ADAM10与共表达siRNA-ADAM10和GRIM-19在肝癌中的表达和功能研究

发布时间：2018-05-23 11:22

本文选题：肝癌 + ADAM10　；参考：《吉林大学》2015年博士论文

【摘要】：肝癌是最常见的恶性肿瘤之一，全球发病率居恶性肿瘤第5位，死亡率居第9位，据估计每年肝癌可导致50余万人死亡。目前肝癌的治疗效果不佳，迫切需要寻求新的治疗手段提高疗效。研究肝癌发生的分子机制，对寻找敏感性和特异性强的分子标记及开发新型治疗手段具有重要意义。ADAMs(Adisintegrin and metalloprotease)家族是一类同时具有解整合素和金属蛋白酶两种结构域的I型跨膜蛋白家族，广泛存在于各种组织细胞中。ADAM10是ADAMs家族的重要成员，研究表明，ADAM10在多种肿瘤中高表达，且促进肿瘤细胞的增殖、侵袭和迁移，但在肝癌中相关研究较少。本研究主要开展以下三部分试验：第一部分利用免疫组化和荧光定量PCR技术检测了ADAM10表达量与肝癌临床病理参数的关系，同时评价了ADAM10在肝癌上的预后价值。第二部分利用一系列体内和体外实验研究了沉默ADAM10基因表达对肝癌细胞的增殖、迁移和侵袭及肿瘤生长的影响。第三部分是在前两部分的基础上，利用基因共表达技术进一步探讨了沉默ADAM10和过表达GRIM-19联合应用对肝癌细胞增殖、细胞周期、细胞迁移、侵袭和凋亡及肿瘤生长的影响。主要研究结果如下： 1．ADAM10表达与肝癌临床病理参数的关系收集98例肝癌患者组织样本，利用免疫组化和荧光定量PCR技术从mRNA和蛋白水平上研究了ADAM10表达与肝脏临床病理参数的关系。在98例肝癌患者中，74(75.6%)人表现ADAM10阳性结果，24人表现ADAM10阴性结果。肿瘤大于5cm组免疫组化阳性比例达到90.63%；肿瘤转移组免疫组化阳性比例达100%；ADAM10免疫染色结果与患者的性别和年龄相关性不显著（P＞0.05）。在TNM分期系统中，TNM分级对ADAM10免疫染色结果影响显著（P＜0.05），肿瘤变异对ADAM10免疫染色结果有显著影响（P＜0.01）。直径大于5cm的肿瘤，其ADAM10免疫染色阳性显著高于直径小于5cm的肿瘤（P＜0.05）。荧光定量PCR结果表明，ADAM10mRNA在肝癌组织中表达水平显著高于癌旁组织（P＜0.01)。ADAM10阳性的患者生存指数明显低于ADAM10阴性的肝癌患者(P＜0.01），上述研究结果提示，ADAM10与肝癌密切相关。 2.沉默ADAM10基因对肝癌细胞增殖、迁移和侵袭及肿瘤生长的影响构建了ADAM10-RNAi质粒，成功转染到肝癌细胞HepG2中。MTT和缺口末端标记实验结果表明，沉默ADAM10基因可显著抑制肝癌细胞的增殖和促进其凋亡(P＜0.01)；western blot实验表明，沉默ADAM10基因可显著下调肝癌细胞中凋亡抑制基因Survivin和Bcl-2的表达水平和AKT和PI3K的磷酸化(P＜0.01)；细胞划痕实验和transwell小室细胞体外侵袭实验结果表明，沉默ADAM10基因可显著抑制肝癌细胞的迁移和侵袭,体内成瘤实验也表明，沉默ADAM10基因可显著抑制肿瘤的生长(P＜0.01)。 3.共表达ADAM10-specific siRNA和GRIM-19对肝癌的影响构建了pSi-ADAM10, pGRIM-19，pSi-ADAM10-GRIM-19质粒，并分别转染到肝癌细胞系HepG2中，荧光定量PCR和western blot实验证明质粒转染成功。与pSi-ADAM10和pGRIM-19处理组相比，共表达GRIM-19和ADAM10特异性ShRNA可显著降低HepG2细胞的增殖能力和S期细胞百分比，提高G1期细胞百分比，上调细胞周期蛋白P21和下调细胞周期蛋白cyclinD1的表达。共表达ADAM10-specific siRNA和GRIM-19可进一步促进肝癌细胞凋亡，且显著上调caspase-3, caspase-8和caspases-9的活性，而下调Bcl-2和Survivin的表达。共表达ADAM10-specific siRNA和GRIM-19处理可显著提高对HepG2细胞迁移和侵袭的抑制，并抑制侵袭相关蛋白MMP-2和MMP-9的表达。体内成瘤实验结果表明，共表达ADAM10-specific siRNA和GRIM-19可导致小鼠肿瘤内ADAM10基因表达显著下调和GRIM-19基因表达显著上调，且诱导细胞凋亡和移植肿瘤生长的效果最好(P0.05)。研究结果提示，与单一治疗相比，在肝癌细胞中共表达ADAM10-specific shRNA和GRIM-19可在体内外显著抑制肿瘤细胞的增殖、迁移和侵袭以及肿瘤的生长。综合上述结果，ADAM10在肝癌中高表达，沉默ADAM10可促进肝癌细胞凋亡，，抑制肝癌细胞肝癌细胞的增殖、迁移和侵袭以及肿瘤的生长，共表达ADAM10沉默质粒和GRIM-19过表达质粒可进一步抑制肝癌细胞的增殖、迁移、侵袭以及肿瘤的生长。
[Abstract]:Liver cancer is one of the most common malignant tumors. The global incidence is fifth of the malignant tumors, and the mortality rate is ninth. It is estimated that liver cancer can cause more than 50 million people to die each year. The treatment effect of liver cancer is not good. It is urgent to seek new treatment methods to improve the curative effect. .ADAMs (Adisintegrin and metalloprotease) family is a class of I type transmembrane protein family with two domains of integrin and metalloproteinase..ADAM10 is an important member of the ADAMs family in a variety of tissue cells. The study shows that ADAM10 is in a variety of swelling. The tumor is highly expressed and promotes the proliferation, invasion and migration of tumor cells, but there are few related studies in the liver cancer. This study mainly carried out the following three parts: the first part, the relationship between the expression of ADAM10 and the clinicopathological parameters of liver cancer was detected by immunohistochemistry and fluorescence quantitative PCR, and the prognosis of ADAM10 in the liver cancer was evaluated. The second part uses a series of in vivo and in vitro experiments to investigate the effects of silent ADAM10 gene expression on the proliferation, migration, invasion and tumor growth of hepatoma cells. The third part is on the basis of the first two parts, using gene co expression technology to further explore the combination of silencing ADAM10 and overexpressed GRIM-19 for hepatoma cells. Proliferation, cell cycle, cell migration, invasion and apoptosis, and tumor growth.
The main results are as follows:
Relationship between 1.ADAM10 expression and clinicopathological parameters of hepatocellular carcinoma
The tissue samples of 98 patients with liver cancer were collected. The relationship between the expression of ADAM10 and the clinicopathological parameters of the liver was studied by immunohistochemistry and fluorescence quantitative PCR. In 98 cases of liver cancer, 74 (75.6%) showed ADAM10 positive results and 24 showed ADAM10 negative results. The positive proportion of immunohistochemistry in group 5cm was greater than that of 5cm group. To 90.63%, the positive proportion of immuno histochemistry in the tumor metastasis group was 100%, and the correlation between the results of ADAM10 immunization and the sex and age of the patients was not significant (P > 0.05). In the TNM staging system, the TNM grading had significant influence on the results of ADAM10 immunostaining (P < 0.05), and the tumor variation had a significant influence on the results of ADAM10 immunization (P < 0.01). The diameter was greater than that of the ADAM10. The ADAM10 immunoreaction of 5cm was significantly higher than that of tumor smaller than 5cm (P < 0.05). The fluorescence quantitative PCR showed that the expression level of ADAM10mRNA in the liver cancer tissues was significantly higher than that of the paracancerous tissues (P < 0.01).ADAM10 positive patients were significantly lower than those of the negative liver cancer patients (P < 0.01). These findings suggest A. DAM10 is closely related to liver cancer.
2. effects of silencing ADAM10 gene on proliferation, migration and invasion, and tumor growth of hepatocellular carcinoma cells
The ADAM10-RNAi plasmid was constructed. The results of successful transfection of.MTT and nick end labeling in HepG2 cells showed that the silence of ADAM10 gene could significantly inhibit the proliferation of hepatoma cells and promote its apoptosis (P < 0.01). The Western blot experiment showed that the silence ADAM10 gene could significantly reduce the apoptosis inhibiting gene Survivin and Bcl-2 in the hepatoma cells. The expression level and phosphorylation of AKT and PI3K (P < 0.01); the results of cell scratch test and in vitro invasion test of Transwell cell cells showed that silent ADAM10 gene could significantly inhibit the migration and invasion of hepatoma cells. In vivo tumorigenesis experiment also showed that silent ADAM10 gene could significantly inhibit the growth of tumor (P < 0.01).
3. the effect of co expression of ADAM10-specific siRNA and GRIM-19 on liver cancer
PSi-ADAM10, pGRIM-19 and pSi-ADAM10-GRIM-19 plasmids were constructed and transfected into the hepatoma cell line HepG2 respectively. The fluorescence quantitative PCR and Western blot experiments proved that the plasmid was transfected successfully. Compared with pSi-ADAM10 and pGRIM-19 treatment group, co expression of GRIM-19 and ADAM10 specific ShRNA significantly reduced the proliferation ability of the cells and the percentage of cell percent. Ratio, increase the percentage of G1 cells, up regulation of cyclin P21 and down regulation of the expression of cyclin cyclinD1. Co expression of ADAM10-specific siRNA and GRIM-19 can further promote the apoptosis of hepatoma cells, and significantly up-regulation the activity of Caspase-3, Caspase-8 and caspases-9, and the expression of Bcl-2 and Survivin in the lower modulation. IRNA and GRIM-19 treatments could significantly increase the inhibition of migration and invasion of HepG2 cells and inhibit the expression of invasion related proteins MMP-2 and MMP-9. In vivo tumorigenesis experiment results showed that co expression of ADAM10-specific siRNA and GRIM-19 could lead to a significant downregulation of ADAM10 gene expression in tumor and significantly up-regulated GRIM-19 gene expression in mice, and induce cells to induce cells. The results of apoptotic and transplanted tumor growth are best (P0.05). The results suggest that the expression of ADAM10-specific shRNA and GRIM-19 in hepatoma cells can significantly inhibit the proliferation, migration and invasion of tumor cells and the growth of tumor cells in vivo and in vivo.
Combined with the above results, ADAM10 is highly expressed in liver cancer. Silencing ADAM10 can promote the apoptosis of hepatoma cells, inhibit the proliferation, migration and invasion of HCC cells, and the growth of tumor. Co expression of ADAM10 silencing plasmids and GRIM-19 overexpressed plasmids can further inhibit the proliferation, migration, invasion and tumor growth of hepatoma cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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