LncRNA PANDAR通过募集Sirt1去乙酰化Ku70促进鼻咽癌放疗抵抗的 分子机理

PANDAR is a long non-coding RNA(lnc RNA) induced by DNA damage. It can modulate cell cycle distribution, proliferation and apoptosis, indicating it might affect tumor radiosensitivity. However, the effect of PANDAR on radiosensitivity and its mechanism are still not clear in nasopharyngeal carcinoma (NPC). Our previous studies found:(1)that PANDAR was up-regulated in the radioresitant-NPC tissues, compared with radiosensitive-NPC ones. Similarly, CNE2-IR, a radioresitant-NPC cell line, also demonstrated a higher expression than the parental cells CNE2 (a more radiosensitive NPC cell line); furtherly, patients with up-regulated PANDAR presented an unfavorable prognosis than their counterparts.(2) the knockdown of PANDAR can enhance radiosensitivity in CNE2-IR, impede the interaction between Sirt1and Ku70, and enhance acetylation level of Ku70.Accordingly, we hypothesized that LncRNA PANDAR might promote nasopharyngeal carcinoma radioresistance by recruiting Sirt1 to reduce acetylation level of Ku70. In order to confirm the hypothesis, we performed these studies as follows. First, we will establish the radiation model of NPC cells by gene knockout and more senior site-directed mutagenesis techniques, and study the effect of PANDAR on radiation–induced DNA damage repair, cell colony formation, and apoptosis of NPC cell in vitro, and radiation–induced tumorgenesis in vivo using the radiation model of xenograft tumor of nude mice in NPC. Next, Co-IP and RNA pulldown assay were used to identify interactive domain between PANDAR and Sirt1 or Ku70.On this basis, finally, we will explore the molecular mechanism underlying to explain how the changed PANDAR promoted radioresistance through PANDAR-Sirt1-Ku70 axis in NPC. Our aim is to clarify the molecular mechanism and implications that lncRNA PANDAR promotes the radioresitance of nasopharyngeal carcinoma by recruiting Sirt1 to reduce acetylation level of Ku70 gene.

PANDAR是一种DNA损伤引起的lncRNA，它调节细胞周期、增殖和凋亡，提示可能影响放疗敏感性。但它对鼻咽癌放疗敏感性影响和机制均不明确。前期研究发现：1）鼻咽癌放疗抵抗组织细胞PANDAR表达高于放疗敏感组织细胞，高表达者预后差；2）在放疗抵抗CNE2-IR细胞，敲低它可降低细胞放疗抵抗，并可阻止Sirt1与Ku70相互作用和增强Ku70乙酰化水平。据此，提出PANDAR通过募集Sirt1去乙酰化Ku70促进鼻咽癌放疗抵抗的假设。为此，通过基因敲除、定向突点等技术建立鼻咽癌细胞模型、裸鼠辐射模型，明确体内外PANDAR对放疗诱导下DNA损伤修复、细胞克隆形成及成瘤能力等影响；通过Co-IP、Pulldown等研究它与这些转录因子互作区域，它通过PANDAR-Sirt1-Ku70轴调节放疗抵抗的分子机理。旨在明确PANDAR通过募集Sirt1去乙酰化Ku70促进鼻咽癌放疗抵抗的分子机理

PANDAR是一种DNA损伤引起的lncRNA，它调节细胞周期、增殖和凋亡，提示可能影响放疗敏感性。但它对鼻咽癌放疗敏感性影响和机制均不明确。前期研究发现：1）鼻咽癌放疗抵抗组织细胞PANDAR表达高于放疗敏感组织细胞，高表达者预后差；2）在放疗抵抗CNE2-IR细胞，敲低它可降低细胞放疗抵抗，并可阻止Sirt1与Ku70相互作用和增强Ku70乙酰化水平。据此，提出PANDAR通过募集Sirt1去乙酰化Ku70促进鼻咽癌放疗抵抗的假设。为此，通过基因敲除、定向突点等技术建立鼻咽癌细胞模型、裸鼠辐射模型，明确体内外PANDAR对放疗诱导下DNA损伤修复、细胞克隆形成及成瘤能力等影响；通过Co-IP、Pulldown等研究它与这些转录因子互作区域，它通过PANDAR-Sirt1-Ku70轴调节放疗抵抗的分子机理。我们研究结果发现：.(1). 临床组织样本及细胞水平证实PANDAR对放疗抵抗性的影响.①在组织水平上，通过扩大样本，临床验证了鼻咽癌放疗抵抗组织细胞PANDAR表达高于放疗敏感组织细胞；②在细胞水平，在放疗抵抗CNE2-IR细胞，敲低之可降低放疗抵抗。说明PANDAR具有促进鼻咽癌组织、细胞的放疗抵抗性功能。.(2). PANDAR功能研究及机制探讨.①研究在敲低和过表达PANDAR情况下检测放疗诱导的γH2AX含量及DNA双链损伤。②敲低和过表达PANDAR情况下检测放疗诱导的同源重组、非同源重组修复能力。PANDAR具有这种功能，与它促进修复和/或抑制凋亡作用有关。③PANDAR介导Sirt1去乙酰化Ku70，改变Ku70乙酰化水平。敲低PANDAR能阻止Ku70的去乙酰化。.(3). 研究了PANDAR表达与鼻咽癌治疗后局部区域复发的关系，也深入研究了鼻咽癌中PANDAR表达在总生存、进展、转移等其他方面的临床意义。.总之，基于以上研究结果，我们初步明确了PANDAR通过募集Sirt1去乙酰化Ku70促进鼻咽癌放疗抵抗的分子机理，将为鼻咽癌放射治疗学提供新的思路和策略。