ARRB1通过H2AX/Caspase-3/NF-κB途径调节DNA损伤应答及肺癌细胞凋亡的机制研究

DNA damage response (DDR) is involved in the occurrence and development of cancers including lung cancer, and it is the basis of radiotherapy and chemotherapy. We found that ARRB1 regulated DNA damage response and apoptosis in lung squamous cell carcinoma induced by chemotherapy. However, few researches were focusing on the impact or mechanism of ARRB1 on DNA damage response or apoptosis..ARRBs (β-arrestins) are negative regulators of GPCR and server as adaptor and signal transducer in scaffolding and modulating intracellular signal networks. Our previous study showed that ARRB1 promoted phosphorylation of H2AX in the process of chemotherapy in lung cancer; ARRB1 could enhance DNA damage response and apoptosis induced by chemotherapy, with Caspase-3/NF-κB pathway involved. Our hypothesis is that ARRB1 plays a pivotal role in DNA damage response and apoptosis, and manipulation of ARRB1 status may improve the efficacy of chemotherapy. .This project intends to explore the influence of ARRB1 on cell cycle arrest, apoptosis and DNA repair, and to explore the mechanism of ARRB1 regulating DDR, and to find the downstream substrates of ARRB1 and the specific functional domain or binding-site, and to study the therapeutic value of ARRB1 in clinical chemotherapy.

DNA损伤应答在肿瘤预防和治疗中发挥重要作用。研究发现ARRB1参与介导DNA损伤应答以及细胞凋亡，但ARRB1调控DDR以及细胞凋亡的具体机制目前不详。.ARRBs是G蛋白偶联受体家族负性调控因子，参与介导多种细胞功能的调控。我们研究发现肺鳞癌化疗中，ARRB1与H2AX结合并促进其磷酸化激活，增加化疗引起的DNA损伤；ARRB1参与介导Caspase-3与NF-κB的调控，促进化疗引起的细胞凋亡。据此我们推测，ARRB1通过H2AX/Caspase-3/NF-κB途径调控DNA损伤应答及肺癌细胞凋亡。.本课题拟研究ARRB1对肺癌细胞周期阻滞、细胞凋亡、DNA损伤修复等的影响，深入探讨ARRB1对DNA损伤应答通路的调控作用；验证下游底物分子，寻找ARRB1特异性结构域或结合位点；在体内实验验证ARRB1在化疗中的作用，为肺癌临床治疗提供新的思路。

背景：ARRB1(β-arretin1)作为支架蛋白参与多种信号通路。先前的研究发现，β-arretin1的过表达增强了辐射诱导的DDR途径激活，以及下游细胞凋亡，而β-arretin1的敲减抑制了DDR过程。然而，进一步的研究发现，随着放疗后修复时间的延长，β-arretin1的过表达反而增加了细胞DNA修复。研究方法：通过GSEA富集分析发现，在肺鳞癌与肺腺癌患者中，ARRB1都可能通过参与P53 TGF-β Smad通路介导UV放射应答，调控DNA损伤修复通路。利用肺腺癌细胞系转染β-arretin1质粒或siRNA并接受放射治疗。CCK8实验测定以评估肿瘤细胞的增殖和活力。应用蛋白质印迹法检测DNA损伤修复途径中的蛋白质表达，其中包括 H2AX，rad50，DNA-PKcs，ku80等。Co-IP评估β-arrestin1和ku80之间的相互作用。通过免疫组织化学（IHC）在肺腺癌组织样本中检测β-arretin1和ku80的共表达情况，并进行生存分析。结果：β-arretin1与ku80结合，并且β-arretin1的过表达增强了DNA损伤修复途径的激活。此外，细胞核内β-arretin1和ku80的共表达提示肺腺癌患者较差的OS和DFS。β-arretin1可能通过介导DNA损伤修复过程，从而参与放疗后期的细胞修复和存活。

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