Chk1介导的CCNB1高表达在结直肠癌中的作用及机制研究

发布时间：2018-09-06 11:29

【摘要】：结直肠癌是严重危害人类健康的主要恶性肿瘤之一,其发病率及病死率在女性恶性肿瘤中高居第二位、男性中高居第三位,仅次于肺癌和乳腺癌。每年全球结直肠癌新发病例超过136万,死亡病例约69万。在我国,其发病率随着人民生活水平的提高、饮食结构向高脂高蛋白低纤维素方向变化而逐年上升,结直肠癌已成为我国近30年来发病率增长最快的恶性肿瘤。随着近年来医学水平的不断进步,人们对于结直肠癌的治疗水平也有了长足的发展,目前关于结直肠癌发生发展及浸润、扩散、转移机制研究方兴未艾。结直肠癌的发生发展是一个极复杂的过程,不仅与环境、饮食及生活习惯等各种外在因素有关,其内在更是一个多阶段、多步骤、多基因参与的复杂过程。尽管在结直肠癌的发生发展过程中有大量的分子事件被发现,这些分子事件在这一过程中发挥着至关重要的作用,但这些分子事件在肿瘤发生发展过程中的具体作用尚未完全清楚。细胞周期素B1 (CCNB1)是细胞周期蛋白家族中一个重要的成员,是细胞G2/M检测点有关的重要细胞周期调控因子。它的一个重要角色就是调节细胞周期蛋白依赖性激酶1(CDK1)并与之形成复合体后使底物磷酸化,启动细胞从G1/S期进入G2/M期,并促进有丝分裂。越来越多的证据表明CCNB1参与了细胞周期检测点的控制,它的功能障碍是肿瘤发生中的早期事件,在人类许多肿瘤中可以观察到其失调控表达,包括乳腺癌、宫颈癌、肺癌等。同时,有证据表明抑制CCNB1的表达可以使乳腺癌细胞对化疗更加敏感。此外,研究表明在肿瘤细胞中野生型P53通过SP1转录因子抑制CCNB1的转录表达。在结直肠癌细胞株HCT116 p53+/+细胞中CCNB1的蛋白水平呈低表达,而在HCT116 p53-/-细胞中呈高表达。这些研究表明CCNB1在肿瘤发展中潜在的关键作用,然而,CCNB1在结直肠癌中的具体功能机制尚未可知。大部分癌症被认为高度依赖于细胞周期检查点激酶1(Chk1)的异常表达,并且CCNB1在以前是被作为预测Chk1抑制剂的疗效的生物标记物。目前对于CCNB1和Chk1各自在肿瘤中的表达机理及临床意义的研究较多,但对二者在结直肠癌中是否存在相关性以及二者通过何种途径共同影响结直肠癌的发生、发展方面的研究仍不清楚。本研究通过检测CCNB1、Chk1在结直肠癌和正常结直肠组织标本中的表达情况、分析其相关性,并通过体外细胞功能实验验证Chk1是CCNB1的正向调控因子,同时结合体内外实验深入探索CCNB1对结直肠癌细胞生物学行为的影响,探讨其发挥生物学效应的具体作用机制。本研究旨在揭示结直肠癌发生发展的新机制,并为探寻结直肠癌防治新策略提供坚实可靠的实验证据和理论支持。第一部分CCNB1在结直肠癌中的表达及功能研究目的：验证CCNB1在结直肠癌组织中表达升高,明确CCNB1对结直肠癌细胞增殖、周期、凋亡等生物学行为的影响。方法：收集结直肠癌组织和癌旁正常组织标本,采用实时荧光定量PCR (quantitative real-time RT-PCR, qRT-PCR)法检测30对结直肠癌组织及癌旁正常组织的CCNB1 mRNA和蛋白的表达水平。分别选取P53-wild HCT116、P53-mutant SW480和 P53-null HCT116细胞株作为实验对象,利用细胞转染技术分别转染CCNB1特异性siRNA或阴性对照siRNA,并采用MTT法检测肿瘤细胞的增殖能力、PI单染检测细胞周期分布、Annexin-V联合PI法检测细胞凋亡、western blot检澳C CNB1调控的下游周期蛋白及凋亡蛋白表达。结果：1. CCNB1在结直肠癌组织中的表达较癌旁正常组织显著升高。2.在结直肠癌细胞株P53-wild HCT116、SW480 和 P53-null HCT116中,抑制CCNB1的表达均能能抑制肿瘤细胞的增殖,并能通过调节细胞周期相关分子CDK1/CDC25C促使细胞周期发生G2/M期阻滞。3.在P53-wildHCT116细胞中抑制CCNB1表达能够通过调节p53/B ax通路促使肿瘤细胞发生早期凋亡,而在P53-null HCT116细胞中抑制CCNB1表达也能通过非p53途径促进肿瘤细胞发生早期凋亡。结论：1. CCNB1在结直肠癌组织中表达显著升高。2.下调CCNB1的表达可抑制结直肠癌细胞增殖、诱导细胞周期G2/M期阻滞和促进肿瘤细胞凋亡,提示CCNB1在结直肠癌发生过程中发挥致癌作用。第二部分结直肠癌CCNBl高表达依赖于细胞周期检查点激酶1(Chkl)的表达目的：确定Chkl是CCNBl的上游调控分子,揭示Chk1在结直肠癌中的作用机制。方法：首先,分别在结直肠癌组织和癌旁正常组织中用qRT-PCR法检测Chk1 mRNA的表达水平,western blot法检测Chkl的蛋白表达水平,分析与CCNB1的组织相关性。其次,利用siRNA特异性干扰结直肠癌细胞中Chkl的表达,western blot检测CCNB1的蛋白表达,MTT法检测肿瘤细胞的增殖能力,明确Chkl在结直肠癌中的作用和对CCNB1的调控作用。结果：1.Chk1在结直肠癌组织中的表达水平均较癌旁正常组织明显增高,并与CCNB1表达水平呈显著正相关。2.干扰结直肠癌细胞Chk1的表达可抑制CCNB1的蛋白水平,细胞增殖能力受阻。结论：Chkl在结直肠癌中靶向调控CCNB1的表达,提示Chkl是CCNB1的上游调控分子。第三部分慢病毒介导的CCNB1 shRNA对结直肠癌裸鼠移植瘤生长抑制作用的研究目的：明确持续抑制CCNB1表达对裸鼠移植瘤生长的调控作用。方法：首先构建含特异性靶向CCNB1基因的短发夹RNA(shRNA)'陧病毒表达载体,将慢病毒分别转导HCT116和SW480细胞株,通过抗生素筛选后得到各自的CCNB1稳定低表达细胞株,western blot法检测CCNB1的表达水平以明确稳筛效率。将上述稳转细胞株及相对应的慢病毒空载体转导的对照组细胞株分别接种于裸鼠皮下,观察成瘤及移植瘤生长情况,免疫组织化学染色方法检测移植瘤中CCNB1的表达,western blot检测移植瘤中与CCNB1的下游分子,包括CDC25C、 CDK1、p53和Bax的表达。结果：1.抑制CCNB1的表达可抑制裸鼠移植瘤的生长速度。2.在移植瘤中抑制CCNB1表达能够抑制CCNB1下游细胞周期相关蛋白CDC25C和CDK1的表达下降,在HCT116移植瘤中也能下调下游凋亡相关蛋白p53 和 Bax的表达。结论：裸鼠体内成瘤实验发现抑制CCNB1可抑制结直肠癌移植瘤的生长,并与体外研究一致。
[Abstract]:Colorectal cancer is one of the major malignant tumors that seriously endanger human health. The incidence and mortality of colorectal cancer rank second in female malignancies, third in male malignancies, second only to lung cancer and breast cancer. With the development of Medical Science in recent years, the level of treatment for colorectal cancer has also been greatly improved. At present, the incidence of colorectal cancer has been related to the occurrence of colorectal cancer. The development and progression of colorectal cancer is a complex process, not only related to external factors such as environment, diet and living habits, but also a complex process involving multiple stages, steps and genes. Cyclin B1 (CCNB1) is an important member of the cyclin family and an important cell related to G2/M detection sites in cells. Cyclin-dependent kinase-1 (CDK1) is an important regulator of cell cycle. It regulates and forms a complex with cyclin-dependent kinase-1 (CDK1) to phosphorylate the substrate, initiates the cell from G1/S phase to G2/M phase, and promotes mitosis. Inhibition of CCNB1 expression has been shown to make breast cancer cells more sensitive to chemotherapy. In addition, studies have shown that wild-type P53 inhibits CCNB1 transcription through SP1 transcription factors in tumor cells. The expression of CCNB1 was low in HCT116 p53+/+ cells and high in HCT116 p53-/-cells. These studies suggest that CCNB1 may play a key role in tumor development. However, the specific functional mechanism of CCNB1 in colorectal cancer remains unknown. Most cancers are thought to be highly dependent on cells. The abnormal expression of cyclic checkpoint kinase 1 (Chk1) and CCNB1 were previously used as biomarkers for predicting the efficacy of Chk1 inhibitors. At present, there are many studies on the expression mechanism and clinical significance of CCNB1 and Chk1 in colorectal cancer, but whether there is a correlation between them and which pathway they share. In this study, we examined the expression of CCNB1 and Chk1 in colorectal cancer and normal colorectal tissues, and analyzed the correlation between them. Chk1 was proved to be a positive regulator of CCNB1 by cell function test in vitro and in vivo and in vitro. The purpose of this study is to reveal the new mechanism of the occurrence and development of colorectal cancer and to provide solid and reliable experimental evidence and theoretical support for exploring new strategies for the prevention and treatment of colorectal cancer. Part I: Expression and function of CCNB1 in colorectal cancer Objective: To verify the elevated expression of CCNB1 in colorectal cancer tissues and to determine the effect of CCNB1 on the proliferation, cycle and apoptosis of colorectal cancer cells. The expression levels of CCNB1 mRNA and protein in normal tissues and adjacent tissues were measured. P53-wild HCT116, P53-mutant SW480 and P53-null HCT116 cell lines were selected as experimental subjects. CCNB1-specific siRNA or negative control siRNA were transfected by cell transfection technique. The proliferation of tumor cells was detected by MTT assay, and the cell cycle was detected by PI single staining. Results: 1. The expression of CCNB1 in colorectal cancer tissues was significantly higher than that in adjacent normal tissues. 2. In colorectal cancer cell lines P53-wild HCT116, SW480 and P53-null HCT116, the expression of CCNB1 was inhibited. Inhibition of CCNB1 expression in P53-wildHCT116 cells can promote early apoptosis of tumor cells by regulating the p53/B ax pathway, and inhibit the expression of CCNB1 in P53-null HCT116 cells. Conclusion: 1. The expression of CCNB1 in colorectal cancer tissues increased significantly. 2. The down-regulation of CCNB1 expression can inhibit the proliferation of colorectal cancer cells, induce G2/M phase arrest of cell cycle and promote apoptosis of tumor cells, suggesting that CCNB1 plays a carcinogenic role in the development of colorectal cancer. High expression of CCNBl in colorectal cancer depends on the expression of cell cycle checkpoint kinase 1 (Chkl). To determine that Chkl is the upstream regulatory molecule of CCNBl and to reveal the mechanism of Chk1 in colorectal cancer. Methods: First, the expression of Chk1 mRNA was detected by qRT-PCR in colorectal cancer tissues and normal tissues adjacent to the cancer, and Western blot was used to detect the expression of Chk1 mRNA. Secondly, siRNA was used to specifically interfere with the expression of Chkl in colorectal cancer cells, Western blot was used to detect the expression of CCNB1 protein, MTT assay was used to detect the proliferation of tumor cells, and the role of Chkl in colorectal cancer and the regulatory role of CCNB1 were clarified. The expression level of CCNB1 in colorectal cancer tissues was significantly higher than that in adjacent normal tissues, and was positively correlated with the expression level of CCNB1. 2. Interference with the expression of Chk1 in colorectal cancer cells could inhibit the protein level of CCNB1 and inhibit cell proliferation. Molecule. Part III. Lentiviral-mediated inhibition of CCNB1 shRNA on colorectal cancer xenografts in nude mice Objective: To investigate the regulatory effect of continuous inhibition of CCNB1 expression on the growth of transplanted tumors in nude mice. HCT116 and SW480 cell lines were screened by antibiotics to obtain stable low-expression CCNB1 cell lines. The expression level of CCNB1 was detected by Western blot to determine the screening efficiency. The expression of CCNB1 was detected by immunohistochemical staining and the expression of CDC25C, CDK1, p53 and Bax was detected by Western blot. Results: 1. Inhibition of CCNB1 expression could inhibit the growth of transplanted tumor in nude mice. 2. Inhibition of CCNB1 expression in transplanted tumor could inhibit the downstream cells of CCNB1. The expression of cyclin-associated proteins CDC25C and CDK1 was down-regulated, and the expression of apoptosis-related proteins p53 and Bax was down-regulated in HCT116 transplanted tumors. Conclusion: Inhibition of CCNB1 in nude mice can inhibit the growth of colorectal cancer xenografts, which is consistent with the study in vitro.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.34

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