以CDK8为靶点抑制结直肠癌肝转移的相关研究

发布时间：2018-07-04 07:56

本文选题：周期蛋白依赖性激酶8 + 结直肠癌　；参考：《广州医科大学》2017年博士论文

【摘要】：结直肠癌是常见的恶性肿瘤之一,肝脏转移是其最常见的远处转移形式,约25%的患者在临床确诊时已有肝转移,结直肠癌的预后与有无远处转移尤其是肝转移密切相关。近年随着技术的进步,以手术为中心的综合治疗提高了结直肠癌肝转移的治疗效果。但对其预后的改善仍不够理想。探求抑制结直肠癌肝转移的新疗法对于改善患者预后及延长生存期非常重要。基因治疗有望成为结直肠癌肝转移的新疗法。周期蛋白依赖性激酶8(CDK8)是结直肠癌的原癌基因,其对结直肠癌肝转移发生发展中的重要信号通路-Wnt/β-catenin通路有调控作用。因而CDK8有望成为结直肠癌肝转移的治疗靶点。第一部分结直肠癌组织中CDK8的表达目的:检测结直肠癌组织CDK8的表达并分析CDK8表达与结直肠癌临床病理特征之间的关系。方法:取结直肠癌石蜡切片标本70例,选取正常结直肠组织20例为对照。免疫组织化学法检测结直肠癌肿瘤组织与正常结直肠组织CDK8表达的差异;分析结直肠癌组织中CDK8表达与临床病理特征的关系。结果:结直肠癌组织CDK8表达阳性率为71.43%,高于正常结直肠组织30%的阳性率(P0.05)。结直肠癌组织中CDK8表达升高与临床分期进展、淋巴结转移、远处转移有关(P0.05);而与患者性别、年龄、肿瘤分化程度、血清CEA水平高低无关(P均0.05)。结论:1、结直肠癌组织中CDK8表达升高;2、CDK8升高与结直肠癌的分期及转移有关。第二部分以CDK8为靶点干预结直肠癌细胞增殖、迁移与侵袭目的:在细胞水平观察沉默CDK8基因对结直肠癌细胞增殖、迁移、侵袭能力的影响。方法:1、制备沉默CDK8基因的重组慢病毒LV-CDK8-sh RNA和对照组空病毒LV-NC,分别感染HCT116细胞;2、荧光定量PCR法检测细胞CDK8基因m RNA,免疫蛋白印迹法检测细胞CDK8蛋白表达;3、MTT实验检测细胞增殖能力;划痕实验检测细胞体外迁移能力;Transwell实验检测细胞侵袭能力。结果:1、成功构建沉默CDK8基因的HCT116细胞(对照组:HCT116-luc-GFP-NC;实验组:HCT116-luc-GFP-sh CDK8);2、HCT116-luc-GFP-sh CDK8细胞CDK8m RNA及蛋白表达均较HCT116-luc-GFP-NC细胞降低(P0.05);3、HCT116-luc-GFP-sh CDK8细胞增殖、迁移、侵袭能力均较HCT116-luc-GFP-NC细胞降低(P0.05)结论:1、LV-CDK8-sh RNA能有效沉默结肠癌HCT116细胞CDK8基因表达;2、沉默CDK8基因表达后,结肠癌HCT116细胞增殖、迁移及侵袭能力均受到抑制。第三部分下调CDK8抑制结直肠癌肝转移机制目的:动物实验观察沉默CDK8基因对结直肠癌肝转移的影响。方法:1、构建结直肠癌肝转移的BLAB/c nu/nu裸鼠实验模型;2、活体成像技术实时观察裸鼠肿瘤生长和转移;3、取裸鼠肝转移灶组织采用蛋白免疫印迹法检测CDK8、β-catenin、E2F1、MMP-7蛋白的表达。结果:1、成功建立裸鼠结直肠癌肝转移模型;2、沉默CDK8基因表达在裸鼠体内能有效抑制结直肠癌肝转移的发生发展;3、沉默CDK8基因表达后,裸鼠结直肠癌肝转移灶内CDK-8,β-catenin和MMP-7的表达减少,而E2F1表达增加。结论:1、沉默CDK8基因表达能有效裸鼠抑制结直肠癌肝转移的发生发展;2、沉默CDK8基因表达可通过抑制Wnt/β-catenin信号通路达到抑制裸鼠体内结直肠癌肝转移的目的。全文结论:沉默CDK8基因表达可以有效的抑制结直肠癌肝转移,CDK8可以作为抑制结直肠癌肝转移的靶点。
[Abstract]:Colorectal cancer is one of the most common malignant tumors. Liver metastasis is the most common form of distant metastasis. About 25% of the patients have liver metastases at the time of clinical diagnosis. The prognosis of colorectal cancer is closely related to distant metastasis, especially liver metastasis. In recent years, with the progress of technology, surgery based comprehensive treatment improves colorectal cancer liver. It is still not ideal for the improvement of the prognosis. A new therapy for inhibiting the liver metastasis of colorectal cancer is very important to improve the prognosis and prolong the survival period of the patients. Gene therapy is expected to be a new therapy for the liver metastasis of colorectal cancer. Cyclin dependent kinase 8 (CDK8) is a proto oncogene of colorectal cancer. -Wnt/ beta -catenin pathway, an important signaling pathway in the development of colorectal cancer, has a regulatory role. Therefore, CDK8 is expected to be a therapeutic target for liver metastasis in colorectal cancer. The expression of CDK8 in colorectal cancer tissue is in the first part: to detect the expression of CDK8 in colorectal cancer tissue and to analyze the relationship between CDK8 and clinicopathological features of colorectal cancer Methods: 70 cases of paraffin section of colorectal cancer were taken and 20 cases of normal colorectal tissue were selected as control. The difference between the expression of CDK8 in colorectal cancer tissues and normal colorectal tissues was detected by immunohistochemistry. The relationship between the expression of CDK8 and the clinicopathological features in colorectal cancer tissue was analyzed. Results: the expression of CDK8 in colorectal cancer tissues was positive. The rate of 71.43% was higher than the positive rate of normal colorectal tissue 30% (P0.05). The increase of CDK8 expression in the colorectal cancer tissues was related to the progression of clinical stages, lymph node metastasis and distant metastasis (P0.05); and the sex, age, the degree of differentiation of the tumor and the level of serum CEA were not related to the patients (P 0.05). Conclusion: 1, the expression of CDK8 in colorectal cancer tissues was elevated; 2, CDK 8 the increase is related to the staging and metastasis of colorectal cancer. The second part interfered with the proliferation, migration and invasion of colorectal cancer cells with CDK8 as the target: the effect of silent CDK8 gene on the proliferation, migration and invasion of colorectal cancer cells at the cell level. Methods: 1, the preparation of recombinant lentivirus LV-CDK8-sh RNA and control group silenced CDK8 gene The empty virus LV-NC, respectively, infected HCT116 cells, 2, the CDK8 gene m RNA was detected by the fluorescence quantitative PCR method, the expression of cell CDK8 protein was detected by immunoblotting; 3, the proliferation ability of the cells was detected by the MTT experiment; the scratch test was used to detect the cell migration ability; the Transwell test was used to detect the cell invasiveness. Results: 1, the HC CDK8 gene of silenced CDK8 was successfully constructed. T116 cells (control group: HCT116-luc-GFP-NC; experimental group: HCT116-luc-GFP-sh CDK8); 2, CDK8m RNA and protein expression in HCT116-luc-GFP-sh CDK8 cells were all lower than HCT116-luc-GFP-NC cells (P0.05); 3, HCT116-luc-GFP-sh CDK8 cells proliferation, migration, and invasion ability were lower than that of 1. Effective silence colon cancer HCT116 cell CDK8 gene expression; 2, after the silence of CDK8 gene expression, colon cancer HCT116 cell proliferation, migration and invasion ability are inhibited. The third part down regulation of CDK8 inhibition of colorectal cancer liver metastasis mechanism: animal experiments to observe the effect of silent CDK8 gene on colorectal cancer liver metastasis. Method: 1, construction of colorectal cancer BLAB/c nu/nu nude mice experimental model of liver metastasis; 2, living body imaging technique was used to observe the tumor growth and metastasis of nude mice in real time. 3, the expression of CDK8, beta -catenin, E2F1, MMP-7 protein was detected by protein immunoblotting in nude mice. Results: 1, the liver metastasis model of nude mice was successfully established, and 2, silent CDK8 gene expression in nude mice. Internal energy effectively inhibited the development and development of liver metastasis of colorectal cancer. 3, after the expression of silent CDK8 gene, the expression of CDK-8, beta -catenin and MMP-7 decreased in nude mice with colorectal cancer, and the expression of E2F1 increased. Conclusion: 1, silent CDK8 gene expression can effectively inhibit the development of liver metastasis in colorectal cancer, and 2, silent CDK8 gene expression can be passed through. The purpose of inhibiting Wnt/ beta -catenin signaling pathway to inhibit the liver metastasis of colorectal cancer in nude mice. Conclusion: the silence of CDK8 gene expression can effectively inhibit the liver metastasis of colorectal cancer, and CDK8 can be used as a target for inhibiting the metastasis of colorectal cancer.
【学位授予单位】：广州医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.34

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