抑制TRIM29的表达与胃癌细胞增殖关系的研究

发布时间：2018-10-24 09:37

【摘要】：背景胃癌是世界范围内因肿瘤死亡的第二大原因,为了更好地诊断与治疗胃癌,确定导致该疾病的发展与恶化的基因及其相关机制至关重要。一些研究显示,TRIM29是胃癌的一个重要致癌基因,并且TRIM29 mRNA的过量表达与不良预后和更低的生存率有关。然而,胃癌中TRIM29的作用机制大部分仍不清楚。RNA干扰技术是近些年来发现的一种特异性高且操作简单的基因RNA转录阻断技术。在本研究中,我们试着通过应用Real time qRT-PCR技术、Western-blot技术以及siRNA干扰技术检测患者胃癌组织中TRIM29基因和蛋白质的水平表达变化,从而为阐释胃癌患者中TRIM29表达与临床和预后的关系提供依据。目的1.探讨TRIM29的表达与胃癌患者临床和预后的关系。2.通过siRNA质粒转染,抑制过表达TRIM29的细胞,分析TRIM29在胃癌细胞中的作用机制。3.在裸鼠实验中,通过分析抑制过表达TRIM29细胞的成瘤情况,探讨TRIM29的表达水平变化对胃癌细胞的影响。方法1.收集2010年3月到2015年1月我院收治的胃腺癌患者38例,分为新辅助化疗组(16例)和直接手术组(22例),收集术前胃镜活检标本、标准D2切除手术的胃癌原发灶标本、淋巴结标本及正常胃黏膜,比较上述标本中TRIM29的1nRNA表达和蛋白质水平的变化,分析其原因。2.在不同胃癌细胞株中检测TRIM29 mRNA和蛋白表达水平,挑选过表达的胃癌细胞株,转染siRNA质粒后,使用Real time qRT-PCR和 Westernblot技术检测TRIM29、P53以及B-catenin的表达水平；MTT法检测细胞倍增时间并绘制生长曲线,流式细胞技术分析干扰前后细胞周期的分布。3.选择TRIM29过表达的胃癌细胞,转染siRNA质粒,移植于裸鼠皮下,观察移植肿瘤的大小与生长情况,并检测TRIM29的表达水平变化。结果1. Real-time qRT-PCR 与 Western-blot结果提示胃癌原发灶标本、阳性淋巴结标本中TRIM29 mRNA和蛋白水平表达显著高于正常胃黏膜、阴性淋巴结标本(P均0.001)。在直接手术组和新辅助化疗组中,TRIM29表达阳性患者的平均生存时间均显著低于TRIM29表达阴性的患者(Log-rank x2=4.068; P=0.044; Log-rank x2=7.487; P=0.006)。TRIM29日性患者新辅助化疗后mRNA的表达水平明显低于化疗前。2.不同胃癌细胞株中TRIM29的nRNA和蛋白质水平均发现有不同程度表达增高。p53的表达在TRIM29阳性表达的不同胃癌细胞株中均出现了明显下调,而B-catenin则出现了异常高表达。RNA干扰后,转染siRNA质粒的胃癌细胞株中TRIM29mRNA和蛋白表达水平降低,显著低于转染空白质粒的细胞。p53基因mRNA及其蛋白表达水平显著高于RNA干扰前,B-catenin基因mRNA及其蛋白表达水平显著低于干扰前。与RNA干扰前相比,TRIM29日性表达的胃癌细胞株在RNA干扰后细胞增殖能力显著降低。3. siRNA质粒转染前TRIM29过表达的胃癌细胞在5只裸鼠皮下生长60天内均出现转移瘤,肿瘤直径大小0.4～1.9cm,而siRNA质粒转染后TRIM29过表达的胃癌细胞在4只裸鼠发现实体肿瘤,直径大小0.1-0.5cm(P0.001),1只未发现肉眼可见实体肿瘤。TRIM29mRNA和蛋白水平在siRNA质粒转染前TRIM29过表达的胃癌细胞在裸鼠皮下形成的转移瘤中的表达显著高于siRNA质粒转染后。结论1.胃癌患者病理组织中TRIM29的mRNA和蛋白表达水平显著升高；TRIM29表达阳性的患者生存时间更短,预后更差。新辅助化疗可在一定程度上影响TRIM29的表达水平。2TRIM29是通过下调p53,提升β-catenin水平来达到增强肿瘤细胞增殖能力的生物学作用。3.通过裸鼠体内实验进一步证明了TRIM29可提升胃癌细胞的增殖能力。
[Abstract]:BACKGROUND OF THE INVENTION Gastric cancer is the second major cause of death in the world. For better diagnosis and treatment of gastric cancer, it is essential to determine the gene and its associated mechanism leading to the development and deterioration of the disease. Some studies have shown that TRIM29 is an important oncogenic gene in gastric cancer, and overexpression of TRIM29 mRNA is associated with poor prognosis and lower survival. However, most of the mechanisms of TRIM29 in gastric cancer are still unclear. RNA interference technique is a highly specific and easy-to-operate gene RNA transcription blocking technique found in recent years. In this study, we tried to detect the expression of TRIM29 gene and protein in gastric cancer tissues by using Real time qRT-PCR, Western-blot and siRNA interference technique, thus providing the basis for explaining the relationship between TRIM29 expression and clinical and prognosis in patients with gastric cancer. Purpose 1. To investigate the relationship between expression of TRIM29 and clinical and prognosis of gastric cancer patients. The effect mechanism of TRIM29 on gastric cancer cells was analyzed by transfection of siRNA plasmid and inhibition of the cells expressing TRIM29. In the nude mouse experiment, the effect of the expression level of TRIM29 on gastric cancer cells was studied by analyzing the tumorigenesis of TRIM29 cells. Method 1. 38 cases of gastric adenocarcinoma treated in our hospital from March 2010 to January 2015 were collected, divided into neoadjuvant chemotherapy group (16 cases) and direct operation group (22 cases). To compare the expression of 1nRNA and the protein level of TRIM29 in the above specimens, the reason was analyzed. The expression levels of TRIM29 mRNA and protein were detected in different gastric cancer cell lines. After transfection of siRNA plasmids, the expression levels of TRIM29, P53 and B-catenin were detected by Real time qRT-PCR and Western blot. The cell doubling time was detected by MTT method and the growth curve was drawn. Flow cytometry was used to analyze the distribution of cell cycle before and after interference. TRIM29 overexpressing gastric cancer cells were selected, siRNA plasmid was transfected, transplanted in nude mice, the size and growth of transplanted tumor were observed, and the expression level of TRIM29 was detected. Result 1. The results of Real-time qRT-PCR and Western-blot showed that the expression of TRIM29 mRNA and protein in the positive lymph node specimen was significantly higher than that of normal gastric mucosa and negative lymph node (P <0.001). In both the direct surgical group and the neoadjuvant chemotherapy group, the mean survival time of TRIM29 expression-positive patients was significantly lower than that of patients with negative expression of TRIM29 (Log-systemx2 = 4.068; P = 0. 034; Log-systemx2 = 7. 487; P = 0. 006). The expression level of mRNA after neoadjuvant chemotherapy in TRIM29 patients was significantly lower than that before chemotherapy. The levels of nRNA and protein in TRIM29 in different gastric cancer cell lines were increased. The expression of p53 was down-regulated in different gastric cancer cell lines with positive expression of TRIM29, while B-catenin showed abnormally high expression. After RNA interference, the expression level of TRIM29mRNA and protein in gastric cancer cell line transfected with siRNA plasmid was decreased, which was significantly lower than that of transfected blank plasmid. The expression level of p53 mRNA and its protein was significantly higher than that before RNA interference, and the expression level of B-catenin mRNA and its protein was significantly lower than that before RNA interference. Compared with RNA interference, the proliferation ability of gastric cancer cell line induced by TRIM29 was significantly decreased after RNA interference. The expression of TRIM29 before transfection of siRNA plasmid showed metastatic tumor within 60 days under the subcutaneous growth of 5 nude mice, and the tumor diameter was 0.4 ~ 1. 9cm. After the siRNA plasmid was transfected, the expression of TRIM29 showed solid tumors in 4 nude mice, and the diameter was 0. 1-0. 5cm (P0.05). The expression of TRIM29mRNA and protein level before siRNA plasmid transfection was significantly higher than that of siRNA plasmid. Conclusion 1. TRIM29 mRNA and protein expression increased significantly in patients with gastric cancer, and the survival time of TRIM29 positive patients was shorter and the prognosis was worse. Neoadjuvant chemotherapy can affect the expression level of TRIM29 to some extent. The ability of TRIM29 to promote the proliferation of gastric cancer cells was further demonstrated by experiments in nude mice.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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