circESRP1作为ceRNA调控TGF-β介导的信号通路参与小细胞肺癌化疗耐药的机制

Chemoresistance is the major cause of poor prognosis in small cell lung cancer (SCLC) patients. Our preliminary study present circESRP1 was down-regulated in multi-drug resistance H69AR cells by circlRNA microarray. circESRP1 knockdown significantly promotes H69 cells chemoresistance to anti-cancer drugs including DDP etc. We also fund circESRP1 may interact with miR-93-5p and upregulate SMAD7 and P21/CDKN1A expression which two of important members of TGF-β mediated signaling pathway. Taken together, we suggest that circESRP1 may be involved in chemoresistance of SCLC by acting as a competitive endogenous RNA of miR-93-5p to modulate TGF-β mediated signaling pathway. In this project, we intend to use the model of small cell lung cancer resistant cells (H69AR and H446DDP) and SCLC tissues to further confirm the role of circESRP1 in SCLC chemoresistance. We also elucidate the mechanism of circESRP1 regulate SCLC chemoresistance by TGF-β mediated signaling pathway using Western blot, luciferase reporter gene and RNA immunoprecipitation (RIP) assay. Through this research project, we will provide the evidence for finding biological marker and the targeting gene of small cell lung cancer chemoresistance.

化疗耐药是导致小细胞肺癌（SCLC）患者预后差的重要因素。我们前期发现circESRP1在SCLC耐药细胞H69AR中显著下调，沉默敏感细胞H69中circESRP1表达，细胞对顺铂等化疗药物产生抗药性，同时还发现circESRP1与miR-93-5p间存在作用关系，影响TGF-β通路中SMAD7和P21/CDKN1A表达。据此我们提出circESRP1可能通过竞争性结合miR-93-5p，调控TGF-β介导下的信号通路，参与SCLC耐药调节的假说。本课题拟利用耐药细胞模型（H69AR和H446DDP）和SCLC患者组织标本，进一步明确circESRP1在SCLC化疗耐药中的作用；运用Western blot、荧光素酶、RIP等技术阐明circESRP1通过ceRNA调控TGF-β介导的信号通路，参与SCLC耐药的机制。通过本项目研究，为寻找SCLC耐药的分子标记物和治疗靶点提供科学依据。

我们前期发现circESRP1与小细胞肺癌（SCLC）化疗耐药关系密切。本课题拟利用耐药细胞模型（H69AR和H446DDP）和SCLC患者组织标本，运用细胞生物学和分子生物学技术，进一步明确circESRP1在SCLC化疗耐药中的作用及机制。主要研究发现：（1）circESRP1在耐药细胞H69AR/H446DDP中表达明显低于敏感细胞H69/H446，上调circESRP1表达，细胞对化疗药物的IC50值增加，细胞凋亡增多，细胞周期G1期阻滞增加，肿瘤生长抑制；反之，下调circESRP1表达，IC50值降低，凋亡增加，细胞周期G1期阻滞减少，促进肿瘤生长；（2）RIP assay及荧光素酶实验等证明cESRP1可与miR-93-5p直接结合，并对miR-93-5p活性有调节作用，FISH确定cESRP1和miR-93-5p存在细胞内共定位；（3）上调或下调细胞中circESRP1表达，TGF-β信号通路中Smad7和p21随之增加或降低；加入外源性TGF-β，cESRP1、Smad7及p21表达均明显增加，而p-Smad2和p-Smad3表达降低，上皮-间质转化相关分子N-cadherin和Vimentin表达也降低；当同时上调mir-93-5p，可阻断cESRP1介导的TGF-β/Smad的抑制。（4）利用裸鼠成瘤模型和PDX模型，发现TGF-β信号通路抑制剂LY2157299联合化疗药物显著抑制肿瘤生长。（5）实时定量PCR结合免疫组化技术，发现circESRP1在癌组织中表达明显低于正常肺组织（P<0.001），且circESRP1表达水平低的患者预后更差，circESRP1表达与p21和smad7的表达有显著的正相关（P＜0.001）。通过本项目研究，进一步丰富SCLC耐药的分子机制，为下一步开展基于circESRP1逆转SCLC耐药的研究提供实验依据。