The Role And Mechanism Of P53 Regulation Of Mitophagy And PGK1 In The Pathogenesis Of Helicobacter Pylori Infection

Background and objectives:Helicobacter pylori（H.pylori）infection is closely related to the occurrence and development of gastric cancer,and is designated as a carcinogen of gastric cancer by WHO,but its exact pathogenic mechanism is still unclear.Tumor cells generate energy primarily through glycolysis and then ferment glucose into lactic acid in the cytoplasm,a process known as the"Warburg effect."Gastric cancer also has the Warburg effect,which is closely related to the occurrence,invasion,metastasis,drug resistance and poor prognosis of gastric cancer.Phosphoglycerate kinase 1（PGK1）,as a key enzyme in the process of aerobic glycolysis,participates in the glycolytic pathway to promote tumor invasion and metastasis.Glycolysis in gastric epithelial cells increases after H.pylori infection,and our previous transcriptome results showed that H.pylori infection can up-regulate the expression of PGK1 in gastric epithelial cells,suggesting that H.pylori infection may increase glucose by up-regulating the expression of PGK1 in gastric epithelial cells Glycolysis is involved in its pathogenic and carcinogenic processes.However,the mechanism by which H.pylori causes these changes is unclear.Mitochondria are the main sites of cellular energy metabolism.The balance of mitochondrial fission/fusion maintains mitochondrial homeostasis.Mitochondrial fission can promote mitophagy,reduce oxidative phosphorylation（OXPHOS）,and upregulate aerobic glycolysis,thereby promoting the occurrence and development of tumors.Mitochondrial reactive oxygen species（ROS）can trigger mitophagy,and melanocyte-inducible transcription factor（MITF）also plays an important role in mitophagy and glycolysis by controlling the autophagy-lysosomal system.H.pylori infection can promote the generation of ROS and mitochondrial ROS in gastric epithelial cells.So,whether H.pylori infection regulates mitophagy by inducing mitochondrial ROS has not yet been reported.MITF is highly expressed in gastric cancer and is involved in the regulation of proliferation,migration and apoptosis of gastric cancer cells.So,is H.pylori,an important causative factor of gastric cancer,involved in this process?Does high expression of MITF cause disease by inducing mitophagy?Neither is currently clear.p53 is a classic tumor suppressor,which not only regulates cell proliferation,apoptosis and cell cycle,but also participates in the regulation of mitochondrial respiration,metabolism,autophagy,mt DNA repair and ROS generation.p53 can inhibit mitochondrial ROS generation and mitophagy,and p53 can also down-regulate PGK1 expression and inhibit glycolysis.H.pylori infection can down-regulate the expression of p53 in gastric epithelial cells,inhibit the anti-tumor function of p53 and promote cell proliferation.Therefore,whether H.pylori infection inhibits the expression of p53 and affects the mitochondrial autophagy and glycolysis of gastric epithelial cells is still unclear.Based on the above research status,this paper carried out the following researches:（1）The effect of H.pylori infection on mitophagy and the role of MITF in it;（2）The regulation and mechanism of H.pylori infection on PGK1 were analyzed;（3）The relationship between p53 and mitochondrial ROS and PGK1 and mitophagy was analyzed;（4）The effect of PGK1 on the biological function of H.pylori-infected gastric epithelial cells.In order to explore the pathogenic and carcinogenic mechanism of H.pylori infection from the new perspective of p53 regulation of mitophagy and PGK1.Materials and Methods:Ⅰ.H.pylori regulates mitophagy in gastric epithelial cells via ROS and MITF1.The effect of H.pylori infection on mitochondrial morphology and mitophagy:A model of H.pylori infection of AGS cells and BGC823 cells was constructed,and the mitochondrial membrane potential and mitochondrial morphology of gastric epithelial cells were analyzed by laser confocal microscopy,high-content cell imaging analysis system was used to detect the expression of mitochondrial ROS（Mito SOX）in gastric epithelial cells.Transmission electron microscopy was used to observe the mitochondrial morphology of gastric epithelial cells.Confocal laser microscopy was used to analyze the localization of mitochondrial fluorescent probe（Mito Tracker）and autophagy marker LC3B,mitochondrial autophagy-related genes were screened with the mitophagy PCR array kit,and the protein expressions of mitochondrial fusion proteins（MFN1,MFN2）,MITF and mitochondrial autophagy markers（p62,LC3Ⅱ）in cells were detected by western blot.2.Regulation of ROS on mitophagy:In the constructed mouse model of H.pylori infection and NAC（ROS inhibitor）treatment,the expressions of MFN1,MFN2 and MITF in mouse gastric tissue were detected by immunohistochemistry.After gastric epithelial cells were treated with NAC,the expression of Mito SOX was detected by high-content cell imaging analysis system,and the expressions of p62 and LC3Ⅱin mitochondria of gastric cancer were detected by western blot.3.Regulation of MITF on mitophagy in gastric epithelial cells:Gastric epithelial cells with knockdown of MITF gene were constructed by si RNA,and the protein expressions of MITF,p62 and LC3Ⅱin mitochondria were detected by western-blot.Ⅱ.Expression of PGK1 in H.pylori-infected gastric epithelial cells and gastric mucosal lesions1.1.The expression of PGK1 in gastric cancer and the possible biological processes involved:24 cases of gastric cancer patients with gastric cancer after surgery and adjacent tissue samples were collected,and the expression of PGK1 was detected by q PCR,western blot and immunohistochemistry.The expression of PGK1in various tumors was analyzed with the TIMER tumor database;the possible biological processes of PGK1 in gastric cancer were analyzed by bioinformatics.2.Expression of PGK1 in different stages of gastric mucosal lesions and its relationship with H.pylori infection:collection of different stages of lesions:mild/severe chronic non-atrophic gastritis（M/SNAG）,atrophic gastritis（AG）,intestinal metaplasia（IM）,dysplasia（Dys）and gastric mucosa（GC）clinical patients with gastric mucosa,immunohistochemical staining to detect the expression of PGK1.3.The effect of H.pylori on the expression of PGK1 in gastric epithelial cells with different PGK1 expression:The stable transfected cell lines and NC cells with low and overexpression of PGK1 were constructed by lentivirus.A co-culture model of H.pylori and gastric epithelium was established,and the expression of PGK1 in gastric epithelial cells was detected by q PCR and western blot.Ⅲ.p53 affects mitophagy and glycolysis in gastric epithelial cells by regulating MITF/PGK11.H.pylori infection affects the glycolysis of gastric epithelial cells through PGK1:H.pylori 7.13 infected stably transfected cell lines with low expression and overexpression of PGK1,and the expression of PGK1 in the cells was detected by q PCR and western blot.The Seahorse XF24 flux analyzer measures the oxygen consumption rate（OCR）and extracellular acidification rate（ECAR）of cells,and simultaneously detects the level of lactate in cells.The co-localization of PGK1 and mitochondria was observed by laser confocal microscopy.The expression of PGK1 in mitochondria was detected by western blot.2.Interaction between p53,MTIF and PGK1 in H.pylori-infected gastric epithelial cells:H.pylori infected gastric epithelial cells overexpressing p53 gene and knocking down MITF gene,and the expression of Mito SOX was detected by high-content cell imaging analysis system.The expressions of PGK1,p53,MITF,p62 and LC3II were detected by western blot and q PCR,and the expression of lactate in gastric epithelial cells was also detected.The expression of PGK1 in gastric mucosa of mice treated with NAC was detected by immunohistochemistry.COIP detects the interaction of p53,MITF and PGK1 proteins in cells.Ⅳ.H.pylori infection regulation of PGK1 affects the biological process of gastric cancer cellsH.pylori infected AGS and BGC823 cells stably transfected with PGK1 gene overexpression and knockdown,and the proliferation of cells was detected by in vitro clone formation assay,Ed U cell proliferation assay and CCK8;the invasion ability of cells was detected by Transwell cell invasion assay;The migratory ability of cells was detected by trace assay and Transwell cell migration assay.Ⅴ.H.pylori infection affects the transcriptional level of gastric cancer cells via PGK1NC-AGS and PGK1^lowAGS were infected with H.pylori 7.13,and then the samples were sent to Shanghai Ouyi Biomedical Technology Co.,Ltd.for transcriptomic analysis.Analysis methods:（1）Analyze transcriptome samples by principal component analysis;（2）The differential genes were analyzed by DESeq2analysis software,and the screening criteria were:Fold Change>1.5,corrected P<0.05;（3）The differential genes were analyzed by GO and KEGG enrichment.Results:Ⅰ.H.pylori regulates mitophagy in gastric epithelial cells via ROS and MITF1.H.pylori infection induces mitochondrial membrane depolarization and mitochondrial fission:After H.pylori infection,the mitochondrial membrane potential of cells is decreased or lost,and mitochondria are extensively fragmented.The protein expressions of MFN1 and MFN2 in gastric epithelial cells infected with H.pylori were significantly lower than those in the uninfected group（P<0.05）in a concentration-dependent manner.The expressions of MFN1 and MFN2 in the gastric mucosa of H.pylori-infected mice were significantly lower than those in the uninfected group（P<0.05）.2.H.pylori regulates mitophagy in gastric epithelial cells through ROS:the expression of Mito SOX in H.pylori-infected gastric epithelial cells was increased（P<0.05）,and the co-localization of LC3B and Mito Tracker was increased.Transmission electron microscopy showed that H.pylori-infected gastric epithelial cells exhibited degenerative changes in mitochondria,loss of mitochondrial cristae membrane and swelling of the matrix,and abnormal mitochondria were seen in autophagosomes,the protein expression of LC3II and p62 was increased in mitochondria.After NAC treatment,the expression of Mito SOX in cells was decreased,and the protein expressions of p62 and LC3II in mitochondria were decreased.3.Screening the key genes of mitophagy and verifying them in vitro and in vivo:PCR array detection showed that H.pylori infection of gastric epithelial cells up-regulated the expression of autophagy-promoting genes MITF,ATF4,ATG13,JUN,BNIP3 and other genes,and the autophagy-inhibiting gene MFN1 and autophagy-promoting genes（AKT1,RHOT2）were expressed.The core gene MITF was selected for verification.The results showed that H.pylori-infected gastric epithelial cells could significantly up-regulate the expression of MITF protein（P<0.05）in a time-and concentration-dependent manner.The expression of MITF in gastric mucosa of H.pylori infected mice was also significantly higher than that of uninfected mice（P<0.05）.4.H.pylori regulates MITF-induced mitophagy in gastric epithelial cells through ROS:After NAC treatment,the expression of MITF in gastric mucosa of mice decreased,and NAC treatment could inhibit the increase of MITF expression caused by H.pylori infection（P<0.05）.After knockdown of MITF gene,the protein expressions of MITF,p62 and LC3BII in mitochondria were decreased.Ⅱ.Expression of PGK1 in H.pylori-infected gastric epithelial cells and gastric mucosal lesions1.Expression of PGK1 in gastric cancer and possible biological processes involved:The expression of PGK1 in gastric cancer tissue was significantly higher than that in adjacent tissue（P<0.05）.Bioinformatics analysis showed that PGK1 may play a role in gastric cancer through p53 signaling pathway,cell cycle,DNA replication,pentose phosphate pathway,galactose metabolism,fructose and mannose metabolism,glycolysis pathway,VEGFA-VEGFR2 signaling pathway and cell cycle.2.The expression of PGK1 in different stages of gastric mucosal lesions:The expression of PGK1 in AG,IM,Dys and GC was significantly higher than that in MNAG（P<0.01）,and with the aggravation of gastric mucosal lesions（AG→IM→Dys→GC）,the expression of PGK1 increased significantly.The expression gradually increased,and the highest expression was in gastric cancer（P<0.05）.3.The relationship between PGK1 and H.pylori infection:In the MNAG,SNAG,Dys,and GC groups,the expression of PGK1 in the H.pylori infection group was significantly higher than that in the uninfected group（P<0.05）.There was no significant difference between the infected group and the uninfected group（P>0.05）.The expression of PGK1 in the gastric mucosa of H.pylori-infected mice was significantly higher than that in the uninfected group（P<0.05）.Whether in PGK1overexpression,knockdown or WT gastric epithelial cells,H.pylori infection significantly up-regulated PGK1 and expression（P<0.05）.Ⅲ.p53 affects mitophagy and glycolysis in gastric epithelial cells by regulating MITF/PGK11.H.pylori infection induces PGK1 mitochondrial translocation to promote cellular glycolysis:PGK1 protein expression was significantly up-regulated in mitochondria of H.pylori-infected gastric epithelial cells,and the co-localization of PGK1 and Mito Tracker mitochondria was significantly increased.In H.pylori-infected gastric epithelial cells,the OCR value was significantly decreased,the ECAR value was significantly increased,and the lactate level was significantly increased（P<0.05）,knockdown of PGK1 could partially reverse the above changes（P<0.05）,while the intracellular lactate level increased significantly after overexpression of PGK1（P<0.05）.2.p53 and ROS can mutually regulate each other during H.pylori infection:the expression of p53 was significantly elevated in gastric epithelial cells treated with ROS inhibitor NAC（P<0.05）;Regardless of the presence or absence of H.pylori infection,p53 gene overexpression could significantly reduce the expression of Mito SOX in gastric epithelial cells（P<0.05）,indicating that p53 and ROS can regulate each other during H.pylori infection.3.H.pylori infection regulates MITF-induced mitophagy through p53:COIP assay found that MITF interacts with p53.The expression of p53 protein in the mitochondria of gastric epithelial cells was negatively correlated with the expression of MITF.H.pylori infection can inhibit the expression of p53 protein,while increasing the protein expression of MITF,p62 and LC3BⅡin WT and p53overexpressed gastric epithelial cells,and p53 overexpression can partially reverse these changes.4.H.pylori infection affects the expression of PGK1 through MITF:COIP assay found that MITF interacts with PGK1,regardless of the presence or absence of H.pylori infection,the expression of PGK1 in gastric epithelial cells decreased after MITF gene knockdown.5.The regulation of PGK1 by p53 and ROS affects the lactate level in gastric epithelial cells:In animal models,NAC treatment can significantly reduce the expression of PGK1 in gastric mucosa of mice with or without H.pylori infection（P<0.05）.At the cellular level,NAC treatment also reduced the expression of PGK1protein in H.pylori-infected and uninfected gastric epithelial cells.COIP assay found that PGK1 interacted with p53 in gastric epithelial cells;Overexpression of p53 can inhibit the expression of PGK1 protein in gastric epithelial cells;H.pylori infection in both WT and p53 overexpressed gastric epithelial cells can significantly increase the intracellular lactate level（P<0.01）,and p53 overexpression can partially reverse this change（P<0.05）.Ⅳ.H.pylori infection regulation of PGK1 affects the biological process of gastric cancer cellsThe proliferation,migration and invasion abilities of H.pylori-infected NC cells,PGK1-overexpressed and knocked-down gastric epithelial cells were significantly higher than those of uninfected cells（P<0.05）.The ability of proliferation,migration and invasion of NC cells was significantly higher than that of NC cells（P<0.05）,while that of PGK1 knockdown cells was the opposite（P<0.05）.Ⅴ.H.pylori infection affects the transcriptional level of gastric cancer cells via PGK11.The effect of PGK1 on the transcriptional level of gastric epithelial cellsThere were 521 differential genes between the NC-PGK1^lowAGS and PGK1^lowAGS.Compared with the NC-PGK1^lowAGS,there were 369 up-regulated genes and 152 down-regulated genes in the PGK1^lowAGS.GO enrichment analysis found that a total of 22 biological processes,9 cellular components and 10 molecular functions were enriched.Differential genes are mainly involved in:growth process,metabolic process,adhesion process,cell killing,reproduction,cell localization and cell movement and other processes,as well as antioxidant activity,catalytic activity,enzyme regulator activity,molecular transducer activity,transporter activity,translation regulator activity and enzyme regulator activity and so on.KEGG mainly enriched 41 signaling pathways,including amino acid metabolism pathway,chemokine signaling pathway,AGE-RAGE signaling pathway,TGF-beta signaling pathway,cytokine-cytokine receptor interaction,viral protein interaction with cytokine and cytokine receptor,TNF signaling pathway,cell adhesion molecule（CAM）,IL-17 signaling pathway,legionellosis,pathways in cancer and so on.2.The role of PGK1 on the transcriptome level of H.pylori-infected gastric epithelial cells（1）There were 502 differential genes between NC-PGK1^lowAGS and NC-PGK1^lowAGS-Hp groups.Compared with NC-PGK1^lowAGS,309 genes were up-regulated and 193 genes were down-regulated in NC-PGK1^lowAGS-Hp.The results of GO enrichment analysis were basically consistent with the results of gastric epithelial cells with low PGK1 expression.KEGG analysis mainly enriched 43 signaling pathways,including DNA replication,mismatch repair,ECM receptor interaction,cell cycle,apoptosis,p53 signaling pathway,glutathione metabolism,central carbon metabolism pathway,arginine and proline metabolism,focal adhesion,TNF signaling pathway and so on.（2）There are 1928 differential genes between PGK1^lowAGS and PGK1^lowAGS-Hp.Compared with PGK1^lowAGS,there are 1053 up-regulated genes and 875 down-regulated genes in PGK1^lowAGS-Hp.KEGG enrichment analysis mainly enriched 43signaling pathways,including central carbon metabolism pathway,DNA replication,mismatch repair,MAPK signaling pathway,HIF-1 signaling pathway,PI3K-Akt signaling pathway,arginine and proline metabolism,apoptosis,Micro RNAs in cancer,cellular senescence and so on.（3）There are 2643 differential genes between NC-PGK1^lowAGS-Hp and PGK1^lowAGS-Hp.Compared with NC-PGK1^lowAGS-Hp,there are 1619 up-regulated genes and 1024 down-regulated genes in PGK1^lowAGS-Hp.KEGG enrichment analysis mainly enriched 39 signaling pathways,including MAPK signaling pathway,Micro RNAs in cancer,PI3K-Akt signaling pathway,pathways in cancer,relaxin signaling pathway,growth hormone synthesis,secretion and action,GABAergic synapses,cytokine-cytokine receptor interactions and so on.To sum up,PGK1 may be involved in H.pylori infection-mediated gastric cancer progression through multiple signaling pathways.Conclusion:1.H.pylori infection promotes mitophagy in gastric epithelial cells by upregulating ROS generation and MITF expression.2.H.pylori infection induces mitochondrial translocation of PGK1 in gastric epithelial cells and promotes glycolysis.3.p53 can inhibit H.pylori-induced mitochondrial ROS generation and the expression of MITF and PGK1,thereby inhibiting mitophagy and glycolysis.H.pylori infection inhibits p53 expression and attenuates the above effects.4.PGK1 is highly expressed in gastric cancer tissue,and H.pylori infection promotes the proliferation,invasion and migration of gastric epithelial cells by up-regulating the expression of PGK1.5.PGK1 may participate in H.pylori-mediated gastric cancer progression by regulating p53,HIF-1α,MAPK and TNF signaling pathways.