MYCN-KCNJ2-ABCC1调节环在PKC/Ras/MEK信号通路调控下参与小细胞肺癌多药耐药机制的研究

Overcoming multi-drug resistance is an important issue for improving poor prognosis of small cell lung cancer (SCLC). Our previous study demonstrated that MYCN, KCNJ2 and ABCC1 were upregulated in multi-drug resistance H69AR cells by cDNA microarray, which was confirmed by RT-PCR. Bioinformatics analysis showed N-Myc could target KCNJ2 promoter and ABCC1 promoter, and down-regulation of MYCN reduces KCNJ2 and ABCC1 expression and sensitizes the cells to chemotherapeutic drugs. And KCNJ2 induces multi-drug resistance of SCLC via interacting with ABCC1. Further inhibiting PKC/Ras/MEK pathway leads to the decrease of MYCN, KCNJ2 and ABCC1 expression. Taken together, we hypothesize that MYCN-KCNJ2-ABCC1 regulatory circuit may induce multi-drug resistance of SCLC under the regulation of PKC/Ras/MEK pathway. In this project, we intend to use the model of small cell lung cancer resistant cells and SCLC tissues to investigate the role of MYCN in SCLC chemoresistance. Also, we further validate that MYCN could directly target KCNJ2 and ABCC1 in SCLC by luciferase reporter gene and ChIP, constituting a regulatory circuit that induces chemoresistance. This study will enrich the molecular mechanism of SCLC multi-drug resistance and provide a potentially novel target for interfering with chemoresistance in SCLC.

小细胞肺癌（SCLC）多药耐药严重影响患者预后。前期通过cDNA基因芯片发现MYCN，KCNJ2和ABCC1在SCLC耐药株中的表达均显著升高。生物信息学预测MYCN与KCNJ2及ABCC1启动子区域均有靶向结合位点，沉默MYCN能降低KCNJ2和ABCC1表达，提高药物敏感性。KCNJ2可调控ABCC1表达参与耐药。同时，抑制PKC/Ras/MEK通路可降低MYCN、KCNJ2和ABCC1的表达。据此我们提出MYCN-KCNJ2-ABCC1形成调节环在PKC/Ras/MEK通路调控下参与SCLC耐药的假说。本课题拟利用耐药细胞模型、临床标本及实验动物，明确MYCN在SCLC多药耐药中的作用；通过荧光素酶报告系统和ChIP等技术手段，确定MYCN靶向调控KCNJ2和ABCC1，三者形成调节环路。通过本项目研究，进一步丰富SCLC多药耐药的分子机制，为SCLC治疗提供可能靶点。

小细胞肺癌(SCLC)是肺癌中恶性程度最高的类型之一，化疗是其主要的治疗方式，然而化疗耐药严重影响患者预后。MYCN作为MYC家族一员，在多种肿瘤中被证实与肿瘤生成、迁移和治疗相关，但在SCLC中的作用尚未知。前期cDNA基因芯片发现MYCN在SCLC耐药细胞株中的表达显著高于敏感细胞株，利用qRT-PCR和WB验证芯片结果。通过获得性和缺失性功能实验进一步发现，降低MYCN的表达可增加化疗敏感性，反之增加MYCN的表达可促进肿瘤生长、加强化疗抵抗性，并且MYCN通过抑制凋亡从而促进SCLC体内和体外的化疗耐药。利用RNA-seq分析，找出1067个MYCN相关的差异表达基因，KEGG通路富集分析发现，Notch通路是相关通路之一，该通路中的成员：HES1、JAG2，当上/下调MYCN表达时，2个基因的表达均出现上/下调。同时，qRT-PCR和WB证实HES1、JAG2在SCLC化疗耐药细胞株中表达显著高于敏感株。进一步通过CHIP-qPCR技术证实MYCN与HES1（Notch通路成员之一）启动子直接结合，并明确结合的具体位点，但MYCN未能与JAG2启动子直接结合。荧光素酶报告基因显示MYCN结合HES1启动子从而调节HES1的转录，MYCN表达与HES1呈正相关。进一步通过NOTCH抑制剂FLI-60抑制HES1的表达，可缓解MYCN介导的化疗耐药。利用公共数据库以及本院42例SCLC病人样本中进一步证实， MYCN在肿瘤组织中的表达高于癌旁组织，同时MYCN在化疗耐药样本中的表达高于化疗敏感样本，并且MYCN表达与HES1呈正相关。K-M生存曲线显示MYCN过表达与不良生存相关。MYCN和HES1可能是SCLC潜在的治疗靶点和预测因子。.此外，我们通过金催化的邻硝基炔与吲哚的级联反应合成2-吲哚基吲哚酮N-氧化物，进一步研究发现这些合成化合物在SCLC中具有很高抗肿瘤活性。