Effect Of 3’UTR Difference Of Glutaminase On The Expression Of Glutaminase In Gastric Cancer Cells

BackgroundNormal cells are powered by ATP produced by mitochondria through glucose metabolism.Tumor cells,however,regardless of the volume of intracellular oxygen,are more dependent on the glycolytic pathway to provide the bulk of energy for cell division,a phenomenon known as the Warburg effect.Simultaneously,they also depend on mitochondria for metabolism,specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate.Glutaminase(GLS)is a key enzyme in glutamine catabolism that converts glutamine to glutamate,which is further catabolized through the TCA cycle to produce ATP.Its high expression is crucial for the metabolic phenotype of tumors.Alternative polyadenylation(APA)is a prevalent mechanism in the regulation of most human genes,with approximately 70% of human genes possessing multiple poly A binding sites that generate transcript subtypes with different 3’ untranslated region(3’ UTR)lengths and contents.Although the protein-coding sequence(CDS)is unaltered,the removal of regulatory elements in the distal 3’ UTRs that may reduce m RNA stability or impair translation efficiency,such as micro RNA(mi RNA)binding domains,affects gene expression.This study intends to explain the post-transcriptional regulatory mechanisms of genes in tumor cells with respect to metabolism and energy utilization through molecular biology,so as to provide experimental basis for the development of new anticancer therapies based on energy metabolism changes in gastric cancer cells.ObjectivesTo explore the APA occurrence of GLS in different gastric cancer cells and the change of GLS expression caused by it,to explore the correlation between GLS 3’ UTR difference and gastric cancer occurrence,and to evaluate the possibility of APA imbalance and GLS differential expression in gastric cancer cells as diagnostic indicators of gastric cancer,so as to provide corresponding theoretical basis for diagnosis of gastric cancer.Methods1.Gastric cancer-related data were obtained in TCGA database,and the expression of GLS was analyzed in 415 gastric cancer tissues and 34 normal tissues in the database.2.The expression of GLS in different gastric cancer cell lines was detected by RT-q PCR,and the expression of GLS protein in gastric epithelial cell lines and gastric cancer cells was detected by Western blot.3.GLS transcripts in different gastric cancer cell lines were amplified,sequenced and analyzed by 3’RACE.4.Bioinformatic analysis was performed on the binding of different GLS 3’UTR and mi RNA.Then mi RNA-23 that binds to most GLS 3’UTR subtypes and mi RNA-302 that only binds to GLS 3’UTR-1 were selected and synthesized,and were transfected into GES,BGC-803,and AGS cells respectively.And the relative expression of GLS in transfected cells was detected by RT q PCR.5.The regulation of mi RNA-23 on different GLS 3’UTR transcripts was evaluated by double luciferase reporter gene experiment.Results1.Compared with normal tissues,the transcriptional level of GLS in gastric cancer tissues was significantly higher.2.Compared with the gastric epithelial cell lines GES,the expression of GLS was up-regulated in gastric cancer cell lines BGC-803,AGS,and MGC-803.3.GLS transcripts obtained from 3’RACE of four kinds of cells could be divided into seven subtypes from long to short,namely 3’UTR-1-7.The types and proportions of3’UTR in different cells were different.GES was dominated by 3’UTR-1,MGC-803 and BGC-803 were dominated by 3’UTR-5,and AGS was dominated by UTR-6.4.Bioinformatic analysis shows that there were two types of mi RNAs that could bind to GLS 3’UTR.One type was highly specific and could only bind to some specific 3’UTR subtype.For example,mi RNA-302 can only bind to 3’UTR-1,and the other type can bind to multiple subtypes.For example,mi RNA-23 can bind to 3’UTR-1-7.When mi RNA-23 was transfected,the relative expression of GLS in gastric mucosal cells,gastric cancer cells BGC-803 and AGS cells was down-regulated compared with the control group and the mimic NC cells;however,when mi RNA-302 was transfected,only the relative expression of GLS in gastric mucosal cells GES was down-regulated,while the relative expression of GLS in gastric cancer cells BGC-803 and AGS cells did not change much.It suggests that the proportion of the longest GLS transcript in gastric cancer cells was extremely low.5.The results of double luciferase reporter gene showed that the fluorescence intensity and Gluc relative enzyme activity in GLS 3’UTR-1 group were significantly lower than those in NC group(P<0.001);the fluorescence intensity and relative activity of Gluc in GLS transcripts GLS 3’UTR-2,GLS 3’UTR-3,GLS 3’UTR-4,GLS 3’UTR-5,GLS 3’UTR-6,and GLS 3’UTR-7 groups were significantly higher than those in GLS3’UTR-1 group(P<0.01).Conclusions1.GLS was transcribed both in gastric cancer tissues and normal gastric mucosa.The transcription level of gastric cancer tissues was higher than that of normal gastric mucosa.The GLS transcription and translation level of gastric cancer cell lines was higher than that of normal gastric epithelial cells GES.2.GLS 3’UTR of gastric cancer cells and normal gastric mucosal epithelial cells could be divided into 7 subtypes from long to short,and gastric cancer cells were mainly short subtypes.Shortening of the transcript of 3’UTR enhanced the mi RNA regulatory response.