The Role Of Deubiquitinating Enzyme USP42 In Regulation Of KLF4 Protein Degradation In Gastric Cancer

BackgroundsGastric cancer is a malignant tumor that seriously affects the lives and health of people in China and around the world.The cause of gastric cancer is not completely clear.Its occurrence and development are a multi-factor and multi-step process,and Helicobacter pylori infection is the key risk factor.Some literatures show that CagA plays the role of bacterial oncoprotein in gastric cancer induced by Helicobacter pylori.In gastric epithelial cells,CagA can activate the expression of oncogenes,and can also cause the inactivation of tumor suppressor genes through hypermethylation of promoters,leading to cancer.CagA can also suppress the immune microenvironment through tumor-associated macrophages to promote the development of cancerKLF4 is a zinc factor-containing transcription factor,which is highly expressed in terminally differentiated gastrointestinal mucosal epithelial cells,and has an inseparable relationship with tumorigenesis and development.There is also a PEST sequence located in the transcription activation domain and the transcription suppression domain,suggesting that KLF4 may be degraded by the ubiquitin-proteasome pathway.KLF4 has a short half-life.It acts as a tumor suppressor gene in gastric cancerThe ubiquitinated proteasome pathway is an important system that regulates protein function and degradation in cells,mainly regulating some structural abnormalities or short-lived proteins in cells.Functional studies on ubiquitination have long focused on the ubiquitination system.However,researches in recent years have shown that the deubiquitinating enzymes DUBs were also actively involved in cellular events in cancer.The DUBs that our group concerned about can cleave the connection between the ubiquitin chain and the substrate protein and the connection between the ubiquitinated chain through cleavage,or can modify the ubiquitin from the distal end of the ubiquitin chain.These activities can potentially Antagonizes intracellular ubiquitinationTherefore,the group treated gastric epithelial cells and gastric cancer cells with proteasome inhibitor MG-132 in the early stage,and confirmed that CagA participates in the proteasome degradation process of KLF4There are many types of deubiquitinating enzymes,of which the USPs superfamily of ubiquitin-specific modification enzymes are the most widely known deubiquitinating enzymes and the most diverse structure.We screened several existing USPs in the laboratory and found that USP42 significantly up-regulated KLF4 protein expression Then we found that with the infection of H.pylori and the transfection of CagA,the expression of KLF4 and USP42 both decreased,this indicates that the regulation of KLF4 by USP42 depends on CagA.Then dose-dependent experiments were used to find that USP42 can increase KLF4 protein expression.Knockdown of USP42 down-regulates KLF4 expression and promotes malignant transformation of gastric epithelial cellsSo how does USP42 participate in regulating deubiquitinating of KLF4?Then,we performed the following experimentsResearch contents1.Expression levels of USP42 and KLF4mRNA in ges-1 and gastric cancer cells were detected.The expression of USP42 and KLF4 proteins and mRNA in gastric cancer tissues of patients with clinical gastric cancer were detected2.CagA plasmids of the same mass and USP42 plasmids of different mass were co-transfected into gastric epithelial cells ges-1 and gastric cancer cells AGS,and the expressions of KLF4,USP42 and CagA were detected by WB to verify whether the down-regulation of KLF4 by CagA could be reversed by the transfection of USP42 plasmids.3.In gastric epithelial cells ges-1 and gastric cancer cells AGS,the USP42 plasmid of the same mass and CagA plasmid of different mass were co-transfected,and the expressions of KLF4,USP42 and CagA were detected by WB to verify whether the upregulation of KLF4 by USP42 could be reversed by CagA plasmid transfection4.The expression of KLF4 and USP42 on the microarray of 75 paired gastric cancer tissues and adjacent tissues was detected,and the correlation between KLF4 and USP42 expression was analyzed5.Statistical analysis of the correlation between KLF4 expression and USP42 expression and clinical indicators such as disease stage and tumor grade6.Establish the mouse model of chronic helicobacter pylori infection:c57 mice were treated by gavage with CagA-positive Helicobacter pylori(PMSS1)and CagA-negative Helicobacter pylori(PMSS1?CagA).Mice were randomly sacrificed at 6 weeks and12 weeks after the last gavage(1)rapid urease experiment was conducted in the pylorus of the stomach of mice,extract gastric mucosal RNA for PCR identification;(2)some tissues were isolated and cultured with Columbia culture medium,and the cultured bacteria were subjected to rapid urease experiment and PCR identification;(3)formalin was used to fix part of the tissue,and paraffin sections were prepared for HE staining and immunohistochemical analysisResearch results1.USP42 and KLF4 were highly expressed in GES-1 cells at mRNA level,and were low expressed in gastric cancer cells.The expressions of USP42 and KLF4 in gastric cancer tissues were lower than those in para-cancer tissues2.WB results showed that CagA down-regulated KLF4 protein expression,which was reversed under the action of the USP42 plasmid in a dose-dependent manner.3.The expression of KLF4 protein was up-regulated by USP42,which was reversed under the action of CagA plasmid in a dose-dependent manner4.Tissue microarray results showed that adjacent non-tumor(NT)tissues had relatively high expression levels of USP42 and KLF4,and there was a significant positive correlation between their expression levels.Abnormal expression of USP42-KLF4 was of clinical significance5.Expression of USP42 and KLF4 in gastric cancer tissues was negatively correlated with age and tumor grade,and down-regulated in advanced tumors There were statistically significant differences in the expression of USP42 and KLF4 in grade ? and ? tumors6.Rapid urease test and PCR results showed that H.pylori colonized the stomach of mice in the irrigation group7.HE staining showed the pathological changes caused by helicobacter pylori infection in the gastric mucosa of mice in the gavage group after 6 weeks Compared with the PMSS1 ?CagA group,the gastric mucosa and submucosa of mice in the PMSS1 group showed certain inflammatory cell infiltration and slight submucosal vascular edema.The expression level of KLF4 in the stomach of both groups was higher.8.HE staining showed gastric mucosa of mice in the gavage group and observed the pathological changes caused by H.pylori infection after 12 weeks.Compared with the PMSS1 ?CagA group,the mice in the PMSS1 group showed more inflammatory cell infiltration in the gastric mucosa and submucosa,obvious dilatation and congestion of small blood vessels and obvious inflammatory changes.PMSS1 ?CagA mice gastric mucosa only scattered lymphocyte infiltration,inflammation change is not obvious.IHC staining of paraffin sections showed that KLF4 expression was lower than that of H.pylori infection at 6 weeks.Conclusions1.The low expression of USP42 caused by H.pylori infection/CagA transfection resulted in the degradation of KLF4 protein in gastric cancer,the abnormal CagA-USP42-KLF4 signaling pathway plays an important role in the development of gastric cancer.2.H.pylori infection/CagA plays an important role in the development of gastric cancer.