lncRNA THAP7-AS1 affects the glycolysis of gastric cancer cells by regulating METTL3 mediated m6A modification / 肿瘤

ABSTRACT

Objective:To explore the effect of long noncoding RNA(lncRNA)THAP7-AS1 on the glycolysis of gastric cancer(GC)cells by regulating methyltransferase-like 3(METTL3)mediated N6-methyladenosine(m6A)modification. Methods:Gene Expression Profiling Interactive Analysis(GEPIA)database was used to analyze the expression levels of THAP7-AS1 and METTL3 in GC tissues and their relationship with the overall survival of GC patients.Real-time fluorescence quantitative PCR and Western blotting were used to analyze the expression of THAP7-AS1,METTL3 mRNA,glucose transporter 1(GLUT1)mRNA,and METTL3 protein in GC tissues and paracancerous tissues samples collected from 80 GC patients in Department of Oncology,The Third Affiliated Hospital of Zunyi Medical University(the First People's Hospital of Zunyi),and the relationship between THAP7-AS1 levels and the clinicopathological characteristics of GC patients was analyzed.Real-time fluorescence quantitative PCR and Western blotting were used to verify the expression of THAP7-AS1,METTL3 mRNA,GLUT1 mRNA,and METTL3 protein in GES-1,BGC-823 and SGC-7901 cells.Lentiviral infection was used to knock-down THAP7-AS1 or overexpress METTL3 BGC-823 and SGC-7901 cells,and real-time fluorescence quantitative PCR and Western blotting were used to examine effect of different treatment on the expression of THAP7-AS1,METTL3 mRNA,GLUT1 mRNA,and METTL3 protein;colorimetry assay was used to detect the m6A modification level in the total RNA;methylated RNA immunoprecipitation(MeRIP)-quantitative PCR(qPCR)was used to detect the GLUT1 m6A modification level;glycolysis stress test kits were used to detect the extracellular acidification rate(ECAR),glucose uptake and lactate production of treated GC cells;Western blotting was used to examine the expression levels of METTL3,GLUT1,M2 type pyruvate kinase(PKM2)and lactic dehydrogenase(LDHA)proteins in treated GC cells;EdU staining,wound healing assay and Transwell invasion assay were used to evaluate the proliferation,migration and invasion of treated GC cells.Finally,a mouse model of subcutaneously transplanted GC tumor was established using nude mice,and the effect of knocking-down THAP7-AS1 was assessed by measuring the tumor volume and weight,as well as the expression levels of METTL3 and GLUT1 proteins in transplanted GC tumor tissues. Results:Analysis of the GEPIA database showed that the expression levels of THAP7-AS1 and METTL3 was higher in GC tissue than those in normal gastric tissues,and the expression levels of THAP7-AS1 and METTL3 are negatively correlated with overall survival of GC patients(P＜0.05).Compared with the paracancerous tissues(or normal gastric epithelial cells),the expression levels of THAP7-AS1,METTL3 mRNA,GLUT1 mRNA and METTL3 protein was significantly increased in GC tissues(or GC cells),and the higher the expression of THAP7-AS1,the higher the TNM stage,the lower the degree of tumor differentiation,and the easier the occurrence of microvascular infiltration and lymph node metastasis(P＜0.05).Knocking-down of THAP7-AS1 down-regulated the expression levels of METTL3 mRNA,GLUT1 mRNA and METTL3 protein,the m6A modification levels in total RNA and GLUT1,the ECAR levels,the glucose uptake,the lactate production,EdU positive rate,scratch healing rate,the number of invaded cells,and the expression levels of glycolysis-related proteins(METTL3,GLUT1,PKM2 and LDHA)in GC cells(P＜0.05).Overexpression of METTL3 could partially reverse these effects of THAP7-AS1 knock-down(P＜0.05).In vivo experiments showed that THAP7-AS1 knock-down can obviously inhibit the growth of transplanted GC tumors(P＜0.05). Conclusion:lncRNA THAP7-AS1 can promote the glycolysis which further promotes the proliferation,migration and invasion of GC cells by regulating METTL3 mediated m6A modification.