MARVELD1影响肺癌细胞基本生物学特征的分子机制

发布时间：2018-09-05 12:57

【摘要】：肿瘤细胞具有多种特性,而恶性肿瘤的基本生物学特征是细胞无限制的增殖和肿瘤细胞的侵袭与转移,后者是肿瘤患者重要的死亡原因之一。目前肺癌的发病率和死亡率已居首位,且严重威胁人类健康。尽管临床诊断和治疗技术不断发展,但患者的5年生存率长期徘徊在30%左右,深入研究其发生及发展的分子机制,将为临床治疗提供新的理论基础,有助于改进肺癌治疗的有效性。MARVELD1是本课题组发现的一个侯选的肿瘤抑制基因,其在在乳腺癌、肝癌等恶性肿瘤组织中表达显著下调,但深入的作用机制尚不明确。因此,本论文采用组织芯片、细胞周期同步化、RNA-seq高通量检测、表达调控分析等技术对MARVELD1在肺癌中的作用及其分子基础开展了深入的研究。研究中首先利用组织芯片免疫组化染色分析MARVELD1在肺癌中的表达,发现MARVELD1在多种组织类型的肺癌中表达明显下调。分析MARVELD1与肺癌病例特征的关系,证实MARVELD1与TNM分期显著负相关,与生存期显著正相关。免疫印迹技术分析MARVELD1在多种不同病理分型的肺癌细胞系中的表达情况,证实了在肺癌细胞中MARVELD1表达的下调。研究进一步利用亚硫酸盐甲基化测序分析技术,揭示了MARVLED1在肺癌细胞中表达下调的分子机制。鉴于恶性增殖是肿瘤的最基本生物学特性,结合MARVELD1在肺癌组织细胞中表达下调的特点,研究进一步分析其对肺癌细胞增殖抑制的能力。分析发现MARVELD1能够抑制A549细胞的体外增殖,抑制细胞周期并阻滞在G1期。通过周期同步化技术分析MARVELD1的表达与周期进程的关系,发现MARVELD1表达伴随着细胞周期进程而增加,其在S期表达较低,G2/M期表达较高。周期同步化和免疫共沉淀技术分析还表明MARVELD1能够与H4K20me1特异性结合,且调控细胞周期G1期进程。分析MARVELD1调控细胞周期G1期进程的分子机制,发现MARVELD1与p53启动子区结合,正调控P53的表达,引发了Cyclin D1的表达下调,从而抑制细胞周期从G1期向S期的进程。转移是恶性肿瘤另一主要基本生物学特征,也是最重要的特性。为探讨MARVELD1对肺癌细胞迁移运动的影响,本研究首先通过扫描电镜和原子力显微镜观察了MARVELD1对细胞表面超微结构及力学特性的影响,发现MARVELD1能够增加细胞表面微绒毛的长度及形态特征,增加细胞的杨氏模量。进一步利用RNA-seq高通量测序技术分析MARVELD1表达对全基因组的影响时,发现MARVELD1可负调控整联蛋白β1和正调控整联蛋白β4的表达。为此,研究通过ChIP、荧光素酶报告基因系统及RNA-IP技术分析了MARVELD1调控其表达的分子机制,由此揭示MARVELD1影响整联蛋白β1和β4在微绒毛尾部的定位。采用细胞铺展实验、细胞划痕愈伤和侵袭小室技术分析,证实MARVELD1具有通过下调整联蛋白β1的表达抑制细胞迁移,通过上调整联蛋白β4表达增强细胞与基底的粘附的双重功能。在以上研究基础上,本文进一步探讨了MARVELD1对肺癌细胞上皮间质转化(Epithelial-mesenchymal transition,EMT)的影响,形态学研究结果表明,MARVELD1具有维持细胞上皮样形态的能力。实时定量PCR和免疫印迹等试验进一步证实MARVELD1能够抑制TGF-β1诱导的上皮间质转化过程。其抑制EMT的分子机制为MARVELD1能够通过调控整联蛋白β1/β4的表达平衡抑制TGF-β1诱导上皮间质转化。综上所述,本文通过对MARVELD1在肺癌中的表达模式分析及对细胞周期及细胞迁移运动影响的研究,表明其在调控细胞周期、抑制细胞运动及上皮间质转化中具有重要生物学功能。鉴于MARVELD1在肺癌中影响了多个网络信号通路,因此MARVELD1在肺癌发生及发展中具有十分重要的作用。
[Abstract]:Tumor cells have many characteristics, and the basic biological characteristics of malignant tumors are unlimited proliferation of cells and invasion and metastasis of tumor cells. The latter is one of the important causes of death in cancer patients. MARVELD1 is a candidate tumor suppressor gene found by our team, which is found in breast cancer, liver cancer and other malignant tumors. The expression of MARVELD1 in lung cancer was significantly down-regulated, but the underlying mechanism was still unclear. In this study, tissue microarrays, cell cycle synchronization, RNA-seq high-throughput detection, expression regulation analysis and other techniques were used to investigate the role of MARVELD1 in lung cancer and its molecular basis. The expression of MARVELD1 in lung cancer was significantly down-regulated in various histological types of lung cancer. The relationship between MARVELD1 and the characteristics of lung cancer was analyzed. It was confirmed that MARVELD1 was negatively correlated with TNM stage and positively correlated with survival time. The expression of MARVELD1 in lung cancer cell lines with different pathological types was analyzed by Western blot. The down-regulation of MARVELD1 expression in lung cancer cells was confirmed. The molecular mechanism of down-regulation of MARVELD1 expression in lung cancer cells was revealed by sulfite methylation sequencing analysis. The results showed that MARVELD1 could inhibit the proliferation of A549 cells in vitro, inhibit cell cycle and block cell cycle in G1 phase. Cycle synchronization and immunoprecipitation analysis also showed that MARVELD1 could specifically bind to H4K20me1 and regulate the G1 phase of cell cycle. To investigate the effect of MARVELD1 on the migration and movement of lung cancer cells, the ultrastructure of the cell surface was observed by scanning electron microscopy and atomic force microscopy. It was found that MARVELD1 could increase the length and morphological characteristics of microvilli on the surface of cells and increase Young's modulus of cells. Further analysis of the effect of MARVELD1 expression on the whole genome by RNA-seq high-throughput sequencing showed that MARVELD1 could negatively regulate the expression of integrin beta 1 and positively regulate the expression of integrin beta 4. The molecular mechanism of MARVELD1 regulating its expression was analyzed by ChIP, luciferase reporter gene system and RNA-IP technique, which revealed that MARVELD1 affected the localization of integrin beta 1 and beta 4 in the tail of microvilli. The expression of white beta-1 inhibits cell migration by up-regulating the expression of connexin beta-4 and enhances cell-substrate adhesion. On the basis of the above studies, the effects of MARVELD1 on the epithelial-mesenchymal transition (EMT) of lung cancer cells were further investigated. Morphological studies showed that MARVELD1 has a dimension. MARVELD1 can inhibit the epithelial-mesenchymal transition induced by TGF-beta 1. The molecular mechanism of MARVELD1 inhibiting EMT is that MARVELD1 can inhibit the epithelial-mesenchymal transition induced by TGF-beta 1 by regulating the balance of expression of integrin beta 1/beta 4. Studies on the expression pattern of MARVELD1 in lung cancer and its effect on cell cycle and migration showed that MARVELD1 has important biological functions in regulating cell cycle, inhibiting cell movement and epithelial-mesenchymal transition. Exhibition plays a very important role.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

【相似文献】