探究CUL4B表达对胃癌细胞的影响

发布时间：2018-07-18 14:01

【摘要】：据统计,胃癌是世界上第五大恶性肿瘤,致死率占世界第三位[1]。胃癌在不同国家和地区之间的发病率存在很大差异,亚洲地区胃癌病例数占世界总病例数的73.6%,其中中国的胃癌患者数占世界的42.7%。在2015年中国确诊的癌症病例中,男性的胃癌发病率占全部癌症发病率的第二位,女性的为第三位。针对胃癌的主要治疗方法包括手术切除、化疗、免疫疗法和定向治疗。虽然近年来诊断和治疗技术都有所提高,但是晚期胃癌病人的生存期仍旧低于1年,临床上缺乏诊断早期胃癌的有效方法,晚期胃癌病人预后很差。CUL4B(Cullin 4B)作为支架蛋白参与构成E3泛素连接酶复合物Cullin 4B-Ring E3 Ligase(CRL4B),参与调节包括细胞周期、细胞信号转导、基因转录和生物体个体生长发育等多项重要生命活动。CRL4B不仅可以通过泛素化降解p53和cyclin E参与调控细胞增殖和衰老过程,还能通过单泛素化H2A蛋白,调节靶基因启动子组蛋白H3的乙酰化以及甲基化等过程,从而在表观遗传水平上调控p16、p21、PTGDS、miRNA-371/372等基因的表达。越来越多的证据显示CRL4B在肿瘤发生发展中发挥重要作用。CRL4B可以通过单泛素化H2AK119,抑制Wnt拮抗因子、PTEN、IGFBP3和miR-194的表达。在宫颈癌、肝癌和非小细胞肺癌等癌症中,CRL4B通过抑制肿瘤抑癌基因的表达发挥癌基因的功能。有趣的是,本实验室还有一项研究结果提示CUL4B可通过调节微环境中MDSC细胞的分化抑制肿瘤的生长。因此,CUL4B在不同环境下在肿瘤的发生和发展发挥不同的功能。截止到目前,CUL4B在胃癌中的作用还未见报道,为了探究CUL4B在胃癌中发挥的功能,我们首先检测了 CUL4B在胃癌组织中的表达水平,并分析了 CUL4B表达对胃癌细胞增殖、迁移侵袭以及对化疗药物敏感性的影响。首先,我们检测了CUL4B在胃癌和癌旁组织中的表达,结果显示,相对于癌旁组织,CUL4B在胃癌组织中表达水平明显上调,并且CUL4B的表达与胃癌的病理分级呈正相关。随后我们选用了三种胃癌细胞—MKN-45、BGC-823和SGC-7901,利用病毒感染,分别构建了三种CUL4B低表达的胃癌细胞系和对应的的空载体对照细胞系,并且构建了MKN-45 CUL4B高表达细胞系和对应的空载体对照细胞系。我们利用MTT实验检测了 CUL4B表达降低后胃癌细胞增殖活性的改变,结果发现CUL4B表达降低抑制胃癌细胞增殖活性。利用流式法检测细胞周期时我们并未发现CUL4B表达降低对胃癌细胞周期存在显著影响,利用细胞动力学分析显示CUL4B不影响胃癌细胞G1/S其以及M/G1期进程,EdU掺入实验也显示CUL4B表达降低对胃癌细胞DNA合成无明显影响,提示CUL4B调控胃癌细胞增殖活性并不是通过对以上过程的调控而实现的。接下来我们利用transwell实验和划痕实验分析CUL4B表达对胃癌细胞迁移侵袭能力的影响,划痕实验结果显示,相对于对照细胞,CUL4B表达降低的细胞迁移能力明显下降。随后,我们采用MTT实验分析发现,相对于对照细胞,低表达CUL4B的胃癌细胞对于同等浓度化疗药物顺铂和吡柔比星的敏感性增加。TUNEL实验分析显示CUL4B低表达胃癌细胞在顺铂处理后凋亡比例更高。这些结果表明CUL4B参与调控胃癌细胞对化疗药物的敏感性。总之,通过本研究我们证实了 CUL4B在胃癌中表达上调,并且CUL4B表达水平与胃癌的病理分级呈正相关;细胞学实验结果显示抑制CUL4B表达会抑制胃癌细胞的增殖活性、侵袭迁移能力并增加胃癌细胞对化疗药物的敏感性,这些结果提示我们CUL4B在胃癌中发挥原癌基因的作用。该结果为进一步深入探究CUL4B功能提供了一定的实验数据,为阐明胃癌的发生机制、筛选新的肿瘤标志基因和研究新的疗法奠定了一定的理论基础。
[Abstract]:According to statistics, gastric cancer is the fifth largest malignant tumor in the world. The mortality rate of the third [1]. gastric cancer in the world is very different in different countries and regions. In Asia, the number of cases of gastric cancer accounts for 73.6% of the total number of cases in the world, of which the number of gastric cancer patients in China accounts for the world's 42.7%. in the 2015 Chinese confirmed cancer cases. The incidence of sexual gastric cancer accounts for second of the total cancer incidence and third in women. The main treatment methods for gastric cancer include surgical resection, chemotherapy, immunotherapy, and directional therapy. Although diagnosis and treatment techniques have improved in recent years, the survival period of patients with advanced gastric cancer is still less than 1 years, and the early diagnosis is lack of early diagnosis. Effective methods of gastric cancer, the prognosis of patients with advanced gastric cancer is poor.CUL4B (Cullin 4B) as a scaffold protein involved in the formation of E3 ubiquitin ligase complex Cullin 4B-Ring E3 Ligase (CRL4B), involved in the regulation of cell cycle, cell signal transduction, gene transcription and individual growth and development of organisms, such as many important activities of.CRL4B can not only pass through the life of.CRL4B. Excessive ubiquitination of p53 and cyclin E participates in the regulation of cell proliferation and senescence, and can also regulate the expression of p16, p21, PTGDS, and miRNA-371/372 by regulating the expression of p16, p21, PTGDS, and miRNA-371/372 through a single ubiquitin H2A protein, regulating the acetylation and methylation of the target gene promoter H3. More and more evidence shows that CRL4B is swollen. .CRL4B can play an important role in the development of tumor. H2AK119 can be used to inhibit the expression of Wnt antagonist, PTEN, IGFBP3 and miR-194. In cancer of cervical cancer, liver cancer and non-small cell lung cancer, CRL4B plays oncogene function by inhibiting the expression of tumor suppressor genes. Interestingly, there is also a research result in this laboratory. It is suggested that CUL4B can inhibit the growth of tumor by regulating the differentiation of MDSC cells in microenvironment. Therefore, CUL4B plays different functions in the occurrence and development of tumor in different environments. To the present, the role of CUL4B in gastric cancer has not been reported. In order to explore the function of CUL4B in gastric cancer, we first detected CUL4B in gastric cancer. The expression level in the tissue, and the effect of CUL4B expression on the proliferation, migration and invasion of gastric cancer cells and the sensitivity to chemotherapeutic drugs. First, we detected the expression of CUL4B in gastric and paracancerous tissues. The results showed that the expression level of CUL4B in gastric cancer tissues was significantly up-regulated compared with the Para cancerous tissues, and the expression of CUL4B and gastric cancer was associated with gastric cancer. Three kinds of gastric cancer cells, MKN-45, BGC-823 and SGC-7901, were then used to construct three kinds of CUL4B low expression gastric cancer cell lines and corresponding unloaded body control cell lines, and the MKN-45 CUL4B high expression cell line and the corresponding empty body control cell line were constructed. MTT test was used to detect the proliferation activity of gastric cancer cells after the decrease of CUL4B expression. The results showed that the expression of CUL4B decreased to inhibit the proliferation activity of gastric cancer cells. We did not find that the decrease of CUL4B expression had a significant effect on the cell cycle of gastric cancer by flow method. The results of cell dynamics analysis showed that CUL4B did not affect the stomach. The cancer cell G1/S and the M/G1 phase process, EdU incorporation test also showed that the decrease of CUL4B expression had no obvious effect on the DNA synthesis of gastric cancer cells, suggesting that CUL4B regulation of the proliferation activity of gastric cancer cells was not realized by the regulation of the above process. Then we used Transwell experiment and scratch test to analyze the migration of CUL4B expression to gastric cancer cell migration. The effect of invasive ability, the results of scratch test showed that the cell migration ability of CUL4B expression decreased significantly compared with the control cells. Then, we used MTT test to find that the sensitivity of gastric cancer cells with low expression of CUL4B to cisplatin and pirirubicin with the same concentration was increased by.TUNEL experimental analysis relative to the control cells. The results showed that the CUL4B low expression of gastric cancer cells had a higher percentage of apoptosis after cisplatin treatment. These results showed that CUL4B was involved in regulating the sensitivity of gastric cancer cells to chemotherapeutic drugs. In conclusion, we confirmed that the expression of CUL4B in gastric cancer was up regulated by this study and that the level of CUL4B expression was positively correlated with the classification of gastric cancer; cytological experimental results showed that Inhibition of CUL4B expression can inhibit the proliferation of gastric cancer cells, invasion and migration and increase the sensitivity of gastric cancer cells to chemotherapeutic drugs. These results suggest that CUL4B plays the role of proto oncogene in gastric cancer. The results provide some experimental data for further exploring the function of CUL4B, in order to elucidate the mechanism of gastric cancer. The selection of new tumor marker genes and the study of new therapies have laid a theoretical foundation.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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