) The frequencies mutation of the 5 SNPs was 42% -60%, wild-type was 2.4%-24.2%, heterozygosis-type was 51.2%-97.6% and mutation-type was 0-34.2% among the 59 cases. No homozygous mutation genotypes were detected in rs2736098 and rs35719940 locus.(4) The average survival time of 41 HCC patients with both of the clinical information and DNA sample was 24 months and the survival rate gradually decreased as the follow-up time accumulates. COX regression analysis showed that regression coefficient(p) of SNP rs2735940 on mortality was 2.044, p=0.018(<0.05), relative risk(Exp(B))= 7.720 (1.427-41.757,95.0% CI).(5) COX regression analysis with rs2735940 and sex, age, family history of cancer, Barcelona staging, treatment type, respectively, showed that:â‘ The pairwise comparison rs2735940 and Barcelona staging, regression coefficient(β) of SNP rs2735940 and Barcelona staging on mortality was 1.164 and 0.991, p=0.084 and 0.006, relative risk(Exp(B))=3.204 (0.854-12.022,95.0% CI) and 2.693 (1.321-5.492,95.0% CI).â‘¡The pairwise comparison rs2735940 and type of treatment, regression coefficient(β) of SNP rs2735940 and type of treatment on mortality was 1.882 and -1.631, p=0.067å'Œ0.013, relative risk(Exp(B))=3.651 (0.916-14.501,95.0% CI) and 0.196 (0.054-0.704,95.0% CI).â‘¢The comparison rs2735940 and sex, age and family history of cancer, respectively, regression coefficient(β) of SNP rs2735940 on mortality was 5.746,2.656 and 6.564, p=0.010, 0.007 and 0.004. Regression coefficient(β) of sex, age and family history of cancer on mortality were 0.012,0.977 and 0.685, p=0.695,0.354 and 0.396.Conclusion(1) The primers designed by us for 10 AS-PCR were specificial for the 5 SNPs mutational locus of telomerase gene, respectively.they are available for AS-PCR typing these locus.(2) The optimizedAS-PCR methods were reliable, economical and easy-operation, which could be used for typing the locus of 5 SNP of telomerase gene.(3) The mutation of rs2735940 3677 Tâ†'C in telomerase promoter region increases the risk of postoperative mortality. |
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