Autophagy Block Induced Activation Of Unfolded Protein Response Upregulated The Expression Of Cd18/integrin β2 In Gastric Cancer Cells To Promote Peritoneal Metastasis Of Gastric Cancer

Objective: Gastric cancer(GC)is one of the top five cancer types worldwide,with high incidence rate and mortality,especially in East Asian countries such as China.The peritoneum is the most common characteristic metastatic site of GC,and the prognosis of GC patients with peritoneal metastasis is very poor.At present,systemic chemotherapy is still the main treatment for peritoneal metastasis of advanced GC.However,peritoneal metastasis is not sensitive to chemotherapy.It is urgent to find effective treatment targets and to enrich treatment methods to improve the prognosis of GC peritoneal metastasis.During the process of GC peritoneal metastasis,GC cells entering the peritoneal cavity interact with monolayer of peritoneal mesothelial cells on the surface of peritoneum and then colonize to form peritoneal metastatic nodules.Up to now,no definite adhesion molecule has been found as a therapeutic target for GC peritoneal metastasis,especially for the adhesion process between GC cells and peritoneal mesothelial cells.Therefore,this study aimed to screen important adhesion molecules participating in the interaction between GC cells and peritoneal mesothelial cells,investigate the potential mechanism of how the expression and function of certain adhesion molecules promote the progress of GC peritoneal metastasis,and explore the possibility of clinical transformation of adhesion molecules as therapeutic targets for GC peritoneal metastasis.Methods: 1.The bioinformatics method was used to screen the differentially expressed genes in gastric cancer samples from patients with peritoneal metastasis in GSE62254 dataset using R software package "limma".2.The Kyoto Encyclopedia of Genes and Genomics(KEGG)pathway enrichment analysis was used to enrich the pathway of highly expressed genes in patients with peritoneal metastasis;Gene ontology(GO)function enrichment analysis was used to enrich the pathway of highly expressed genes in MKN45P3 cells.3.In vitro tumor cells-human peritoneal mesothelial cells adhesion assay was used to detect the adhesion ability of gastric cancer cells to peritoneal mesothelial cells.4.Cell viability assay was used to detect cell proliferation rate.5.Transwell assay was used to detect the migration and invasion ability of tumor cells.6.STRING database was used to construct protein-protein interaction network.7.Real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect m RNA level.8.Western Blot was used to detect the protein expression.9.Small interfering RNA(si RNA)transfection technology was used to instantaneously knock down the target gene expression.10.The lentivirus short hairpin RNA(sh RNA)transfection technology was used to construct a stable knockout cell line.11.Cellular immunofluorescence assay was used to detect the localization of proteins.12.Flow cytometry was used to detect the expression of cell surface antigen.13.Nuclear protein and plasma protein were extracted by protein nucleoplasma separation experiment for further Western Blot test to detect protein localization.14.The peritoneal metastasis model was established by intraperitoneal injection in BALB/c-nude mice.15.Statistical analysis: All statistical analyses were performed using Graph Pad Prism software and R studio software.All the experiments were conducted at least three independent repeated experiments,and the experimental results were showed as mean ± standard deviation(SD).Student’s t-tests were used to evaluate the differences between the two groups.P < 0.05 was considered statistically significant.Results: 1.The enhanced cell adhesion promoted the peritoneal metastasis of gastric cancer,and the high peritoneal metastasis gastric cancer cell line MKN45 P3 had a stronger ability of adhering to human peritoneal mesothelial cells.Through the analysis of GSE62254 dataset,the highly expressed genes in gastric cancer patients with peritoneal metastasis were highly enriched in cell adhesion-related pathways such as Focal Adhesion,Cell Adhesion Molecules,and ECM-Receptor Interaction.In vitro tumor cells-human peritoneal mesothelial cells adhesion test showed that compared with the parent cell line MKN45,the high peritoneal metastasis gastric cancer cell line MKN45P3 had stronger ability of adhering to human peritoneal mesothelial cells HMr SV5.2.CD18/integrin β2 was a highly expressed adhesion molecule in MKN45 P3 cells.The STRING database was used to construct the protein-protein interaction network for the differentially expressed genes in cell adhesion pathways,suggesting that the integrin family may be an important adhesion molecule family that promoted peritoneal metastasis of gastric cancer.RT-q PCR results showed that compared with parental cell lines,CD18/integrin β2,member of integrin family,was highly expressed in MKN45 P3 cells.RT-q PCR and Western Blot verified CD18/integrin β2 were highly expressed in MKN45P3 cells at both m RNA and protein level.Immunofluorescence and flow cytometry showed that the expression of CD18/integrin β2 on the surface of MKN45 P3 cells was significantly higher than on the surface of MKN45 cells.3.CD18/integrin β2 promoted the adhesion of gastric cancer cells to peritoneal mesothelial cells.Subsequently,in MKN45 P3 cells CD18/integrin β2 was instantaneously knocked down using si RNA,and Western Blot and flow cytometry verified that the total and surface expression of CD18/integrin β2 were significantly downregulated.Knock down of CD18/integrin β2 significantly inhibited the adhesion of MKN45 P3 cells to HMr SV5 cells.Neutralizing antibody α-CD18 was used to block cell surface CD18/integrin β2,and the adhesion of MKN45 P3 cells to HMr SV5 cells was significantly inhibited.Overexpression of CD18/integrin β2 significantly improved the adhesion of MKN45 cells and SNU-216 cells to HMr SV5 cells.4.In vivo experiment confirmed that CD18/integrin β2 promoted gastric cancer peritoneal metastasis.In vivo peritoneal metastasis model was established by intraperitoneal injection of MKN45P3 cells into BALB/c-nude mice.Compared with negative control cells,stably CD18/integrin β2 knockout MKN45 P3 cells led to significantly fewer peritoneal metastatic nodules and less bloody ascites,resulting in a lower degree of weight loss in nude mice.5.CD18/integrin β2 transcription was enhanced in MKN45 P3 cells,and s XBP1 from UPR pathway is a potential transcription factor of CD18/integrin β2.We detected CD18/integrin β2 protein expression using Western Blot after treatment of actinomycin(CHX),MG132 or chloroquine(CQ),and the results showed that there was no significant difference in protein degradation rate between the two cells,suggesting that the difference in CD18/integrin β2 expression mainly came from the transcription level.PROMO website was used to predict potential transcription factors of CD18/integrin β2,combined with the GO function enrichment results of MKN45 P3 and MKN45 RNA-seq,suggesting that the key transcription factor s XBP1 from the Unfolded Protein Response(UPR)may be a potential transcription factor of CD18/integrin β2.IRE1α-s XBP1 axis in UPR pathway was upregulated at protein level in MKN45 P3 cells,and the spliced-XBP1 m RNA also significantly increased.The results of protein nucleoplasm separation showed that the expression of transcription factor s XBP1 in the nucleus of MKN45 P3 was higher than that in MKN45.JASPAR website predicted that there was an s XBP1 binding site in the promoter region of CD18/integrin β2.In MKN45 P3 cells,knockdown of XBP1 significantly downregulated the transcription level of CD18/integrin β2.6.s XBP1 upregulated CD18/integrin β2 expression and promoted the adhesion of gastric cancer cells to peritoneal mesothelial cells.XBP1 knockdown was performed in MKN45 P3 cells.Western Blot results showed that CD18/integrin β2 protein expression was decreased,and the adhesion of MKN45 P3 cells to peritoneal mesothelial cells significantly decreased after knocking down XBP1 in MKN45 P3 cells.After stable knockout of CD18/integrin β2,instantaneous knockdown of XBP1 in MKN45 P3 cells were then performed but could not further inhibit the adhesion to peritoneal mesothelial cells.7.Autophagy flux block promoted UPR activation and s XBP1 expression.To find out the cause of UPR activation in MKN45 P3 cells,we first detected whether there was a block in the autophagic flux of cells.Western Blot was used to detect the expression of LC3 B and cargo receptor P62 in MKN45 and MKN45 P3 cells.It was found that the ratio of LC3B-II/I in MKN45 P3 cells was significantly increased,and the expression of autophagic cargo receptor P62 protein increased,suggesting that autophagic flux was blocked.In MKN45 cells,after treatment with CQ to inhibit autophagy-lysosomal fusion to simulate block of autophagic flux,s XBP1 m RNA expression was significantly increased,and protein level was upregulated.In MKN45 cells,knockdown of ATG5 inhibited the extension of autophagic membrane and the formation of autophagic body to simulate block of autophagic flux,and the expression of s XBP1 m RNA and protein were significantly increased.8.Autophagy flux block promoted CD18/integrin β2 expression and promoted the adhesion of gastric cancer cells to peritoneal mesothelial cells.After blocking autophagy flux by CQ treatment or ATG5 knockdown in MKN45 cells,CD18/integrin β2 m RNA level was significantly upregulated and protein expression was increased.After CQ treatment or the knockdown of ATG5 in MKN45 cells,further knockdown of XBP1 significantly inhibited the m RNA and protein expression of CD18/integrin β2.The adhesion of MKN45 cells to peritoneal mesothelial cells was significantly enhanced after ATG5 knockdown.In vitro tumor cells-human peritoneal mesothelial cells adhesion test showed that knockdown of CD18/integrin β2 or using neutralizing antibody α-CD18 to block surface CD18/integrin β2 expression after knockdown of ATG5 in MKN45 cells significantly reversed the effect of autophagic flux block on cell adhesion.Conclusion: 1.Bioinformatics analysis showed that integrin family was significantly associated with peritoneal metastasis of gastric cancer;in vitro experiments confirmed that MKN45 P3,a cell line with high peritoneal metastasis potential,had stronger adhesion ability to human peritoneal mesothelial cells than its parent cells,and CD18/integrin β2,a member of integrin family was a highly expressed adhesion molecule in MKN45 P3 cells.2.CD18/integrin β2 promoted the adhesion of gastric cancer cells to peritoneal mesothelial cells,and gene knockout or neutralizing antibody blocking significantly inhibited the adhesion of gastric cancer cells to peritoneal mesothelial cells.3.The high expression of CD18/integrin β2 in MKN45 P3 cells mainly came from the increase of transcription level.The activation of UPR pathway led to the transcription factor s XBP1 entering the nucleus and promoting CD18/integrin β2 transcription.4.Autophagic flux in MKN45 P3 cells was significantly blocked,which was the possible reason for the activation of UPR pathway in MKN45 P3 cells.The autophagic flux block promoted CD18/integrin β2 transcription by upregulating the expression of s XBP1,thus promoting the adhesion of gastric cancer cells to peritoneal mesothelial cells.