背景:肌腱干/祖细胞(TSPCs)在肌腱修复中起着至关重要的作用,再生和体内平衡。然而,TSPCs衰老的具体机制尚不清楚。
目的:本研究旨在探讨HPF1在TSPCs衰老中的作用及其分子机制。
方法:从3至4和24-26个月大的Sprague-Dawley雄性大鼠中获得年轻和老年的TSPCs(Y-TSPCs和A-TSPCs),TSPCs(Y-TSPCs和A-TSPCs)进行衰老相关β-半乳糖苷酶(SA-β-Gal)染色和端粒酶活性检测,p16,p21,Scx,Tnmd,通过Westernblot或逆转录定量聚合酶链反应(RT-qPCR)检测Col1,Col3HPF1和PAPR1的表达水平,使用相互免疫共沉淀(co-IP)来探索HPFl和PARP1之间的相互作用。核糖核蛋白免疫沉淀(RNP-IP)用于分析HuR与衰老标记基因mRNA的结合,IP用于进行HPFl对HuR的PARA化,检测到p16和p21的半衰期。最后,我们建立了一个体内模型,并对肌腱组织进行苏木精和曙红(HE)和马尾松三色染色,以及ColI和TNMD的免疫组织化学分析。
结果:与Y-TSPCs相比,A-TSPCs能显著增强细胞衰老,显著降低肌腱分化能力,并显著上调了HPF1和PARP1的表达。此外,HPF1和PARP1相互作用并协调TSPCs的衰老和分化,HPF1还可以调节p21和p21的表达,p16或p21与HuR的相互作用,以及PARP1对HuR的聚ADP核糖基化。HPFl过表达和siHuR共转染显着降低了p16和p21的半衰期,并且HPFl和PARP1通过HuR调节p16和p21的mRNA水平。最后,体内实验表明,HPF1或PARP1过表达既能抑制肌腱分化能力,又能促进细胞衰老。
结论:HPF1通过PARP1介导的聚ADP核糖基化促进TSPCs衰老,抑制TSPCs肌腱分化。
Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear.
This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs.
Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated β-galactosidase (SA-β-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson\'s trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD.
Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence.
HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.