Molecular Mechanism Of HJURP Inhibiting P21 Expression In Promoting Hepatocellular Carcinoma Proliferation

Background:Hepatocellular carcinoma(HCC)accounts for about 75%of liver cancer and is a malignant tumor with a high mortality rate.Although there are many treatments for hepatocellular carcinoma,surgical resection and liver transplantation are still the radical strategies.At present,high recurrence and mortality rate after operation are important causes of low 5-year overall survival rate for HCC patients(only 18%).In order to improve the therapeutic effect of HCC,apart from the continuous improvement of medical diagnosis and treatment techniques,it is also important to understand the mechanism of occurrence and development of the disease and to find novel intervention targets.At the same time,uncontrolled proliferation of tumor cells is one of the important reasons for the progression of hepatocellular carcinoma.In summary,the study of key molecular mechanisms in the process of proliferation in HCC is expected to provide a new theoretical basis and therapeutic strategy for the treatment of HCC.Objectives:The aim of this study is to investigate the molecular mechanism of HJURP in promoting the proliferation of hepatocellular carcinoma.This investigation is to verify the expression level of HJURP in hepatocellular carcinoma cell lines and clinical specimens and to explore its clinical significance.Firstly,the cell models of HJURP stable knockdown and overexpression were constructed in vitro.Secondly,the biological function of HJURP in regulating the proliferation of HCC cells was detected at the in vitro and in vivo.Finally,the specific signaling pathways and mechanisms of HJURP in inhibiting p21 expression were further elucidated.Material and methods:1.219 pairs of liver cancer and matched para-cancerous tissue samples were collected from the No.l hospital of Zhejiang University,and relevant clinicopathological information were collected from the medical record system.Real-time quantitative PCR and immunohistochemistry were used to detect HJURP expression in HCC and adjacent tissues.The expression level of HJURP was correlated with clinicopathological features and prognosis of HCC patients;2.Download the expression data of HJURP in HCC and adjacent tissues from Oncomine database for further statistical analysis;3.Construct HJURP stable knockdown cell lines using HCC-LM3 and Huh7 by lentiviral transfection,construct HJURP overexpressing cell line with MHCC-97H cells.The role of HJURP in promoting hepatocellular carcinoma proliferation was detected by CCK-8,flow cytometry cell cycle analysis,clone formation and subcutaneous tumor formation in nude mice and cell cycle related proteins were detected by western blotting;4.In the cell model of HJURP stable transfection,western blotting and immunofluorescence were used to verify that HJURP regulates the nucleoplasmic shuttle of the tumor suppressor molecule p21 and the level of ubiquitination of p21 was detected by protein immunoprecipitation.The expression of HJURP was negatively correlated with the expression of p21 in tissue samples;5.In HJURP stably transfected hepatoma cell lines,western blotting was used to verify that HJURP regulates p21 nuclear expression by inhibiting MAPK/ERK1/2 and activating AKT/GSK3? signaling pathways;6.In HCC cell lines,the rescue assay revealed that the key E3 ubiquitination ligase that involved in p21 ubiquitination degrradation is SKP2.Results:1.In 219 pairs of liver cancer specimens,the results of immunohistochemistry and real-time quantitative fluorescent PCR confirmed that the expression of HJURP in tumor tissues was significantly higher than that in adjacent tissues,and HJURP is high expressed in 74.54%of tumor tissues.Accordingly,the results of western blotting suggested that HJURP expression is also higher in six common hepatoma cell lines;2.In vitro and in vivo experiments showed that HJURP knockdown significantly reduced the CCK-8 absorbance,the abilities of colony formation in hepatocellular carcinoma cells and the ability of subcutaneous tumor formation in nude mice.Besides,depletion of HJURP caused G1 phase arrest in HCC.After overexpression of HJURP,the proliferative capacity of HCC cells was significantly enhanced;3.The results of western blotting showed that HJURP inhibited p21 at the post-translational level and HJURP promoted p21 protein from the nucleus to the cytoplasm.The results of co-immunoprecipitation confirmed that the HJURP promoted the level of ubiquitination of p21,mainly by E3 ubiquitination ligase SKP2;4.We found that p21 was mainly expressed in the nucleus in HJURP knockdown HCC cells and the expression of p-ERK1/2 and p-GSK3? was increased,while expression of p-AKT was decreased.Similarly,the expression of p-ERK1/2 and p-GSK3? were enhanced when HJURP was overexpressed.In addition,ERK1/2 pathway inhibitor U0126 and AKT pathway agonist SC-79 were co-cultured with HJURP knockdown cells,the expression of p-ERK1/2 and p-GSK3? was restored,and the proliferation ability of liver cancer cells was also rescued;5.The results of immunohistochemistry in clinical specimens of liver cancer showed that 69.5%HJURP high expression specimens showed low expression of p21,while 66.7%specimens with low HJURP expression showed high expression of p21;6.By analyzing the clinicopathological data of clinical specimens,the results showed that the overall survival rate of HCC patients with high HJURP expression was significantly lower than that of patients with low HJURP expression.And the high expression of HJURP was significantly correlated with tumor size and AJCC stage of tumor.Conclusions:1.HJURP is highly expressed in HCC tissues and is associated with the proliferative abilities of hepatocellular carcinoma and poor prognosis of HCC individuals;2.Knockdown and overexpression of HJURP can respectively reduce and increase the proliferation ability of hepatocellular carcinoma.HJURP promotes p21 from the nucleus to the cytoplasm and is degraded by E3 ubiquitination ligase SKP2;3.MAPK/ERK1/2 and AKT/GSK3? signaling pathways are involved in the biological process by which HJURP inhibits p21 expression.The MAPK/ERK1/2 and AKT/GSK3? signaling pathways are potential mechanism for HJURP-promoted hepatocellular carcinoma proliferation.