| Background: About 2 billion people worldwide are currently or have been infected with hepatitis B virus(HBV), and about 240 million people are chronic HBV surface antigen carriers. It is estimated that 650,000 people died of chronic hepatitis B complication every year, and about 130,000 people died of acute hepatitis. On the whole, HBV infection accounts for about 45% of liver cancer cases, accounting for about 30% of cases of cirrhosis. Liver cancer ranks among the top three causes of death among male population, especially in Southeast Asia. Hepatocellular carcinoma(HCC) is one of the most common malignancies with a high morbidity and mortality worldwide. Its morbidity ranks the fifth and the mortality ranks the third in the malignant tumor. The morbidity and mortality of HCC are consistently increasing in the world per year. Due to the very high metastasis and recurrence rates, the patients with HCC present a poor prognosis. In china, there are nearly 100 million chronic carriers of hepatitis B virus and chronic persistent HBV infection is an independent risk factor for HCC. Therefore, there is a huge high risk population for HCC. However, the precision pathogenesis of HCC is unclear and the effective prevention and treatment are lack. Although the radical resection of HCC was performed, 60-70% of patients remain recurrent during 5 years. Moreover, the use of new anticancer drugs for HCC treatment(such as sorafenib) brings new hope for HCC patients, but the significant side effects of sorafenib make HCC treatment face great challenges. The etiology of HCC is involved in viral hepatitis, alcoholic hepatitis, metabolic diseases and so on, and its pathophysiology is very complex. With the in-depth study of HCC-related gene and molecular levels, an increasing number of genes and proteins were reported to be associated with malignant characteristics, histological classification and tumor staging of HCC. Every step during HCC occurrence and development may Furthermore, the recurrence and metastasis of HCC is closely related to every step of the occurrence and development of HCC. The gene and molecular in these processes will be molecular targets. Thus, studuing the molecular mechanism of HCC proliferation and metastasis, accurately predicting prognosis for patients, preventing HCC metastasis, and taking effectively preventive measures could be a breakthrough to improve the efficacy of treatment and prognosis. Meanwhile, these studies are also meaningful for the diagnosis of cancer metastasis, and the founding of new effective therapeutic measure. Cyclin B1(also named CCNB1, a mature promoting factor for CCNBD) is a structural protein. Its expression varies periodically throughout the cell cycle, beginning to synthesize in the cytoplasm in S phase, increaseing in the G1 phase, and reaching the peak in G2 / M after entering into the nucleus. After the post-conversion, the expression of Cyclin B1 disappeared regulated by ubiquitin degradation. When in M phase of the cell cycle, the activity of MPF reached the highest and promoted the connection between ubiquitin and CCNB1, and then the complex could be identified and degraded by proteasome. Following this process, MPF is inactive and M phase is complete. The N-terminal of CCNB1 protein has a sequence named as degradation box which plays a crucial role in CCNB1 related degradation. A series of factors can control the cell cycle by promoting CCNBl synthesis or degradation to regulate MPF kinase activity. In normal cells, there are a variety of molecules to the maintenance of genome integrity and stability. However, the disorder of cell cycle happened sometimes, resulting in chromosomal abnormalities, uncontrolled cell proliferation, or apoptosis obstacles and eventually tumors formation. Moreover, there exist some tumor suppressor gene which product cyclin-dependent kinase inhibitor(CKI). cyclins are the product of some proto-oncogene. Thus, the proto-oncogenes, oncogenes and tumor suppressor genes are the genes which are associated with cell cycle regulation. Recent studies have confirmed the expression of CCNB1 in tumor tissue was abnormal, which was overexpression and abnormal localized in the cytoplasm rather than the nucleus. Porter et al found that the inhibition of cytoplasmic transport for could induce apoptosis and the accumulation of CCNB1 in the nucleus is necessary for CCNB1 dependent apoptosis. They also found that the overexpression of CCNB1, especially in the cytoplasm, was related to the occurrence and development of tumor. the prognosis of esophageal squamous cell carcinoma and non-small cell lung cancer is closely related to CCNB1 expression. Gttnsky et al found CCNB1 gene in tumor tissue can be used as a predictor of resistance, local or distant metastasis, recurrence, survival for cancer patients to guide clinical treatment and evaluation. In tumor tissues, high expression of CCNB1 induced the disorder regulation of MPF. Thus, CCNB1 could be as a target of anti-tumor therapy by inhibiting the function of MPF, resulting in the difficult to cross the G / M checkpoint and the inhibition of mitosis proliferation. Currently, the research targeted directly CCNB1 still in the initial stages, and there are still many problems. Thus, there need further in-depth researches on the CCNB1 associated signal transduction pathway to find more effective anti-cancer target and the anti-tumor research targeting CCNB1 will certainly have broader clinical application. Micro RNA(ie miRNA) is an endogenous small non-coding RNA. Human genome contains thousands of micro RNA. Each micro RNA can regulate two one hundred target genes, and nearly 33 % of protein-coding genes are regulated by micro RNA in human body. These data suggested that micro RNA had an important role in the regulation of human genes. Recent studies have found that about 50% of known miRNAs were associated with human tumors, suggesting the crucial role of miRNAs in tumorigenesis. Studies have indicated that miRNAs, regulating gene expression intracellular signaling pathways in the post-transcriptional level, could be served as oncogenes or tumor suppressor genes involved in tumor development and progression. Tumor-associated miRNA is called tumor miRNA genomics(Onco-miR). The overexpression of oncogene nature miRNA promotes tumorigenesis by promoting cell proliferation, inhibition of apoptosis, unlimited replication potential, angiogenesis, invasion and metastasis. Micro RNA can participate in many cellular processes such as proliferation, senescence, apoptosis and autophagy. When miRNA inhibits the expression of oncogenes, it will function as a tumor suppressor gene. When micro RNA inhibits the expression of tumor suppressor gene, it becomes carcinogenic molecules. However, the mechanism of miRNA in cancer is very complex and the regulatory networks are extensive. The expression of a same miRNA in different cancers may be different, even there exists distinguish in different stages or type of in a same cancer. Further studies on particular miRNA in particular cancer are needed which could contribute to a better understanding of the pathogenesis of cancer, and find the appropriate treatment. Recently, a series of studies on the roles of miRNA on HCC have promoted the understanding of HCC. Numerous studies have indicated that miRNAs play an important role in the progressof HCC, and are directly involved in the differentiation, apoptosis, invasion and distant metastasis. The expressions of miRNAs are closely related with the degree of malignancy, stage, grade and other clinicopathological features, suggesting that miRNAs may serve as not only markers for diagnosis and individualized treatmen, but also as prognostic markers. Therefore, miRNA might be considered as therapeutic targets to provide a new approach for the treatment of HCC. Recently, miR-122 was identified as a liver-specific miRNA, abundant in normal liver, low expression in HCC tissues. Other studies have found miR-122 could target cyclin G1, Bcl-2 gene and facilitate the hepatitis C virus(HCV) replication. Moreover, other miRNAs, such as let-7family, miR-101, miR-221, miR-34 a, miR-224 and miR-222 also play an important role in the development of HCC. These results suggest that the regulation of miRNAs is an important part of the HCC progression. Thus, the the relationship between the miRNA and HCC is the focus of many researchers currently. In this study, we firstly analyzed the TCGA and GEO database and found that showed that the expression of CCNB1 was abnormally elevated and closely related to the malignant phenotype. Moreover, CCNB1 expression could be an independent prognostic factor of overall survival in HCC. Kaplan-Meier analysis showed that the survival time of patients with low CCNB1 was significantly better than patients with high CCNB1, suggesting that the expression of CCNB1 was closely related to the prognosis of HCC patients. While further studies confirmed that cell proliferation, migration and invasion and tumorigenicity of HCC cell lines decreased after CCNB1 knockdown significantly increased after CCNB1 knockdown, suggesting that CCNB1 played an important role in the development of HCC. In order to study the molecular mechanisms CCNB1 based on abnormally high expression in HCC, we first screened the targeted inhibition of CCNB1 by miR-144. Then we demonstrated that miR-144 can be targeted inhibition CCNB1 expression in vitro. Consistently, we found the expression of miR-144 significantly decreased in HCC, and the survival time of patients with low miR-144 expression was significantly worse in patients with high miR-144 expression, suggesting that expression of miR-144 was closely related with clinical prognosis and miR-144 may be a tumor suppressor gene. After overexpression of miR-144 in HCC cell lines, the proliferation, migration and invasion and tumorigenic ability decreased, presenting a similar effect after CCNB1 knock out, which suggested that miR-144 / CCNB1 pathway may play an important role in the development of hepatocellular carcinoma. Moreover, in vivo experiments of subcutaneous tumor formation further verified the role of miR-144-CCNB1 pathway in HCC,which provided more sufficient evidence for miR-144-CCNB1 pathway being potential therapic target in HCC treatment. In conclusions, the expression of CCNB1 was significantly increased in HCC, and was closely related with metastasis, recurrence and clinical prognosis of patients. CCNB1 played a role of cancer-promoting gene in the occurrence and development of HCC. CCNB1 could be regulated by miR-144 in HCC. Meanwhile, the expression of miR-144 in HCC was significantly reduced, and survival time of the patient with low expression of miR-144 was significantly shorter than that with high expression of miR-144. Overexpression of CCNB1 in HCC cell lines in vivo and in vitro significant decreased the proliferation, migration and invasion of HCC, resulting in a significant decreased of tumorigenic capacity. Mi R144, serving as tumor suppressor, played a inhibit role in the occurrence and development of HCC by negative regulation of CCNB1, thereby contributing to the progress of HCC. Mi R-144/CCNB1 pathway played an important role in the development of HCC, thus it may become a new target for treatment of HCC.The first part: the expression and biological function of CCNB1 in hepatocellular carcinomaObjective: Confirm the expression of CCNB1 in HCC tissues and the biological function in the occurrence and development of HCCMethod: 1. Western Blot was used to detect the expression of CCNB1 in next eight pairs of HCC tissues and adjacent tissues. 2. Based on bioinformatics, microarray analysis software(BRB-Arraytools), TCGA and GEO public databases were used to analyze the mRNA expression of CCNB1 in HCC tissues and normal liver tissues. 3. TCGA public databases and its follow-up data of HCC microarray data were used to analyze the relationship between the mRNA expression of CCNB1 in tissues and clinical phenotype of HCC. 4. The relationship of mRNA and protein expression of CCNB1 and clinical characteristics and prognosis of HCC patients were analyzed. 5. Immunohistochemistry and tissue microarray technology were used to determine the protein expression of CCNB1 in HCC tissues, as well as analyzed the relationship between the degree of CCNB1 expression and clinical features and prognosis of HCC patients. 6. RNA interference gene were used to silence the gene of CCNB1 in HepG2 cells and 7721, and proliferation(MTT), apoptosis(Annexin V staining), migration(scratch test) and invasion(transwell) were performed to investigate the functions of CCNB1 in HCC cell lines.Result: 1. Compared with the normal liver tissue, the expression of CCNB1 in the mRNA(TCGA and GEO gene database analysis) and protein levels(HCC tissue microarray) in HCC tissues have an significant increase(P <0.05).2. There were a positive correlation between the expression of CCNB1 and the prognosis(P = 0.001), as well as metastasis(P = 0.000), TNM stage(p = 0.006), recurrence(0.041) of HCC patients, while no correlation were found between the expression of CCNB1 and age, sex, hepatitis B, tumor size, AFP level. The high expression of CCNB1 indicated a poor prognosis(P <0.01), and CCNB1 is an independent risk factor in overall survival of patients with HCC. Kaplan-Meier analysis showed that the survival time of patients with low CCNB1 was significantly better than patients with high CCNB1, suggesting that the expression of CCNB1 was closely related to the prognosis of HCC patients. 3. Further studies confirmed that cell proliferation, migration and invasion and tumorigenicity of HCC cell lines decreased after CCNB1 knockdown significantly increased after CCNB1 knockdown, suggesting that CCNB1 played an important role in the development of HCC.The second part: Screening and identifying the miRNA gene targeting CCNB1 and investigating the biological behavior of the miRNA gene in HCC cell linesObjective: Bioinformatics techniques were used to Screening and identified the miRNA gene targeting CCNB1.Method: 1. The miRNA gene targeting CCNB1 were screened by Targetscan, miRwalk and miRand. And then micro RNA expression profiling of 425 patients in TCGA database with HCC were used to find the miRNA(miR-144) which was low expression in HCC tissue 2. RT-PCR and Western blot were used to detect the change CCNB1 mRNA and protein levels after over-expression or inhibition of miR-144 3. Luciferase reporter was used to verify the negative regulatory role of miR-144 targeting for the CCNB1.4. The relationship between miR-144 expression and clinical phenotype in patients with HCC were analyzed by micro RNA microarray expression data combined with its follow-up data in TCGA data. 5. Cell proliferation, migration and invasion and tumorigenicity of HCC cell lines were determined by overexpression or inhibition of miR-144 in HCC cell lines.Result: 1. Firstly, we found that CCNB1 may be one of the target genes of miR-144 by bioinformatics prediction analysis. And then luciferase reporter experiments confirmed that miR-144 can inhibit the activity of fluorescent reporter gene expression on CCNB1, while the mutant and control plasmid had no this effect. Overexpression or inhibition of miR-144 had a significant effect on CCNB1 level. 2. The expression of miR-144 in HCC tissues was significantly decreased(p <0.0001). Kaplan-Meier analysis showed a significant correlation between prognosis and the expression of miR-144 in HCC patients(p = 0.017). The survival time of patients with low miR-144 expression was significantly worse in patients with high miR-144 expression, suggesting that expression of miR-144 was closely related with clinical prognosis and miR-144 may be a tumor suppressor gene. 3. After overexpression of miR-144 in HCC cell lines, the proliferation, migration and invasion and tumorigenic ability decreased, presenting a similar effect after CCNB1 knock out, which suggested that miR-144 could inhibite the development of hepatocellular carcinoma by negative the expression of CCNB1. Moreover, in vivo experiments of subcutaneous tumor formation further verified the role of miR-144-CCNB1 pathway in HCC,which provided more sufficient evidence for miR-144-CCNB1 pathway being potential therapic target in HCC treatment.The third part: the effect of miR-144 on biological behavior of HCC in vitroThe effect of miR-144 on biological behavior of HCC in tumor-bearing mice Objective:Method: 1. Using a lentivirus was constructed overexpression of miR-144 in the 7721 cell line and RT-PCR and Westernblot were used to identify successful overexpression of miR-144 in the 7721 cell line. 2. Mice were transfected with over-expression of miR-144 and empty 7721 cells. 3. The survival time of mice and tumor growth curve were observed and recorded to investigate the effect of miR-144 in vivo(Kaplan-Meier).Result: 1. RT-PCR and Western blot were used to identify successful overexpression of miR-144 in the 7721 cell line. 2. The mice were transfected with 7721 cells of miR-144 over-expression had a slow growth and a long survival time compared with that empty 7721 cells. 3. Immunohistochemistry confirmed that the expression of CCNB1 and Ki67 was decreased in tumor after transfected with 7721 cells of miR-144 over-expression.The full text of the conclusions 1. The expression of CCNB1 was significantly increased in HCC, and was closely related with metastasis, recurrence and clinical prognosis of patients. CCNB1 played a role of cancer-promoting gene in the occurrence and development of HCC. 2. CCNB1 could be regulated by miR-144 in HCC. Meanwhile, the expression of miR-144 in HCC was significantly reduced, and survival time of the patient with low expression of miR-144 was significantly shorter than that with high expression of miR-144. 3. Overexpression of CCNB1 in HCC cell lines in vivo and in vitro significant decreased the proliferation, migration and invasion of HCC, resulting in a significant decreased of tumorigenic capacity. Mi R144, serving as tumor suppressor, played a inhibit role in the occurrence and development of HCC by negative regulation of CCNB1, thereby contributing to the progress of HCC. Mi R-144/CCNB1 pathway played an important role in the development of HCC, thus it may become a new target for treatment of HCC. |
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