miR-592在非小细胞肺癌中的生物学功能及其基本机制探讨

发布时间：2018-02-16 10:30

本文关键词： 非小细胞肺癌 microRNA microRNA-592 SOX9　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【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。
[Abstract]:Lung cancer especially non-small cell lung cancer (non-small cell lung cancer, NSCLC) is a kind of malignant tumor with high mortality. Although some progress in clinical study and oncology, but non-small cell lung cancer patients is still not optimistic, 5 years survival rate is less than 16%. in the treatment of non small cell lung cancer is difficult to view the genetic changes of genetic heterogeneity and non-small cell lung cancer. Therefore, the urgent requirements for NSCLC, to study the molecular mechanism of the development, to improve the diagnosis of the disease, prevention and treatment effect of.Micro RNAs (miRNAs) is a class of endogenous length is about 18-25 nucleotides, encoding non small single stranded RNA but, with the target m RNA molecular 3'non encoding region of the translation is limited. More and more studies have confirmed that miRNAs may be involved in cell proliferation, cell cycle regulation, cell differentiation, apoptosis and other biological Process. In addition, other research shows that miRNAs is involved in a variety of tumors including NSCLC, occurrence, development, in-depth study of the related molecular mechanism of the diagnosis and treatment of NSCLC have important significance of.Micro RNA-592 (miR-592) as a member of the miRNAs family, has been in colon cancer, liver cancer, shows its unique advantages in diagnosis and for the treatment of prostate cancer and other cancers. The study showed that the expression of miR-592 in NSCLC cells was inhibited, but the incidence of NSCLC and the molecular role of the development process and the corresponding mechanism is not clear. The experimental study on the biological function of miR-592 in the development of NSCLC and the molecular mechanism of the corresponding and for the diagnosis and treatment of miR-592 in NSCLC, to provide data support. The specific process is as follows: the experimental objective: To study the effect of miR-592 on NSCLC cell biological function, explore the related Molecular mechanism. To find new targets for the diagnosis and treatment of NSCLC. Methods: the experiment using quantitative real-time PCR (real time quantitative RT-PCR, RT-q PCR) were compared in lung cancer tissues and cancer adjacent tissues miR-592, expression differences between normal cells and lung cancer cell lines. The expression of miR-592 and tumor metastasis staging difference each other the relationship between tumor and lymph node. In order to further study the effect of miR-592 on NSCLC cell biological function, this experiment will miR-592 mimic into A549 cells by CCK8 experiment, clone formation assay, cell scratch assay, Transwell invasion assay, A549 cell proliferation on the expression of miR-592 mimic metastasis ability analysis to invasion. The mechanism of inhibition of miR-592 thorough elucidation of non-small cell lung cancer, the target gene of miR-592 was predicted by target Scan and miRanda software, and the target gene The function is verified. Finally, we tested the effect of miR-592 on mice of cancer growth in mice. Results: We used the expression of RT-q PCR on miR-592 in 40 lung cancer tissues and adjacent tissues were analyzed. The results showed that: miR-592 in lung cancer tissues was significantly lower than that of adjacent tissues. Analysis of differential expression and clinical miR-592 staging and lymph node metastasis and the relationship between the results showed that the expression of miR-592 III-IV in lung cancer tissue was significantly lower than that of I-II in patients with lung cancer; expression of miR-592 lymph node metastasis in patients with lung cancer tumor tissue were significantly lower than that in non metastatic patients. In lung cancer cell lines (A549. H1299, SPCA1, H358) and normal lung cell line (BEAS-2B) miR-592 expression level in the study showed that the expression level of miR-592 in lung cancer cell lines were all significantly lower than that in normal lung fine The expression level of the cell. In order to further study the effect of miR-592 overexpression on NSCLC cell biology function, this experiment will be transformed into miR-592 mimic.CCK8 results in A549 cells transfected with miR-592 showed that the cell activity of mimic group and control group (transfected with miR-Ctrl) compared inhibited significantly; clone formation assay indicated that the expression of miR-592 in mimic group the A549 cell clone formation rate was significantly lower than the control group transfected with miR-Ctrl; cell scratch assay and Transwell invasion assay showed that transfection of miR-592 mimic group with the control force than the metastasis and invasion of A549 cells was significantly inhibited. This experiment adopts the software of miR-592 target genes were predicted, the results showed that miR-592 with SOX9 gene 3 'region (SOX9 3 -UTR) combined. In order to verify the SOX9 3 -UTR is a direct target of miR-592, this experiment will be inserted into the Wild type and mutant SOX9 (WT) SOX9 (Mut) luciferase expression plasmid and miR-592 or miR-Ctrl were transfected into A549 cells. The results showed: to SOX9 (WT) expression of fluorescein fluorescence intensity was significantly lower than the control group. To study the function of SOX9, we use RT-q PCR technology to analyze the expression of SOX9 40 tumor samples and in the adjacent tissues. Results showed that the expression level of SOX9 in lung cancer tissues was significantly higher than that in paracancerous tissues, and by using the Spielman rank correlation analysis to analyze the data ("Spearman s rank test), was negatively related to the expression results showed that the expression level of miR-592 and SOX9 in 40 tumor tissue samples. In order to verify. Over expression of miR-592 activity in A549 cells, inhibit the migration and invasion effect is achieved by inhibition of the SOX9 gene. The miR-592-mimic and P VAX1-SOX9 (not including the 3 '-UTR region), miR- 592-mimic and P VAX1 miR-Ctrl P VAX1 and empty vector and empty vector were transfected into A549 cells. The results showed that miR-592-mimic and P VAX1-SOX9 (not including the 3 '-UTR region) alleviated the inhibitory effect of miR-592 on the expression of SOX9; at the same time, the overexpression of SOX9 gene can effectively alleviate the miR-592 on the proliferation of tumor cell metastasis inhibition the role of cell invasion experiments. Animal experiments show that compared with miR-Ctrl over expression of miR-592 can effectively reduce tumor volume, reduce the tumor weight. At the same time, we also analyzed the effect of overexpression of miR-592 on the expression of SOX9 gene in tumor tissue. The results showed that in tumor tissues with the expression of miR-592 SOX9 gene expression was significantly increased. The inhibition of miR-592. In conclusion, by inhibiting the expression of SOX9 to inhibit tumor growth. In conclusion, the present study shows that the expression of miR-592 in non small cell Lung cancer tissues and cell lines was significantly lower, miR-592 and low expression of TNM staging and tumor metastasis negative correlation; overexpression of miR-592 inhibits the proliferation of lung cancer cells in vitro, colony formation, migration and invasion in vivo, inhibit tumor growth by inhibiting SOX9 protein target to achieve in a certain extent, these studies suggest that miR-592 can be used as a tumor suppressor gene is a potential therapeutic target.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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