Regulation Mechanism Of Orphan Nuclear Receptor Nurr1 And Histone Methyltransferase SETDB1 To Mediate Gastric Malignant Transformation

BackgroundGastric cancer(GC)is a major disease that seriously affects the health of Chinese people.The 5-year survival rate of GC patients is low,due to late diagnosis and the lack of effective treatment.Therefore,revealing the mechanism of gastric malignant transformation is an important scientific problem for early intervention of GC.Helicobacter pylori(H.pylori)is a primary carcinogen.H.pylori infection could induce gastritis and gastric mucosal damage and persistent inflammation increases the risk of malignant transformation.Although the research on H.pylori in GC has made some progress,the mechanism of H.pylori promoting malignant transformation still requires further exploration.This study mainly explored the regulation mechanisms of orphan nuclear receptor Nurrl and histone methyltransferase SETDB1 in Helicobacter pylori-associated gastric malignant transformation.The nuclear receptor family is one of the most abundant transcriptional regulators in human.It plays a crucial role in cell differentiation,proliferation,apoptosis,metabolism and homeostasis maintenance.The human genome contains 48 nuclear receptor superfamily,including a large number of orphan nuclear receptors.Orphan nuclear receptors could be activated by external stress stimuli,affecting physiological and pathological processes by regulating abnormal genes expression.In our study,we analyzed the expression of nuclear receptor genes in human specimens.We found that Nurrl upregulation was most significantly in GC specimens.Earlier studies on Nurrl mainly focused on its regulatory mechanism for neuron development.In recent years,researches have focused on its effect on various cancers.However,the role of Nurrl in the progression of GC is still unclear.Epigenetic molecules are involved in the occurrence and development of multiple cancers.Many inhibitors of histone modifying-associated enzymes were recognized as targeted drugs in cancer therapy and have been used in the clinic.Therefore,the exploration of epigenetic molecules that play an important role in the development of GC can provide a basic theoretical basis for the treatment of GC.We analyzed the expression of histone methyltransferase and histone demethylase in two GEO databases and TCGA database.We found that SETDB1 expression was significantly increased in tumor tissues when compared with their normal counterparts.In addition,we analyzed Kaplan-Meier Plotter database and found that high expression of SETDB1 was associated with poor prognosis in GC patients.SETDB1 belongs to the histone methyltransferase subfamily.SETDB1 could regulate early embryonic development and embryonic stem cells proliferation.Moreover,SETDB1 could promote tumorigenesis of multiple cancers and it is considered as a potential therapeutic target for many tumors.However,function and mechanism of SETDB1 in the initiation and progression of Helicobacte pylori-associated GC have not been fully elucidated.Aims1.Clarify the function and mechanism of orphan nuclear receptor Nurrl in Helicobacter pylori-associated gastric carcinogenesis.2.Clarify the regulatory mechanism of histone methyltransferase SETDB1 in the occurrence and progression of Helicobacter pylori-associated gastric cancer.Methods and Results1.Orphan nuclear receptor Nurrl accelerates gastric carcinogenesis by promoting transcriptional activation of cell cycle regulator CDK4.H.pylori infection could trigger the PI3K/AKT signaling pathway and increasing the expression of the transcription factor SP1.SP1 could bind to Nurrl promoter and promote Nurrl expression.(1)Nurrl expression is elevated in GC and increased Nurrl level predicts poor prognosisGene differential expression analysis was performed and found that Nurrl expression was obviously upregulated in GC samples with respect to the AG samples.IHC staining found that Nurrl expression was gradually increased during the progression from gastritis to GC.RT-PCR showed that Nurrl mRNA expression was increased in human GC samples than that in AG samples.GEO database(GSE30727,GSE54129)analysis and Western blot showed that Nurrl expression was higher in human GC tissues than the adjacent normal tissues.Survival analysis was performed in three GEO databases(GSE51105,GSE62254,GSE14210)and found that high expression of Nurrl predicted poor prognosis.(2)Nurrl promotes GC cells proliferation both in vitro and in vivoColony formation and CCK8 assays showed that Nurrl knockdown inhibited GC cells proliferation,and GC cells proliferation ability was enhanced when Nurrl was overexpressed.Next,generated GC cells with stable Nurrl suppression and the matched control cells.These cells were injected subcutaneously into nude mice.The growth rate was significantly attenuated in the group with the injection of Nurrl suppressed cells.The harvested tumor sizes were smaller and tumor weights were lighter in the Nurrl suppression group as compared to the controls.These data further supported the pro-proliferative role of Nurrl both in vitro and in vivo(3)Nurrl enhances GC cells proliferation by directly targeting CDK4RT-PCR was performed to analyze expression of genes with Nurrl binding sites within their promoters and found that CDK4 could be positively regulated by Nurrl.Western blot assay showed that Nurrl knockdown suppressed the expression of CDK4 and downstream genes,while Nurrl overexpression had an opposite effect.Luciferase reporter system and ChIP(Chromatin Immunoprecipitation)assays found that Nurrl directly bound to the CDK4 promoter and promoted the transcriptional activity of CDK4.Colony formation and CCK8 assays revealed that CDK4 could promote GC cells proliferation in vitro.Xenograft model in nude mice confirmed that CDK4 could promote tumorigenesis of GC cells.(4)H.pylori upregulates Nurrl expressionPT-PCR and Western blot assays found that H.pylori infection could induce Nurrl expression which was dose and time dependent.RT-PCR and IHC staining assays showed that Nurrl expression was higher in H.pylori-positive AG samples compared with that in H.pylori-negative samples.(5)H.pylori upregulates Nurrl expression by the PI3K/AKT pathwayIn order to determine how H.pylori infection upregulated Nurrl expression,five key signaling pathways inhibitors were added before infection with H.pylori in BGC-823 cells.Western blot assays revealed that the upregulation of Nurrl protein expression induced by H.pylori infection was blocked by PI3K/AKT inhibitor(LY294002)strongly.RT-PCR showed that the induction of Nurrl mRNA expression by H.pylori was also blocked by LY294002 treatment.Western blot also confirmed the protein changes of AKT,P-AKT,SP1 were consistent with that of Nurrl.Our results indicated that infection of H.pylori increased Nurrl expression through activation of the PI3K/AKT pathway.(6)Transcription factor SP1 directly targets Nurrl and regulates the expression of Nurrl and downstream genesRT-PCR and Western blot analysis showed that Nurrl expression could be positively regulated by SP1.There were three SP1 binding sites in the promoter of Nurrl identified by PROMO 3.0.Luciferase reporter system and ChIP analysis verified that Nurrl was a direct target gene of SP1.RT-PCR and Western blot assays showed that H.pylori could regulate Nurrl and its targets expression through SP1.(7)Nurrl expression was positively correlated with SP1,CDK4 and Ki67 expression in animals and clinical specimensIHC staining assay found that the expression of Nurrl,SP1,CDK4 and Ki67 was significantly increased in H.pylori infection group.The expression of Nurrl,SP1,CDK4 and Ki67 was increased notably in GC tissues with respect to adjacent normal tissues.IHC staining assays showed that the Nurrl expression was positively correlated with SP1,CDK4 and Ki67.2.SETDB1 expression was unregulated in GC and its abnormal expression facilitated the occurrence and progression of GC.SETDB1 interacted with ERG and bound to the CCND1 and MMP9 promoters,promoting their expression and mediating the progression of GC.H.pylori increased SETDB1 expression through the transcription factor TCF4 in GC cells.TCF4 bound to the SETDB1 promoter to enhance its transcription activity.(1)SETDB1 is upregulated in GC and high SETDB1 expression predicts poor prognosis of GC patients.To investigate genes closely related to GC,gene expression profiles of GC and their corresponding non-tumorous tissues were analyzed from two GEO databases(GSE13911 and GSE79973).Analyses showed that five histone methyltransferases(SETDB1,PRMTI,PRMT3,PRMT5 and SMYD5)and one histone demethylase(MINA)were upregulated in GC samples than that in normal samples on both two GEO databases.Next,we analyzed the expression of these six genes in the TCGA database.The data showed that the expression of SETDB1 was notably elevated in tumor tissues when compared with their normal counterparts.The results of Western blot were consistent with that of the above databases.IHC staining showed that SETDB1 expression was relatively low in superficial gastritis(SG),mildly upregulated in atrophic gastritis(AG)and intestinal metaplasia(IM),moderately increased in dysplasia(DYS)and strikingly upregulated in GC tissues.RT-PCR assays found that SETDB1 mRNA expression was significantly increased in human GC tissues when compared with those in AG tissues.Kaplan-Meier Plotter analyses showed that high expression of SETDB1 significantly correlated with the poor overall survival of GC patients.(2)SETDB1 promotes GC cells proliferation and tumor developmentColony formation and CCK8 analysis in vitro showed that SETDB1 could increase GC cells proliferation.Xenograft tumor model in nude mice in vivo showed that SETDB1 could promote tumorigenicity.IHC staining showed that Ki67 expression was significantly reduced in SETDB1 knockdown tumors.(3)SETDB1 facilitates the invasion and metastasis of GC cells.Transwell penetration and wound healing assays showed that SETDB1 could promote GC cells metastasis which did not depend on its methyltransferase function.To examine the role of SETDB1 in tumor metastasis,we further introduced SETDB1 knockdown GC cells into nude mice by tail vein injection.Notably,suppression of SETDB1 expression markedly reduced the metastasis of GC in the lungs of nude mice.HE staining showed that SETDB1 knockdown reduced the number of metastatic nodules in the lungs.(4)CCND1 and MMP9 are downstream effectors of SETDB1-mediated functions.To investigate the mechanisms of proliferation and invasion induced by SETDB1 in GC cells,RT-PCR was performed to analyze the expression of genes related to cells proliferation and metastasis.We found that SETDB1 knockdown markedly reduced the mRNA expression of CCND1 and MMP9.Next,RT-PCR and Western blot assays revealed that SETDB1 knockdown inhibited CCND1 and MMP9 expression,while SETDB1 overexpression increased their expression.(5)SETDBI promotes the transcription of CCND1 and MMP9 by physically associating with ERG.Endogenous and exogenous IP assays showed that SETDB1 could interact physically with ERG.Luciferase reporter assays showed that SETDB1 could enhance the transcriptional activity of CCND1 and MMP9.ChIP assay found that SETDB1 bound to the promoters of CCND1 and MMP9 in GC cells.Colony formation and Transwell assays revealed that CCND1 and MMP9 were downstream targets of SETDB1,and the biological functions of SETDB1 were largely dependent on CCND1 and MMP9 activities.(6)TCF4 enhances SETDB1 expression in GC cells.As predicted by PROMO3.0,SETDBI promoter contains a classical TCF4 binding motif CAAAG.RT-PCR and Western blot analyses showed that TCF4 could regulate mRNA and protein expression of SETDB1 in GC cells.Luciferase reporter assays revealed that TCF4 knockdown could suppress SETDBI promoter luciferase activity,while TCF4 overexpression had the opposite effect.ChIP assays showed that TCF4 directly bound to the SETDB1 promoter and regulated transcription of SETDB1.(7)H.pylori upregulates SETDB1 expression via TCF4.RT-PCR and Western blot assays showed that both SETDB1 mRNA and protein expression were significantly increased by H.pylori infection,in a dose and time-dependent manner.RT-PCR showed that SETDB1 mRNA expression was higher in H.pylori-positive human AG tissues than those in H.pylori-negative tissues.IHC staining showed that SETDB1 protein expression was consistent with the mRNA expression in human AG samples.We constructed a gastritis mouse model infected with H.pylori to evaluate the expression of SETDB1.IHC staining showed that SETDB1 expression was higher in H.pylori infection group when compared with the non-infected control group.RT-PCR and Western blot results revealed that H.pylori could promote SETDB1 expression via TCF4.(8)SETDB 1 expression is correlated with GC pathogenesisIHC staining analyses showed that SETDB 1 expression was increased in the MNU(N-methyl-N-nitrosourea)-treated gastritis mouse model with H.pylori infection.Moreover,we detected SETDB 1 expression in human normal samples,GC samples and metastatic samples.SETDB 1 expression was weak in normal samples,but obviously upregulated in GC samples.A higher SETDB 1 expression was observed in metastasis samples when compared with GC samples.ConclusionsIn this study,we found that the expressions of orphan nuclear receptor Nurrl and histone methyltransferase SETDB 1 were increased in GC,and their high expressions were associated with shorter survival of GC patients.Nurrl and SETDB1 could affect the expression of target genes through transcriptional regulation and promoting the occurrence and development of GC.At the same time,we found that H.pylori infection could up-regulate the expression of Nurrl and SETDB1.This study revealed a new regulatory mechanism of the occurrence and development of Helicobacter pylori-associated GC,suggesting that targeted Nurrl and SETDB 1 regulatory pathway is of great significance in the intervention of GC.