The Mechanism Of FBXO7 In The Occurrence And Development Of Gastric Cancer Mediated By Helicobacter Pylori

BackgroundGastric cancer is one of the most common malignant tumors in the world.According to the world cancer data in 2018,the prevalence rate of gastric cancer ranks fifth in the world,and the mortality rate ranks third in malignant tumors.More and more studies show that Helicobacter pylori plays an important role in the occurrence and development of gastric cancer.Therefore,it is urgent to reveal the specific mechanism of Helicobacter pylori mediated gastric cancer occurrence and development,and to explore drug treatment targets based on pathogenic mechanism.Ubiquitination is an important post-translational modification of proteins.Ubiquitin proteasome system(UPS)is the main pathway of intracellular protein degradation,which plays a biological role through protein activating enzyme E1,ubiquitin binding enzyme E2 and ubiquitin protein ligase E3.Ubiquitin ligase E3 is the key factor to select and specifically degrade substrate proteins.Related studies have reported that MTA1 ubiquitination pathway plays an important role in the development and metastasis of gastric cancer.E3 ubiquitin ligase Cbl-b is associated with maintaining epithelial phenotype and inhibiting cell migration in gastric cancer.Low expression of E3 ubiquitin ligase CHIP enhances the migration and invasion of gastric cancer AGS cells.SCF,also known as skpl-cullinl-F-box(SCF),is a representative member of CRL(Cullin ring ligase)family.Through domain interaction,F-box protein is recruited as a component of SCF E3 ubiquitin ligase to target protein ubiquitination.Studies have shown that some F-box protein family members can be used as substrates of ATM(ataxia telangiectasia mutant gene)to participate in DNA damage response;In addition,some F-box proteins such as Skp2,FBXW7,FBXO15 and FBXO39 are closely related to tumor metabolism.FBXO7 is a member of the protein family containing the F-box domain,which can bind to Skp 1.Our data suggest that mutations in the FBXO7 gene can lead to Park15(Parkinson’s pyramidal syndrome).In addition,FBXO7 can directly bind to p27 and Cdk6,and increase the level of cyclin d-cdk6 complex.Its overexpression can induce the transformation of immortalized fibroblasts in a Cdk6 dependent manner,indicating that FBXO7 is involved in the process of malignant transformation.Relevant research results show that some members of F-box family are related to the invasion,metastasis,differentiation and clinical stage of gastric cancer cells,but there is no research on the role of FBXO7 in gastric cancer.In this study,we first explored the role and molecular mechanism of FBXO7 in the occurrence and development of gastric cancer mediated by Helicobacter pylori,further elaborated the mechanism of gastric cancer occurrence and development,and provided new candidate targets for early intervention of gastric cancer.AimsObjective to explore the mechanism of Helicobacter pylori regulating the expression of FBXO7 and influencing the occurrence and development of gastric cancer,and to clarify the important role and molecular mechanism of FBXO7 in the occurrence and development of gastric cancer induced by Helicobacter pylori,so as to provide potential targets and ideas for early clinical diagnosis and intervention.Experimental Methods1.FBXO7 expression level detection in gastric cancer tissuesWe detected the protein level of FBXO7 by immunohistochemical staining technique in paraffin sections of gastric cancer tissues and para-carcinoma tissues.At the same time,we detected the mRNA levels of FBXO7 in 86 pairs of human gastric cancer and para-cancerous tissues by QRT PCR assay,in.order to evaluate whether it could be a marker for the diagnosis of gastric cancer in the early stage or a target for treatment in the later stage.2.Effects of FBXO7 on gastric cancer cell proliferation,migration and invasion in vitroWe transfected FBXO7 small interfering RNA and overexpression plasmids into gastric cancer cells BGC823 and MGC803.After that we used QRT-PCR and Western blot to detect the effect of small interfering and overexpression plasmids.We detected its effect on the proliferation ability of gastric cancer cells by clonogenic and CCK8 cell viability experiments,on the cell migration ability by cell scratch assay and Transwell cell migration assay,and on the invasion ability of gastric cancer cells by Transwell Matrigel assay.3.Effects of FBXO7 on gastric cancer cell proliferation,migration and invasion in vivoWe examined the mRNA and protein levels of FBXO7 in stable cell lines transfected with lentivirus using QRT-PCR and Western blot.We subjected transfected control cells and FBXO7 stable interference group cells to mouse subcutaneous tumorigenesis assays and tail vein metastasis assays,thus to examine the effects of FBXO7 in vivo on cell functions such as proliferation and migration of gastric cancer cell.4.Screening of FBXO7 signaling pathway by immunoprecipitation and protein mass spectrometryThe protein molecules directly or indirectly bound to FBXO7 were collected by immunoprecipitation,and then detected by protein mass spectrometry.The protein molecules related to cancer were screened out from the obtained mass spectrometry results,and then enriched and analyzed on the website of metascape(website:http://Metascape.org)To screen out the key molecules and pathways that affect cell proliferation,migration and invasion,P38MAPK signaling pathway.After that,we further confirmed that FBXO7 is involved in this signaling pathway through inhibitors.Firstly,FBXO7 siRNA was transfected into BGC823 cells and p38MAPK pathway inhibitor(SB203580)was added.Then,the effects of p38MAPK inhibitor on migration and proliferation of gastric cancer cells were detected by Transwell and CCK-8 experiments,so as to determine the regulatory mechanism of FBXO7 in gastric cancer cells.5.The specific mechanism of FBXO7 involved in p38MAPK regulation in gastric cancer by cell molecular biology experimentFirst,we verified the reciprocal binding interaction of FBXO7 with p38 α by Co-IP in BGC823.We then transfected small interfering,wild-type,and mutant plasmids of FBXO7(Deletion of 335-372 amino acid sequence)in MGC803 and BGC823,and performed Western blot experiments on extracted total protein to examine changes in protein levels of p38 as well as pp38.After that,we extracted proteins after 0,1,2,3,4,5,and 6h incubation with Cyclohexamide to detect the changes of p38 α as well as pp38 protein half-life in the experimental and control groups of BGC823 cells stably interfering with FBXO7.After BGC823 cells were transfected with mutant and wild-type plasmids,the total protein level of p38 α was examined after the addition of MG 132.After FBXO7 was stably interfered in BGC823 cells,MG132 reagent was incubated in the experimental group and the control group for 12h,and then CO-IP assay was used to detect the ubiquitination of p38 α.In order to further verify whether FBXO7 is involved in regulating p38MAPK signaling pathway to affect the function of gastric cancer cells,we first analyzing the correlation between p38 α and HSF1,STAT1,c-myc on gepia website(http://gepia.cancer-pku.cn/).After analyzing the correlation between p38 α and HSF1,STAT1,c-myc in gastric cancer,we transfected FBXO7 mutant plasmid and wild-type plasmid into BGC823 cells and detected HSF1,STAT1,c-myc expression by QRT-PCR.6.Regulation of Helicobacter pylori infection on FBXO7 and p38MAPK in gastric cancer cellsWe infected gastric cancer cells BGC823 with two strains of Helicobactor pylori,Hp11637 and Hp26695,at MOI=1:100,using PBS as a control.We collected cells at 1.5h,3h,6h and 9h post infection.After that we used QRT-PCR and Western blot to detect the mRNA level and protein level of FBXO7.After that,we infected BGC823 gastric cancer cells with Hp26695 and Hp11637,two strains of Helicobactor pylori at MOI=50,MOI=100 and MOI=150.The cells were collected 12 h later.We used QRT-PCR and Western blot to detect the changes of mRNA levels and protein levels of the target molecule FBXO7.At the same time,5-Aza-2’-deoxycytidine was added to gastric cancer cells infected by Helicobacter pylori.QRT-PCR and Western blot were used to detect the mRNA and protein levels of FBXO7.Experimental result1.FBXO 7 is lowly expressed in human gastric cancer tissuesImmunohistochemistry results showed that the protein expression level of FBXO7 was lower in gastric cancer tissues compared with para-cancerous tissues.The mRNA expression levels of FBXO7 of 86 pairs of clinical gastric cancer tissues and para-cancerous tissues were detected by qRT-PCR experiments,and it was found that the mRNA expression level of FBXO7 in gastric cancer tissues was lower than that in para-cancerous tissues.2.Down regulation of FBXO7 expression in vitro enhanced gastric cancer cell proliferation,invasion and migration,and up regulation of FBXO7 expression inhibited gastric cancer cell proliferation,invasion and migrationWe transfected FBXO7 specific siRNA into gastric cancer cells MGC803,BGC823 and found down-regulation of both mRNA and protein levels of FBXO7 in the experimental group compared with the control group.Clonogenic assay and CCK8 cell viability assay the clonogenic capacity and cell proliferation of the two gastric cancer cell lines,MGC803 and BGC823,were enhanced after down-regulation of FBXO7,and the results of Transwell migration assay and cell scratch assay showed that the migration ability of the two gastric cancer cells,MGC803 and BGC823,was significantly enhanced after down-regulation of FBXO7;The results of Transwell Matrigel invasion assay showed that the invasion abilities of two gastric cancer cells,MGC803 and BGC823,were enhanced after interfering with FBXO7.Transfection of FBXO7 mutant plasmids and overexpression plasmids into the two gastric cancer cells MGC803 and BGC823 described above,cell clonogenic assay,CCK8 cell viability assay,Transwell assay and cell scratch assay showed that the ability of proliferation,invasion and migration were significantly inhibited in both gastric cancer cells after transfection with FBXO7 wild-type plasmid.However,no significant difference was observed in the ability of the two types of gastric cancer cells to proliferate,invade and migrate after transfection with FBXO7 mutant plasmids.3.Increased proliferation and metastasis ability of gastric cancer cells in vivo after interference with FBXO7First QRT-PCR and Western blot were performed to determine that the mRNA level and protein level of FBXO7 in BGC823 cells stably disrupted with FBXO7 were significantly decreased compared with the control cells.Subsequently,cell injection was performed in the axilla of the mice,and the mice were observed,measured,and recorded after the tumors grew out,and after the recordings were completed,the mice were sacrificed,and the tumors were removed for weighing and photographed.Our results showed that the proliferation ability of gastric cancer cells in mice was significantly enhanced after stable interference with FBXO7.Gastric cancer cells were injected into the tail vein of nude mice,and 31 days later,live imaging of the mice was performed,and the results showed that the fluorescence intensity of the interference group was obviously enhanced compared with that of the control group.The nude mice were sacrificed and dissected,the results showed that the fluorescence signal of liver and lung in the interference group was stronger than that in the control group.Gross observation showed that the number of nodules in the interference group was significantly more than that in the control group,and the lung weight in the interference group was significantly greater than that in the control group.The results of H&E staining showed that the number of tumor metastasis in the interference group was significantly more than that in the control group.4.FBXO7 regulates MAPK signaling pathwayThe results of protein profiling of FBXO7 were intersected with 6243 cancer disease-related molecules and the selected molecules were analyzed in metascape website,which showed that FBXO7 was associated with p38 MAPK signaling pathway,suggesting that in gastric cancer FBXO7 may affect the occurrence and development of gastric cancer by participating in regulation of p38 MAPK signaling pathway.After that,p38 MAPK pathway inhibitor(SB203580)was added after transfection of small interfering of FBXO7 in BGC823 cells,the results of Transwell migration assay showed that after interfering with FBXO7,the cell migration ability was up-regulated obviously,but the up regulation of cell migration ability was inhibited after adding inhibitor drugs.The same was true for the CCK-8 proliferation assay,thus establishing that FBXO7 affected cell functional behavior through the regulation of p38 MAPK signaling pathway in gastric cancer cells.5.FBXO7 regulates degradation of pp38 through ubiquitinationTo further define the regulation of p38 MAPK signaling by FBXO7,we first performed coimmunoprecipitation experiments in the BGC823 cell line,which showed that FBXO7 binds p38 a protein,after that we did fluorescence colocalization experiments between FBXO7 and pp38 in MGC803 and BGC823 cells,and the results showed that the localization of FBXO7 and pp38 in the cells was basically consistent.After that,we transfected FBXO7 small interfering RNA,wild-type and mutant plasmids into BGC823 and MGC803 cells.Western blot results showed that after FBXO7 interference,p38 a protein level was up-regulated in BGC823 cells,and there was no significant change in p3 8 a protein level in MGC803 Cells,but pp38 protein level was up-regulated in both cell lines.After transfection of FBXO7 mutant and wild-type plasmids,the mRNA and protein levels of FBXO7 were up-regulated,but the protein levels of p3 8 α and pp38 did not change significantly compared with the control group.However,after transfection of FBXO7 wild-type plasmids,the protein levels of p38 α and pp38 were down regulated in BGC823 cells,and there was no significant change in the protein level of p38 α in MGC803 cell,pp38 protein level was down regulated.The results suggest that FBXO7 may regulate pp38 degradation through ubiquitinationUpon inhibition of protein synthesis by the addition of Cyclohexamide to the BGC823 cell line that stably interferes with FBXO7,p38 α and pp38 protein degradation becomes slower and protein half-life is prolonged.Transfection of both mutant and wild-type plasmids with the addition of the proteasome inhibitor MG 132 showed that the decrease in the total protein level of p38 α due to FBXO7 overexpression was upregulated by the addition of MG132.In BGC823 cells stably transfected with FBXO7,MG 132 treatment for 12 h,CO-IP experiments were performed,and the ubiquitination of p38 α was decreased in the experimental group compared with the control group.Above results clarified that FBXO7 could regulate p38 MAPK signaling pathway through a ubiquitination dependent manner.In order to further clarify the regulation of p38MAPK signaling pathway by FBXO7 and its downstream key molecules,we analyzed the positive correlation between p38 α and HSF1,STAT1,c-myc in gastric cancer through gepia website.Then,FBXO7 mutant plasmid and wild-type plasmid were transfected into BGC823 cells.QRT-PCR results showed that the mRNA levels of HSF1,STAT1 and c-myc were significantly increased after transfection with FBXO7 wild-type plasmid.However,the mRNA levels of HSF1,STAT1 and c-myc did not change significantly after transfection of FBXO7 mutant plasmid.The results suggest that FBXO7 regulates pp38 protein level through ubiquitination to affect some key downstream molecules,and then regulate the function of gastric cancer cells.6.Helicobacter pylori infection down regulates the expression of FBXO7 and up regulates the protein levels of p38 α and pp38QRT-PCR and Western blot showed that the gastric cancer cell BGC823 was infected with Hp26695 and Hp11637 at an MOI=100,and the cells were charged at 0h,1.5h,3h,6h and 9h post infection,and the mRNA levels and protein levels of FBXO7 were gradually downregulated.The gastric cancer cells BGC823 and MGC803 were infected with Hp26695 and Hp11637 at MOI=50,MOI=100 and MOI=150,respectively,and the mRNA and protein levels of FBXO7 were downregulated after 12h.Meanwhile,the gastric cancer cell line BGC823 was infected by Hp26695 and Hp11637 in the same way as above,and the extracted proteins were collected.Western blot showed that p38 αprotein level and pp38 protein level were up-regulated.In addition,QRT-PCR and Western blot results showed that the addition of 5-Aza-2’-deoxycytidine inhibited the down-regulation of FBXO7 mRNA and protein levels,suggesting that Helicobacter pylori infection may regulate the expression of FBXO7 by affecting the methylation of FBXO7 promoter.Conclusion1.FBXO7 can inhibit the proliferation,migration and invasion of gastric cancer cells in vitro and in vivo.2.The low expression of FBXO7 in human gastric cancer suggests that FBXO7 may be a potential diagnostic marker for gastric cancer.3.FBXO7 can regulate p38MAPK signaling pathway by degrading pp38 through ubiquitination,affecting the proliferation,migration and invasion of gastric cancer cells.4.Helicobacter pylori infection can down regulate the expression of FBXO7 through methylation modification,and then up regulate the protein expression of pp38,suggesting that Helicobacter pylori infection may affect the occurrence and development of gastric cancer by affecting p38MAPK signaling pathway through FBXO7.