The Involvement Of TIPE And CUL4A In The Development Of Hepatocellular Carinoma And The Biological Functions

Background:In the latest half century, as a significant health concern to human beings, malignant tumour is dangerous to the human healthy and survival. Its incidence, morbidity and mortality are increasing year by year. As indicated, the mortality of hepatocellular carcinoma has been at the second place in China. Its treatment is always the focus of most clinical research workers. However, the insidious onset and slow progression of symptoms usually result in delayed diagnosis with the missing of the best opportunity for surgical operation and chemotherapy treatment. Therefore, based on the novel discovery about cellular and molecular mechanisms of HCC, gene therapy has become popular as a promising therapy for HCC.Tumor necrosis factor (TNF)-alpha-induced protein8(TNFAIP8or TIPE), is one member of TIPE family which is a newly identified protein family consistied of four members--TIPE, TIPE1(TIPEL1), TIPE2(TIPEL2), and TIPE3(TIPEL3). All of the TIPE family member have high homology in molecular structure. TIPE, also known as SCC-S2, GG2-1and MDC-3.13, was the first identified member of this family,. TIPE was originally discovered to be highly expressed in a metastatic HNSCC-derived cell line, as compared to its matched primary cell line and then was named as the SCC-S2. Further study showed that TIPE was ubiquitously expressed in many human organs and tissues, especially highly expressed in the spleen, lymph nodes, thymus, the thyroid, bone marrow, placenta tissues,but lowly expressed in spinal cord, ovary, lung, adrenal gland, heart, brain, skeletal muscle. Moreover, TIPE was also expressed in a variety of tumors and was closely associated with carcinogenesis.However, it is still unknown about the roles of TIPE in the development of Hepatocellular Carcinoma(HCC). Therefore, the present study was designed to characterize the expression pattern of TIPE in the HCC and to explore its possible roles in the development and progression of HCC.Objectives:1To explore the expression of TIPE at the different clinical stages of hepatoma tissues and and investigate its roles in the development and progression of HCC2To elucidate the molecular mechanisms for the involvement of TIPE in the pathogenesis of HCC.Methods:1To study the expression pattern of TIPE in HCC tissues and to analyze its clinical significance1.1The expression pattern of TIPEmRNA and TIPE protein in HCC tissuesWe collected16specimens of fresh HCC tissue samples. TRIZOL method was employed to extract total RNA and the TIPE mRNA level was detected by RT-PCR. At the same time, we collected145specimens of HCC tissue samples and the TIPE protein level was detected by IHC and the correlation between TIPE expression..1.2Analysis of the correlation between the expression of TIPE protein and it’s clinical indicatorsAnalyse the the correlation between the expression of TIPE protein and the clinical indicators, including tumor size, tumor pathological grade, TNM stages, tumor diameter, blood vessels and lymphatic metastasis and survival period.2The effects of TIPE on the growth of HCC cells in vitro and in vivo2.1To detect the TIPE expression in various hepatocellular carcinoma cell linesTotal RNA from the SMMC7721, QSG7701, HepG2.2.15, HepG2, BEL7402extract by using Trizol method and TIPE mRNA expression level was detected by using RT-PCR method..2.2The effects of TIPE on the growth of HCC cells in vitroTIPE siRNA or NC siRNA were transfected into BEL7402and HepG2cell lines respectively. For overexpression assay, pcDNA3-TIPE or pcDNA3was transfected into HepG2. Then CCK8method was used to monitor the cell growth everyday and the growth curve was drawn. The difference between groups were statistically analyzed.2.3The effects of TIPE on the growth of HCC in vivoH22cells were transplanted subcutaneously into the BALB/c mice. The mice were divided into2groups at random and injected with pRK5-mTIPE or pRK5respectively. Tumor size was monitored by measuring two diameters perpendicular to each other with calipers, the volume of tumor was calculated, and the tumor growth curve was drawn. The difference between the two groups was statistically anzelyzed. At last, the mice were euthanized, and the tumor were isolated and weighted.3The effects of TIPE on the metastasis and the EMT process of HCC cells3.1The effect of TIPE on the invasion and migration of HCC cells in vitroTIPEsiRNA or NCsiRNA were transfected into BEL7402and HepG2respectively. Cells were separated with trypsin and plated in the upper chamber of transwell (treated or untreated with matrigel). After12h or24h incubation, the number of cells that had passed through the filter was counted per microscopic field.3.2The effects of TIPE on the expression of EMT related molecules and transcription factorsAccording to literature reports, the transformation of epithelial mesenchymal (EMT) play a key role in the invasion and metastasis of hepatocellular carcinoma. Therefore, we tested the EMT-related molecules and transcription factors.TIPE siRNA or NC siRNA were transfected into BEL7402and HepG2respectively. For overexpression assay, pcDNA3-TIPE or pcDNA3were transfected into HepG2.48hrs later, the expression of EMT molecules and their corresponding transcription factors, including E-cadherin, N-cadherin, Vimentin, Slug, Snail, Twist, was tested by WB.4To study the role of Racl on the involvement of TIPE in HCC pathogenesis4.1The influence of the TIPE on the Racl activationBEL7402cell was transfected with TIPE siRNA or NC siRNA, or co-transfected with H-Ras12V plasmid. Racl-GTP levels were detected by the Pull-down assay.4.2The role of Racl in the effects of TIPE on the growth of HCCHepG2and BEL7402cells were cotransfected with TIPE siRNA and/or Rac1siRNA or NC siRNA and the growth curve was detected by CCK8method.4.3The involvement of Racl in the effect of TIPE on the metastasis of HCCHepG2and BEL7402cells were cotransfected with TIPE siRNA and/or Racl siRNA or NC siRNA. The cell migration and invasion ability was detected by transwell assay.Results:1Decreased expression of TIPE in HCC with significant clinical significances1.1Down-regulated expression of TIPE in HCCWe analyzed TIPE mRNA expression in16HCC patients. RT-PCR analysis showed that69%(11/16) of patients exhibited decreased TIPE mRNA expression in cancerous tissues when compared with their corresponding paracancerous tissues.Isotype control indicates that the TIPE antibody can be used for further experiments. TIPE was highly expressed in liver hemangioma. The average staining intensities of TIPE in HCC tissues is significantly lower than those of the adjacent paracancerous tissues.1.2The expression of TIPE protein is correlated with pathological indicatorsBy analyzing the relationship between the expression of TIPE and clinical pathology indices, we found that the expression of TIPE was negatively correlated with tumor diameter, pathologic grade, TNM stage, and lymphoid or vascular invasion. Moreover, the overall5-year survival rate of the HCC patients with low TIPE expression was significantly higher than that of the patients with high TIPE expression.2TIPE markedly inhibits the growth of liver tumor cells in vitro and in vivo2.1The different expression level of TIPE in HCC cell linesThe TIPE mRNA was highly expressed in QSG7701, HepG2.2.15, cell line BEL7402, while it was moderately expressed in HepG2cells. We selected HepG2, BEL7402to knockdown the expression of TIPE, meanwhile, TIPE overexpression assay was also performed in HepG2cells.2.2TIPE inhibits the growth of HCC cell line in vitroTIPE siRNA or NC siRNA were.transfected into BEL7402and HepG2cell lines respectively. Cell growth curve displayed that TIPE siRNA significantly increased the cell proliferation ability compared to the control group. It implies that TIPE can inhibit cell proliferation ability in HCC.2.3TIPE significantly inhibits the growth of subcutaneously xenograft tumorSubcutaneously transplanted tumor model showed that TIPE overexpression significantly limited the speed of tumor growth when compared with control group. RT-PCR demonstrated that TIPE was significantly overexpressed in pcDNA-TIPE injected group.3TIPE inhibits the mobility of HCC cells though Epithelial-to-Mesenchymal Transition3.1TIPE ameliorates the invasion and migration ability of HCC cellsTIPE siRNA significantly increased cell invasion ability (p<0.005) and migration ability (p<0.05) of BEL7402and HepG2cell lines. It indicated that TIPE has an inhibitory effect on cell invasion and migration. 3.2TIPE regulates Epithelial-to-Mesenchymal Transition processWestern blot showed that TIPE siRNA significantly downregulated the expression of E-cadherin, while up-regulated the expression of N-cadherin and Vimentin. Furthermore, the expression of well-known EMT transcription factors slug, snail, twist was significantly down-regulated by TIPE siRNA. All results indicated that TIPE can inhibit the EMT progress.4Regulation of Racl activity is involved in the effects of TIPE on cell growth, migration and invasionRac1belongs to the Ras superfamily members, who participate in the regulation of a variety of biological activities, and also plays an important role in tumor development. Therefore, we tested the influence of TIPE on the Rac1activity and the role of Racl in TIPE-mediated inhibition on the growth and metastasis of liver cancer.4.1TIPE inhibits Racl activityPull-down showed that TIPE siRNA significantly increased the endogenous Racl activity and the Rac1activity induced by H-Ras mutant overexpression. These results indicated that TIPE could inhibit the Racl activity both at the endogenous and in over-activated condition.4.2Racl knockdown abrogated the effects of TIPE on cell growthTIPE siRNA and/or Rac1siRNA were transfected into BEL7402and HepG2cell lines respectively. Cell growth curve assay showed that TIPE knowdown promoted the growth of HCC, which was almost abrogated by Racl knockdown. All these results implies that Racl is largely responsible for the effects of TIPE on cell growth.4.3Racl knockdown ameliorated the effects of TIPE on cell migration and invasionTIPE siRNA and/or Rac1siRNA were transfected into BEL7402and HepG2cell lines respectively. Transwell assay showed that TIPE knowdown promotes the migration and invasion of HCC, which was almost reversed by Rac1knockdown.Conclusions:1. TIPE expression at both mRNA and protein levels is significantly lower in HCC tissues than that of adjacent noncancerous tissues. The levels of TIPE expression is correlated with tumor stage, metastasis and the patient survival of HCC.2. TIPE has an inhibitory effect on the proliferation of HCC cells.3. TIPE markedly inhibits migration and invasion ability of liver cancer cells and reverses the process of EMT conversion. 4. TIPE inhibits Racl activation, which is involved,in the regulation of cell proliferation and metastasis of TIPE in HCC.Significances:1. This study firstly demonstrates that TIPE played anti-tumor role in HCC by inhibiting both cell growth and migration.2. This work provides new insights into the biological role of TIPE in carcinogenesis and provided a new candidate target for clinical therapy of HCC. Background:Hepatocellular carcinoma (HCC) occurs commonly and with increasing frequency in China. For the high incidence, high malignancy, low cure rate and poor prognosis, HCC has a serious impact on human health and the quality of life. With deeper cognition about molecular mechanisms of disease, gene therapy has become one of the most important research fields. Ubiquitin-proteasome system based on the degradation of proteins, play an important role in cell apoptosis, proliferation and differentiation, cell cycle process, transcription regulation, immune response, and the process of inflammation. But in recent years, more and more findings demonstrate that CUL4A is involved in tumorogenesis and development, Cullin family belongs to E3ligase, is highly conservative, and contains seven protein molecules CUL1,CUL2, CUL3, CUL4A, CUL4B,CUL5, CUL7. As the member of Cullin family, Cu14A and CUL4B has similarity on structure and function. By mediating the ubiquitination and degradation of tumor suppressor proteins P21and P27and P53, CUL4A plays the role in the development and progression. In squamous cell carcinoma, adrenal cortical adenoma, children medulloblastoma, malignant pleural stromal tumor, CUL4A showed high expression.Objectives:In this study, we observed the expression and of CUL4A in human hepatocellular carcinoma(HCC) and investigated its role in the development of HCC, which would be helpful for understanding the biological behavior of HCC and provide theoretic basis for the clinical diagnosis and treatment of HCCMethods:1To study the expression of CUL4A in human hepatocellular carcinoma (HCC) and its significance1.1The detection of the CUL4A protein expression level in HCC tissuesWe collected57specimens of HCC tissue samples and immunohistochemistry stain was applied to determine the changes of the expression of HCC and its adjacent tissues.1.2Analysis of the correlation between the expression of TIPE protein and clinical indicatorsThe correlation between the expression of CUL4A protein and it’s clinical indicators (age,gender, differentiation, TNM stage, tumor diameter, lymphatic invasion and venous invasion) was statistically analyzed.2The effects of CUL4A on the growth and cell cycle of HCC cells in vitro2.1The effects of CUL4A on the growth of liver cancer cell in vitroCUL4A siRNA and NC siRNA were transfected into BEL7402and HepG2cell lines respectively. The cell growths were continuously detected for five days by CCK-8on every24h2.2The effects of CUL4A on of cell cell cycle process of HCC cellsCUL4A siRNA or NC siRNA were transfected into BEL7402and HepG2cell lines respectively. The cell cycle distribution was measured by flow cytometry and the expression of cell cycle-related proteins was detected by western blot.3The effects of CUL4A on the metastatic potential of HCC cells in vitro and the molecular mechanism3.1The effects of CUL4A on the ability of migration and invasion of HCC cellsCUL4A siRNA or NC siRNA were transfected into HepG2and BEL7402cells respectively. We detect The effects of CUL4A on the ability of migration and invasion of HCC cells by transwell assay.3.2The effects of CUL4A on the expression of EMT related molecules and transcription factorsThe expression of EMT molecules and its related transcription factors in BEL7402or HepG2cells transfected with TIPE siRNA or NC siRNA was detected by western blot.Results1Increased expression of CUL4A in HCC has significant clinical significance1.1Increased expression of CUL4A protein in HCC tissuesThe average staining intensities of in HCC tissues was significantly higher than that of the corresponding adjacent tissues.1.2The expression of CUL4A protein is correlated with pathological indicatorsAnalysis the relationship between the expression of CUL4A and clinical pathology indexs showed that CUL4A expression in HCC tissues was negatively correlated with tumor diameter, pathologic stage, TNM stage, or lymphoid tissue of vascular invasion, but positively correlated with the overall5-year survival rate of the HCC patients. 2CUL4A plays an important role in promoting the growth of HCC cells2.1CUL4A enhances the ability growth of HCC in vitroCUL4A siRNA or NC siRNA were transfected into BEL7402and HepG2cell lines respectively and the cell proliferation ability significantly lower in TIPE siRNA transfected group than that of control group. It indicates that CUL4A can promote cell proliferation ability in HCC.2.2CUL4A accelerates cell cycle progression of liver cancer cellsFlow cytometry analysis showed that CUL4A siRNA significantly knock-downed CUL4A expression in BEL7402and HepG2cell lines and the percentage of cell in S phase was also decreased in CUL4A siRNA transfected cells compared with that of control group. It indicates that CUL4A can accelerate cell cycle process in HCC.2.3CUL4A knockdown modulates the expression of cell cycle proteinWestern blot assay showed that CUL4A siRNA down-regulated the expression of cell cycle proteins CyclinA,CyclinBl.3CUL4A increases the mobility of HCC cells by modulating Epithelial-to-Mesenchymal Transition3.1CUL4A increases the invasion and migration ability of HCC cellsCUL4A siRNA in in BEL7402and HepG2cell lines significant inhibited cell invasion (p<0.005) and migration (p<0.05). It indicates that CUL4A promotes the invasion and migration ability of HCC cells..3.2CUL4A regulates Epithelial-to-Mesenchymal Transition processCUL4A knockdown in BEL7402and HepG2cells up-regulated the expression of E-cadherin, while down-regulated N-cadherin and vimentin. Furthermore, the expression of transcription factors slug,snail,twist were down-regulated. It implies that CUL4A can accelerate the progress of EMT transition.Conclusions:1. CUL4A protein is significantly up-regulated in HCC tissues. The increased CUL4A expression was correlated with pathological stage, differentiation grade and patient survival.2. CUL4A knockdown inhibits the growth of liver cancer cells. Moreover, CUL4A siRNA impairs cell cycle progression and downregulated the expression of cell cycle related genes.3. CUL4A protein increases the invasion and migration of HCC cells.4. CUL4A promotes epithelial-to-mesenchymal transition.Significances:1. We have proved the key role of the CUL4A in regulation of growth and metastasis of liver cancer cell. 2. Our study demonstrates that CUL4A is a therapeutic target in liver cancer, and underscores the value of CUL4A as a potential biomarker of HCC, this is also providing a theoretical basis for anti-cancer drugs.