【作者】 任晓帅；

【导师】 张必良；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 肺癌是全球范围内因癌症导致死亡最重要的原因,在我国,肺癌的发生率和死亡率都呈明显上升的趋势,其5年生存率只有20%左右。其主要原因是大多数肺癌患者在确诊时癌细胞已经发生转移。microRNAs(miRNAs)是一类存在于真核生物中,内源性的,长度约为22nt的小分子RNA,通过靶向mRNA的3’UTR结合序列,降解mRNA或抑制其翻译。miRNA通过参与基因转录后水平调控,在个体发育,细胞增殖,分化及凋亡等生命过程中起重要作用。越来越多的证据表明,1miRNA的表达和功能失调与肿瘤的发生,发展密切相关,在肿瘤中发挥着类似于癌基因或抑癌基因的功能。MicroRNA-449a (miR-449a)的表达下调常见于多种肿瘤中,但在肺癌中的生物学功能尚未明确。在本论文中,我们通过一系列的细胞和分子生物学实验技术,研究了miR-449a在肺癌中的表达及生理,病理学作用。首先我们利用qRT-PCR的方法检测了miR-449a在156例肺癌组织(主要是腺癌)以及肺癌细胞系中的表达,结果发现miR-449a在肺癌组织及肺癌细胞系中表达明显下调。同时我们采用x2分析miR-449a的表达水平与患者的性别,年龄,吸烟史,病理类型,复发情况,死亡情况等临床特征的相关性,结果显示miR-449a的表达与肺癌病人的性别,复发和死亡情况密切相关。为了进一步明确miR-449a的诊断价值,我们分析了miR-449a的表达和预后的关系,Kaplan-Meier生存分析显示,miR-449a低表达组病人生存时间要明显比高表达组病人短(p=0.019, log-rank test)。在体外miR-449a能明显降低肺癌细胞系的生长活力,抑制细胞的增殖,促进细胞的衰老和凋亡,而且能明显抑制肺癌细胞在小鼠体内成瘤的能力。双荧光报告基因系统,qRT-PCR和western blot实验表明,过表达lniR-449a能够降低E2F33’UTR报告载体的荧光素酶活性,抑制E2F3mRNA和蛋白水平的表达,并且miR-449a能明显降低E2F3基因的转录激活活性,证明E2F3是miR-449a的一个直接的作用靶点。在肺癌细胞中利用si-RNA沉默E2F3的表达可产生和过表达miR-449a类似的细胞周期阻滞和细胞衰老现象,而过表达E2F3,则可以恢复由过表达miR-449a所引起的细胞活力的下降。此外,miR-449a也显著抑制了CCND1,CDK6蛋白的表达。本论文的研究结果揭示了miR-449a在肺癌发生过程中和细胞周期调控中的重要作用,提示miR-449a可以作为肺癌诊断和预后的一个重要的标志物,并且在肺癌的治疗中具有潜在的应用价值。更多还原

【Abstract】 Lung cancer is the leading cause of cancer deaths worldwide, and has a rapidly increasing incidence and mortality in our country. Five-year survival rate of lung cancer is only20%. A major reason for poor outcomes in the lung cancer patients is that have spread beyond the primary site at the time of diagnosis. MicroRNAs (miRNAs) are endogenous, small noncoding RNAs about22nt long that are found in eukaryotes, and can regulate the translation or degradation of messenger RNA (mRNA) through pairing with complementary nucleotide sequences in the3’-untranslated region (3’-UTR) of target mRNA. miRNA negative regulates the gene expression in a posttranscriptional manner. To date, miRNAs have been involved in a wide range of cellular processes, such as development, cellular proliferation, differentiation, and apoptosis. Recently, growing evidence has indicated that deregulation of miRNAs contributes to tumorigenesis. miRNAs can function as oncogenes or tumor suppressors involved in cancer developmemt.Dysregulation of microRNA-449a (miR-449a) has been detected in various types of human cancers. However, the biological function of miR-449a in lung tumorigenesis remains largely unclear. In the present study, we aimed to identify the expression and pathophysiologic significance of miR-449a in lung cancer. Using quantitative RT-PCR (qRT-PCR), we analyzed the expression of miR-449a in156human lung cancer tissues (predominantly adenocarcinoma) and lung cancer cell lines. Our results showed that compared with paired normal tissues, miR-449a expression was significantly downregulated in156lung cancer tissues (p<0.001) and the lung cancer cell lines examined. We also investigated whether miR-449a downregulation is related to clinicopathological features of lung cancer patients, including sex, age, smoking history, histologic type, recurrence and death.χ2tests revealed a significant relationship between low miR-449a expression and patient sex, recurrence and death. To determine whether the expression of mature miR-449a was related to the prognosis of lung cancer patients, miR-449a was used for further survival analysis. Kaplan-Meier survival analysis revealed that lung cancer patients with reduced miR-449a expression had shorter survival than did patients with high miR-449a expression (p=0.019by log-rank test). The transient introduction of miR-449a into lung cancer cells caused cell cycle arrest, cell senescence, and cell apoptosis in vitro. miR-449a also suppressed tumor formation in vivo in nude mice. Further studies (Luciferase reporter assay, qRT-PCR, western-blot) revealed that overexpression of miR-449a significantly suppressed the luciferase activity of reporter plasmids containing the3’-untranslated sequence of E2F3, and also decreased E2F3mRNA and protein levels. Moreover, introduction of miR-449a significantly repressed the transactivation of E2F3. These results suggested that E2F3is a direct target of miR-449a. Moreover, silencing of E2F3by siRNA also inhibited cell proliferation and induced a senescent phenotype in lung cancer cells. E2F3overexpression rescued the suppression of cell viability caused by miR-449a. Overexpression of miR-449a also significantly decreased protein levels of CCND1and CDK.6. Taken together, our results suggest that miR-449a plays an important role in the tumorigenesis of lung cancer and might be a predictor of cancer recurrence and survival in lung cancer patients.It might have the potential therapeutic applications.更多还原