mTOR/HIF-1α/SOX2通路正向调控非小细胞肺癌分化细胞去分化致放疗抵抗的机制研究

The studies have shown that the tumor stem cells are closely related with the tumor treatment resistance. The resent studies have found that the cancer stem cells in the radioresistance of non-small cell lung cancer (NSCLC) are increased, but the mechanism is not very clear. Our preliminary data have demonstrated that the radiotherapy induced the differentiated cells of NSCLC expressed the surface markers of cancer stem cells, and activated the mTOR/HIF-1α pathway. Inhibition of mTOR/HIF-1α pathway, the expression of cancer stem cells markers was significantly inhibited after radiotherapy. As a transcription factor, HIF-1α regulated SOX2. Thus, we hypothesized that mTOR/HIF-1α/SOX2 pathway may be involved in the regulation of NSCLC dedifferentiation induced by radiotherapy. Based on our preliminary data, through the detection of clinical samples by Next Generation Sequencing and the circulating tumor cells, liquid biopsy, we will verify the relationship between the activation of mTOR/HIF-1α/SOX2 pathway by radiotherapy and the dedifferentiation of NSCLC. As the object of the NSCLC cells differentiated cells (ALDH-CD133-), we will verify that the radiotherapy induce the differentiation cells of NSCLC to become the dedifferentiation cells. We will elucidate the molecular mechanism that the radiotherapy activates the mTOR/HIF-1α/SOX2 pathway to control the dedifferentiation of cells. At last, we will inhibit the mTOR/HIF-1α/SOX2 pathway to reverse the radioresistance of NSCLC in vitro. Our research will elucidate the sources and the molecular mechanisms of the treatment enrichment of the cancer stem cells, and discover the new drug targets.

肿瘤干细胞与肿瘤治疗抵抗密切相关。研究发现放疗抵抗的非小细胞肺癌中癌干细胞比例明显升高，但机制不清。我们前期发现：放疗诱导非小细胞肺癌分化细胞表达癌干细胞标志和特性，mTOR/HIF-1α通路激活；抑制mTOR/HIF-1α，放疗后癌干细胞标志表达明显抑制；并发现HIF-1α作为转录因子可能调控SOX2。由此我们推测mTOR/HIF-1α/SOX2正向调控非小细胞肺癌分化细胞去分化致放疗抵抗。本课题拟在前期基础上：通过液体活检技术研究临床放疗活化mTOR/HIF-1α/SOX2通路与非小细胞肺癌去分化的关系；以癌分化细胞为研究对象明确放疗诱导肺癌分化细胞去分化导致放疗抵抗，阐明放疗通过mTOR/HIF-1α/SOX2通路调控癌分化细胞去分化的分子机制；最后干预mTOR/HIF-1α/SOX2逆转放疗抵抗。本研究将揭示癌干细胞治疗性富集的重要细胞来源及调控机制，为发现新的药物靶点提供依据。

背景：治疗在非小细胞肺癌患者的治疗中占据重要地位，然而放疗抵抗的产生极大程度地限制了放疗疗效，导致放疗失败以及肿瘤的复发转移。目前认为肿瘤干细胞是促进放疗抵抗的主要因素。SOX2是肿瘤干细胞特异性表达的一种转录因子，参与肿瘤的发生发展和细胞的干性维持，但其与非小细胞肺癌放疗抵抗之间的关系尚不清楚。本研究主要验证了非小细胞肺癌放疗抵抗细胞中SOX2的表达水平，探讨SOX2对非小细胞肺癌放疗敏感性和细胞干性表达等生物学性状的影响以及可能的作用机制。.方法：1.选取H460细胞株为研究对象，对H460亲本细胞（H460-PT）给予单次 6Gy 的X 线照射，每周一次，经多次照射（总剂量 72Gy）诱导形成放疗抵抗细胞（H460-RR），通过克隆形成实验检测细胞放疗敏感性变化，通过western blot和免疫荧光检测细胞内γH2AX表达水平从而验证放疗抵抗细胞株的放疗损伤修复能力。2.采用western blot和qRT-PCR技术分别检测 H460-PT 与 H460-RR 中肿瘤干细胞标志物 CD133, ALDH 和 SOX2 在蛋白和 mRNA 水平的表达情况，并通过成球实验检测两组细胞的成球能力。3.利用细胞划痕和Transwell 实验检测H460-PT和H460-RR的迁移运动能力。4.使用慢病毒转染 H460-PT 构建 SOX2过表达模型，检测SOX2上调后细胞放疗敏感性的变化。5.使用慢病毒转染 H460-RR 构建SOX2 敲减模型，检测SOX2表达下调后细胞放疗敏感性的变化。6.通过 western blot，qRT-PCR和成球实验检测SOX2表达水平的上调与下调对细胞干性标志物表达以及成球能力的影响，通过划痕和Transwell 检测SOX2 表达水平改变对细胞迁移能力的影响。.结果：1.成功构建放疗抵抗细胞株：累计照射剂量达72Gy 的 H460 细胞表现出更强的放疗耐受能力，即为本研究预期的放疗抵抗细胞 H460-RR（p<0.05）。2.放疗抵抗细胞株呈现去分化特征：H460-RR 中CD133，ALDH 和SOX2 的表达量在蛋白和mRNA水平均显著升高，成球能力也明显增强（p<0.05）。3.放疗抵抗细胞株迁移能力显著增强（p<0.05）。4. SOX2 参与放疗抵抗的调控：过表达 SOX2 增强 H460-PT 放疗抗性和 DNA

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