长链非编码RNA-NCRNA00173与hnRNPA2B1/hnRNPI互作调控CHK2/P53通路参与小细胞肺癌耐药的机制研究

Small cell lung cancer (SCLC) is characterised by poor prognosis since chemoresistance develops rapidly and is followed by significant disease relapse. Our previous study demonstrated that a long noncoding RNA NCRNA00173(Linc00173) was upregulated in H69AR, a chemoresistant SCLC cell line, by a lncRNA microarray analysis. NCRNA00173 promoted chemoresistance of SCLC cells, affected cell cycle, and suppressed cell apoptosis. RNA pulldown and mass spectrum experiments were performed to find the RNA-binding proteins hnRNPA2B1 and hnRNPI that interact with NCRNA00173 and NCRNA00173 may bind and direct hnRNPA2B1 and hnRNPI to interact with the mRNA of cell cycle factor CHK2 and affect the expression of CHK2 and the downstream factor P53 was further identified.Therefore, I propose a hypothesis that NCRNA00173 modulates chemoresistance of SCLC by binding hnRNPA2B1 and hnRNPI to regulate CHK2/P53 pathway. Chemoresistant SCLC cell lines, tumor xenograft models and SCLC tissues will be used to further elucidate the mechanism that NCRNA00173 interacts with RNA-binding proteins to affect downstream signaling pathways and promote chemoresistance in SCLC. The molecular mechanism of chemoresistance in SCLC is further enriched by this study, which might provide a potential novel target to reverse chemoresistance of SCLC.

小细胞肺癌（SCLC）多药耐药是患者预后不良的重要原因之一。申请人前期利用lncRNA表达谱芯片发现NCRNA00173在小细胞肺癌耐药细胞H69AR中高表达，能促进细胞对化疗药物产生耐药性。通过RNA-Pulldown结合质谱分析发现NCRNA00173与RNA结合蛋白hnRNPA2B1和hnRNPI有结合关系，并进一步发现NCRNA00173可能通过结合并引导hnRNPA2B1和hnRNPI与CHK2结合，影响CHK2和下游P53表达。申请人由此提出NCRNA00173通过与hnRNPA2B1和hnRNPI结合互作，抑制CHK2/P53通路，参与调节SCLC化疗耐药的科学假说。本项目拟利用SCLC耐药细胞模型、实验动物及临床样本，进一步阐明NCRNA00173与RNA结合蛋白互作调控下游信号通路促进SCLC耐药性的机制。通过本研究，丰富SCLC耐药的分子机制，为逆转耐药提供潜在的靶点。

化疗多药耐药是小细胞肺癌（SCLC）患者预后不良的重要原因之一，探明小细胞肺癌化疗耐药的分子机制是目前肺癌临床治疗学的重要研究课题之一。申请人前期通过表达谱芯片发现NCRNA00173在小细胞肺癌耐药细胞中高表达，能促进细胞对化疗药物产生耐药性。本课题利用小细胞肺癌患者临床标本，小细胞肺癌耐药细胞模型、实验动物（裸鼠皮下成瘤）明确NCRNA00173通过影响下游因子参与诱导小细胞肺癌的多药耐药。主要研究结果：（1）NCRNA00173可与hnRNPA2B1和hnRNPI互作调控小细胞肺癌耐药：①通过RIP、RNA-Pulldown及Co-IP实验明确了NCRNA00173 与hnRNPA2B1和hnRNPI可直接结合及作用位点；②通过干扰、过表达后行qPCR、WB及免疫荧光、核浆分离等实验明确 NCRNA00173 与 hnRNPA2B1 和 hnRNPI 可相互调节；③通过干扰及过表达hnRNPA2B1及hnRNPI后行CCK8实验及流式细胞学实验，明确 hnRNPA2B1 和 hnRNPI 可调节小细胞肺癌耐药；（2）NCRNA00173可与 hnRNPA2B1 和 hnRNPI 共同调控 CHK2/P53 通路参与小细胞肺癌耐药：①通过RIP实验、干扰及过表达NCRNA00173、hnRNPA2B1、hnRNPI后行qRT-PCR、WB等实验明确 NCRNA00173 与 hnRNPA2B1 和 hnRNPI 调控 CHK2/P53 通路；②通过免疫组化及qRT-PCR分析临床样本中 NCRNA00173 与 CHK2 的表达密切相关及CHK2的低表达与小细胞肺癌患者的化疗敏感性、预后密切相关。（3）CHK2可参与调控小细胞肺癌的药物敏感性：①行回复实验证实CHK2可作为NCRNA00173的下游因子影响细胞周期及凋亡并调节化疗药物敏感性。②通过裸鼠皮下成瘤实验明确CHK2下调可体内导致小细胞肺癌耐药；本课题的研究进一步丰富了小细胞肺癌耐药的分子机制，为寻找小细胞肺癌耐药患者的治疗靶点及耐药相关分子标志物提供实验依据。

内容获取失败，请点击重试