The Effects And Mechanisms Of JUN-PPP1R15A/GADD34 For Resisting The Glucose Deprivation-Induced Energy Stress In Tumor Microenvironment And Promoting Gastric Cancer Progression

BackgroundGastric cancer is one of the most frequent and common tumors in the world,ranking fifth among all malignant tumors in terms of incidence and fourth in terms of mortality in 2020.The early symptoms of gastric cancer are not typical and the disease is relatively insidious,with most patients being diagnosed at an advanced stage.Recurrence after stomach cancer surgery is a major issue and is often the ultimate cause of death.Glucose metabolism plays an important role in tumor progression.When glucose intake is insufficient and tumor growth outpaces its energy supply,tumor cells typically maintain high levels of glycolysis in response to energy stress.Overcoming the compensatory mechanism of energy stress is crucial for the survival of tumor cells.The survival mechanism of tumor cells under energy stress may be related to tumor recurrence or metastasis.This study aims to understand the changes in key gene expression of gastric cancer cells under energy stress based on an energy stress model induced by glucose deprivation and explore the potential mechanisms by which cells survive under the energy stress environment.This will help to further discover new therapeutic targets and prognostic markers of tumors.Part Ⅰ The effects of energy stress on gastric cancer cells and regulation of key gene expressionsObjectiveConstructing an in vitro gastric cancer cell glucose-depriving energy stress model,screening key genes and studying their expression regulation.Methods1.Gastric cancer cells were induced by glucose starvation(0mM,1mM)to establish the energy stress model.The expression levels of glucose metabolism-related genes were detected by RT-qPCR to verify the model.The effects of energy stress on the proliferation,apoptosis,and cell cycle of gastric cancer cells were detected by CCK-8,MTS,and flow cytometry.Transcriptome sequencing analysis(RNA-seq)was performed on the glucose concentration 0mM and 25 mM groups to understand the gene expression changes in gastric cancer cells under energy stress(0mM).Combined with the GSE13548 dataset,key genes were screened using Cytoscape software,and Western blotting verified the expression of key genes c-Jun and PPP1R15A(This gene alias is also known as GADD34,and PPP1R15A is used in this article)under energy stress.2.ALGGEN-PROMO and JASPAR websites were used to predict the binding relationship and binding region of the PPP1R15A promoter to transcription factor cJun.ChIP-PCR was used to verify the binding of the transcription factor c-Jun to the PPP1R15A promoter,and dual luciferase reporter gene assay was used to verify the regulation of PPP1R15A by c-Jun.Western blotting was used to examine the expression level of PPP1R15A after overexpression of c-Jun.ResultsThe energy stress model of gastric cancer cells was successfully established.The expression levels of G6 PD,GSS,HK1,LDHA,PDK1,PFKM,PGM1,PKM,SLC2A1,and SLC16A1,which are related to the glucose metabolism pathway,were significantly increased in gastric cancer cells(P<0.01).The proliferation ability of gastric cancer cells decreased,apoptosis increased and the proportion of the G0/G1 phase cells increased under energy stress.Screening of the genes indicated that the expression levels of JUN and PPP1R15A were significantly increased under energy stress.The transcription factor c-Jun was bound to the PPP1R15A promoter region(CGTGACGTCAGC)and regulated PPP1R15A expression.Overexpression of c-Jun increased the PPP1R15A expression.ConclusionsGlucose deprivation inhibited gastric cancer cell proliferation and promoted apoptosis and G1/S cell cycle arrest.The expression levels of JUN and PPP1R15A were elevated and JUN positively regulated the expression of PPP1R15A under energy stress.JUN-PPP1R15A may be involved in important biological processes under energy stress.Part Ⅱ The Expression and clinical significance of JUN-PPP1R15A in gastric cancerObjectiveTo study the expression levels of JUN and PPP1R15A in gastric cancer and their relationship with prognosis.Methods1.246 cases of gastric cancer treated by surgery in a single center were retrospectively collected.Immunohistochemical staining was applied to detect the expression levels of JUN and PPP1R15A in gastric cancer tissues and paracancerous controls,and semi-quantitative analysis was performed.The gastric cancer cohort was followed up periodically to collect survival data.R software(4.2.1)was used to perform the proportional risk hypothesis test and fit survival regression,etc.2.375 cases of gastric cancer and 32 control tissues from the TCGA gastric cancer database were analyzed individually or in combination with 359 normal tissues from the GTEx database for differences in PPP1R15A expression levels between gastric cancer and control tissues.The relationship between PPP1R15A expression level and prognosis was analyzed in 875 cases of gastric cancer patients collected in the GEO database.ResultsIn the clinical gastric cancer cohort,the expression levels of JUN and PPP1R15A in gastric cancer tissues were higher than those in paracancerous control tissues(P<0.001).There were differences in positive vascular invasion(81.3% vs 75.6%),negative lymph nodes(14.6% vs 22.8%),and higher TNM stage(82.9% vs 78.0%)between high and low JUN expression groups,but the difference was not statistically X significant.The risk of death in the group with high JUN expression was 1.60 times higher than that in the group with low JUN expression(HR: 1.60,95% CI: 1.15-2.21).There were differences in positive vascular invasion(82.1% vs 74.8%),distant metastasis(10.6% vs 8.9%),and larger tumor diameter(60.2% vs 52.0%)between high and low PPP1R15A expression groups,but the difference was not statistically significant.The risk of death in the group with high expression of PPP1R15A was 1.58 times higher than that in the group with low expression of PPP1R15A(HR: 1.58,95%CI: 1.14-2.20).The co-expression of PPP1R15A and JUN was associated with the overall survival of gastric cancer patients(P<0.05): Compared with JUN-PPP1R15A concurrent low expression group(LL),the HR(Hazard Ratio,HR)in JUN-PPP1R15A concurrent high expression group(HH),JUN low-PPP1R15A high expression group(HL)and JUN high-PPP1R15A low expression group(LH)was 2.32(95% CI: 1.47-3.65),2.22(95%CI: 1.33-3.69)and 2.21(95% CI: 1.35-3.65)respectively.In addition,there were differences among the four groups(HL,HH,LH,LL)with positive vascular invasion(80.8% vs 83.1% vs 78.8% vs 71.8%)and distant metastasis(11.5% vs 9.9% vs 9.6%vs 8.5%),but the differences were not statistically significant.The combined analysis of the TCGA gastric cancer database and the GTEx database suggested that the expression level of PPP1R15A in gastric cancer was higher than that in the normal group(P=0.026).In the GEO database,the median survival of the high PPP1R15A expression group was 23.6 months,and that of the low PPP1R15A expression group was 75.5 months.The prognosis of patients in the high PPP1R15A expression group was poorer(P<0.001).ConclusionsJUN and PPP1R15A were highly expressed in gastric cancer tissues.The high expression of both genes was associated with poor prognosis in patients with gastric cancer,and both were independent risk factors for gastric cancer prognosis.The high expression of JUN-PPP1R15A may be related to the progression of gastric cancer.Part Ⅲ Study of PPP1R15A in resisting energy stress and promoting gastric cancer progressionObjectiveTo study the expression and function of PPP1R15A in gastric cancer cells.Methods1.Cell experiments: RT-qPCR and Western blotting were used to detect the expression of PPP1R15A in normal gastric mucosal epithelial cells GES-1 and gastric cancer cell lines(AGS,BGC-823,HGC-27,SGC-7901).The effects of PPP1R15A knockdown and overexpression on cell proliferation,apoptosis,and cell cycle were detected by CCK-8,colony formation assay,and flow cytometry.Gastric cancer cell lines with stable knockdown and overexpression of PPP1R15A were constructed using lentiviral vectors.CCK-8 and flow cytometry were used to detect the effects of stable knockdown of PPP1R15A on cell proliferation,apoptosis,and cell cycle during energy stress.2.Animal experiments: The effect of overexpression of PPP1R15A on the proliferative ability of gastric cancer cells in vivo was examined in the nude mouse transplantation tumor experiment.ResultsThe mRNA and protein levels of PPP1R15A were higher in gastric cancer cell lines than in normal gastric mucosal epithelial cells.PPP1R15A knockdown resulted in decreased proliferative ability,increased apoptosis,and less cell cycle G0/G1 proportion in gastric cancer cells,contrary to the results of PPP1R15A overexpression.Knockdown of PPP1R15A under energy stress still resulted in decreased cell proliferation ability,increased apoptosis,and fewer cells in the G0/G1 phase.The nude mouse transplantation tumor experiment suggested the subcutaneous tumor-bearing volume and weight of gastric cancer cells in the PPP1R15A overexpression group were higher than those in the control group(P<0.05).ConclusionsThe results of in vivo and in vitro experiments suggested that PPP1R15A promoted gastric cancer cell proliferation,inhibited apoptosis,and promoted G1/S cell cycle XII arrest.PPP1R15A knockdown promoted increased apoptosis caused by energy stress,also inhibited cell proliferation,and affected the cell cycle.PPP1R15A resisted the negative effects of energy stress and promoted the progression of gastric cancer.Part Ⅳ Study on the mechanism of PPP1R15A regulating autophagy under energy stressObjectiveTo study the regulatory effects of PPP1R15A on autophagy under energy stress.Methods1.Under non-energy stress conditions,stable PPP1R15A overexpression gastric cancer cell line HGC-27 was subjected to transcriptome sequencing(mRNA-seq)and metabolite sequencing(Metabolite-seq),and differential genes and differential metabolites were analyzed separately for pathway enrichment and co-analyzed for two omics sequencing.2.With the non-energy stress state as the control,the effects of PPP1R15A knockdown on the expression of autophagy pathway-related proteins were detected under energy stress.Transmission electron microscopy was used to observe the effects of PPP1R15A on autophagosomes and autolysosomes.mRFP-GFP-LC3 tandem fluorescent protein adenovirus infected cells were used to observe the effects of PPP1R15A on autophagy flux using the laser scanning confocal microscope.The relationship between the expression levels of PPP1R15A and autophagy pathwayrelated proteins was verified in the TCGA gastric cancer database.ResultsmRNA-seq identified a total of 6857 differentially expressed genes,and the enrichment analysis found that PPP1R15A overexpression might affect cellular autophagy and other related pathways.Metabolite-seq identified a total of 5588 differentially expressed metabolites,and the enrichment analysis suggested that PPP1R15A overexpression may affect lysosome and autophagy-related cellular responses.A joint analysis of the two omics sequencing identified a total of 98 intersections of enriched pathways,including autophagy and lysosomes.PPP1R15A knockdown resulted in lower expression levels of autophagy-related proteins ATG5,Beclin1,and LC3 B II,higher P62 expression,reduced number of autolysosomes observed by transmission electron microscopy,and reduced number of autophagosome and autolysosome observed by laser scanning confocal microscope,which was contrary to the results of PPP1R15A overexpression and energy stress.At the same time,knockdown of PPP1R15A under energy stress still resulted in decreased expression levels of autophagy-related proteins ATG5,Beclin1,and LC3 B II,increased P62 expression,decreased number of autolysosomes and autolysosomes.In the TCGA database,the expression level of autophagy pathway-related genes was higher in the high PPP1R15A group(P<0.01).ConclusionsIn gastric cancer cell lines,knockdown of PPP1R15A inhibited autophagy,and overexpression of PPP1R15A promoted autophagy.Gastric cancer cells promoted autophagy by increasing the expression level of PPP1R15A under energy stress.The PPP1R15A gene can directly regulate the autophagy of gastric cancer cells,which may be one of the mechanisms of PPP1R15A to resist energy stress and promote gastric cancer progression.