Molecular Mechanism Of BANCR/miR-145-5p Axis Regulating Glucose Metabolism And Tumor Stemness In Gastric Cancer Cells

Part One Clinical study on the correlation between glucose metabolism and expression of BANCR,miR-145-5p,Reg3 A and DMBT1 in tumor cells of patients with gastric cancerObjective: To explore the correlation between glucose metabolism and the expression of BANCR,miR-145-5p,Reg3 A and DMBT1 in tumor cells of patients with gastric cancer.Methods: The clinical and pathological data of 36 patients with gastric cancer who underwent surgical treatment were analyzed.Collection of patients with clinical pathological information,postoperative tissue samples,observe the cell glucose intake and lactic acid production and gastric cancer cell Warburg effect(p-AKT,GLUT1,HK2,LDHA protein),the application of RT-PCR method to detect BANCR transcription,miR-145-5p,using ELISA method to detect Reg3 A and DMBT1 expression,statistics and the prognosis of patients with outcomes,using the linear correlation analysis of glucose metabolism and BANCR,miR-145-5p,Reg3 A and DMBT1 expression of correlation coefficient.Results: Among the 36 patients included in the study,the proportion of male patients and female patients were 66.67% and 33.33% respectively,of which the proportion of patients with age < 60 and ≥ 60 were 55.56% and44.44% respectively,the proportion of patients with tumor size ≥ 5.0 cm and < 5.0 cm were 41.67% and 58.33% respectively,the proportion of patients with tumor invasion depth T1,T2,T3 and T4 were 11.11%,27.78%,36.11%and 25.00% respectively,and the stages of lymph node metastasis were N0,N1,N2 The proportion of patients with N3 was 41.67%,25.00%,13.89% and19.44% respectively.The proportion of patients with TNM stage I,II,III and IV was 25.00%,13.89%,36.11% and 25.00% respectively.The proportion of patients with low differentiation,medium differentiation and high differentiation was 58.33%,30.56% and 11.11% respectively.The prognosis of patients with different stages was compared,and the difference was statistically significant(P<0.05).TNM stage from stage I to stage IV,the number of deaths in patients gradually increased,especially in stage IV patients.The infiltration,lymph node metastasis and histological differentiation of gastric cancer cells with different TNM stages were observed.With TNM staging from stage I to stage IV,glucose intake and lactic acid production gradually increased,Warburg effect was significant,the transcript level of BANCR was increased,the transcript level of miR-145-5p and the expression levels of Reg3 A and DMBT1 were decreased,and the number of patients died gradually increased.According to the correlation analysis,the multiple factors related to glucose metabolism included the expression levels of BANCR,miR-145-5p,Reg3 A and DMBT1.The multiple factors related to the prognosis of gastric cancer patients included Warburg effect,Reg3 A and DMBT1 expression levels.Conclusion: Glucose metabolism was positively correlated with the expressions of BANCR in tumor cells of patients with gastric cancer,and a negatively correlated with the expressions of miR-145-5p,Reg3 A and DMBT1 in tumor cells of patients with gastric cancer.Part Two Reg3 A exerts tumor suppressor effect by targeting DMBT1 in gastric cancerObjective: Regeneration family member 3α(Reg3A)belongs to a pancreatic secreted protein,which may be involved in cell proliferation or differentiation.However,the role and downstream regulatory mechanisms of Reg3 A in gastric cancer(GC)are still unclear.This study aimed to elucidate the role and mechanism of Reg3 A in regulating cell proliferation in GC.Methods: The expression levels of Reg3 A in GC patients and cells were investigated by PCR and Western blot.The correlation of Reg3 A with clinicopathological features of GC was explored using the TCGA dataset and clinical samples.Cell viability,colony formation,and xenograft tumor assays were performed to examine the effect of Reg3 A on cell proliferation.In addition,we predicted Reg3A-related genes by analyzing the TCGA dataset,and further explored the downstream regulatory mechanism of Reg3 A in GC.Results: Reg3 A was significantly down-regulated in both in vitro and in vivo experiments(P<0.05).The expression level of Reg3 A in gastric cancer was negatively correlated with TNM classification(P<0.001)and lymph nodes(P<0.001).Reg3 A significantly inhibited the proliferation of GC cells(P<0.05).Bioinformatics analysis and experimental results confirmed that Reg3 A positively regulates the expression of malignant brain tumor 1(DMBT1)deletion(P<0.05).In addition,both Reg3 A and DMBT1 could prolong overall survival(OS,P<0.01),post-progression survival(PPS,P<0.05)and first progression survival(FP,P<0.01).In GC,DMBT1 si RNA could reverse the function of Reg3 A on cell proliferation inhibition(P<0.05).Conclusion: Reg3 A may become a novel tumor suppressor by promoting the expression of DMBT1,which may be a potential therapeutic target for GC patients.Part Three Study on the mechanism of BANCR/miR-145-5p axis regula- ting glucose metabolism in gastric cancer cellsObjective: To explore the mechanism of BANCR/miR-145-5p axis regulating glucose metabolism of gastric cancer cells.Methods: AGS,a gastric cancer cell line,was purchased from Wuhan China typical Culture Preservation Center.The cells were incubated at 37°C in a humid atmosphere containing 20% O2 and 5% CO2.Cells were transfected with BANCR mock transfectant and miR-145-5p transfectant.The cells were divided into control group,BANCR transfection group,and miR-145-5p transfection group.Luciferase reporter gene assay was used to detect the targeting effect of BANCR and miR-145-5p.The glucose consumption and lactic acid production of cells were detected by the kit and spectrophotometry respectively.Seahorse Bioscience XF24 extracellular flux analyzer was used to measure mitochondrial oxygen consumption rate and extracellular acidification rate.The protein expression of p-AKT,GLUT1,HK2 and LDHA was analyzed by Western Blot.The cell proliferation was detected by CCK-8kit.Transwell analysis was used to detect cell migration and invasion.Results: after the co-transfection of wild-type BANCR 3’-UTR and miR-145-5p mimics,the luciferase intensity decreased(P<0.05).The luciferase of mutant BANCR 3’-UTR did not change(P>0.05).Compared with the miR-145-5p transfection group and the control group,the BANCR transfection group had higher glucose consumption and lactate production(P<0.05),and the miR-145-5p transfection group had lower glucose consumption and lactate production than the control group(P<0.05).The ECAR and OCR of the BANCR transfection group were higher than those of the control group and miR-145-5p transfection group(P<0.05),and the ECAR and OCR of the miR-145-5p transfection group were lower than the control group(P<0.05).The protein expression of p-AKT,GLUT1,HK2 and LDHA in the BANCR transfection group was higher than that in the control group and miR-145-5p transfection group(P<0.05).The miR-145-5p transfection group was p-The protein expression of AKT,GLUT1,HK2 and LDHA decreased(P<0.05).There was no difference in cell proliferation on day 1(P>0.05).On day 2 to 5,cell proliferation in BANCR transfection group was higher than that in control group and miR-145-5p transfection group(P<0.05),miR-145-The cell proliferation of the 5p transfection group was lower than that of the control group(P<0.05).Compared with the control group and miR-145-5p transfection group,the number of cell migration and invasion increased in the BANCR transfection group(P<0.05),and the number of cell migration and invasion decreased in the miR-145-5p transfection group compared with the control group(P<0.05).Compared with the control group and the miR-145-5p transfection group,the BANCR transfection group had higher IGF-I,HIF-1α and PDK-1 protein expressions(P<0.05).The miR-145-5p transfection group was higher than the control group IGF-I,HIF-1α and PDK-1 protein expression decreased(P<0.05).Conclusion: BANCR-miR-145-5p acts as the regulating axis of glucose energy metabolism in gastric cancer cells,reduces BANCR or promotes the transcriptional expression of miR-145-5p gene,inhibits glycolysis pathway,and reduces the occurrence of tumor stemness in gastric cancer cells.Part Four Validation test of BANCR/miR-145-5p axis regulating the glucose metabolism and tumor stemness of gastric cancercells by interfering with the expression of Reg3 A and DMBT1Objective: To investigate the validation test of BANCR/miR-145-5p axis regulating the glucose metabolism and tumor stemness of gastric cancer cells by interfering with the expression of Reg3 A and DMBT1.Methods: The gastric cancer cell line MKN-45 was obtained from the Beiner Collection Center.The cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum,and male BALB/c nude mice(4weeks old)were used for tumor xenotransplantation experiments.According to the experimental requirements,the cells were divided into a control group,a BANCR transfection group,and a miR-145-5p transfection group.Tumor xenotransplanted mice were divided into A group,B group and C group according to the different injection of transfected MKN-45 cells.The m RNA expression of Reg3 A and DMBT1 was analyzed by RT-PCR.The dual luciferase reporter gene determines the target relationship between BANCR and miR-145-5p.The glucose concentration at 48 h and 72 h was detected by an automatic biochemical analyzer.The lactic acid determination kit and pyruvate determination kit were used to detect cell lactic acid and pyruvate and their concentration.The migration and invasion ability of each group of cells was analyzed by transwell.Cell viability and apoptosis were detected by CCK-8 and flow cytometry.The expression of apoptotic protein caspase-3 and PARP1 was analyzed by Western blot.MKN-45 cells were transfected by tumor injection,a subcutaneous xenograft tumor model was established in nude mice,and the tumor volume was measured.Results: The m RNA expression of Reg3 A and DMBT1 in the BANCR transfection group was lower than that in the miR-145-5p transfection group and the control group(P<0.05),and the m RNA expression of Reg3 A and DMBT1 in the miR-145-5p transfection group was higher than that of the control group(P<0.05).miR-145-5p inhibited the luciferase activity in MKN-45 cells transfected with wild-type BANCR 3’-UTR(P<0.05),but did not affect the activity of mutant BANCR 3’-UTR luciferase(P>0.05).Compared with the miR-145-5p transfection group and the control group,the BANCR transfection group had higher glucose consumption and lactate and pyruvate metabolite concentrations(P<0.05).The miR-145-5p transfection group had higher glucose consumption and lactate consumption than the control group.And the concentration of pyruvate metabolites decreased(P<0.05).Compared with the control group and the miR-145-5p transfection group,the BANCR transfection group had more cell migration and invasion,higher cell viability,and lower apoptosis rate(P<0.05).The miR-145-5p transfection group was higher than the control group The number of cell migration and invasion decreased,cell viability decreased,and cell apoptosis rate increased(P<0.05).The expression of caspase-3 and PARP1 protein in the BANCR transfection group was lower than that of the miR-145-5p transfection group and the control group(P<0.05),and the caspase-3 and PARP1 protein expression of the miR-145-5p transfection group was higher than that of the control group(P<0.05).On the 20 th day,the tumor volume of group B was larger than that of group C and A(P<0.05).On day 25 and 30,the tumor volume of group B was larger than that of group C and A(P<0.05).Group C Compared with group A,the tumor volume was smaller(P<0.05).Conclusion: The LBANCRmiR-145-5p axis can interfere with the expression of Reg3A/DMBT1 to regulate the occurrence of gastric cancer cells and the glucose metabolism pathway,thereby affecting the tumor stemness of gastric cancer cells.