TRIM3基因在原发性肝癌中表达及功能的初步研究

发布时间：2018-05-27 02:18

本文选题：TRIM3 + 肝癌　；参考：《广东药学院》2015年硕士论文

【摘要】：研究背景及目的肝癌(hepatocellular carcinoma,HCC)是一种全球常见的恶性肿瘤,在所有实体肿瘤中发病率居第五位,肿瘤相关死亡率居于第四位。我国为原发性肝癌的高发区,近几年呈现发病率上升的趋势。尽管目前肝癌的治疗手段有了很大程度的进步,但是肝癌预后并不理想,五年生存率只有25%-39%。导致肝癌预后差的主要原因是恶性程度高、血管侵犯及肝内转移。肝癌的发病过程是多种因素共同作用的结果,其中遗传因素中最主要的是癌基因激活和抑癌基因失活。为了改善肝癌患者预后,有必要寻找新的早期诊断和有效治疗肝癌的分子靶点。TRIM3蛋白又称BERP蛋白,最初是作为作为α-辅肌动蛋白4结合蛋白而被克隆出来,但是它的具体功能目前并不是十分明确。TRIM家族蛋白在结构上均有功能相似的3个结构域(靠近N端的锌指结构域、位于中间的1-2个B-box结构域、C端的卷曲螺旋结构域),也因此而得名。近年来有多项研究证实TRIM家族蛋白成员与肿瘤的发生发展密切相关,有研究证实在恶性胶质瘤中TRIM3是一个新的抑癌基因,TRIM3发挥其抑制细胞生长的作用是通过抑制p21蛋白表达而起作用。TRIM3表达下调能促进细胞中p21的表达增加,并且能促进细胞增殖和小鼠肿瘤的生长和血管形成。过表达TRIM3可使细胞在细胞周期G0/1期累积,导致细胞生长阻滞。因此认为TRIM3可能是一个潜在的抑癌基因,然而,到目前为止,TRIM3在肝癌中的作用仍不清楚。本课题的目的是通过检测TRIM3基因在肝癌中的表达,分析其与肿瘤患者临床参数及预后的关系,并初步研究TRIM3在肝癌的发生过程当中的生物学功能。研究方法实时荧光定量RT-PCR、Western Blotting和免疫组织化学技术检测肝癌细胞系及肝癌组织标本中TRIM3的表达情况。通过转染TRIM3基因片段,使TRIM3蛋白在Hep G2和Bel-7402中过表达,研究TRIM3的表达对肝癌细胞生长(增殖、克隆形成)、细胞周期、细胞凋亡、细胞转移(迁移及侵袭)等生物学功能的影响。统计方法:配对t检验主要用于肝癌组织与癌旁正常组织TRIM3转录和蛋白表达的差异比较;卡方检验主要用于高表达组和低表达组临床参数之间的差异比较;生存分析用于比较高表达组和低表达组生存时间的比较;Cox回归分析主要用于研究与肝癌术后总体生存时间相关的影响因素。结果1.肝癌组织中TRIM3 m RNA和蛋白的表达水平显著低于配对的癌旁组织(p0.05)。与肝正常细胞系LO2相比,TRIM3在肝癌细胞株中的表达下调,尤其是在Hep G2和Bel7402这两株肝癌细胞中。2.免疫组织化学法检测了129例石蜡包埋组织中TRIM3蛋白的表达情况,发现TRIM3蛋白主要表达在细胞浆中,相对于癌组织的低表达,癌旁的肝组织为高表达;TRIM3表达与肿瘤大小(P=0.034)、病理分级(P0.001)、TNM分期(P=0.021)和血清AFP水平(P=0.025)显著相关;生存分析表明TRIM3低表达的患者预后不良,可作为肝癌患者独立的预后指标。3.构建过表达TRIM3的慢病毒表达载体,感染Hep G2和Bel7402细胞,发现过表达TRIM3能抑制肝癌细胞的增殖、克隆形成、以及迁移侵袭能力,诱导细胞周期阻滞于G1期,促进肿瘤细胞的凋亡。结论TRIM3在肝癌的发生发展中具有重要作用,可以作为肝癌分子靶向治疗的靶点和肝癌预后的一个独立指标。
[Abstract]:Background and objective hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The incidence of cancer is fifth in all solid tumors and fourth in tumor related mortality. China is a high incidence area of primary liver cancer in China. The prognosis of liver cancer is not good. The prognosis of liver cancer is not ideal. The main cause of poor prognosis of the five year survival rate is high malignancy, vascular invasion and intrahepatic metastasis. The pathogenesis of liver cancer is the result of a variety of factors, of which the most important of the genetic factors is the activation of oncogene and the inactivation of tumor suppressor genes. The prognosis of good liver cancer patients, it is necessary to find new early diagnosis and effective treatment of liver cancer molecular target.TRIM3 protein called BERP protein, which was originally cloned as alpha cofactor 4 binding protein, but its specific function is not very clear about the 3 knots of the.TRIM family protein in structure. The domain (the domain of the zinc finger near the N, 1-2 B-box domains in the middle, the curl spiral domain in the C end) is also named. In recent years, a number of studies have confirmed that the members of the TRIM family protein are closely related to the development of the tumor. Studies have shown that TRIM3 is a new tumor suppressor gene in the malignant glioma, and TRIM3 plays its inhibition. The role of cell growth is to inhibit the expression of p21 protein by inhibiting the expression of.TRIM3, which can promote the increase of p21 expression in cells, and promote cell proliferation and tumor growth and angiogenesis in mice. Overexpression of TRIM3 can cause cells to accumulate in the G0/1 phase of cell cycle, leading to cell growth block. Therefore, TRIM3 may be a possible one. Potential tumor suppressor genes, however, the role of TRIM3 in liver cancer is still unclear so far. The purpose of this study is to analyze the relationship between the expression of TRIM3 gene in liver cancer, analyze the relationship with the clinical parameters and prognosis of the cancer patients, and preliminary study the biological function of TRIM3 in the process of liver cancer. Quantitative RT-PCR, Western Blotting and immunohistochemical technique were used to detect the expression of TRIM3 in liver cancer cell lines and hepatocellular carcinoma tissue specimens. By transfecting TRIM3 gene fragments, TRIM3 protein was overexpressed in Hep G2 and Bel-7402, and the expression of TRIM3 was studied for the growth of hepatoma cells (proliferation, clone formation), cell cycle, cell apoptosis and cell transformation. The effects of biological functions such as migration and invasion. Statistical methods: the paired t test was used to compare the difference in the TRIM3 transcription and protein expression between the liver cancer tissue and the paracancerous normal tissues; the chi square test was mainly used to compare the differences between the high expression group and the low expression group, and the survival analysis was used to compare the high expression group and the low expression group. Comparison of survival time; Cox regression analysis was mainly used to study the influence factors related to the overall survival time after liver cancer. Results 1. the expression level of TRIM3 m RNA and protein in liver cancer tissues was significantly lower than that of the paired paracancerous tissue (P0.05). The expression of TRIM3 in the hepatocellular carcinoma cell lines was down, especially in Hep G, compared with the normal liver cell line LO2. The expression of TRIM3 protein in 129 paraffin embedded tissues was detected by.2. immunohistochemistry in 2 and Bel7402 hepatoma cells. The expression of TRIM3 protein was mainly expressed in the cytoplasm. The expression of the liver tissue near the cancer tissue was highly expressed, the expression of TRIM3 and the size of the tumor (P=0.034), the pathological grade (P0.001), and the TNM staging (P=0). .021) was significantly related to serum AFP level (P=0.025); survival analysis showed that the patients with low expression of TRIM3 had poor prognosis, and could be used as an independent prognostic indicator of liver cancer patients.3. to construct a lentivirus expression vector expressing TRIM3, infected with Hep G2 and Bel7402 cells, and that overexpressed TRIM3 could inhibit the proliferation, clone formation, and migration invasion of hepatoma cells. The ability to induce cell cycle arrest in G1 phase and promote the apoptosis of tumor cells. Conclusion TRIM3 plays an important role in the development and development of liver cancer. It can be used as a target for the target therapy of liver cancer and an independent indicator of the prognosis of liver cancer.
【学位授予单位】：广东药学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7

【参考文献】