Tumor Necrosis Factor ?-induced Protein 1 Inhibits Hepatocellular Carcinoma Progression By Blocking CSNK2B-dependent Nuclear Factor-?B Pathway

Hepatocellular carcinoma(HCC)has become the second leading cause of cancer related death in the world.According to Chinese cancer statistics from 2015,HCC accounts for one-fifth of the incidence of malignant tumors in China.It poses a serious threat to people's health and quality of life.More than 90% of HCC is primary HCC,which can be induced by chronic HBV or HCV,alcoholism,metabolic syndrome related to diabetes and obesity.Surgical resection,liver transplantation,radiotherapy and drug therapy are used to treat HCC,but surgical resection is still the most effective mean to treat this disease.In fact,due to the highly metastatic potentials of HCC and the violation of neovascularization,less than 20% of patients can be treated by surgical operation in the clinic.Besides,the high recurrence rate of HCC results in the five-year survival rate is less than 30% after operation.This phenomenon has become a severe challenge for the HCC treatment.At present,it is believed that the high metastasis and the abnormal activation of angiogenesis are major factors causing the high recurrence rate and low survival rate of HCC.Therefore,to elucidate the molecular mechanisms underlying HCC metastasis and abnormal angiogenesis and explore new targets for treating HCC are important and significant.Tumor necrosis factor alpha inducible protein 1(TNFAIP1),alsoknown as B12 or bacurd2,can be induced by TNF ? and LPS.Previous studies showed that TNFAIP1 involves in tumor proliferation,apoptosis and migration;it also plays an important role in DNA synthesis,immunity,neuron development and Alzh-eimer's disease.However,the role and the underlying mechanisms of TNFAIP1 in HCC are rarely reported.In order to study the expression and the function of TNFAIP1 in HCC,we designed the following experimental programs: Immunohistochemistry,western blot and RT-qPCR were used to analyze the expression of TNFAIP1 in HCC tumor tissues and cell lines;MHCC97H cells were infected with Control and TNFAIP1 lentiviral particles to generate MHCC97H-Control and MHCC97H-TNFAIP1 stable cell lines,SMMC7721 cells were infected with shControl and shTNFAIP1 lentiviral particles to generate SMMC7721-shControl and SMMC7721-shTNFAIP-1 stable cell lines;cell counting kit-8(CCK8)was used to analyze the effects of TNFAIP1 on the proliferation of MHCC97 H stable cells and SMMC7721 stable cells;the nude mouse tumorigenicity assay was used to identify the effects of TNFAIP1 on tumor growth;dead End Fluorometric TUNEL system and western blot were used to analyze the effects of TNFAIP1 on the apoptosis in MHCC97 H stable cells and SMMC7721 stable cells;transwell migration and invasion assay and lung metastasis assay were used to analyze the effects of TNFAIP1 on the invasion and metastasis of MHCC97 H stable cells and SMMC7721 stablecells in vitro and in vivo;Tube formation assay was used to analyze the effects of TNFAIP1 on angiogenesis in HUVECs-Control,HUVECs-TNFAIP1,HUVECs-shControl and HUVECs-shTNFAIP1 stable cell lines;western blot,RT-qPCR,immunohistochemistry,Liquid chromatography tandem mass spectrometry(LC-MS/MS),immunoprecipitation(CO-IP),GST-pull down and ubiquitin experiments were used to study the molecular mechanism.We found that: 1)TNFAIP1 expression was reduced in HCC tissues and cell lines,and was negatively correlated with the increase of HCC histological grade;2)Overexpression of TNFAIP1 inhibited HCC cells proliferation,metastasis,EMT and angiogenesis in vitro and in vivo,and promoted HCC cells apoptosis.Conversely,knockdown of TNFAIP1 showed opposite effects;3)Overexpression of TNFAIP1 down-regulated the expression of NF-?B target genes including: Bcl-2,Bcl-xL,CCND1,MMP2,MMP9,TWIST1 and VEGF.Whereas,knockdown of TNFAIP1 showed opposite effects;4)Overexpression of TNFAIP1 decreased the expression of p-p65(ser536),p-p65(ser529),P-I?B?(ser32),p-IKK?/?(ser176/180)and inhibited NF-?B trans-activation.In contrast,knockdown of TNFAIP1 resulted in the increased levels of p-p65(ser536),p-p65(ser529),p-I?B?(ser32),p-IKK?/?(ser176/180)and enhanced NF-?B trans-activation;5)Overexpression of TNFAIP1 reduced the expression of HIF-1? and HIF-1? and inhibited HIF-1 pathway,knockdown of TNFAIP1 resulted in the increased expression of HIF-1 ?and HIF-1? and activated HIF-1 pathway;6)TNFAIP1 interacted with protein kinase casein kinase II ?(CK2?,CSNK2B)and promoted its ubiquitin-mediated degradation with Cul3,causing attenuation of CSNK2B-dependent NF-?B trans-activation in HCC cell;7)CSNK2B enhanced the transcription activation of NF-?B;8)Overexpression of CSNK2 B counteracted the inhibitory effects of TNFAIP1 on the HCC cells proliferation,migration and angiogenesis;9)TNFAIP1 was negatively correlated with CSNK2 B in HCC tissues.Conclusion: overexpression of TNFAIP1 selectively down-regulates CSNK2 B through ubiquitin mediated degradation pathway and blocks the transcription activation of NF-?B,and then decreases the expression of NF-?B target genes,including CCND1,Bcl-2,Bcl-XL,MMP2,MMP9,TWIST1 and VEGF,inhibits proliferation,invasion and metastasis,EMT and angiogenesis of HCC cells and promotes HCC cells apoptosis.Our results support that TNFAIP1 can act as a tumor suppressor of HCC by modulating TNFAIP1/CSNK2B/NF-?B pathway,which implies that TNFAIP1 may represent a potential marker and a promising therapeutic target for HCC.