The Regulation And Function Of ETS1 And L-plastin In Helicobacter Pylori-associated Gastric Diseases

BackgroundsHelicobacter pylori(H.pylori)is a Gram-negative spiral pathogen and colonized in gastric mucosa.At present,more than 50%of the world’s population is infected with the pathogen.Since the discovery of H.pylori in 1982,a large number of subsequent studies have shown that H.pylori infections are closely related to the occurrence and development of many gastric diseases,including chronic gastritis,gastric ulcers and gastric cancer(GC).Antibiotics are still the main way to eradicate H.pylori.However,recent studies have shown that the drug resistance rate is increasing.At present,H.pylori infection has the characteristics of high infection rate and wide range,leads to many gastric diseases and increases drug resistance rate.Hence,it is of great clinical significance to deeply study the pathogenic mechanism of H.pylori infection.H.pylori gastritis is the basis of H.pylori-associated diseases.Gastric epithelial cells(GECs)are the first site of contact between H.pylori and the host,and the interaction between them has been confirmed to play an important role in H.pylori gastritis.Transcription factors(TFs)can significantly affect the homeostasis of GECs.ETS1(ETS proto-oncogene 1,transcription factor)is a member of the ETS(E26 transformation specific)transcription factor family and is involved in the occurrence and progression of a variety of inflammatory diseases.Studies have found that ETS1 is not expressed in normal GECs,but is expressed in GC cells,and is closely related to the invasion and metastasis.At present,the expression and function of ETS1 in H.pylori gastritis have not been reported.Our previous study found that H.pylori infection could induce GECs to express ETS1.Therefore,elucidating the regulatory mechanism and functional role of ETS1 in GECs is helpful to understand the pathogenesis of H.pylori gastritis,and can provide new theoretical basis for the treatment of H.pylori-associated diseases.H.pylori-associated GC has caused a great social burden.According to the WHO Global Cancer Database,in china,the number of H.pylori-associated cancer cases accounts for approximately 40%of the cancer cases attributable to H.pylori infection in the world.However,the pathogenic mechanism of H.pylori infection promoting the occurrence and development of GC has not been well explained.Metastasis is the main cause of poor prognosis.Tumor metastasis requires dynamic rearrangement of the actin skeleton,which is also an important feature of host cells after H.pylori infection.The dynamic rearrangement of the Actin skeleton is regulated by a variety of actin-binding proteins(ABPs).L-plastin is an important ABP that cross-links and stabilizes F-actin.When actin polymerization occurs,L-plastin inserts into the newly synthesized F-actin and stabilizes these structures.Based on this,L-plastin plays an important role in cell-to-cell interactions,integrin adhesion,and cell migration.Under normal physiological conditions,L-plastin is only expressed in hematopoietic cells.However,L-plastin is also ectopic expressed in various cancer cells of non-hematopoietic origin.At present,the expression pattern,regulation mechanism and function of L-plastin in H.pylori-associated GC are still unknown.To elucidate the molecular mechanism and functional role of L-plastin in GC cells is helpful to enrich the pathogenic mechanism of H.pylori infection and provide new ideas and theoretical basis for preventing the progression of GC.Objectives1.To investigate the expression pattern,regulation mechanism and function of ETS1 in H.pylori gastritis.2.To investigate the expression pattern,regulatory mechanism and functional of L-plastin in H.pylori-associated GC.MethodsPartⅠ:The expression pattern,regulation mechanism and function of ETS1 in H.pylori gastritis1.The expression pattern of ETS1 in H.pylori gastritis(1)The expression of ETS1 in human gastric mucosa was detected by qRT-PCR,WB and IHC assay.(2)The animal model of H.pylori infection(C57 mice)was established,and the expression of ETS1 in the gastric mucosa of mice was detected by qRT-PCR,WB and IHC assay.(3)GEO database microarray and RNA-seq assay were used to analyze the effects of H.pylori infection on the expression of ETS transcription factor family molecules.(4)The expression of ETS1 in H.pylori infected cells was detected by qRT-PCR and WB assay.2.The regulatory mechanism of ETS1 in GECs during H.pylori infection(1)The H.pylori Traswell infection model andΔcag A infection model was constructed and the ETS1 expression was detected by qRT-PCR and WB.(2)H.pylori NCTC 11637 infected the BAY 11-7082 pre-processed AGS cells;and then the expression of ETS1 was analyzed by qRT-PCR and WB.(3)Dual luciferase reporter assay was used to detect the effect of H.pylori infection on ETS1 promoter activity.(4)The ETS1 promoter was analyzed by PROMO website.The direct regulation of p65on ETS1 was explored by Ch IP assay.(5)Inflammatory cytokines(IFN-γ,IL-17A,IL-22,IL-6,IL-12,IL-23,IL-1β,and TNFα)with H.pylori infection or stimulate cells alone,and then the expression of ETS1 was analyzed by qRT-PCR and WB.3.The function of ETS1 in ETS1 in GECs during H.pylori infection(1)Through the experiment siRNA interference assay and RNA-seq assay,the target genes of ETS1 regulated in the state of H.pylori infection were analyzed;and then the role of the ETS1 was analyzed by Gene-ontology.(2)According to the H.pylori-infected gastric samples’histopathological evaluation and ETS1 expression,the relationship between ETS1 expression and the severity of gastritis was evaluated.PartⅡ:The expression pattern,regulation mechanism and function of L-plastin in H.pylori-associated GC1.The expression pattern of L-plastin in H.pylori-associated GC(1)KEGG database,GEPIA website,expression profile microarray and IF double staining assay were used to screen important ABPs in H.pylori-associated GC.(2)qRT-PCR and WB assay were used to detect the expression of L-plastin in H.pylori infected GC cell lines and primary GC cells(Ep CAM~+).(3)The expression of L-plastin in hman GC tissues was analyzed by qRT-PCR,WB,IHC and IF double staining assay.2.The regulatory mechanism of L-plastin in GC cells during H.pylori infection(1)The H.pylori Traswell infection model andΔcag A infection model was constructed;and then the L-plastin expression was detected by qRT-PCR and WB.(2)Using U0126,JNK inhibitor II,SB203580,Wortmannin,AG490 or BAY 11-7082 to pre-process GC cells,then the cells were infected with H.pylori NCTC 11637 orΔcag A;then,the expression of L-plastin and signal pathway key proteins were analyzed by qRT-PCR and WB.(3)Transiently expressed cag A(cag A-pc DNA3.1)in GC cells;then,the expression of L-plastin was analyzed by qRT-PCR and WB and the signal pathway key proteins were analyzed by WB.(4)Dual luciferase reporter assay was used to detect the effect of H.pylori infection on L-plastin promoter activity.(5)The L-plastin promoter was analyzed by PROMO website.The direct regulation of SP1 on L-plastin was explored by siRNA interference assay and Ch IP assay.3.The function of L-plastin in GC cells during H.pylori infection(1)The lentivirus-mediated stable overexpression L-plastin cell lines(LV5-L-plastin)and their control cell lines(LV5-NC)were constructed;then,the expression of L-plastin was analyzed by WB.(2)The lentivirus-mediated stable knockdown L-plastin cell lines(LV3-sh L-plastin)and their control cell lines(LV3-NC)were constructed;then,those cells were infected with H.pylori NCTC 11637;next,the expression of L-plastin was tested by WB.(3)Transwell assay was used to assess the migration ability of stable overexpression L-plastin cells(LV5-L-plastin)and gentamycin treated H.pylori NCTC 11637 infected stable knockdown L-plastin cells(H.pylori NCTC 11637 infected LV3-sh L-plastin).(4)The pulmonary metastasis model was constructed by stable overexpression L-plastin cells(LV5-L-plastin)or gentamycin treated H.pylori NCTC 11637 infected stable knockdown L-plastin cells(H.pylori NCTC 11637 infected LV3-sh L-plastin);then,the effect of L-plastin facilitates GC metastasis was evaluated.ResultsPartⅠ:The expression pattern,regulation mechanism and function of ETS1 in H.pylori gastritis1.The expression pattern of ETS1 in H.pylori gastritis(1)The results of qRT-PCR,WB and IHC showed that ETS1 was increased in the gastric mucosa of H.pylori-infected patients.Meanwhile,IHC results showed that the expression of ETS1 was upregulated in GECs.(2)The results of qRT-PCR showed that the expression of ETS1 in the gastric mucosa of H.pylori infected mice changed dynamically,and reached the peak at 12 weeks after infection.The qRT-PCR,WB and IHC assay showed that ETS1 were increased in the gastric mucosa of H.pylori infected mice.Meanwhile,IHC results also showed that the expression of ETS1 was upregulated in GECs.(3)The results of GEO database microarray and RNA-seq showed that after H.pylori infection,the expression of ETS1 increased most.(4)The results of qRT-PCR and WB displayed that the expression of ETS1 increased with the extension of H.pylori infection time(0 h,6 h,12 h and 24 h)or the increase of MOI(0,50,100 and 200).2.The regulation mechanism of ETS1 in GECs during H.pylori infection(1)The results of Traswell infection assay showed that the direct contact of H.pylori with cells could significantly induce the expression of ETS1.Δcag A infection assay showed:ETS1 levels only increased following infection with H.pylori wild-type(H.pylori NCTC11637)but not withΔcag A.(2)The inhibitor assay showed that inhibition of the NF-κB pathway using BAY11-7082 remarkably suppressed ETS1 expression during H.pylori infection.(3)In contrast to the uninfected control,infection with H.pylori significantly enhanced luciferase activity in AGS cells.Furthermore,enhanced luciferase activity was also cag A dependent.(4)The PROMO website showed that ETS1 promoter contains two p65 binding sites(binding site 1:GCTTTTCCCAG(-1568 to-1558);and binding site 2:GACTTTCCCGA(-373 to-363)).Ch IP assay showed that H.pylori infected cells increased p65 binding to the ETS1 promoter.Moreover,the binding was attenuated by BAY 11-7082.(5)The IFN-γ,IL-17A,IL-22,IL-6,IL-12,and IL-23 had no effect on modulating ETS1expression during H.pylori infection.Notably,IL-1βand TNFαexerted a synergistic effect on H.pylori-mediated ETS1 expression.The expression of ETS1 was suppressed by using BAY 11-7082 in IL-1βor TNFαstimulated AGS cells.3.The function of ETS1 in GECs during H.pylori infection(1)siRNA assay and RNA-seq assay showed that 75 transcripts can be positively regulated by ETS1 during H.pylori infection.Among those transcripts,39 proteincoding genes were founded.Top thirty GO terms of Gene-ontology analysis of target genes revealed that the ETS1 is participate in various biological processes,including positive regulation of cell motility,cell migration,cellular component movement,inflammatory response,leukocyte migration,and locomotion.(2)The levels of ETS1 expression is significantly correlated with the severity of gastritis.PartⅡ:The expression pattern,regulation mechanism and function of L-plastin in H.pylori-associated GC1.The expression pattern of L-plastin in H.pylori-associated GC(1)Based on KEGG database,found 193 ABPs.The GEPIA website revealed that 48ABPs were markedly up-regulated in GC tissues.The 48 up-regulated ABPs in H.pylori NCTC 11637 infected AGS cells were screened by microarray and found that L-plastin expression increased the most.IF double staining assay results showed that L-plastin expression was increased after H.pylori infection and co-localized with actin.(2)The results of qRT-PCR and WB assay showed that H.pylori infected GC cell lines(AGS and HGC-27)and primary GC cells(Ep CAM+)significantly up-regulated the expression of L-plastin,meanwhile,the expression of L-plastin increased with the extension of H.pylori infection time(0 h,6 h,12 h,24 h and 36 h)or the increase of MOI(0,10,20,50,100 and 200).(3)The results of qRT-PCR,WB and IHC assay showed that L-plastin expression in H.pylori-infected GC tissues was greatly increased.Meanwhile,IHC results showed that the expression of L-plastin was upregulated in GC cells.The results of IF double staining experiment showed that there was co-localization between L-plastin and Ep CAM in GC tissues with H.pylori infection.2.The regulatory mechanism of L-plastin in GC cells during H.pylori infection(1)The results of Traswell infection assay showed that the direct contact of H.pylori with cells could significantly induce the expression of L-plastin.Δcag A infection assay showed:L-plastin levels only increased following infection with H.pylori wild-type(H.pylori NCTC 11637)but not withΔcag A.(2)The results of qRT-PCR and WB assay showed that inhibition of the ERK pathway using U0126 remarkably suppressed L-plastin expression.WB results showed that compared withΔcag A infection,H.pylori NCTC 11637 infection can significantly induced phosphorylation of ERK1/2.(3)The results of qRT-PCR and WB assay showed that the plasmid cag A-pc DNA3.1could significantly activate the ERK signaling pathway and up-regulate the expression of L-plastin after transfection in GC cells.(4)The promoter activity analysis showed that L-plastin promoter(-100/0)mediate transcription responsiveness to H.pylori.(5)The PROMO website showed that L-plastin promoter(-100/0)contains a SP1binding site(GGGGCGGGGA).siRNA assay and Ch IP assay shwed that H.pylori can effectively accelerate the SP1 to L-plastin promoter region to regulate the expression of L-plastin.3.The function of L-plastin in GC cells during H.pylori infection(1)WB assay showed that compared with LV5-NC,LV5-L-plastin cells could overexpress L-plastin.(2)WB assay showed that compared with the control,the expression of L-plastin in H.pylori NCTC 11637 infected LV3-sh L-plastin cells was significantly reduced.(3)Transwell assay showed that compared with LV5-NC,the number of cell migration was significantly increased after L-plastin overexpression;meanwhile,the number of cell migration was greatly reduced in H.pylori NCTC 11637-infected L-plastin knockdown GC cells.(4)Pulmonary metastasis model showed that compared to control cell(LV5-NC)recipients,mice received LV5-L-plastin cells showed increased metastatic nodules in the lungs;in addition,the prevalence of lung metastases was decreased in mice received H.pylori NCTC 11637-infected LV3-sh L-plastin cells compared to control recipients(H.pylori NCTC11637-infected LV3-NC).Conclusions and SignificancesPartⅠ:The expression pattern,regulation mechanism and function of ETS1 in H.pylori gastritis.1.H.pylori induces ETS1 expression in GECs.2.H.pylori cag A activated NF-κB pathway mediates ETS1 expression;and IL-1βand TNFαhave synergistic effects on ETS1 expression during H.pylori infection.3.ETS1 positively regulates inflammatory response in H.pylori gastritis.4.This study suggests that ETS1 may be involved in H.pylori mediated"inflammation-cancer"transformation,making ETS1 a potential target to inhibit the progression of H.pylori gastritis.PartⅡ:The expression pattern,regulation mechanism and function of L-plastin in H.pylori-associated GC.1.H.pylori induces L-plastin expression in GC cells.2.H.pylori induces L-plastin expression via the cag A-ERK-SP1 pathway3.H.pylori-induced L-plastin promotes GC metastasis.4.This study indicated that L-plastin can sever as a potential therapeutic target against H.pylori-associated GC.