The Effect And Mechanism Of PID1 On Insulin Resistance And Hepatocarcinoma Induction

BackgroundAccumulated epidemiologic studies reveal that obesity,insulin resistance(IR)and nonalcoholic fatty liver disease(NAFLD)are risk factors for cancer,including liver cancer.In these conditions,numerous molecular events potentially contribute to tumor-promoting activity including activation of cancer-promoting pathways,induction of chronic inflammation,modification of epigenetic,provocation of dysbiosis,and regulation of lipid metabolism.However,there still lacks effective targets for insulin resistance-induced liver cancer.PID1 is a new obesity related gene found highly expressed in abdominal adipose tissue of obeses.Existing studies have focused on its role and mechanism of insulin resistance.Our previous study demonstrated that PID1is highly expressed in tumor cells compared to normal cells,which prompted us that PID1 may be a potential oncogenic gene.In our previous study on ob/ob mouse model,we identified that knockdown of PID1 can significantly decrease mouse body weight,reduce fat volume and weight,and improve fatty liver and insulin resistance.ObjectiveBased on the preliminary work,our study will inveatigate the function and molecular mechanism of PID1 on the development of hepatocarcinoma,further explore the mechanism of PID1 interference on improving fatty liver and insulin resistance of ob/ob mice,and then identify the role of PID1 on liver cancer promoting in insulin resistance.MethodsFor the first,we detected the expression of PID1 on cells,tissue chips and clinical samples of liver cancer.And we employed chromatin immunoprecipitation(ChIP)to identify the histone modification of PID1.Then we established a liver cancer xenograft model to identify the effect of PID1 knockdown on tumor growth and immune conditions.We subsequently constructed liver-specific overexpression of PID1 mouse model by hydrodynamic injection,to clarify the effect of PID1 on liver pathological changes,glycolysis and lipid metabolism conditions,and EGFR/KRAS/ERK signaling pathway expressions.Besides,we used co-immunoprecipitation(Co-IP)to determine the protein interacting effect of PID1 with EGFR.Morever,we applied shEGFR plasmid or EGFR inhibitor to measure the essentiality of EGFR on the tumorigenic function of PID1.For the second,we used flow cytometry to quantitate the ratio of liver immune infiltrating T cells,macrophages,bone marrow-derived suppressor cells(MDSCs),regulatory T cells(Tregs),and the phenotype and function of dendritic cells,on PID1liver-specific overexpressing mouse model.At the same time we detected mRNA levels of inflammatory factor-related genes on PID1 mice liver,as well as the cytokines secretion of serum.We also screened the percentage of T cells,macrophages,bone marrow-derived suppressor cells of spleen from PID1 mice.Subsequently,we applied liquid chromatography-mass spectrometry combined with immunoprecipitation to determine the interacting proteins of PID1.Additionally,we measured the immune condition of PID1 mice treated with shEGFR plasmid or EGFR inhibitor.Eventually,we sorted the liver infiltrated MDSCs to identify the the impact of PID1 overexpression on MDSCs.For the third,we measured the expression of liver PPAR?and FABP1,fat PPAR?and FABP4 on PID1 knockdown ob/ob mice.Then we used electrophoretic mobility shift assay to determine the transcription activity of liver PPAR?.Whereafter,we quantitated the protein expression of FABP1 on cytoplasm and nucleus from liver,respectively.As well,we also employed immunofluorescence to view the distribution of FABP1.We then detected the influence of PID1 konckdown on mouse normal liver cells,and the effect of insulin on cell proliferation.Afterwards,we constructed an insulin resistance cell model to determine cell proliferation,viability,and the expression of proliferation-associated antigen,proto-oncogene an tumor stem cell related genes.ResultsFirstly,we found that PID1 was highly expressed in cells,tissue chips and clinical samples of liver cancer compared to non-tumor controls.The expression of PID1 was decreased by the methyltransferase inhibitor MI-2 and regulated by H3K4me3modification.While liver cancer cells and tissues demonstrated increased H3K4me3modification.PID1 interference inhibited tumor growth of H22 xenograft model.Also,PID1 increased the proportion of spleen CD3~+,CD4~+and CD8~+T cells,decreased the ratio of MDSCs and Tregs,promoted the secretion of IL-6 MCP-1,TNF-?,IFN-?,together with the inhibition of protooncogene and cancer stem cell gene.Additionlly,PID1 induced partial neoplastic lesions in liver,with increased Ki67 expression,up-regulated serum level of ALT,AST,NAFE,TG and TCHO,down-regulated serum level of HDLC.PID1 also promoted insulin secretion and enhanced glycolytic-related gene expression,while decreased expression of PID1 attenuated glycolysis of mouse hepatoma cells.Further,PID1 overexpression resulted in the upregulation of cancer stem cells markers,tumorigenesis-related genes and proliferation markers,as well as the activation of EGFR/KRAS/ERK pathway.Also,PID1 can interact with EGFR,and suppression of EGFR,either by liver specific knockdown or systemic inhibition,suppressed the cancer-promoting effects of PID1 in liver.Secondly,we found that PID1 markedly increased the recruitment of MDSCs,while exhibited little influence on other immune cell subtypes,such as CD3~+,CD4~+T cells,Tregs,macrophages,and DCs.Furthermore,overexpression of PID1 in the liver induced a general immunosuppressive environment,marked by pronounced reductions in the numbers of CD3~+,CD4~+,and CD8~+T cells,noticeable differentiations of Tregs,and recruitment of MDSC in the spleen.These data were also confirmed by decreased serum cytokines levels of IL6,MCP-1,IFN-?,TNF-?,IL12 and increased IL10,as well as the mRNA expression levels of pivotal cytokines in PID1-induced liver tumors.Moreover,PID1 can also interactwith ARG1,the marker of MDSCs activation.Overexpression of PID1 in liver MDSCs also increased expression of immunosuppressive molecules,decreased expression of immune-promoting molecules,and enhanced T cells inhibition ability of MDSCs.Besides,overexpression of PID1combined with either shEGFR or Erlotinib still exerted immunosuppression effect,characterized by reduced functional T cells,and increased MDSCs and Tregs in liver and spleen.Thirdly,we found that PID1 up-regulated liver FABP1,PPAR?expression,fat PPAR?expression,and down-regulated fat FABP4 expression.Knockdown of PID1enhanced liver PPAR?transcriptional activity.Moreover,knockdown of PID1increased FABP1 expression in nucleus,decreased FABP1 expression in cytoplasm,which was also confirmed by immunofluorescence of liver FABP1.In addition,reduced expression of PID1 inhibited the growth of hepatocytes AML12.The administration of insulin promoted the clone formation of HepG2 cells,accompanied by significantly upregulation of PID1.Insulin resistance model of HepG2 and L02 cells exhibited stronger clone formation ability,higher mitochondrial membrane potential,higher expression of tumor proliferation-associated antigen,cancer promoting genes and cancer stem cell related genes.ConclusionsFirst of all,the highly expression of PID1 is regulated by H3K4me3 modification in liver cancer cells and clinical liver cancer samples.Liver overexpression of PID1promotes tumorigenesis partly via the interaction with EGFR to activate the EGFR/KRAS/ERK pathway,and the regulation of liver metabolism.Next,liver overexpression of PID1 interacts with ARG1 to activate MDSCs,promotes the T cell inhibitory function of MDSCs,thus inducing an immunosuppression environment to accelerate tumor formation.Moreover,the immunosuppression effect of PID1 is neither dependent on the EGFR/KRAS/ERK pathway nor a consequence of the neoplasic event.Eventually,konckdown of PID1 can on the one hand directly regulate lipid metabolism by promoting the expression of FABP1,and on the other hand enhance the transcriptional activity of PPAR?by promoting the binding of FABP1 to PPAR?,thereby enhancing the transcription of lipid metabolism-related enzymes,thus improving fatty liver and insulin resistance of ob/ob mice.Insulin and insulin resistance can promote liver cells proliferation by activating the expression of PID1,which is in part dependent on the H3K4me3 modification.