| BackgroundPrimary hepatocellular carcinoma(HCC)is one of the most frequently malignant tumors,and a leading cause of cancer-related deaths in humans.Of the HCC patients,more than 50%of the world's HCC patients occur in China.At present,the prevalence of HCC is still growing,and it is the main public health problem facing the world in the 21st century,especially China.The occurrence and development of hepatocellular carcinoma is an extremely complex biological process,involving many environmental and genetic risk factors,such as activation and inactivation of molecular and cellular signaling pathways,disordering of oncogenes and anti-cancer genes,cancer stem cell differentiation,etc.Traditional treatments such as surgical resection,interventional embolization,chemotherapy and radiation therapy have a low cure rate and a high rate of recurrence and metastasis;Due to the complexity of molecular pathogenesis and the heterogeneity of HCC,targeted medicines,such as polytyrosine kinase inhibitors and programmed cell death protein-1 antagonists,are not able to achieve universal results,so new therapeutic targets based on specific molecular signaling pathways in HCC become more and more urgently in recent years.Innate immunity is a natural immune defense function gradually formed by organisms during long-term phylogenetic evolution.It recognizes risk-related factors including danger-associated molecular patterns(DAMPs),or pathogen-associated molecular patterns(DAMPs)through germline-encoded pattern recognition receptors(PRRs)and becomes the first-line defense barriers of host against various invading pathogens.Meanwhile,it is also involved in the development of many pathophysiological processes including various cancers.Initiation and regulation of the innate immune response is mediated by PRRs,like Toll-like receptors(TLRs),retinoic-acid-inducible gene I(RIG-I)-like receptors(RLRs),nucleotide-binding oligomerization domain(Nod)-like receptors(NLRs),absent in melanoma 2(AIM2),AIM2-like receptors(ALRs),C-type lectin receptors(CLRs)or other receptors.PRRs and their regulatory factors play a role in tumor suppression or cancer promotion under different tissues,different cell types and different pathophysiological conditions.Some PRRs can induce an anti-cancer immune response to inhibit tumor development.While,the out-of-control innate immune signaling pathway is an important mechanism for cancer cell proliferation and evasion of immune surveillance.Chronic inflammatory response is an important driver of cancer development and progression,and activation of the innate immune response through the innate immune signaling pathway is a bridge between chronic inflammation and cancer.Researchers have found that PRRs associated innate immunity play a key roles in the development and progression of various tumors,including HCC.As a protein of DNA receptor,HIN-200 family induces formation of inflammasome complexes involving in regulation of cell differentiation,proliferation,senescence,and apoptosis through sensing pathogen DNA or damaging DNA to exert inhibitive role in tumor progression.IFI16 is an interferon-inducible member of the human HIN-200 nuclear protein family.It functions as a complicated factor in tumors,since it either has been regarded as a transcriptional regulator of tumor suppression in most tumors,or oncogenic,including HCC.Besides,IFI16 has been identified to forming atypical inflammasome complex in virus infection-associated cancer,including Kaposi sarcoma associated herpes virus,lymphoma or nasopharyngeal carcinoma associated EB virus.Therefore,elucidating the molecular mechanisms of innate immunity and the role of PRRs and their signaling pathways in HCC,will provide references to new therapeutic targets and therapeutic strategies for the treatment of HCC.ObjectiveAccording to literatures,IFI16 can act as a tumor suppressor or oncogenic factor in HCC.The underlying mechanism of IFI16 in HCC is still need to be fully understood.Therefore,this study intends to detect the expression of IFI16 in hepatocarcinoma tissues and hepatocellular carcinoma cell lines,and to confirm that IFI16 mediating formation of inflammatory complexes in the pathogenesis of HCC.In this study,series of experiments were performed to find out the signal transduction pathways associated with molecular mechanism of HCC pathogenesis to elucidate the role IFI16 in HCC.Material and methods1.Twenty pairs of hepatocellular carcinoma and para-cancerous tissues from patients were collected,and hematoxylin and eosin staining(H&E staining)and immunohistochemistry were performed to identify the expression,positioning of IFI16 in hepatocarcinoma tissues and adjacent tissues.Expression of IFI16 on mRNA and protein level evaluated using quantitative real-time polymerase chain reaction(qRT-PCR)and Western blotting assay.2.Expression of IFI16 on mRNA and protein level were assessed using qPCR and Western blot in cultured human hepatoma cell lines,including HepG2,HL-7702,Huh7,Bel-7402,SMMC772 and human normal liver cell line,L-02.3.Hepatoma cell lines(Huh-7 and SMMC-7721)with relatively low expression of IFI16 were selected for overexpression experiments.The two hepatocellular carcinoma cell lines were transfected with pcDNA3.1-IFI16 plasmid or empty plasmid respectively.Then IFI16 expression were determined by qRT-PCR and Western blot.4.Cell viability of IFI16-overexpressed Huh-7 and SMMC-7721 cells were measured by Cell counting Kit-8(CCK8).5.Cell migratory ability,invasion,and proliferation were measured in cells after IFI16 overexpressing,treated with p53 inhibitor(PFT?),and inflammasome inhibitor(AC-YVAD-CMK)using colony formation,wound healing,and Transwell assay respectively.6.Cell apoptosis rate was determined by flow cytometry in IFI16-overexpressed,PFT a treated,and AC-YVAD-CMK treated Huh-7 and SMMC-7721 cells.7.Western blot assay was used to determine the expression of proteins related to p53 signaling pathway(p53,p-p53ser15,p21),inflammasome complex(ASC,pro-caspase-1,caspase-1,pro-IL-1 ?,IL-1 ?,pro-IL-18,IL-18)in cells treated with pcDNA3.1 IFI16 plasmid and AC-YVAD-CMK.8.ELISA assay and LDH release assay were used to assess the level of IL-1?,IL-18 and LDH in cultural environment of IFI16-overexpressed Huh-7 and SMMC-7721 cells after treated with PFT?,AC-YVAD-CMK at 12h,24h and 48h after treatments.9.Balb/c nude mice were used to subcutaneously inject hepatoma cell line to establish a xenograft animal model.Afterwards,p53 inhibitor and inflammatory complex inhibitor were used in rats.H&E staining and immunohistochemical assay were used to determine the expression of IFI16,p53 and Ki67 in tumors under different conditions.Results1.The expression of IFI16 protein was mainly located in the nuclei of hepatocytes,and the expression level of IFI16 in liver cancer tissues was significantly lower than that in adjacent tissues(p<0.01,p<0.001).Similarly,the expression level of IFI16 in hepatocellular carcinoma cell lines was significantly lower than that in normal liver cell lines(P<0.05;P<0.01).2.Compared with the blank group or the negative control group,the cell viability was significantly decreased in the Huh-7 and SMMC-7721 cell lines 72 hours after cells transfected with the IFI16 pcDNA3.1 vector(p<0.05).Results of clonal formation assay,cell scratch assay and Transwell assay showed that the colony formation ability,migration and invasion ability were significantly decreased in IFI16-overexpressed cells(p<0.05,p<0.01).3.Compared with the blank group and the negative control group,cells transfected with IFI16-overexpressed vector for 48h showed a significant promotive role in increasing apoptosis levels(p<0.05).4.The expression levels of p53,p-p53ser15 and p21 were significantly increased at 72 h after transfection of IFI16 compared with the blank group and the negative control group(p<0.05).The hepatoma cell line with p53 inhibitor PFTa in overexpressed IFI16 showed a relative lower apoptosis level compared with IFI16 hepatocellular carcinoma cell line,and the ability of colony formation,migration and invasion of liver cancer cells was significantly enhanced(p<0.05).5.Results of Western blotting assay showed that expression of inflammasome-related proteins,ASC,pro-caspase-1,caspase-1 and downstream proteins of inflammasome,pro-IL-1?,IL-1?,pro-IL-18 and IL-18 were significantly up-regulated(p<0.05).While these proteins in cells treated with AC-YVAD-CMK were reversed significantly compared with IFI16-overexpressed cell lines(p<0.05).6.Apoptosis level presented a relative higher tendency in IFI16-overexpressed cells compared with Blank group(p<0.01).While this up-regulated apoptosis level induced by IFI16 overexpression was reversed by further treatment of AC-YVAD-CMK significantly(p<0.05).Meanwhile,results of clonal formation assay,cell scratch assay and Transwell assay showed that IFI16 overexpression significantly suppress cell proliferation,migration and invasion ability compared with blank group(p<0.05,p<0.01);However,it was reversed by treatment of AC-YVAD-CMK,characterized by upregulation of cell proliferation,migration and invasion ability in IFI16-overexpressed cell lines(p<0.05).7.IL-1?,IL-18 and LDH were relative higher in IFI16-overexpressed cell lines 48 h after transfection.However,these evoked IL-1?,IL-18 and LDH level were then reversed by AC-YVAD-CMK(p<0.01 or p<0.05).8.IFI16 overexpression presented a significantly suppressive role in tumor volume and volume growth rate(p<0.05)compared with control.While,tumor volume and volume growth rate were recovered significantly by treatment of PFTa(p<0.05),a p53 inhibitor and inflammasome inhibitor AC-YVAD-CMK.Results of immunohistochemical assay showed that expression of IFI16 and p53 were significantly higher in tumor induced by IFI16-overexpressed cell(p<0.05),accompanying with ki67 expression decreasing(p<0.01)while PFT? and AC-YVAD-CMK suppressed p53 and IFI16-inflammasome pathway in tumor according to immunohistochemical assay,with increasing of Ki67 expression(p<0.05).Conclusions1.The expression of IFI16 was down-regulated in hepatocellular carcinoma tissues and human hepatocellular carcinoma cell lines,suggesting that downregulation of IFI16 expression or dyfunction may be involved in the pathogenesis of HCC.2.In hepatocellular carcinoma(HCC)cells,IFI16 can inhibit tumor migration,invasion and proliferation by activating the inflammasome complex activity of tumor cells,and this inhibitive effects can be significantly reversed by the presence of inflammatory complex inhibitors,AC-YVAD-CMK.3.In hepatocellular carcinoma(HCC)cells,IFI16 mediates tumor inhibition by stimulating inflammasome complex signaling via p53/p21 pathway,and p53 inhibitor can reverses IFI16-mediated tumor suppression.4.In vivo,overexpression of IFI16 can inhibit the growth of HCC tumors,and the presence of p53 inhibitors and inflammasome complex inhibitors can block the anti-tumor effect induced by IFI16.IFI16 plays an important role in the regulation of hepatocarcinogenesis and development by activating inflammasome complex via p53/p21 pathway. |
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