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目的:构建端粒保护蛋白TPP1短发夹RNA表达载体,探讨抑制TPP1表达后对端粒DNA损伤及细胞增殖的影响.方法:体外构建端粒保护蛋白TPP1shRNA逆转录病毒表达载体shTPP1-01,shTPP1-02,与表达外源性TPP1的TPP1-HA质粒共同转染293T细胞,Western Blot检测shTPP1抑制外源性TPP1蛋白表达;RT-PCR检测shTPP1抑制小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)内源性TPP1mRNA的表达.再用shTPP1分别感染ATM+/+MEF和ATM-/-MEF细胞后,分别采用细胞生长曲线和BrdU掺入法检测细胞增殖,免疫荧光/端粒肽核酸荧光原位杂交法(IF/PNA-FISH)检测端粒功能障碍诱导损伤灶(TIF)的形成.结果:shTPP1-01和shTPP1-02均能明显抑制外源性TPP1蛋白和内源性TPP1mRNA的表达,shTPP1-01和shTPP1-02作用于原代培养的MEFs后细胞生长缓慢,BrdU阳性细胞数量分别占8.0%和8.7%,显著低于对照细胞的29.3%(P<0.01,P<0.01);而细胞表现为体积变大,形态扁平等类似细胞衰老的形态学改变;IF/PNA-FISH法检测结果显示,shTPP1作用于ATM+/+MEF细胞后导致端粒DNA损伤标志物-磷酸化的H2AX(γ-H2AX)和53BP1损伤灶(TIF)的形成,定量结果显示约50%的ATM+/+细胞中出现至少5个TIF;而在shTPP1作用后的ATM-/-细胞中,端粒γ-H2AX和53BP1形成的TIF数量显著减少,出现5个TIF的细胞数量不足5%,与对照组细胞相比无显著性差异.结论:抑制TPP1表达后诱发端粒ATM依赖的DNA损伤反应,进而抑制细胞增殖,促进细胞衰老.
OBJECTIVE: To construct the short hairpin RNA (RNAi) vector of telomere protective protein TPP1 and investigate its effect on the DNA damage and proliferation of human telomere after TPP1 expression.Methods: To construct the TPP1shRNA retroviral vector shTPP1-01 and shTPP1 -02, transfected 293T cells with TPP1-HA plasmid expressing exogenous TPP1, and shTPP1 detected by Western Blot inhibited the expression of exogenous TPP1 protein; RT-PCR detection shTPP1 inhibited mouse embryonic fibroblast (MEF ) Endogenous TPP1mRNA expression.And then infected with shTPP1 ATM + / + MEF and ATM - / - MEF cells, respectively, by cell growth curve and BrdU incorporation assay of cell proliferation, immunofluorescence / telomere nucleic acid fluorescence in situ The formation of TIF induced by telomere dysfunction (TIF) was detected by hybridization assay (IF / PNA-FISH) .Results: Both shTPP1-01 and shTPP1-02 inhibited the expression of exogenous TPP1 protein and endogenous TPP1 mRNA, while shTPP1 -01 and shTPP1-02 slowed down the growth of primary cultured MEFs. The number of BrdU positive cells was 8.0% and 8.7% respectively, which was significantly lower than that of control cells (29.3%, P <0.01, P <0.01) Performance for the larger, flat and similar morphology of cells The results of IF / PNA-FISH showed that shTPP1 could induce the telomere DNA damage markers-phosphorylated H2AX (γ-H2AX) and 53BP1-lesion (TIF) , And quantitative results showed that at least 5 TIFs appeared in about 50% of ATM + / + cells; while in ATM - / - cells after shTPP1 treatment, the number of TIFs formed by telomere γ-H2AX and 53BP1 decreased significantly The number of TIF cells was less than 5%, which was not significantly different from that of control cells.Conclusion: Inhibiting the expression of TPP1 induces telomere ATM-dependent DNA damage response, thereby inhibiting cell proliferation and promoting cell senescence.