【作者】 李志宏；

【导师】 邵国光；

【作者基本信息】 吉林大学 ， 外科学， 2016， 博士

【摘要】 肺癌特别是非小细胞肺癌(non-small cell lung cancer,NSCLC)是一种死亡率较高的恶性肿瘤。尽管在临床学和肿瘤学的研究上取得一定的进展,但非小细胞肺癌预后仍不容乐观,患者5年生存率低于16%。治疗非小细胞肺癌的困难在于非小细胞肺癌的遗传异质性和表观遗传的改变。因此,迫切要求对NSCLC发生、发展的分子机制进行研究,以提高对该疾病的诊断、预防和治疗效果。Micro RNAs(miRNAs)是一类长度约为18-25个碱基的内源性、非编码单链小分子RNA,可与目标m RNA分子3’端非编码区结合,最终使其翻译受限。越来越多的研究证实miRNAs可参与细胞增殖、细胞周期调控、细胞分化、细胞凋亡等多个生物学过程。此外,另有研究显示,miRNAs参与包括NSCLC在内的多种肿瘤的发生、发展过程,深入研究其相关的分子机制对NSCLC的诊断和治疗有重要意义。micro RNA-592(miR-592)作为miRNAs家族的一员,已在结肠癌、肝癌、前列腺癌等多种癌症的诊断和治疗中显示出其独特的优势。研究显示,miR-592在NSCLC细胞中的表达受到了显著抑制,但其在NSCLC发生和发展过程中扮演的角色和相应的分子机理尚不清楚。本实验研究了miR-592在NSCLC发展过程中的生物学作用及其相应的分子机制,为miR-592应用于NSCLC的诊断和治疗中,提供数据支持。具体实验过程如下:实验目的:研究miR-592对NSCLC细胞生物功能的影响,探讨其相关的分子机制。为NSCLC的诊断和治疗寻找新的靶点。试验方法:本实验采用实时定量PCR(real time quantitative RT-PCR,RT-q PCR)比较了miR-592在肺癌组织与癌旁组织,正常细胞与肺癌细胞系中的表达差异。分析miR-592表达差异与肿瘤分期和肿瘤淋巴转移相互关系。为了进一步研究过miR-592对NSCLC细胞生物功能的影响,本实验将miR-592 mimic转化入A549细胞。采用CCK8实验、克隆形成实验、细胞划痕实验、Transwell侵袭实验对过表达miR-592 mimic的A549细胞增殖能力、转移能力,侵袭能力进行分析。为了彻底阐明miR-592抑制非小细胞肺癌的作用机理,采用target Scan和miRanda软件对miR-592可能的靶基因进行了预测,并对靶基因的功能进行验证。最后,我们在小鼠体内检测了miR-592对小鼠癌症增长的影响。实验结果:我们采用RT-q PCR对40份肺癌组织和癌旁组织中miR-592的表达情况进行了分析,结果显示:miR-592在肺癌组织中的表达情况显著低于癌旁组织。我们分析了miR-592的差异表达与临床分期和淋巴转移之间的关系,结果显示:III-IV肺癌患者肿瘤组织中miR-592的表达量显著低于I-II肺癌患者;发生淋巴转移的肺癌患者肿瘤组织中miR-592的表达量显著低于未发生转移的患者。对肺癌细胞系(A549,H1299,SPCA1、H358)与正常肺细胞系(BEAS-2B)中miR-592表达水平研究显示:miR-592在所有的肺癌细胞系的表达水平均显著低于在正常肺细胞的中的表达水平。为了进一步研究过表达miR-592对NSCLC细胞生物学功能的影响,本实验将miR-592 mimic转化入A549细胞中。CCK8结果显示转染miR-592 mimic组的细胞活性与对照组(转染miR-Ctrl)相比受到了显著的抑制;克隆形成实验显示转染miR-592 mimic组的A549细胞株克隆形成率显著低于转染miR-Ctrl的对照组;细胞划痕实验和Transwell侵袭实验结果显示转染miR-592 mimic组与对照相比A549细胞的转移和侵袭能力都受到了显著的抑制。本实验采用软件对miR-592的靶基因进行了预测,结果显示miR-592可与SOX9基因的3’区域(SOX9 3’-UTR)进行结合。为了进一步验证SOX9 3’-UTR是否是miR-592的直接靶点,本实验将插入野生型SOX9(WT)和突变SOX9(Mut)的荧光素表达质粒与miR-592或miR-Ctrl共转入A549细胞中。结果显示:转入SOX9(WT)荧光素表达质粒的荧光强度显著低于对照组。为进一步研究SOX9的功能,我们采用RT-q PCR技术分析了40肿瘤样本与癌旁组织中SOX9的表达情况,结果显示在肺癌肿瘤组织中SOX9的表达水平显著高于癌旁组织,并采用斯皮尔曼等级相关分析对数据进行分析(Spearman’s rank test),结果显示在40份肿瘤组织样品中SOX9的表达水平与miR-592的表达呈负相关。为了验证过表达miR-592对A549细胞活性、迁移和侵袭作用的抑制是通过对SOX9基因的抑制实现的。本实验将miR-592-mimic和p VAX1-SOX9(不含3′-UTR区域)、miR-592-mimic和p VAX1空载体、miR-Ctrl和p VAX1空载体共转入A549细胞中。结果显示:miR-592-mimic和p VAX1-SOX9(不含3′-UTR区域)缓解了miR-592对SOX9表达的抑制作用;同时,过表达SOX9基因可以有效缓解miR-592对肿瘤细胞增殖、细胞转移、细胞侵袭实验的抑制作用。动物实验显示与miR-Ctrl相比过表达miR-592可以有效减小肿瘤的体积,降低肿瘤的重量。同时,我们还分析了肿瘤组织中过表达miR-592对SOX9基因表达的影响。结果显示:在肿瘤组织中随着miR-592表达的提高,SOX9基因表达受到显著的抑制。综上所述,miR-592是通过抑制SOX9的表达来实现对肿瘤生长的抑制的。结论:综上所述,当前的研究表明miR-592表达在非小细胞肺癌组织和细胞系明显下调,低表达的miR-592与TNM分期和肿瘤转移负相关;过表达miR-592在体外抑制肺癌细胞增殖,克隆形成,迁移和侵袭,在体内抑制肿瘤增长,在一定程度上通过抑制靶蛋白SOX9来实现,这些研究建议miR-592能够作为抑癌基因,是一个潜在的理疗目标。更多还原

【Abstract】 Lung cancer, predominantly non-small cell lung cancer(NSCLC), is one of the leading causes of cancer-related mortality worldwide. Despite recent advances in clinical and experimental oncology, the prognosis of NSCLC remains unfavorable and the 5-year survival rate of patients with NSCLC is less than 16%. The difficulties of curing NSCLC are mainly due to an unclear elucidation of the heterogeneous genetic and epigenetic changes of NSCLC. Therefore, there is an urgent need to elucidate the molecular mechanisms underlying carcinogenesis, progression in NSCLC for improving the diagnosis, prevention, and treatment of this disease.Micro RNAs(miRNAs) are class of endogenous, small non-coding RNA molecules with a length of 18–25 nucleotides that negatively regulate m RNA stability and/or repress m RNA translation by binding to the 3’-untranslated region(3’-UTR). Increasing evidence has been suggested that miRNAs play significant roles in diverse biological processes, such as cell proliferation, cycle, differentiation, apoptosis and metastasis. Deregulation of miRNAs has been widely reported to be involved in the development of various cancers, including NSCLC, which may provide a new and promising way to treat NSCLC.Micro RNA-592(miR-592), an important miRNAs, has been proposed to be a new prognosis predictor and a new prospective target for several types of cancer. Although recently a report showed that the expression of miR-592 was reduced in NSCLC cell lines and compared to normal cells using microarray data sets. However, the biological roles and underlying mechanism of miR-592 in NSCLC remains unclear. Therefore, the aim of the present study was to investigate the biological function and the potential mechanisms of miR-592 on cell growth and metastasis in NSCLC,which may provide a new approach for NSCLC therapeutics. Objective:The aim of the present study was to investigate the biological function and the potential mechanisms of miR-592 on cell growth and metastasis in NSCLC,which provide a new target for NSCLC therapeutics. Methods:To determine the expression levels of miR-592 in human NSCLC specimens and NSCLC cell lines, q RT-PCR analysis was performed. we also analyzed miR-592 expression levels among different clinical stages and histological grade of the patients. To examine the biological role of miR-592 on growth of human NSCLC, A549 cells were transfected with miR-592 mimic. The CCK8 assay, colony formation assay, wound healing assay and transwell invasion assay was performed to investigate whether up-regulation of miR-592 had a direct influence on NSCLC cell proliferation, migration and invasion. To fully understand mechanism of miR-592 in inhibiting human NSCLC procession, Target Scan search program was used to predict targets of miR-592 and the function of the target gene was studied. In addition, the effect on tumor growth of miR-592 in vivo were investigated in nude mice. Results:To determine the expression levels of miR-592 in human NSCLC specimens, q RT-PCR analysis was performed in 40 pairs of NSCLC specimens and matched adjacent non-tumor tissues(ANT). The results revealed that miR-592 expression levels in NSCLC tissues were significantly lower than those in adjacent non-tumor tissues. we also analyzed miR-592 expression levels among different clinical stages, and found that the expression levels of miR-592 in advanced TNM stage(III-IV) were significantly downregulated compared with those in low TNM stage(I and II). In addition, miR-592 levels were markedly lower in the patients with lymph node metastases than those in the patients without lymph node metastases. We also examined miR-592 expression level in four NSCLC cell lines(A549, H1299, SPCA1 and H358) and a normal lung cell line(BEAS-2B), and found that miR-592 was significantly downregulated in all NSCLC cell lines, as compared to normal lung cell line(*P<0.05).To examine the biological role of miR-592 on growth of human NSCLC, A549 cells were transfected with miR-592 mimic. The CCK8 assay indicated that cell proliferation was significantly impaired in A549 cells transfected with miR-592 mimic compared to cells transfected with miR-Ctrl. The colony formation assays revealed that the ration of colony formation was significantly downregulated in A549 cells transfected with miR-592 mimic compared to cells transfected with miR-Ctrl. The wound healing assay and transwell invasion assay showed that migration and invasion were attenuated in A549 cells transfected with miR-592 mimic compared to cells transfected with miR-Ctrl.Target Scan and miRandasearch program was used to predict targets of miR-592. As the result showed, one predicted binding site in the SOX9 3’-UTR with a perfect complementarity to the seed region of the miR-592 was observed. To explore whether miR-592 targets SOX9 by binding to its 3′-UTR region, A549 cells were co-transfected with the wild type(WT) or mutant(Mut) SOX9 luciferase reporter vector and miR-592 or miR-Ctrl. Dual-luciferase reporter assay that luciferase activities were obviously decreased in the cells transfected with the wild type SOX9 reporter plasmid, but not in the cells with the mutant type SOX9 reporter plasmid. The TR-q PCR results showed that the expression levels of SOX9 were significantly higher in NSCLC tissues than those in the non-tumor tissues. In order to further study the function of SOX9,the results showed that the expression levels of SOX9 were significantly higher in NSCLC tissues than those in the non-tumor tissues. Using Spearman’s rank correlation analysis, we found that the expression levels of SOX9 and miR-592 were inversely correlated in 40 human NSCLC specimens.To investigate whether the tumor suppressor role of miR-592 on NSCLC cell proliferation, migration and invasion is mediated by inhibiting the expression of SOX9, A549 cells were co-transfected with miR-592 mimic and SOX9 overexpression plasmid without 3′-UTR, miR-592-mimic and p VAX1 empty vector, miR-Ctrl and p VAX1 empty vector. The RT-q PCR results showed that miR-592-induced SOX9 downregulation was rescued following cotransfection. Moreover, overexpression of SOX9 also reversed the inhibition effect on cell proliferation, colony formation, migration and invasion in A549 cells induced by miR-592 overexpressed.The animal studies showed that the tumor sizes and weights in miR-592 group were markedly decreased compared to miR-Ctrl group. In addition, we also measured miR-592 expression and SOX9 expression in tumor tissues. Consistent with in vitro data, levels of miR-592 expression was upregulated, whereas SOX9 expression was downregulated in tumor tissues from miR-592 group compared to miR-Ctrl group.Taken together, these results suggest that miR-592 inhibits tumor growth of NSCLC in vivo by repressing SOX9. Conclusion:In summary, the present study provide evidence that miR-592 expression was significantly down-regulated in NSCLC cell lines and tissues, and its expression was negative associated with advanced tumor/nodes/metastasis(TNM) classification stages and lymph node metastasis, and that restoration of miR-592 expression in NSCLCs inhibited cell proliferation, colony formation, migration and invasion, as well as suppressed tumor growth in vivo by targeting SOX9. These results suggested that miR-592 function as tumor suppressor in NSCLC by repressing SOX9, and might serve as a promising therapeutic target for NSCLC treatment.更多还原