摘要
目的个体的端粒长度受到遗传和环境因素的共同调控,遗传度为35%～80%。人类端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)是端粒酶活性的限速成分,hTERT基因的序列变异与人类衰老相关的疾病风险密切相关,本研究拟探讨hTERT基因启动子区遗传变异对转录活性影响的功能研究。方法根据生物信息学预测和文献分析,本研究选择hTERT基因启动子区的两个单核苷酸多态性位点rs2735940(-1327T/C)和rs2853669(-190T/C),构建含有4种单体型的荧光素酶报告载体,包括野生型T-T,突变型T-C,C-T,C-C。由于rs2853669位于转录因子Ets2和c-Myc的结合位点,与c-Myc、Ets2真核表达载体共转染Hela细胞,检测荧光素酶活性变化,比较不同单体型对hTERT基因启动子转录活性的影响。结果研究结果表明,hTERT启动子区rs2853669携带C等位基因的单体型荧光素酶活性较低。与野生型T-T比较,单体型T-C的荧光素酶活性降低31%,单体型C-C的荧光素酶活性降低43%(P<0.001)。转录因子Ets2和c-Myc协同作用,显著增强hTERT基因启动子的活性(P<0.001)。结论 hTERT基因启动子区单体型遗传变异C-C(rs2735940-rs2853669)通过影响与转录因子Ets2和c-Myc的结合效率,降低该基因的转录活性和表达。
Objective Telomere length is determined by both genetic and environmental factors,with estimated heritability between 35% and 80%.Human telomerase reverse transcriptase（hTERT） is the rate-limiting catalytic subunit of the telomerase enzyme.The hTERT expression is strictly regulated at the transcription machinery,and thus it may be speculated that sequence changes in the hTERT promoter region interfere with the activation/depression of hTERT gene.To date,there is limited data available concerning the influence of Ets binding site mutation or polymorphism on hTERT expression.Methods Two single nucleotide polymorphisms,rs2735940（-1327T/C） and rs2853669（-190T/C）,were genotyped by the PCR-restriction fragmental length polymorphism method.To determine whether different haplotypes of the 2 variants（rs2735940 and rs2853669） affects the transcription activity of hTERT gene,a series of pGL3 luciferase reporter plasmids carrying rs2735940T-or C-and rs2853669T-or C-sequence were constructed.We further constructed plasmids for expression of pcDNA3.1-Ets2 and pcDNA3.1-c-Myc.Hela cells were co-transfected with these two kinds of plasmids.The hTERT gene promoter activities were assessed by measuring relative luciferase activity.Results The results showed that hTERT promoter haplotype carrying the at-risk rs2853669C allele had significantly lower luciferase activity than did that with the wild-type T-T haplotype,with 31% decrease for the T-C haplotype and 43% decrease for the C-C haplotype（P0.001）,respectively.The transcriptional factors Ets2 and c-Myc had a combined effect by enhancing the promoter activity of hTERT gene（P0.001）.Conclusion Sequence containing the hTERT promoter haplotype C-C（rs2735940 and rs2853669） has a lower transcription activity,via interfering with the binding efficiency with transcription factors Ets2 and c-Myc.
出处
《中国分子心脏病学杂志》
CAS
2012年第3期178-181,共4页
Molecular Cardiology of China
基金
国家自然科学基金(81070172)
北京协和医学院青年科研基金(2010-XHJ2)
