miR-363调控肺腺癌干细胞促进肿瘤转移的分子机制研究

Tumor metastasis is a pivotal factor on lung adenocarcinoma(AC) prognosis. Epithelial and mesenchymal transition(EMT) has been thought to be an important driver of cell invasive of AC, while cancer stem cell(CSC) has been thought to be a determined factor of AC colonization and outgrowth in a distant organ. In our previous study, we have screened out miR-363，the top miRNA marker which is highly expressed in metastatic AC by laser capture microdissection（LCM）and Agilent miRNA assay. We also confirmed that miR-363 was able ro negatively regulate the EMT, proliferation and local invasion of AC. In this project, wo want to continuously explore the effects of miR-363 on expressions, subcellular localization, recyclings and transcriptional activities of Numb, E-cadherin and beta-catenin using AC stem cells, as well as surgical tissues from clinic. Furthermore, we want to elucidate the regulatory roles of miR-363 in tumor metastasis of AC via Wnt/beta-catenin and Notch signaling pathways, for establishing a theoretical basis of AC prognosis and therapeutics based on miR-363.

肿瘤转移是影响肺癌预后的关键因素。在肺癌侵袭转移链中，EMT (epithelial mesenchymal transition)是肺癌转移的重要驱动力，而CSC(cancer stem cell)是肺癌转移的决定性因素。前期工作我们通过激光捕获显微切割从转移组及非转移组肺腺癌组织中分离纯净肿瘤细胞，进行Aglient miRNA芯片分析,筛选出表达显著升高且居首的miR-363，且证实miR-363在肺腺癌EMT、侵袭及转移中起着关键作用。本项目拟以肺腺癌干细胞及临床肺腺癌标本为研究对象，进一步观察miR-363对Numb、E-cadherin和β-catenin分子表达、亚细胞定位、代谢及转录功能变化，探讨miR-363对Wnt/β-catenin及Notch信号通路传导的影响及其在肺腺癌干细胞促进肿瘤转移中的作用，为基于miR-363的肺腺癌转移分子预测及靶向治疗奠定理论基础。

本项目经过4年努力,已完成大部分实验研究。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。.通过激光捕获显微切割从转移组及非转移组肺腺癌组织中分离纯净肿瘤细胞，进行Aglient miRNA芯片分析,筛选出表达显著升高且居首的miR-363，且证实miR-363在肺腺癌EMT、侵袭及转移中起着关键作用。肺腺癌干细胞及临床肺腺癌标本中进行体内外实验，研究了miR-363对E-cadherin和β-catenin分子表达及转录功能变化，正在进一步对miR-363在肺腺癌转移中信号通路的作用机制进行研究。.在肺癌组织miRNA筛选及鉴定的基础上，运用106例肺癌血浆芯片筛选出显著差异表达的miRNA，并于565例training cohort进行验证， 选出13个显著差异表达的miRNA，进一步于416例肺癌血浆进行验证，由此建立两个miRNA数学模型进行鉴别诊断。A：由6个microRNAs (miR-17, miR-190b, miR-19a, miR-19b, miR-26b, and miR-375)组成的panelA能准确鉴别肺癌及高危人群(AUC 0.873 and 0.868). SCLC与NSCLC（miR-29a与miR-375）和SQ与AC（miR-34a与miR-205）。B：由3个microRNAs (miR-17, miR-190b, and miR-375) 组成的panelB能准确鉴别SCLC）及NSCLC..项目期间，作为第一作者发表基金标注SCI论文3篇。并多次于国内、国际会议做关于miRNA与肺癌研究机制发言。课题培养研究生2名。