抑制TPP1表达对端粒保护蛋白1在染色体端粒定位及其功能的影响

TPP1 confers telomere end protection by recruiting Pot1a and Pot1b to telomeres

目的观察TPP1受到抑制后,对端粒保护蛋白1a(Pot1a)和端粒保护蛋白1b(Pot1b)在端粒上的定位及其对端粒保护功能产生的影响。方法利用逆转录病毒介导的RNA干扰技术抑制小鼠胚胎成纤维样细胞内源性TPP1的表达后,采用免疫荧光/端粒肽核酸荧光原位杂交法检测外源性Pot1a和Pot1b在端粒上的定位,通过端粒末端脱氧尿嘧啶转移酶法检测TPP1降低后对染色体末端保护功能的影响,SA-βgal染色检测细胞衰老,Western blot检测磷酸化的P53水平和P21含量。结果TPP1受到抑制后大约65%的MEF细胞中Pot1a和Pot1b不能定位在端粒上,显著高于空载体对照组的5%(t=10.96,P<0.01);TdT-FITC检测结果显示,大约50%的细胞在TPP1受到抑制后出现TdT-FITC阳性,其中90%的TdT-FITC阳性信号出现在端粒上,而在空载体对照组TdT-FITC阳性细胞为4.7%(t=14.37,P<0.01),位于端粒上的TdT-FITC阳性信号<3%(t=17.59,P<0.01);β-gal染色(细胞衰老的特征性标志)结果显示,在TPP1受到抑制后β-gal阳性细胞率分别达25.7%和22.0%,显著高于对照组细胞的1.7%(t=18.23,P<0.01;t=16.9,P<0.01);进一步研究表明,TPP1受到抑制的细胞导致P53ser18磷酸化,P21表达明显增强。结论TPP1是Pot1a和Pot1b定位在端粒上必需的,抑制TPP1导致染色体末端失去保护功能并诱导P53依赖的细胞衰老。

Objective To characterize the effects of TPPI knockdown on Potla and Potlb localization at telomeres and on the telomere end protection. Methods Knockdown of endogenous TPPI in mouse embryonic fibroblasts （MEFs） with the retrovirus vector encoding shRNA targeting TPPI, IF/PNA-FISH was performed to determine the localization of Potl a and Potl b at telomeres, and TdT-FITC was applied to characterize the effects on the function of telomere end protection, cellular senescence was analyzed by SA-beta gal staining, and phosphorylated p53^ser18 and p21 were examined by Western blotting. Results Potla and Potlb were unable to localize at telomeres in about 65% of MEFs with TPPI knockdown, while that was found in less than 5% of MEFs without TPPI knockdown （t = 10.96, P 〈 0.01 ）. TdT-FITC positive cells were detected in 50% of TPPI knockdown MEFs, as compared with that of 4.7% in the control MEFs （t = 14.37, P 〈0.01 ）. Furthermore, 90% of TdT signals appeared to co-localize with telomeres in TPPI knockdown cells, which was significantly higher than that of 3% in the control MEFs （t = 17.59, P 〈0.01 ）. The SA-beta gal positive cells, a hallmark of cellular senescence, were found in 25.7% and 22.0% of TPPI knockdown cells by shTPP1-1 or shTPP1-2, paralleled with marked induction of p53^ser18 phosphorylation and p21 expression in those cells.Whereas, cellular senescence occurred only in 1.7% of the control MEFs （t = 18.23, P 〈0.01; t = 16.9, P 〈0.01）, and increased p53%^ser18 phosphorylation and p21 expression were not detected. Conclusion TPP1 is required for Potl a and Potl b localization at telomeres, and knockdown of TPP1 results in telomere end accessible and elicits p53-dependent cellular senescence.