microRNA-7在非小细胞肺癌中的表达、功能及新的作用机制研究

Lung cancer is the leading cause of death from cancer in the world, about 80% of which are non-small cell lung cancers (NSCLC). NSCLC has become the number one cancer killer in the worldwide. Despite improvements in early diagnosis and treatment responses by emerging technologies and newly developed chemo/targeted therapies, the high mortality has not been markedly changed. The overall 5-year survival for NSCLC patients is still low (15%) and the recurrence rate is too high, even in early-stage groups. Although attempts have been made to identify important genes and pathways in contribution to tumorigenesis of NSCLC, most of the studies are focused on the molecular network of protein-coding genes. However, knowledge of genomic aberrations associated with non-coding genes such as microRNA and their contributions to NSCLC is relatively limited.. MicroRNAs represent an abundant group of short, non-coding RNAs with the potential to target and silence multiple genes across diverse biological processes, such as cell differentiation, proliferation, growth, mobility, and apoptosis. Tons of exciting evidences have assigned an important regulatory role for miRNAs in several human diseases including cancer. MiRNAs are directly involved in cancer initiation and progression by repressing the expression of important cancer-related genes and thereby function as tumor suppressors or oncogenes. We reported recently that miR-7 induced apoptosis and inhibited cell proliferation of A549 cells. Besides, miR-7 has been indicated to be a potential tumor suppressor in several human cancers, such as brain cancer, breast cancer, and pituitary adenoma. Over-expression of miR-7 could inhibit the expression of its target genes like EGFR, IRS1, PAK1, and thus negatively regulated cancer cells growth. However, the expression of miR-7, as well as its biological function and potential novel target genes in NSCLC remain to be further elucidated. . In view of this, this project is designed to analyze the expressions of miR-7 in NSCLC tissues and NSCLC cell lines, and to investigate its function in NSCLC derived cells. Furthermore, the potential new targets of miR-7 as well as their biological function will be further studied by employing bioinformatics-associated technologies and experimental verification assays including differential proteomics analysis, cell proliferation and apoptosis, cell colony formation, real-time PCR, western blot, report gene luciferase assay, invasive and shift assay in vitro, and tumor formation in vivo. By elucidating the expression, function and mechanisms of miR-7 in NSCLC, this study will provide new clues and basic rationales for the diagnosis, treatment and prognosis of NSCLC.

microRNA（miRNA）是基因表达调控机制体系中新的热点分子，广泛参与调控细胞增殖、分化、凋亡等一系列生物学事件。近年来的研究表明，肿瘤的发生发展与miRNA的表达与调控功能异常密切相关。我们前期研究发现miR-7可明显抑制非小细胞肺癌来源的A549肿瘤细胞的生长，诱导细胞凋亡。然而，在非小细胞肺癌肿瘤组织中，miR-7的表达及作用如何尚不清楚；miR-7是否存在与非小细胞肺癌发生、发展关系更为密切的新靶基因、这些靶基因在非小细胞肺癌中的表达及作用如何，等等，均是有待研究解决的具有实际意义的科学问题。鉴于此，本课题拟在研究分析非小细胞肺癌组织中miR-7表达水平差异的基础上，进一步研究其对非小细胞肺癌来源的肿瘤细胞的作用方式及作用机制，并利用蛋白组学与生物信息学技术对其靶基因进行科学筛查、实验验证及功能研究，以期为非小细胞肺癌的诊断、治疗、预后乃至靶向药物设计提供新的思路和依据。

肺癌是全球发病率和死亡率最高的肿瘤，既往研究主要集中在蛋白编码基因及相关信号通路在肺癌发生发展中的作用，而与基因表达异常关系密切的非编码序列如microRNAs (miRNAs) 的有待加强。.miRNAs是一类微小非编码RNA，其通过靶向沉默诸多与细胞增殖、细胞分化、细胞凋亡、细胞运动以及细胞生长等参与肿瘤发生与发展。近期研究报道，miR-7在脑胶质瘤、垂体瘤以及乳腺肿瘤组织中的表达较癌旁对照组明显下调，参与肿瘤发生。然而，miR-7在NSCLC肿瘤组织中的表达及作用机制如何？有待深入研究和探索。.鉴于此，我们收集了41对肺癌手术切除的新鲜NSCLC肿瘤组织及其癌旁对照组织标本， realtime PCR方法检测了miR-7在NSCLC肿瘤组织中的表达情况；通过改变NSCLC来源的肿瘤细胞中miR-7表达水平，研究miR-7对非小细胞肺癌细胞的作用方式及其作用机制；利用生物信息学、双荧光素酶活性实验、realtime PCR以及Western blot发现和验证miR-7新的靶基因；正向及负向调控等实验验证研究miR-7新的靶基因对非小细胞肺癌细胞的作用机制，并对miR-7及其新的靶基因与临床相关性进行统计分析，阐明miR-7在NSCLC发生、发展中的作用及其机制，为非小细胞肺癌诊断、靶向药物设计提供理论和实验依据。.本项目研究首次发现：miR-7在NSCLC组织表达明显下调；PA28gamma作为新发现的miR-7靶基因，其表达受miR-7靶向负性调控；miR-7表达与PA28gamma表达存在负性相关，NSCLC组织中，PA28gamma基因的高表达可能和miR-7低水平表达调控密切相关。miR-7不但可以在体外抑制非小细胞肺癌细胞的生长，还可抑制其在裸鼠体内的肿瘤形成。同样，沉默PA28gamma基因可以诱导出类似miR-7对NSCLC细胞的作用；另外，miR-7可以通过负性调控PA28gamma的表达，影响细胞周期相关蛋白p21基因、Cyclin D1基因的表达，进而诱导细胞周期阻滞，负性调节NSCLC肿瘤细胞的生长。.我们的研究结果提示NSCLC中，miR-7靶向调控PA28gamma基因表达，参与了肿瘤的发生与发展。miR-7与 PA28gamma可望成为NSCLC潜在的治疗靶点，可能具有潜在的临床应用价值。