The Mechanisms Of Semaphorin 7A R148W Functional Mutation In Liver Inflammation And Programmed Cell Death

BackgroundSemaphorins are a protein family characterized by a conserved extracellular amino‐terminal SEMA domain.Semaphorin 7A（SEMA7A）is classified as the VII semaphorin due to its unique glycosylphosphatidylinositol（GPI）anchorage on the cell membrane.SEMA7A plays an essential role in both the nervous and immune systems which mainly mediated by its receptors,including Integrins and Plexins.Accumulating evidence has shown that the SEMA7A^R148W mutation(Sema7a^R145W in mice)is a gain-of-function mutation and a risk factor involved in progressive familial intrahepatic cholestasis（PFIC）and metabolic associated fatty liver disease（MAFLD）.Inflammation is an important immune response involved in the occurrence and development of cholestasis and fatty liver disease.Therefore,the molecular mechanism of the SEMA7A^R148W mutation in liver inflammation needs to be studied and clearly described.Inflammation underlies a broad range of physiological and pathological processes and is closely associated with cholestasis and MAFLD.Under sustained hepatic inflammation circumstances,the liver parenchyma will be damaged and generally exhibit a higher cancer incidence.Thus,the exploration of the molecular mechanisms of chronic inflammation could represent attractive therapeutic targets with a reduced risk of tumour progression.Nuclear factor kappa B（NF-κB）,an important transcription factor in the inflammatory response,plays an essential role in chronic liver disease.The NF-κB protein family consists of p50,p52,p65（also known as Rel-A）,C-Rel and Rel-B.NF-κB p50 mostly arises from the precursor protein NF-κB p105（NFKB1）and forms heterodimers with p65.In stimulated cells,NF-κB p50/p65 heterodimers are released from the inhibitory complex and phosphorylated immediately.After all these processes,NF-κB p50/p65 enters the nucleus,binds to DNA,and activates gene transcription.Proinflammatory cytokines such as TNF-αand IL-1βare principally encoded by target genes of the NF-κB p50/p65 canonical pathway.The existence of chronic liver inflammation can damage the normal hepatocytes,inspiring the liver fibrosis,inducing liver cirrhosis,even turning to liver cancer.The programmed cell death which including apoptosis,pyroptosis,necroptosis,ferroptosis and autophagy can regulate homeostasis in different kinds of cells and tissues by eliminate damage,infectious and aged cells.The activating pathway of apoptosis included intrinsic pathway which related to mitochondria and extrinsic pathway which related to tumor necrosis factor receptor（TNFR）,TNF-αetc.As for pyroptosis activating,the inflammasome NLRP3 pathway is the most classic way.And NLRP3 is one of the target gene which upregulated by NF-κB p65 signalling.Objective（1）To determine the molecular mechanisms of SEMA7A^R148W mutation and SEMA7A^WThigh expression in liver inflammation and hepatocellular carcinoma（HCC）.（2）To explore the relationship between SEMA7A^R148W mutation and programmed cell death.Methods（1）Designed and generated heterozygous/homozygous Sema7a ^R145W mutant mice（C57BL/6J background）to determine the mutation effects and corresponding molecular mechanisms in liver inflammation.（2）Establish diethylnitrosamine（DEN）/CCl4 induced hepatocellular carcinoma mousemodel to explore the effects of semaphorin 7A protein in pro-inflammation.（3）Using Haematoxylin–eosin（H&E）-staining experiment to evaluate the level ofimmunoinflammation cell infiltration in homozygous Sema7a ^R145W mutant mice and wild type mice.（4）Using immunofluorescence（IF）experiment to determine the expression and cellularlocalization of p-NF-κB p65 Ser529.（5）Evaluate expression levels of classical NF-κB pathway related proteins andpro-inflammation cytokines by Real-time quantitative reverse transcription-polymerase chain reaction（Real-Time q RT-PCR）experiment and Western blotting experiment.（6）Construct corresponding plasmids and using coimmunoprecipitations（co-IP）experiment to determine the protein-protein interaction among integrinβ1 and NF-κB p105.（7）Use wound healing assay and cell counting kit-8（CCK-8）assay to analyze HCCcell migration and proliferation rate in SEMA7A overexpression model.（8）Using TUNEL staining experiment to evaluate the apoptosis level in liver tissue ofSema7a^R145Wheterozygous and homozygous mouse.（9）Using Western blotting experiment and Real-Time q RT-PCR experiment to evaluatethe level of different kinds of programmed cell death in liver tissues and primary hepatocytes from Sema7a^R145Wheterozygous and homozygous mice.Results（1）The inflammatory infiltration in liver sections of Sema7a^R145W homozygous mice is higher than those from the WT group,and phosphorylated proteins in NF-κB p50/p65pathway are increased in Sema7a^R145W homozygous mice.Together,Sema7a^R145W mutation might play a critical role in inflammation via the NF-κB p50/p65 pathway.（2）Vivo and vitro experiment results displayed that integrinβ1 interacts with NF-κB p105,promoting NF-κB p105 into p50 and activating the downstream pathway.（3）The SEMA7A^R148W mutation was not found in HCC patients,however,the m RNA levels of SEMA7A^WT and integrinβ1 were significantly increased in the tumour group and Sema7a/integrinβ1/NF-κB p105 signalling pathway is activated in hepatocellular carcinoma.（4）Subsequent wound healing and cell proliferation assays proved that upregulated SEMA7A^WT could accelerate the migratory and proliferation capability of Hep G2 cells.（5）Apoptosis level and pyroptosis level are both increased in Sema7a^R145Wheterozygous and homozygous mice liver,whearese necroptosis level is depressed and autophagy level is not activated.ConclusionIn this study,in a genetic Sema7a^R145W homozygous mouse model,we first identified that integrinβ1 could interact with the C-terminal domain of NF-κB p105 to promote p50generation and stimulate the NF-κB p50/p65 canonical pathway,consequently rendering hepatocytes more susceptible to inflammation.Furthermore,we discovered that the protein level of Sema7a^WT(SEMA7A^WT)is increased in HCC patients,HCC mouse model and HCC cell lines.The ectopic expression of SEMA7A^WT in vitro also activated NF-κB p50/p65 and upregulated proinflammation cytokines.In addition,we observed ascending cell migration and proliferation in SEMA7A^WT transfected Hep G2 cells.Together,these results suggest that both the Sema7a^R145W(SEMA7A^R148W)mutation and high Sema7a^WT(SEMA7A^WT)protein expression displayed similar proinflammatory signalling.Sema7a^WT(SEMA7A^WT)is correlated with HCC,which of extensive clinical significance and might be a therapeutic target in inflammation and tumorigenesis.Also,in this study,we firstly found that apoptosis and pyroptosis can be activated in Sema7a^R145Wheterozygous and homozygous mice liver via extrinsic pathway and NLRP3 inflammasome pathway,respectively.Further exploring the molecular mechanisms of this mutation in programmed cell death are with profound significance.