The Effect And Molecular Mechanism Of Nucleotide-binding Oligomerzation Domain Proteins In Hepatocellular Carcinoma

ObjectiveHepatocellular carcinoma(HCC)is one of the most common malignancies worldwide with increased morbidity and mortality in recent years.Most HCC patients were diagnosed at advanced stages without the opportunity of surgical resection.Chemotherapy is the only option for many late stages HCC patients.Until now,the overall prognosis of HCC is far from satisfactory.It is urgent to find new molecular targets to inhibit the progression of HCC and enhance response to chemotherapeutic drugs for HCC treatment.The nucleotide-binding oligomerization domain(NOD)proteins,NOD1 and NOD2,the founding members of the intracellular NOD-like receptor family,sense conserved motifs in bacterial peptidoglycan and induce pro-inflammatory and anti-microbial responses.Based on recent reports showing that several innate immune sensors play pivotal roles in cancer,it is plausible to expect that NODI and NOD2 also play a role in the development of HCC.It is reported that NODI is associated with altered risk of gastric,colorectal,liver and other kinds of malignant tumors;and NOD2 gene polymorphisms have been associated with increased risk of lymphoma,colorectal,gastric,breast,ovarian,lung and laryngeal cancers.However,the biological function and involved molecular mechanism of NOD1 and NOD2 in HCC are far from being clarified.This study aims to detect the expression level of NOD1 and NOD2 in HCC clinical specimens and to verify the regulation effect of NODI and NOD2 on the progression of HCC and the sensitivity of HCC cells to chemotherapy,and finally to clarify the molecular mechanism of the function of NODI and NOD2.Therefore,this study will provide new clues to clarify the molecular mechanism of HCC disease progression and response to chemotherapy drugs,and provide new therapy strategy for HCC treatment.Part ? The effect and molecular mechanism of NOD1 in the progression ofhepatocellular carcinomaMaterials and Methods1.To detect the function of NOD1 in the regulation of HCC progression1.1 To detect NOD1 expression in clinical HCC specimens120 pairs of HCC tissues and matched non-cancerous liver tissues(including 30 pairs of the junction of liver cancer and non-cancerous tissues)were used for immunohistochemistry(IHC)staining to detect their localization and expression level of NOD1 in HCC.The IPP6.Plus image analysis software was used to further analyze the expression difference of NOD1 in HCC tissue and matched non-cancer liver tissue.Western blot was used to detect the NODI protein level in 64 pairs of HCC tissues and corresponding non-cancer liver tissues.Image J software was used to quantitatively analyze the NODI expression bands in HCC tissue and corresponding non-cancerous liver tissues,and Graph Pad Prism5.0 software was used to statistically analyze the gray levels of the bands.1.2 To construct xenograft tumor model to clarify the regulatory effect of NODI on HCCTo construct xenograft tumor model,0.1 ml of 107 HUH7 cells were subcutaneously injected to both flanks of the nude mice.When visible tumors appeared,the tumors at the left flanks were injected with Myc-NOD1 plasmid while tumors at the right flanks were injected with Myc-vector plasmid.The mice were sacrificed on day 28 after the first plasmid injection,and the tumors were separated from both flanks of the mice for further analysis.Western blot was used to detect the successful overexpression of NOD1 in tumor tissue.Compare and analyze the volume and weight of the tumor between the experimental group and the control group.3.To detect the regulatory effect of NOD1 on the proliferation of HCC cellsHepG2 and HUH7 cells were used to construct the loss-of-function cellular model by transfection with specific SiRNAs against NOD1,and the successful knockdown of NODI was verified by western blot.CCK-8 assay and colony formation assay were used to detect the proliferation ability and colony formation ablity of the NOD1 loss-of-function cellular model.HUH7 cells and HepG2 cells were transfected with Myc-NOD1 plasmid to construct the gain-of-function cellular model,and the successful overexpression of NOD1 was verified by western blot.CCK-8 assay and colony formation assay were used to detect the proliferation ability and colony formation ability of the NOD1 gain-of-function cellular model.Flow cytometry assay was used to detect the changes of cell cycle after knockdown or overexpression of NOD1 expression.Besides,western blot was used to detect the expression of P21,p-CyclinD,CyclinD,p-Cyclin E,CyclinE,p-CDC2 and CDC2 after overexpression of NOD1;western blot was also used to detect the expression changes of cell cycle related proteins including P21,p-Cyclin D and Cyclin D after knockdown of NOD1 expression.4.To explore the molecular target of NOD1Co-Immunocrecipitation(Co-IP)was used to test several potential interacting partners that might interact with NOD1,including P53,TSC1,P65,ERK,SRC and LKB1.HepG2 and HUH7 cells were co-transfected with Myc-NOD1 and HA-SRC.24h after the transfection,Co-IP was performed to detect the interaction between Myc-NOD1 and HA-SRC.HepG2 cells were transfected with Myc-NOD1 and HA-SRC plasmids and further cultured for 24h.Immunofluorescence assay was performed to verify the co-localization between Myc-NOD1 and HA-SRC.NOD1 protein and SRC protein were synthesized by an in vitro translation system.Co-IP assay was performed to detect the direct interaction between NODI and SRC.The truncated mutants of SRC were constructed,and were further co-transfected with Myc-NOD1 plasmid into HUH7 cells.24h after the transfection,Co-IP assay was performed to determine which domain of SRC interacted with NODI.The truncated mutants of NOD1 were constructed,and were further co-transfected with HA-SRC into HUH7 cells.24h after the transfection,Co-IP assay was used to verify which domain of NOD1 interacted with SRC.4.To explore the mechanism of NODW regulating the proliferation of HCC cellsHCC cells were transfected with Myc-NOD1,and Myc-vector transfected cells acted as a mock control.24h after the transfection,western blot assay was performed to detect the levels of p-SRC and SRC.HCC cells were transfected with Si-NOD1,and Si-NC transfected cells acted as a mock control,48h after the transfection,western blot assay was performed to detect the levels of p-SRC and SRC.HUH7 cells were transfected with Si-NOD1 to knockdown the expression of NOD1,and PP2(100nM)was added to inhibit the activation of SRC signaling.48h after the transfection,western blot assay was performed to detect the levels of p-SRC,SRC,P21,p-Cyclin E,Cyclin E,p-CDC2 and CDC2.The colony formation assay of these transfected cells was further performed.HepG2 and HUH7 cells were transfected with Myc-NOD1,and Myc-vector transfected cells acted as a mock control.24h after the transfection,the expression levels of MAPK pathway associated proteins including p-P3 8,P3 8,p-JNK,JNK,p-ERK and ERK were detected by western blot.HepG2 cells and HUH7 cells were transfected with two Si-NOD1 sequences,and Si-NC sequence transfected cells acted as a mock control.Western blot assay was performed to detect the levels of p-P38,P38,p-JNK,JNK,p-ERK and ERK.HepG2 cells were transfected with Si-NOD1 to knockdown the expression of NOD1,and MEK inhibitor U0126(1?M)was added to inhibit the activation of MAPK pathway.Western blot was performed to detect the expression of p-ERK and ERK.Colony formation assay of these transfected cells was further performed.HepG2 cells were co-transfected with Si-NOD1 and Si-SRC,and being further cultured for 48h before western blot assay was performed to detect the levels of SRC,p-SRC,p-ERK and ERK in these transfected cells.Protein extracted from the in vivo xenograft tumor model was used for western blot assay to detect the level of SRC-MAPK axis associated protein.5.To detect the regulatory effect of NOD1 on the chemosensitivity of HCC cells5.1 To detect the regulatory effect of NOD1 on the chemosensitivity of HCC cells to 5-FUHepG2 cells were plated in 96-well plate at the density of 1X104 cells/well.After being cultured overnight,the cells were transfected with Myc-NOD1 or mock control.The transfected cells were further treated with different dosages(0.01?g/ml,0.1 ?g/ml,1?g/ml,10?g/m,100?g/m and 1000?g/ml)of 5-FU.48h after the treatment,CCK-8 assay was performed to detect the cell viability.HepG2 cells were plated in 96-well plate at the density of 1X104 cells/well.After being cultured overnight,the cells were transfected with different dosages(0.5?g/ml,1?g/ml and 2?g/ml)of Myc-NOD1 plasmid and treated with different dosages(5?g/ml,10?g/ml and 20?g/ml)of 5-FU.Cell viabilities were detected by CCK-8 assay after the cells were treated with NOD1 and 5-FU for 48h.Combination index(CI)values were calculated by CalcuSyn software.HepG2 cells were transfected with Myc-NOD1 or mock control,followed by further treatment with 5-FU(10?g/ml)for 48h.Western blot assay was performed to detect the expression levels of p-SRC,SRC,p-ERK,ERK and P21.0.1ml of HUH7 cells were subcutaneously injected to both flanks of the nude mice at the density of 1x108cells/ml.When visible tumors appeared,the tumors were divided into 4 groups with different treatment,including Myc-vector transfected group with PBS treatment,Myc-vector transfected group with 5-FU(50mg/kg)treatment,Myc-NOD1 transfected group with PBS treatment,and Myc-NOD1 transfected group with 5-FU(50mg/kg)treatment.The tumor growth curve was recorded.The tumor volumes and tumor weights of the indicated groups were analyzed and compared.Protein extracted from the tumors was used for western blot assay to detect the expression levels of Myc,p-SRC,SRC,p-ERK,ERK,P21,p-Cyclin D and Cyclin D.5.2 To detect the regulatory effect of NOD1 on the chemosensitivity of HCC cells to sorafenibHepG2 cells and HUH7 cells were plated in 96-well plate at the density of 8 x103 cells/well.After being cultured overnight,the cells were transfected with Myc-NOD1 or mock control.The transfected cells were further treated with different dosages(0.01?M,0.1?M,1?M,10?M,100?M and 1000?M)of sorafenib.48h after the treatment,CCK-8 assay was performed to detect the cell viabilities of the treated HepG2 cells and HUH7 cells.HUH7 cells were plated in 96-well plate at the density of 8x103 cells/well.After being cultured overnight,the cells were transfected with different dosages(0.5?g/ml,1?g/ml and 2?g/ml)of Myc-NOD1 plasmid and treated with different dosages(5?M,10?M and 20?M)of sorafenib.Cell viabilities were detected by CCK-8 assay after the cells were treated with NOD1 and sorafenib for 48h.Combination index(CI)values were calculated by CalcuSyn software.HepG2 cells were plated in 6-well plate before being transfected with Myc-NOD1 or mock control.6h after the transfection,cells were treated with sorafenib(5?M),and the cells with the treatment of the same volume of DMSO acted as a vehicle control.24h after the treatment,cells were collected to perform the colony formation assay.Sorafenib was added to the cells every 72h,and colony number was counted 8 days after the transfection.HepG2 cells were transfected with Myc-NOD1 or mock control,followed by further treatment with sorafenib for 48h.Western blot assay was performed to detect the levels of p-SRC,SRC,p-ERK and ERK.Results1.NOD1 significantly suppressed HCC progression1.1 NOD1 was significantly downregulated in HCC tissuesIHC assay and western blot assay from pairs of HCC tissues and matched non-cancerous liver tissues indicated NODI was significantly downregulated in HCC tissues compared with corresponding non-cancerous liver tissues.The clinical investigation showed significant downregulation of NOD1 in HCC tissues,which indicated that NOD1 might play a role in the development of HCC.1.2 Exogenous overexpression of NOD1 significantly inhibited tumorigenesis in mice modelTo further verify whether loss of NOD1 expression contributed to HCC progression,we detected the effect of NOD1 on xenograft tumor model in nude mice by subcutaneously injecting HUH7 cells to both flanks of the nude mice.When visible tumors appeared,tumors at the left flanks were injected with 20?g of Myc-NOD1 plasmid while tumors at the right flanks were injected with 20?g of Myc-vector plasmid every other day until the mice were sacrificed on day 28 after the first injection.Tumors were isolated from both flanks of the mice for further analysis.Western blot was performed to verify the successful overexpression of NOD1 in Myc-NOD1 transfected group.We showed that both tumor sizes and volumes of Myc-NOD1 injected group were significantly smaller than the Myc-vector injected group.Besides,the tumor weights of Myc-NOD1 transfected group were much less than those of the Myc-vector transfected group.Altogether,the clinical investigation together with the in vivo animal experiment indicated that NOD1 participated in the development of HCC.2.NOD1 significantly inhibited the proliferation of HCC cells via inducing cell cycle arrest2.1 Loss of NOD1 expression promoted the proliferation of HCC cellsHepG2 and HUH7 cells were used to construct the loss-of-function cellular model by transfection with specific SiRNAs against NOD1,and the successful knockdown of NOD1 was verified by western blot.After we knocked down NOD1 expression in HCC cells by two Si-NOD1 sequences,the proliferation status was detected by CCK-8 assay.The Si-NOD1 transfected group showed much higher growth rate than the Si-NC transfected group,which indicated that absent expression of NOD1 promoted proliferation of HCC cells.Colony formation assay further verified that knockdown of NOD1 expression increased the colony formation capability of HCC cells.2.2 Overexpression of NOD1 suppressed the proliferation of HCC cellsTo further define the effect of NOD1 in HCC cells,HUH7 cells and HepG2 cells were transfected with Myc-NOD1 plasmid to construct the gain-of-function cellular model.CCK-8 assay together with colony formation assay demonstrated that exogenous overexpression of NODI significantly suppressed the proliferation and colony formation capabilities of HCC cells.2.3 NOD1 suppressed the proliferation of HCC cells via inducing cell cycle arrestThe analysis of cell cycle progression showed that knockdown of NODI significantly induced cell cycle into mitosis phase,while overexpression of NOD1 dramatically induced cell cycle arrest at G1 phase in HCC cells.Besides,our data showed that overexpression of NOD1 in HCC cells significantly upregulated the expression of P21,while it significantly inhibited the levels of p-Cyclin D,Cyclin D,p-Cyclin E,Cyclin E,p-CDC2 and CDC2 in HCC cells.On the contrary,knockdown of NOD1 expression significantly suppressed the P21 level while upregulated the levels of p-Cyclin D and Cyclin D.Thus,these data further verified that NOD1 inhibited the proliferation of HCC cells by inducing cell cycle arrest at G1 phase.3.NODI exerted its effect by directly interacting with SRCCo-IP assay showed that NOD1 could stably bind with SRC protein.Immunofluorescence(IF)staining and laser confocal photographing further defined that NODI and SRC co-localized in the cytoplasm of HCC cells.To further identify whether the binding between NOD1 and SRC was a direct interaction,we did Co-IP assay with the in vitro synthesized NOD1 and SRC proteins by an in vitro protein translation system,and our data further confirmed the direct interaction between NOD1 and SRC.All these data together demonstrated that NOD1 exerted its function by directly interacting with SRC.To further determine which domain of SRC is responsible for the interaction with NOD1,we constructed a series of truncated mutants of SRC,and Co-IP assay indicated SRC interacted with NOD1 via its SH3 domain.Next,we constructed the truncated mutants of NOD1 to identify the binding domain of NOD1 with SRC,and Co-IP assay indicated that NOD1 interacted with SRC via its CARD domain.These data further indicated that NOD1 directly interacted with SRC in HCC cells.4.NOD1 acted as a tumor suppressor via inhibiting SRC activation in HCCOur western blot data showed overexpression of NODI significantly decreased the phosphorylation level of p-SRC at Tyr419 site,while knockdown of NOD1 dramatically increased the phosphorylation of this site.These data suggested NOD1 effectively inhibited the activation of SRC signaling in HCC cells.Our data showed that after blocking SRC signaling by its specific inhibitor PP2,the expression of p-CyclinE,CyclinE,p-CDC2 and CDC2 was significantly decreased while the P21 expression was dramatically increased in Si-NOD1 transfected cells,which indicated that suppression of SRC signaling was responsible for the cell cycle arrest induced by NOD1.Furthermore,the colony formation capability of Si-NOD1 transfected HCC cells was also significantly decreased after block of SRC signaling.All these data verified that NODI inhibited cell cycle progression of HCC cells by inhibiting SRC signaling.5.NOD1 significantly inhibited the activation of SRC-MAPK axis in HCCOur investigation indicated overexpression of NOD1 effectively inhibited the activation of MAPK signaling pathway,while absent expression of NOD1 significantly activated the MAPK signaling pathway.To further verify that NOD1 exerted its anti-tumor effect through MAPK pathway,we treated the Si-NOD1 transfected HCC cells with MAPK inhibitor U0126 and further detected their malignant behaviors.Our data showed that after blocking MAPK pathway,the colony formation capability of Si-NOD1 transfected cells was significantly inhibited,which indicated that NOD1 inhibited HCC cells via MAPK pathway.Western blot data further showed that block of SRC signaling by Si-SRC could significantly rescue the activation of MAPK pathway by transfection with Si-NOD1,which indicated that MAPK pathway was downstream of SRC signaling.Besides,western blot assay of the protein extracted from the xenograft tumors in nude mice further validated that NOD1 inhibited the activation of MAPK pathway via suppressing the activation of SRC signaling.All these data verified that NODI suppressed HCC progression by inhibiting SRC-MAPK axis.6.NOD1 significantly enhanced the response of HCC cells to 5-FUDrug sensitivity tests showed that NOD1 could significantly increase response of HCC cells to 5-FU treatment.Besides,the combination of NOD1 and 5-FU significantly inhibited the proliferation of HepG2 cells.Further investigation indicated that NOD1 synergistically promoted the therapeutic effect of 5-FU on HCC cells.Western blot assay further validated that NOD1 synergistically promoted the effect of 5-FU by inhibiting SRC signaling and MAPK pathway,which was also accompanied with upregulating P21 expression level.These in vitro data indicated that NOD1 significantly enhanced chemosensitivity of HCC cells to 5-FU treatment.Our in vivo data demonstrated that the Myc-NOD1 transfected group with 5-FU treatment showed the slowest growth rate compared with other groups.After 5-FU treatment,the volumes and weights of the NOD1 transfected tumors were both significantly smaller than the other three groups.Western blot assay showed that NOD1 transfected tumors with 5-FU treatment had the lowest levels of p-SRC,p-ERK and p-CyclinD,whereas tumors in this group had the highest level of P21 expression,which was in consistence with our in vitro data showing NOD1 significantly enhanced the chemosensitivity of HCC cells to 5-FU via inhibiting the activation of SRC-MAPK axis.7.NOD1 significantly enhanced the response of HCC cells to sorafenibDrug sensitivity tests showed that NOD1 significantly increased the chemosensitivity of HCC cells to sorafenib treatment,and the combination of NOD1 and sorafenib can significantly inhibit the proliferation of HCC cells.Further investigation showed that NOD1 synergistically promoted the therapeutic effect of sorafenib.Colony formation further validated NOD1 dramatically inhibited HCC proliferation after sorafenib treatment.Western blot assay further validated that NOD1 synergistically promoted the effect of sorafenib by inhibiting SRC signaling and MAPK pathway.All these data demonstrated that NOD1 significantly enhanced the chemosensitivity of HCC cells to chemotherapeutic treatment.Part ? The effect and molecular mechanism of NOD2 in the progression ofhepatocellular carcinomaMaterials and Methods1.To detect the regulatory effect of NOD2 on the progression of HCC in vivo1.1 To detect the regulatory effect of NOD2 on the progression of HCC by DEN/CC14 induced HCC mice modelNOD2-/-mice(n=7)and WT mice(n=8)were injected with DEN(100mg/kg,i.p.)at the age of 6 weeks followed by 12 injections of CC14(0.5ml/kg,i.p.).The mice were sacrificed at 24 weeks after DEN injection.Formed tumors in livers were separated from the NOD2-/-mice and WT mice.NOD2 expression of the isolated livers from NOD2-/-and WT mice was detected by western blot assay.Tumor numbers,liver body ratios,the largest tumor diameters and body weights of the NOD2-/-and WT mice were analyzed and compared.HE staining was performed to detect the differentiation status of indicated tissues.1.2 To detect the effect of NOD2 on the progression of HCC by xeno-grafted tumor model in nude mice1 x 107 HUH7 cells were subcutaneously injected to the right flanks of the nude mice(n=15).When visible tumor appeared,the mice were randomly divided into three groups.Two groups of tumors(n=5 for each group)were injected with two independent siRNAs against NOD2(Si-NOD2),and the other group(n=5)was injected with nonsense sequence(Si-NC)as a control.The mice were sacrificed on day 38 after the first transfection,and the formed tumors were statistically analyzed.Tumor volumes and tumor weights of these three groups of tumors were measured and statistically analyzed.Relative NOD2 mRNA expression in these groups was detected by qRT-PCR.The relative NOD2 protein expression was detected by western blot.1 x 107HUH7 cells were subcutaneously injected to both flanks of the nude mice(n=11).When visible tumor appeared,the tumors in the left flanks were transfected with NOD2 plasmid while tumors in the right flanks were transfected with mock plasmid.The mice were sacrificed and the tumors were isolated on day 21 after the first injection of the plasmid.Tumor volumes and tumor weights of NOD2 and mock plasmid transfected groups were statistically analyzed and compared.qRT-PCR and western blot were performed to detect the relative NOD2 expression level in NOD2 plasmid and mock plasmid transfected group.2.To detect the regulatory effect of NOD2 on the progression of HCC in vitroWestern blot was used to detect the basic expression level of NOD2 in HCC cell line,HCC cells with high NOD2 expression was used to construct NOD2 loss-of-function cellular model,while HCC cells with low NOD2 expression was used to construct NOD2 gain-of-function cellular model.Three interference RNAs against NOD2 were synthesized and tested in HUH7 cells for their blocking efficiency.HUH7 cells were transfected with the mixture of Si-NOD2-1 and Si-NOD2-2,and the proliferation of HCC cells was detected at Oh,24h,48h and 72h.HUH7 cells were transfected with the mixture of Si-NOD2-1 and Si-NOD2-2,and the invasion and colony formation capabilities of these transfected cells were detected and compared.HepG2 cells were transfected with NOD2 plasmid or mock plasmid,western blot was performed to verify the successful overexpression of NOD2.The proliferation of HCC cells was detected at Oh,24h,48h and 72h after the transfection.Invasion and colony formation capabilities of these transfected cells were detected and statistically analyzed.HUH7 cells were plated in 96-well plate at the density of 6x103 cells/well.After being cultured overnight,the cells were treated with MDP(10?g/ml)or control.48h after the treatment,CCK-8 assay was performed to detect the cell viability.HUH7 cells were treated with the MDP(10?g/ml)or control,and the invasion and colony formation capabilities of these transfected cells were detected and compared.Cells were treated with MDP every 24 hours.3.To detect the regulatory effect of NOD2 on the progression of HCC by clinical specimen165 pairs of HCC tissues and their corresponding non-cancerous liver tissues were used for IHC assay to detect the location and relative expression of NOD2.NOD2 expression by IHC assay in cancerous and non-cancerous liver tissues was further quantitatively analyzed by IPP6 software.NOD2 expression in different TNM stages by IHC assay was statistically analyzed.Expression levels of NOD2 mRNA in 64 HCC tissues and matched non-cancerous liver tissues were detected and statistically analyzed.Western blot was performed to detect the relative protein levels of NOD2 in paired HCC samples.The relative band densities from all of the detected patients were analyzed by Image J software and normalized by P-actin.The relative NOD2 protein expression in well-differentiated tumors and poor-differentiated tumors were statistically analyzed.4.To detect the signal transduction pathway of NOD2 in HCCHepG2 cells were transfected with NOD2 plasmid or mock control plasmid,and further cultured for 24h.Activation status of AMPK pathway,including p-LKB1,p-AMPKa and p-AMPK? were detected by western blot.HUH7 cells were transfected with Si-NOD2 or Si-NC,and the activation status of AMPK pathway was detected by western blot.HepG2 cells were transfected with NOD2 plasmid or mock control plasmid,and the activation status of mTORC1 pathway,including p-S6K1,p-S6 and p-4E-BP1 were detected by western blot.HUH7 cells were transfected with the mixture of two SiRNAs against NOD2(Si-NOD2),and the cells transfected with random sequences(Si-NC)acted as a mock control.Activation status of mTORC1 pathway was detected by western blot.HepG2 cells were transfected with NOD2 plasmid,and further cultured for 6h before compound C(10?M)was added to inhibit the activation of AMPK pathway.Western blot assay was performed to detect the expression of NOD2,p-mTOR,p-AMPKa,p-S6K1,p-S6 and p-4E-BP1 after the transfection.Colony formation and invasion of the NOD2-transfected HCC cells with compound C treatment were analyzed.The protein from tumor tissues of DEN/CCl4 HCC model and xenografted tumor models in nude mice were extracted respectively.Relative expression of the related signaling transduction pathway proteins,including AMPK pathway related proteins and mTORC1 pathway related proteins were detected by western blot.5.To detect the effect of NOD2 on autophagy and apoptosis of HCC cellsHepG2 cells were transfected with NOD2 plasmid and the autophagy related markers were detected by western blot.HUH7 cells were transfected with Si-NOD2,and the autophagy related markers including Beclin-1,ATG12,ATG7,ATG5,ATG3,ATG16L1 and LC3 were detected by western blot.HepG2 cells were transfected with NOD2 plasmid and further cultured for 24h before apoptosis assay by Annexin V-PI staining.HepG2 cells were transfected with NOD2 plasmid and further cultured for 24h.Activation of Caspase9 and Caspase3 were detected by western blot.HUH7 cells were transfected with Si-NOD2 and further cultured for 24h before western blot assay of Caspase9 and Caspase3.NOD2 transfected HepG2 cells were co-transfected with Si-ATG5 to block autophagy in NOD2 overexpressed cells.Autophagy and apoptosis levels of the transfected cells were detected by western blot.The protein from tumor tissues of DEN/CC14 HCC model and xenografted tumor models in nude mice were extracted respectively.Western blot assay was performed to detect the activation of autophagy and apoptosis.6.To detect the regulatory effect of NOD2 on the chemosensitivity of HCC cellsHepG2 cells were plated in 96-well plate at the density of 1x104 cells/well.After being cultured overnight,the cells were transfected with Myc-NOD2 plasmid or mock control plasmid.The transfected cells were further treated with different dosages of sorafenib or lenvatinib or 5-FU.48h after the treatment,CCK-8 assay was used to detect the cell viability.HepG2 cells were plated in 6-well plate before transfected with NOD2 plasmid or mock control plasmid.HCC cells were treated with sorafenib or lenvatinib or 5-FU,and the cells treated with the same volume of DMSO acted as a control group.Annexin V/PI staining was used to detect cell apoptosis after the treatment with sorafenib for 48h.HepG2 cells were transfected with Myc-NOD2 plasmid or mock control plasmid,followed by further treatment with sorafenib(5?M)or lenvatinib(10?M)for 48h.Western blot assay was performed to detect the activation of AMPK pathway and mTORCl pathway.Apoptosis and autophagy related markers of the sorafenib or lenvatinib treated HCC cells were also detected by western blot.7.To explore the mechanism of NOD2 regulating AMPK signal pathwayHUH7 cells were cultured for 24h before cell lysate was obtained for Co-IP assay.The endogenous interaction between NOD2 and LKB1/AMPKa,LKB1 and NOD2/AMPKaas well as AMPKa and NOD2/LKBlwere detected by IP assay by using the indicated antibodies.Immunofluorescence assay was performed to verify the co-localization between NOD2 and AMPKa/LKB1.NOD2,AMPKa and LKB1 proteins were obtained from the in vitro translation system.NOD2 protein was mixed with AMPKa or LKB1 protein,and the direct binding between NOD2 and AMPKa,NOD2 and LKB1 were detected by IP assay.Results1.NOD2 deficiency promoted hepatocarcinogenesis in NOD2 null miceTo verify whether NOD2 played a role in HCC progression,we constructed HCC animal model by injecting N-Nitrosodiethylamine(DEN)and carbon tetrachloride(CC14)to NOD2-/-and WT mice multiple times.The mice were sacrificed 24 weeks after the induction,and the tumor formation status of WT and NOD2-/-mice were analyzed and compared.NOD2 deficiency in NOD2-/-mice was confirmed by western blot.Compared with the WT mice,the tumor numbers,liver body ratios,and tumor diameters in the NOD2-/-mice were significantly increased,whereas the body weights of the NOD2-/-mice were significantly decreased.HE staining showed that tumors in the NOD2-/-mice were less differentiated compared with the WT mice.Furthermore,the NOD2-/-mice presented more metastasis loci in the mesentery and the diaphragm than those in the WT mice,and these metastatic loci were further confirmed by pathological analysis by two professional pathologists.These data collectively indicated that NOD2 deficiency facilitated hepatocarcinogenesis and exacerbated HCC development.2.NOD2 suppressed malignancy of HCC cells in vivoBoth the volumes and weights of the formed tumors were dramatically increased in the Si-NOD2 transfected groups compared with the Si-NC transfected group.Real-time polymerase chain reaction(PCR)and western blot assay demonstrated successful knockdown of NOD2 in the Si-NOD2 transfected groups,which verified that loss of NOD2 expression exacerbated hepatocarcinogenesis.Our results showed that NOD2 plasmid had significantly smaller tumor volumes and lower tumor weights than those tumors transfected with the mock plasmid.Real-time PCR and western blot assay verified successful overexpression of NOD2 in the NOD2 plasmid transfected group.Thus these in vivo data indicated that NOD2 inhibited the xenograft tumor growth in HCC mice models.3.NOD2 suppressed malignancy of HCC cells in vitroWe constructed gain-of-function cellular model by transfection of NOD2 plasmid to HepG2 cells which had a lower level of NOD2 expression,and we further constructed loss-of-function model by transfection of Si-NOD2 to HUH7 cells which had a higher level of NOD2 expression.Our data showed that after knockdown of NOD2,the proliferation,invasion and colony formation capabilities of HCC cells were significantly increased,while overexpression of NOD2 in HCC cells inhibited these malignant behaviors.Besides,our data showed that in agreement with the effect of exogenous NOD2 overexpression,MDP treatment could also inhibit the malignant behaviors of HCC cells.Altogether,these in vitro data further supported our data from animal models that NOD2 acted as a tumor suppressor in HCC progression.4.Absent expression of NOD2 contributed to clinical hepatocellular carcinoma progressionIHC assay indicated that NOD2 expression was significantly decreased in HCC tissues compared with matched non-cancerous liver tissues,average optical density analysis showed the same outcome.Further analysis of the IHC staining showed that NOD2 expression was dramatically decreased in HCC patients with advanced tumor node metastasis(TNM)stages.In consistence with the IHC data,quantitative real time PCR(qRT-PCR)and western blot assay also showed that NOD2 expression was either co...