The Roles And Underlying Mechanism Of NPAS2 In Growth Of Hepatocellular Carcinoma Cells

【Background】 The incidence of hepatocellular carcinoma(HCC)was reported to be increasing in recent years.HCC has become the world’s second leading cause of tumor-related death globally,and was responsible for about 800,000 deaths each year.Our country is the world’s highest incidence of HCC and the largest number of deaths in the world wide.The current treatment of HCC is divided into radical dissection,palliative care and symptomatic treatment.Although the 5-year survival rate of early radical dissection can reach 75%,but the majority of patients with HCC were found to be unable to undergo radical dissection.This leads to a very low survival rate of 5-years in patients with HCC.However,the sensitivity and specificity of current markers for diagnosing HCC are not very well.Therefore,it is urgent to find a diagnostic marker of HCC with high specificity and sensitivity,especially molecular markers of HCC.In addition.In addition,effectiveness of targeted therapy in HCC patients are very limited,and targeted drugs are only effective for some HCC patients including sorafeni.Thus,there are still many problems in the early diagnosis and treatment of HCC.It is of great significance for the prevention and treatment of HCC in our country to further explore the mechanism of the occurrence and development of HCC,and to find the molecular markers and the new biological therapy of early HCC.Circadian rhythm regulates various metabolic and physiological functions of the body.It is found that circadian rhythm is generated by core circadian genes through transcription,translation of negative feedback loop.Until now,ten core circadian genes have been found: NPAS2,CLOCK,BMAL1,PER1,PER2,PER3,CRY1,CRY2,TIM and CKI.These core circadian genes are involved in regulating numerous life activities of cells,including cell proliferation,cell apoptosis,cellular metabolism and cellular immunity.There is growing evidence that tumorigenesis are directly related to circadian gene.NPAS2 is an important member of core circadian genes.It participates in the DNA damage repair,inflammatory response,hormone secretion,cell metabolism and many other physiological activities.NPAS2 is closely related to a variety of diseases,including hypertension,fetal liver metabolism,nonalcoholic fatty liver disease,winter depression,chronic fatigue syndrome and tumors.Our preliminary results suggest that the expression of NPAS2 is significantly increased in HCC.At present,the understanding of NPAS2 is not comprehensive,and the role of NPAS2 has not been reported in HCC.Therefore,it is great significance to explore the possible role and molecular mechanism of the survival-promoting role of NPAS2 in HCC.【Aims】 1.To determine the expression of NPAS2 in HCC,and to analyze the relationship between NPAS2 expression and the prognosis of HCC patients.2.To analyze the role of NPAS2 in HCC cells.3.To elucidate the specific molecular mechanism of the role of NPAS2 in HCC cells.【Methods】 1.The collected samples of HCC tissue and public datasets were used as the research object.QRT-PCR,Western Blot and immunohistochemistry staining were used to detect the expression of NPAS2 in HCC.The expression level of NPAS2 was analyzed with the prognosis and clinical parameters.2.The effects of NPAS2 on the growth of HCC cells was analyzed by MTS,cloning,Ed U,flow cytometry and Western Blot.3.The effect of NPAS2 on tumor growth in vivo was analyzed by subcutaneous tumorigenesis in nude mice.4.The molecular mechanism of NPAS2 on tumor growth was analyzed by q RT-PCR,Western Blot,IHC,dual-Luciferase reporter gene system,Chip-PCR and Co-IP.5.Whether BMAL1 was involved in the survival-promoting role of NPAS2 was analyzed by cell growth phenotype.【Results】1.The expression of NPAS2 was significantly up-regulated in HCC tumor tissues when compared with paired nontumor tissues.Moreover,our data indicated that high expression of NPAS2 was significantly associated with high AFP and larger tumor size,implying that NPAS2 may play a tumor-promoting role in HCC.Finally,Kaplan-Meier analysis revealed that HCC patients with high expression of NPAS2 had significantly poorer overall survival and recurrence-free survival than those with low expression.2.NPAS2 promotes cell survival in vitro and in vivo mainly by inducing cell proliferation and inhibiting cell apoptosis.3.The survival-promoting role of NPAS2 was mediated via transcriptional upregulation of the CDC25 A phosphatase in HCC.NPAS2 promotes G1 to S phase transition of cell cycle by CDC25A-mediated activation of CDK2/4/6 and inhibits cell apoptosis by CDC25A-mediated dephosphorylation of Bcl-2 at Thr69.4.NPAS2 controls the expression of CDC25 A by forming heterodimer with BMAL1 to promote HCC cell survival.【Conclusion】Through the detection of clinical tissue samples,bioinformatics analysis,in vitro and in vivo experiments,we found for the first time that the expression of NPAS2 in HCC tissues was increased and negatively correlated with the prognosis of HCC patients.We found that the cell proliferation-promoting and cell apoptosis inhibiting role of NPAS2 was mediated via transcriptional upregulation of the CDC25 A phosphatase.Moreover,another core circadian gene BMAL1 was also found to be associated with the NPAS2-mediated tumor cell survival in HCC.Therefore,NPAS2 may be a new molecular marker of prognosis and a new biomedical therap target y of HCC.