CDYL通过调控hMLH1启动子区组蛋白H3K27三甲基化促进小细胞肺癌耐药的研究

Reversing the chemoresistance of SCLC（Small Cell Lung Cancer, SCLC）is an important problem to be solved. Some studies preliminarily showed that CDYL can regulate the expression of H3K27 trimethylation catalyzed by EZH2（enhancer of zeste homologue 2，EZH2）. However, the relationship between CDYL and chemoresistance is still unknown. Our previous study showed that the expression of transcriptional repressor CDYL（Chromodomain Y-like, CDYL）in the SCLC drug resistance cell H69/AR was higher than that in the sensitive cell H69. Furthermore, down-regulation of CDYL in the H69/AR cell reversed the chemoresistance, and inhibited the expression of H3K27 trimethylation, but promoted the expression of drug sensitivity gene hMLH1. Besides, over-expression of hMLH1 reduced the chemoresistance of SCLC. Thus, we propose a hypothesis that CDYL promotes the chemoresistance of SCLC by regulating the H3K27 trimethylation in the promoter region of hMLH1. To test our hypothesis, we formulate the following studies. Firstly, the relationship between CDYL and chemoresistance in the clinical samples is to be analyzed. Secondly, whether CDYL promotes the chemoresistance is to be verified in the SCLC chemoresistance cells and animal experiments. Then, the experiments such as luciferase assay and Chip experiment are performed to investigate whether CDYL promotes the chemoresistance of SCLC by regulating the H3K27 trimethylation in the promoter region of hMLH1. We may provide a new method to reverse the chemoresistance of SCLC by targeting the expression of CDYL.

逆转小细胞肺癌（SCLC）耐药是临床亟需解决的重要问题。有研究初步提示转录抑制因子（CDYL）有可能通过募集甲基转移酶EZH2调控H3K27三甲基化（H3K27me3），但并未见其参与调节肿瘤耐药的报道。我们前期首次发现：CDYL在SCLC耐药细胞株中高表达，下调其表达可降低肿瘤细胞耐药性；同时H3K27me3减少，药物敏感基因hMLH1表达增高；过表达hMLH1可降低耐药。据此，我们认为CDYL有可能通过EZH2调控hMLH1启动子区H3K27me3，进而促进SCLC耐药。为验证假设，本项目拟进一步在SCLC临床标本中证实CDYL与耐药的相关性；然后，利用体内外实验明确CDYL可促进SCLC耐药；最后，通过荧光素酶/ChIP等实验，在SCLC中重点探讨CDYL通过调控hMLH1启动子H3K27me3抑制其转录的机制。本项目有望为SCLC耐药逆转机制研究提供新思路。

【研究背景】.小细胞肺癌（small cell lung cancer, SCLC）是一种高度恶性的肺癌，化疗容易耐药，进而导致治疗失败，然而其潜在的耐药机制却未阐明。.【主要研究内容、重要结果】.1、化疗耐药SCLC患者肿瘤样本的CDYL强阳性表达率显著高于化疗敏感SCLC患者肿瘤样本，CDYL表达水平和SCLC患者的临床分期、预后相关。.2、上调化疗敏感SCLC细胞的CDYL表达，显著增加了细胞对化疗药物的IC50值，减少了化疗药物诱导的细胞凋亡和G1期阻滞；而下调化疗耐药SCLC细胞的CDYL表达，则显著降低了细胞对化疗药物的IC50值，增加了化疗药物诱导的细胞凋亡和G1期阻滞。.3、上调CDYL表达减少了化疗药物对裸鼠移植瘤的生长抑制，而下调CDYL表达则增加了化疗药物对裸鼠移植瘤的生长抑制。.4、CDYL直接结合并抑制CDKN1C基因（cyclin-dependent kinase inhibitor 1C，周期蛋白依赖性激酶1C）；化疗耐药SCLC细胞的CDKN1C表达显著低于化疗敏感SCLC细胞，构建CDKN1C表达上调和下调的SCLC细胞模型，上调化疗耐药SCLC细胞的CDKN1C表达降低了细胞对化疗药物的IC50值，增加了化疗药物诱导的细胞凋亡和G1期阻滞，而下调化疗敏感SCLC细胞的CDKN1C表达则增加了细胞对化疗药物的IC50值。.5、CDYL直接募集组蛋白甲基转移酶EZH2来催化CDKN1C基因启动子组蛋白H3K27三甲基化（H3K27me3），进而抑制CDKN1C转录。.6、EZH2抑制剂GSK126不但逆转了CDYL对CDKN1C的表达抑制，而且在体内和体外均可降低CDYL介导的SCLC化疗耐药。.【结论和意义】.CDYL通过EZH2-H3K27me3途径抑制CDKN1C转录，进而促进SCLC化疗耐药；EZH2特异性抑制剂GSK126降低了CDYL介导的SCLC化疗耐药。.综上：本研究揭示了CDYL/EZH2/H3K27me3/CDKN1C通路介导了SCLC化疗耐药，为临床上降低SCLC化疗耐药提供了潜在的治疗靶点。