小细胞肺癌相关的miRNA-375靶基因Runx1的鉴定和功能研究

In this study，we aim to identify the target gene Runx1 of microRNA-375（miR-375） and explore its function in small cell lung carcinoma（SCLC）. We have screened out miR-375，the miRNA marker which is closely related to SCLC and show over-expression by laser capture microdissection（LCM）and Agilent miRNA assay. Furthermore, we sceened out miR-375 target genes by miRecords and KEGG database analysis, checked them by quantitive RT-PCR in 50 cases of SCLC tissue acquired by LCM, and then confirmed six tumor associated target genes including Runx1. In this project, we will first determine target gene Runx1 by quantitive RT-PCR, Western-blot,and luciferase activity assay combined with transfection. The specific functions of miR-375 on SCLC cell lines, and the definite functions of Runx1 on SCLC cell lines transfected miR-375, will be studied by proliferation, apoptosis, invasion and migration assay, and also the expression of Runx1 studied by RT-PCR and Western Blot. Furthermore，we will detect the mRNA and protein expression of tumor associated genes in Stat3 signal pathway in SCLC cell lines which express Runx1 in different degrees to clarify the possible relationship of Runx1 and Stat3 pathway in SCLC. Through the project, the function of target gene Runx1 of miR-375 will be elucidated, and the project will provide theoretical basis for new target research in SCLC treatments.

我们前期已通过激光捕获显微切割（LCM）及miRNA 芯片技术，筛选出与小细胞肺癌（SCLC）显著差异表达的microRNA分子标志物miR-375；通过运用数据库分析及对LCM收集的50例SCLC组织进行靶基因的筛选和qRT-PCR验证，确定了包括Runx1在内的6个癌相关靶基因。本研究拟通过与转染相结合的qRT-PCR、Western-blot和荧光素酶活性检测法对Runx1进行鉴定；通过增殖、凋亡、侵袭、迁移等实验，检测miR-375对SCLC细胞株的具体生物学作用，并检测Runx1的表达变化；同时利用转染miR-375的SCLC细胞株分析Runx1的具体生物功能；在差异表达Runx1的SCLC细胞株中检测Stat3信号通路相关肿瘤因子的mRNA和蛋白表达，初步探讨SCLC中Runx1与Stat3信号通路的关系。为SCLC的治疗新靶点提供分子理论依据。

本项目经过3年努力,已顺利完成实验研究。通过激光显微分离技术从82例肺癌中分离出纯净的肺癌细胞,运用Agilent芯片技术进行全基因组miRNA差异表达分析, 筛选出22个在不同类型肺癌中显著差异表达的miRNA。通过miRecords及KEGG数据库分析,我们初步筛选出miR-375的37个候选靶基因可能参与调节SCLC的已知相关信号通路; 运用qRT-PCR法,我们对37个基因在50例显微切割的SCLC标本中进行了组织水平的验证。结果显示,包括Runx1、ITPKB在内的6个基因为SCLC中miR-375的目的靶基因。通过与转染实验相结合的荧光定量RT-PCR、Western-blot和荧光素酶活性检测实验进行功能研究，预测并验证了miR-375的靶基因ITPKB。通过细胞增殖实验证实miR-375可通过靶基因ITPKB促进细胞增殖。结合已报道的ITPKB在细胞调控中的多种作用,提示miR-375在SCLC发病分子机制中具有重要作用,并有望成为SCLC诊断与治疗的潜在靶点。.项目期间，作为第一作者发表SCI论文2篇，其中直接相关SCI论文1篇 (PLOS ONE,影响因子3.234分)。2016年被评为第一届全国卫生产业企业管理协会实验医学专家委员会病理专业委员会委员; 课题培养硕士研究生1名。