Chk1-induced CCNB1 Overexpression Promotes Cell Proliferation And Tumor Growth In Human Colorectal Cancer

Colorectal cancer is one of the major causes of cancer-related morbidity and mortality, ranking as the third most common malignancy in the worldwide, with an estimated incidence of 1 360 000 new cancer cases and over 600 000 deaths occurring each year. As the increase of living standard and the change of diet structure, the incidence rates of Colorectal cancer have been increasing in our country. With the standard of medical increasing, more and more studies have been made about Colorectal cancer occurrence, development and metastatic mechanism in recent years.The development of colorectal cancer is a multistep process characterized by accumulation of epigenetic and genetic events, and influenced by lifestyle. Despite a number of molecular events having been discovered, the process of tumor initiation and progression is still unclear and molecules that play a crucial role in this process remain to be identified.CCNB1, an important member of cyclin family, is a key initiator and rigorous quality control step of mitosis. It has a pivotal role in regulating cyclin-dependent kinase 1 (CDK1) and forming complex with it, which phosphorylates their substrates to promote the transition of cell cycle from G2 phase to mitosis. Increasing evidence demonstrates that CCNB1 is involved in checkpoint control, whose dysfunction is an early event in tumorigenesis, and that its deregulated expression is observed in a number of different human cancers including breast cancer, cervical cancer, lung cancer. In parallel, evidence has showed that inhibition of CCNB1 expression renders breast cancer cells more sensitive to chemotherapy drug. Furthermore, it has been revealed that wild-type p53 represses the transcription of CCNB 1 through Spl transcription factor in tumor cells, and the protein level of CCNB1 is undetectable in HCT116 p53+/+cells, while overexpressed in HCT116 p53-/-cells. These data highlight the potential pivotal roles of CCNB 1 in the tumor development. However, detailed functional characteristics of CCNB 1 are still unknown in colorectal cancer to date.In mammalian cells, a large proportion of human cancers are thought to be highly reliant on the checkpoint kinase 1 (Chkl) expression, and CCNB1 was previously suggested to serve as a biomarker predictive of the efficacy of Chkl inhibitors. Despite a number of the mechanism of expressed and clinical significance about CCNB1,Chkl respectively in tumor having been discovered, but for those correlation in colorectal cancer as well as through what way to affect the occurrence and development of colorectal cancer research is less.Here, we validated Chkl was a right regulator of CCNB1 and observe the influence of modulating CCNB1 expression on biobehaviors of colorectal cancer cells and growth of transplanted tumor through different modern molecular biology techniques, accordingly elucidate the key role and mechanism of CCNB1 overexpression in promoting colorectal tumorigenesis. The aim of our study is to provide insight of new mechanism of the development of colorectal cancer and obtain experimental evidence for exploring new strategy for colorectal cancer therapy and prevention.Part Ⅰ The expression and role of CCNB1 in colorectal cancerObjectiveTo verify the overexpression of CCNB1 in colorecal cancer tissues; and to investigate the biological actions of CCNB1 in colorectal cancer cells.MethodsExpression of CCNB1 was examined between colorectal cancer tissues and their pair-matched adjacent normal colorectal tissues from 30 individual patients using quantitative real-time RT--PCR (qRT-PCR) and western blot. We then examined the expression of CCNB1 in a panel of five human colorectal cancer cell lines (named HCT116, RKO, HT29, SW480, and SW620) by western blot, and chose HCT116 and SW480 cell lines, where CCNB1 was expressed respectively lower and higher among the five cell lines, for further investigation. siRNA or negative control was transfected into two human colorectal cancer cells lines, HCT116 and SW480 cells. Cell proliferation experiments were carried out by MTT assay. Cell cycle and apoptosis analysis were conducted by PI kit and Annexin-V&PI kit, respectively. Western blot was used to detect the downstream targets of CCNB1.Results1. Expression of CCNB1 was frequently overexpressed in colorectal cancer tissues compared with adjacent normal colorectal tissues.2. Repression of CCNB1 suppressed cell proliferation, induced apoptosis, blocked G2/M transition through regulates cell cycle factors CDK1/CDC25C in both two colorectal cancer cells HCT116 and SW480 cells.3. Ectopic inhibition of CCNB1 might induce apoptosis enhancement through activating p53 and Bax in p53-wild HCT116 cells.4. Inhibition of CCNB1 suppresses cell proliferation, blocks cell cycle progression, and induces apoptosis in p53-null HCT116 cells.Conclusions1. CCNB1 is overexpressed in human colorectal cancer tissues.2. Inhibition of CCNB1 expression suppresses cell proliferation, blocks cell cycle progression, and induces apoptosis in certain colorectal cancer cells.Part Ⅱ Elevated CCNB1 expression participate in the progression of colorectal cancer is dependent on checkpoint kinase 1(Chk1) expressionObjectiveTo verify Chk1 might be the right regulator of CCNB1 in colorectal cancer and to reveal the mechanism of Chkl in olorectal cancer progression.MethodsFirstly, qRT-PCR was used to detect Chkl mRNA level, and western blot was used to detect Chkl protein level between colorectal cancer tissues and their pair-matched adjacent normal colorectal tissues. And the association of Chkl and CCNB1 expression in colorectal cancer patients were analyzed. Transfected specific siRNA targeting Chkl to HCT116 and SW480 cells. Western blot was used to detect the downstream targets of CCNB1. Cell proliferation experiments were carried out by MTT assay. To validate Chkl might be the right regulator of CCNB1 in colorectal cancer.Results1. Expression of Chkl was frequently overexpressed in both mRNA and protein levels in colorectal cancer tissues compared with adjacent normal colorectal tissues and correlated positively with CCNB1 protein expression.2. Downregulation of Chkl suppressed CCNB1 protein expression in both two cell lines. Moreover, Inhibition of Chkl significantly decreased the growth rate of both colorectal cancer cell lines.ConclusionsElevated CCNB1 expression in colorectal cancer is dependent on Chkl expression.Part III Effects of CCNB1 on the tumorigenicity of colorectal cancer in vivoObjectiveTo validate the effect of CCNB1 suppression on the growth of colorectal cancer tumors in vivo.MethodsFirst, we engineered HCT116 and SW480-based cells, which were stably expressing short hairpin RNA (shRNA) for CCNB1 by lentiviral transduction. To know the knockdown efficiency, endogenous expression of CCNB 1 protein was examined by western blot. The HCT116-shCCNBl, HCT116-shCtrl, SW480-shCCNB1, and SW480-shCtrl cells were subcutaneously inoculated into BALB/c nude mice to observe tumor growth. The expression of CCNB1 in xenograft were detected by immunohis-tochemical method. Western blot was used to detect the downstream targets of CCNB1 in xenograft.Results1. Inhibition of CCNB1 expression could suppress the growth of xenograft from colorectal cancer nude mice obviously.2. Repression of CCNB1 downregulates cell cycle factors CDC25C/CDK1 and elevated p53 and Bax in p53-wild HCT116 nude mice.ConclusionsEvidence from animal models suggests that Inhibition of CCNB1 expression suppresses the tumorigenicity in vivo.