肺癌中lncRNA HOTAIR通过表观遗传学修饰细胞周期相关基因在EGFR-TKIs耐药逆转中的作用研究

This is a series of studies about the current hot spots “molecular mechanism of acquired resistance of EGFR-TKIs and resistance reversal in lung cancer treatment”. Based on the previous studies from the two funds supported by the National Natural Science Foundation of China (30972963, 81172233), we found that Rb-E2F1 signaling pathway and its regulated cell cycle abnormalities played an important role in gefitinib-acquired drug resistance in lung cancer treatment, and therefore we proposed a new theory of "cell cycle inhibitor + molecular targeting drug" to reverse the acquired resistance of EGFR-TKI in lung cancer treatment. Further studies also found that lncRNA (Long non-coding RNA) HOTAIR (Homeobox Transcript Antisense RNA) regulated Rb-E2F1 signaling pathway and cell cycle progression through the epigenetic modification, and then decreased the sensitivity of lung cancer cells to EGFR-TKIs (such as Gefitinib) treatment. In this proposal, we will plan to investigate the molecular mechanisms of HOTAIR that regulate cell cycle progress through the epigenetic modification to the promoters of P21, P16, Rb and E2F1 genes via IP and CHIP techniques. We will further explore the mechanism of HOTAIR to promote the acquired resistance of EGFR-TKIs using gene transfection, MTT and FACS techniques in lung cancer cells and mice models. Furthermore, CRISPR / Cas9 technique will be used to reverse drug resistance by targeting HOTAIR and related genes that regulate the tumor growth. We also explore the relationship between HOTAIR and its related cell cycle abnormality with the acquired resistance of the third generation of EGFR-TKIs and other TKIs, which will further enrich and perfect this new theory of "cell cycle inhibitor + molecular targeting drug", and will provide new ideas and new methods for molecular targeted therapy and acquired drug-resistance reversal in lung cancer treatment.

这是针对肺癌分子靶向治疗获得性耐药机制的系列研究。在前两个国家自然科学基金的资助下，我们发现Rb-E2F1及所调控的细胞周期异常在吉非替尼获得性耐药中发挥重要作用, 提出了“细胞周期抑制剂＋分子靶向药”逆转肺癌靶向耐药新理论。进一步发现LncRNA HOTAIR通过表观遗传学修饰，上调Rb-E2F1通路及细胞周期，使肺癌细胞对EGFR-TKIs的敏感性下降。本研究拟通过IP和CHIP等技术，探讨HOTAIR通过对P21，P16，Rb等的表观遗传学修饰，调控细胞周期的机理；通过基因转染，MTT等技术，在肺癌细胞及小鼠模型中，探讨HOTAIR促进TKI耐药的机制；筛选HOTAIR及其调控耐药的关键靶点，应用CRISPR/Cas9技术干预并逆转耐药；探讨HOTAIR及细胞周期异常与三代和其他TKIs耐药的关系，丰富完善“细胞周期抑制剂＋分子靶向药”理论，为肺癌分子靶向治疗耐药逆转提供新思路和方法

EGFR-TKIs获得性耐药是肺癌靶向治疗中的关键问题，也成为困扰全世界的难题。我们的前期研究表明，长链非编码RNA HOTAIR可能通过调控细胞周期蛋白来影响肺癌细胞对EGFR-TKI的敏感性。我们在前期工作的基础上，深入研究HOTAIR通过PRC2复合物，介导H3K27me3表观遗传修饰细胞周期蛋白如p16和p21，从⽽调控肺癌吉非替尼耐药的分子机制。首先，我们发现，HOTAIR SNPs rs920778和rs1899663 与吸烟、男性和中国肺癌人群中的鳞状细胞癌相关；其次，我们发现HOTAIR显著抑制了p16、p21等蛋白，调控EGFR-TKI耐药细胞的细胞周期进程。 HOTAIR能与EZH2 共同作用，通过H3K27me3介导p16和p21启动子区域的招募，沉默p16和p21 的表达。.基于以上的结果，我们利用EZH2抑制剂结合EGFR-TKI，研究逆转 EGFR 野生型肺癌细胞对吉非替尼的耐药策略，EZH2 抑制剂与吉非替尼联合应用可显著抑制A549和H1299 细胞的增殖； 另外，我们研究还发现，EGFR-TKIs诱导所产生的保护性自噬是EGFR-TKIs获得性耐药的重要原因。我们利用组蛋白去乙酰化酶抑制剂SAHA结合EGFR-TKI进行处理，减少EGFR-TKIs所产生的保护性自噬，探讨EGFR-TKIs获得性耐药的逆转策略。该研究为肺癌分⼦靶向治疗和耐药逆转提供新思路和新⽅法。