SOX2-OT作为ceRNA调控SOX2/Hedgehog轴介导肺癌干细胞增殖和耐药的研究

Hedgehog pathway-mediated proliferation of cancer stem cells (CSCs) is a major cause of drug resistance development in lung cancer therapy. Recent studies have shown that dysregulation of long non-coding RNAs (lncRNAs) is closely associated with drug resistance. Our group found that lncRNA SOX2-OT which is aberrantly expressed in lung CSCs and drug resistant cells reciprocally and positively interacts with GLI1, a key transcription factor of the Hedgehog cascade. Fluorescent in situ hybridization (FISH) indicated that SOX2-OT transcripts are mainly located in the cytoplasm and bioinformatics analysis suggested that SOX2-OT, as a ceRNA could upregulate expression of SOX2, a molecule upstream of GLI1. Thus, we propose that SOX2-OT plays an important role in lung CSCs-mediated drug resistance through regulating the SOX2/Hedgehog signaling axis. This project is designed to dissect the molecular mechanism of SOX2-OT/Hedgehog positive loop by employing ChIP, RIP and other target analysis techniques; to correlate the expression level of SOX2-OT with prognosis of patients; and to validate regulatory roles of SOX2-OT in CSC proliferation, drug resistance and expression of relevant markers in vitro and in vivo. We aim to take novel insight into molecular mechanisms of drug resistance development in lung cancer and provide novel therapeutic targets for clinic.

Hedgehog通路异常激活导致的肿瘤干细胞增殖是肺癌耐药形成的重要原因。近期研究表明，长链非编码RNA（lncRNA）的异常调控与耐药产生密切相关。本课题组发现，lncRNA SOX2-OT在肺癌干细胞和耐药细胞中高表达且与Hedgehog通路关键转录因子GLI1存在正反馈调控效应；FISH染色显示SOX2-OT主要位于细胞浆，生物信息分析提示其作为ceRNA调节GLI1上游调控分子SOX2表达。因而，我们推测SOX2-OT通过SOX2/Hedgehog信号轴调控肺癌干细胞介导的耐药性。本项目拟采用ChIP、RIP和报告基因定点突变等方法深入解析SOX2-OT与Hedgehog通路形成正反馈环路的分子机理；分析SOX2-OT表达水平与患者预后的相关性；在体内外评估SOX2-OT对肺癌干细胞增殖、耐药性及相关标记物表达水平的影响。旨在丰富肺癌耐药形成的分子机理并为临床提供新的治疗靶点。

项目背景：肺癌是我国发病率和死亡率最高的恶性肿瘤，多药耐药和早期转移的发生是治疗失败的重要原因。Hedgehog信号通路的异常激活是肿瘤干细胞增殖和干性维持的驱动因素之一。课题组前期研究揭示该通路关键组份SMO活化导致的终端转录因子GLI1和GLI2激活对肺癌干细胞恶性行为学特征有重要调控作用。然而，据报道在40%的肺癌样本中GLI1/2激活却没有检测到SMO表达，提示SMO非依赖式的GLI1/2活化方式在肺癌发生发展中扮演了重要角色。.研究内容：本项目探索了肺癌中异常激活的长链非编码RNA SOX2OT和MIR31HG以SMO非依赖的非传统模式激活GLI1/2的分子机理，及其对肺癌细胞多药耐药性、转移能力及免疫逃逸等恶行行为学特征的重要作用，通过对信号环路关键组份SOX2-OT、SOX2和GLI1进行基因修饰或药物干预，评估靶向抑制该通路在肺癌治疗中的潜在价值。从分子、细胞和整体水平上探求干扰肺癌干细胞介导耐药、转移和免疫逃逸的新策略。.重要结果：Hedgehog通路关键组份GLI1和GLI2通过调控SOX2，ABCG2，PD-L1等干细胞、耐药、转移和免疫抑制等相关基因促进肿瘤的发展，半枝莲和去甲斑蝥素等传统中药通过靶向该通路在体内外抑制肿瘤的生长和转移；长链非编码RNA SOX2OT通过METTL3/14/IGF2BP2介导的m6A修饰在转录后层面激活GLI1并形成正反馈环路，促进肺癌细胞生长及干性；长链非编码RNA MIR31HG通过引导WDR5/MLL3/P300复合物在GLI2增强子区域富集并形成活化域，通过促进GLI2转录导致肺癌细胞耐药、转移和免疫逃逸，靶向MIR31HG的核酸药物在体内外有效抑制肿瘤发展。.科学意义：本项目从表观遗传层面揭示了Hedgehog通路中GLI1/2非传统式的新型激活方式，该类激活方式不依赖于其上游SMO活化和GLI1/2本身的基因突变，进一步阐明了肺癌耐药、转移和免疫逃逸产生的分子机理。为SOX2OT和MIR31HG核酸药物的研发及与现有治疗方案的联合应用提供理论基础。