Study On The Mechanism Of Jianpi Qingre Huoxue Prescription Regulating The Occurrence And Development Of Gastric Cancer By LncRNA DLGAP1-AS2/HuR/c-Myc Axis

ObjectiveGastric cancer(GC)is the fifth most common malignancy worldwide and one of the leading causes of cancer-related death.Because the symptoms of GC patients are often not detectable in the early stage,most patients are diagnosed in the late stage of the cancer,therefore,it is of great significance to further study the mechanism of GC occurrence and development and carry out relevant drug intervention.The purpose of this study was to further explore whether Jianpi Qingre Huoxue prescription could regulate DLGAP1-AS2/HuR/c-Myc axis to inhibit the occurrence and development of GC,and to provide scientific basis for the application of Jianpi Qingre Huoxue prescription to the prevention and treatment of GC.Methods1.The effect of DLGAP1-AS2/HuR(ELAVL1)/c-Myc axis on the occurrence and development of GC(1)Bioinformatics analysis was used to identify DLGAP1-AS2,analyze its expression in different datasets and pan-cancer,and its prognostic role among GC patients.Meanwhile,the relationship between the expression of DLGAP1-AS2 and different clinical features was analyzed.(2)The expression of DLGAP1-AS2 in GC tissues,tissues at different pathological stages(chronic non-atrophic gastritis,CNAG;chronic atrophic gastritis,CAG;precancerous lesions of gastric cancer,PLGC;gastric cancer,GC)and different GC cell lines were detected by real-time quantitative PCR(RT-qPCR).(3)DLGAP1-AS2 interference siRNA was constructed to transfect GC cells,and the transfection efficiency was detected by RT-qPCR.The effects of silencing DLGAP1-AS2on the proliferation,apoptosis,cell cycle,migration and invasion ability of GC cells were detected by CCK-8,flow cytometry and Transwell migration and invasion assays,respectively.Stable lentivirus transfected cells knocked down by DLGAP1-AS2 were constructed.Tumor formation model in BALB/c nude mice was used to observe tumor growth in nude mice after silencing DLGAP1-AS2.(4)Binding of DLGAP1-AS2 with HuR protein was predicted by ENCORI and RPISeq databases,the correlation between DLGAP1-AS2 and HuR expression was analyzed by GEPIA database.The binding of DLGAP1-AS2 with HuR protein in GC cells was detected byRNA binding protein immunoprecipitation(RIP).(5)After transfected with HuR siRNAs in GC cells,CCK-8,flow cytometry,and Transwell migration and invasion assay were used to detect the effects of silencing HuR on proliferation,apoptosis,migration and invasion of GC cells,respectively.ENCORI and RPISeq databases were used to predict the binding of HuR protein to c-Myc,GEPIA database was used to analyze the correlation between HuR protein with c-Myc expression,RIP was used to detect the binding of HuR protein with c-Myc in GC cells.(6)The expression of c-Myc and its downstream molecules were detected by RT-qPCR after DLGAP1-AS2 overexpression or empty plasmid were transfected separately,or DLGAP1-AS2 overexpression and HuR siRNA were co-transfected.After treating GC cells with actinomycin D,the attenuation degree of c-Myc mRNA was measured at different time points.2.Regulation mechanism of the prescription of Jianpi Qingre Huoxue on DLGAP1-AS2/HuR(ELAVL1)/c-Myc molecular network(1)CCK-8 assay was used to detect the proliferation of GC cells after treated with different concentrations of Jianpi Qingre Huoxue prescription,and Probit regression analysis was used to calculate the drug concentration under half maximal inhibitory concentration(IC50).(2)GC cells treated with Jianpi Qingre Huoxue prescription at concentration of IC50,CCK-8 and flow cytometry were used to detect the effects of the Jianpi Qingre Huoxue prescription on the proliferation,apoptosis and cell cycle of GC cells,respectively.(3)GC cells treated with Jianpi Qingre Huoxue prescription at concentration of IC50,RT-qPCR was used to detect the expression of DLGAP1-AS2,HuR,c-Myc,and the expression of downstream molecules of c-Myc.(4)The tumor volume and weight were investigated in nude mouse tumor model after the intervention of Jianpi Qingjie Huoxue prescription.Results1.The effect of DLGAP1-AS2/HuR(ELAVL1)/c-Myc axis on the occurrence and development of GC(1)Based on cut-off of|FC|>2.0 and adjusted P<0.05,differently expressed lncRNAs were identified in GSE70880,GSE19826,GSE13911 and TCGA-STAD datasets,respectively,and only lncRNA DLGAP1-AS2 expression was consistently upregulated in these four datasets.The expression of DLGAP1-AS2 was analyzed in GEPIA database,and the results showed that DLGAP1-AS2 was up-regulated in other tumors as well.Kaplan Meier database showed that the overall survival,progression-free survival and post-progression survival of patients with high DLGAP1-AS2 expression were significantly shorter than those in the low expression group(logrank P<0.01).The GSE15945 dataset(n=197)in Kaplan Meier database was used to analyze the relationship between the expression of DLGAP1-AS2 and different clinicopathological factors.The results showed that the patients with high expression of DLGAP1-AS1 had shorter overall survival time in advanced GC patients(stage III+IV)(logrank P_{stage III}=0.0083,logrank P_{stage IV}=0.0048)and progression-free survival(Logrank P_{stage III}=0.0454,logrank P_{stage IV}=0.019),but had no effect on survival time of patients with early GC(stage I+II,logrank P>0.05);patients with high DLGAP1-AS2 expression had shorter overall survival time(logrank P=0.0164)and progression-free survival time(logrank P=0.0116)in GC patients with lymph node metastasis(N1+2+3),but had no significant effect on the survival time of GC patients without lymph node metastasis(N0,logrank P>0.05).(2)RT-qPCR showed that the DLGAP1-AS2 expression was up-regulated in GC tissues compared with adjacent tissues(P<0.05).Compared with chronic non-atrophic gastritis group,the expression of DLGAP1-AS2 was up-regulated in atrophic gastritis,precancerous lesions and gastric cancer tissues(P<0.05).In addition,compared with GES-1 cells,DLGAP1-AS2 was significantly up-regulated in AGS,SGC-7901 and MGC-803 GC cell lines(P<0.05).(3)Cell proliferation was reduced,apoptosis rate was increased,cell cycle was stopped,and migration and invasion abilities were weakened after silencing of DLGAP1-AS2 in AGS and SGC-7901 cells(P<0.05).The tumor size and mass of the nude mice were decreased after DLGAP1-AS2 inhibition(P<0.05).(4)ENCORI and RPISeq databases predicted DLGAP1-AS2 can bind with HuR protein,GEPIA database suggested that DLGAP1-AS2 expression was positively related to and HuR expression(P<0.05,R>0),RIP found that DLGAP1-AS2 can bind with HuR protein(P<0.05).(5)Cell proliferation decreased,apoptosis rate increased,and migration and invasion ability decreased after HuR silencing in AGS and SGC-7901,(P<0.05);ENCORI database and RPISEQ database predicted DLGAP1-AS2 can bind with HuR protein,GEPIA database suggested that HuR expression was positively related to and c-Myc expression(P<0.05,R>0),RIP found that HuR protein can bind with c-Myc(P<0.05).(6)The expression of c-Myc was increased after transfection with DLGAP1-AS2overexpression plasmid compared with the empty plasmid transfection control group in AGS and SGC-7901 cells(P<0.05).There was no significant difference in the expression of c-Myc after transfection with DLGAP1-AS2 overexpression plasmid+HuR siRNA compared with the empty plasmid transfection control group(P>0.05).When pc DNA-DLGAP1-AS2 vector was co-transfected with HuR siRNA,HuR silencing could significantly reduce the half-life of c-Myc increased by DLGAP1-AS2(P<0.05).In addition,transfected GC cells using DLGAP1-AS2 overexpression plasmid and its empty plasmid,CCNB1 and CDK4 expression were increased,P21 and GADD4 expression were decreased in DLGAP1-AS2 overexpression group compared with control group(P<0.05),while transfected with DLGAP1-AS2 overexpression plasmid+HuR siRNA,CCNB1,CDK4,P21 and GADD45 expression had no significant difference between empty plasmid transfection group and co-transfected group(P>0.05).2.Regulation mechanism of Jianpi Qingre Huoxue prescription on DLGAP1-AS2/HuR(ELAVL1)/c-Myc molecular network(1)Probit regression analysis showed that the IC50 of Jianpi Qingre Huoxue prescription on AGS cells was 4.236mg/m L at 24h,3.645 mg/m L at 48h and 3.963 mg/m L at 72h,and the average IC50 at three time points was 3.948mg/m L.Therefore,the concentration of 4mg/m L of lyophilized powder solution was selected for subsequent cell experiments.The IC50 of lyophilized powder solution on SGC-7901 cell at 24h,48h and72h was 4.125mg/m L,3.882 mg/m L and 3.924 mg/m L,respectively.The mean IC50 at three time points was 3.977mg/m L.Therefore,the concentration of lyophilized powder solution at 4 mg/m L was selected for subsequent cell experiments.(2)Jianpi Qingre Huoxue prescription could effectively inhibit the proliferation of AGS and SGC-7901 cells,promote the apoptosis of AGS and SGC-7901 cells,and induce cell stagnation in S phase(P<0.05).(3)RT-qPCR results showed that Jianpi Qingre Huoxue prescription could inhibit the expressions of DLGAP1-AS2,HuR and c-Myc in AGS and SGC-7901 cells(P<0.05).In addition,the prescription of Jianpi Qingre Huoxue could inhibit the expressions of CCNB1and CDK4 in AGS and SGC-7901 cells,and promote the expressions of P21 and GADD45.(4)After the intervention of Jianpi Qingre Huoxue prescription,it was found that the tumor volume and mass in high-dose,medium-dose and low-dose groups and5-fluorouracil(5-Fu)positive control group were decreased compared with the control group(P<0.05).There was no significant difference in tumor volume and mass between the high,medium and low dose groups and the positive control group(P>0.05).ConclusionThis study successfully identified lncRNA DLGAP1-AS2,and found DLGAP1-AS2/HuR(ELAVL1)/c-Myc axis'effect on GC occurrence and development by bioinformatics and relevant experiments.Moreover,Jianpi Qingre Huoxue prescription can inhibit GC cells proliferation,promote apoptosis and S phase of stagnation,and can inhibit DLGAP1-AS2,HuR,c-Myc expression,and can inhibit or promote the expression of c-Myc downstream genes,which suggested that the Jianpi Qingre Huoxue prescription can inhibit the occurrence or progression of GC by DLGAP1-AS2/HuR/c-Myc axis,which can provide a scientific basis for the application of the Jianpi Qingre Huoxue prescription in the treatment of PLGC and GC.