| BackgroundGastric cancer is a common malignant tumor of the digestive system,which ranks fifth in incidence and third in mortality worldwide,and in China,it ranks second in incidence and mortality,with a very serious disease burden.Abnormally expressed lncRNAs in gastric cancer are involved in various aspects of the malignant biological behavior of gastric cancer cells,including proliferation,migration,invasion,and metastasis,and are closely related to the clinicopathological characteristics and prognosis of patients.m6A modifications,the most abundant type of modifications in human RNA,are also present in lncRNAs,which can affect the processing,transport,and degradation of lncRNAs,as well as their interaction with DNA,RNA,and protein.Aerobic glycolysis is one of the important features that distinguish malignant tumors from normal tissue and is likewise a critical factor in the proliferation,invasion,and metastasis of tumor cells.We found through our preliminary study that lncRNA OIP5-AS1 was aberrantly highly expressed in gastric cancer and involved in proliferation,apoptosis,and cell cycle regulation in gastric cancer.Further bioinformatics analysis and literature search indicated that m6A modification may exist in OIP5-AS1 and is closely related to tumor aerobic glycolysis.Therefore,this study aimed to investigate the effects of m6A-modified OIP5-AS1 on the proliferation,migration,invasion,and glycolysis of gastric cancer and its mechanism of action.Methods1.The expression of OIP5-AS1 in pan-cancer and gastric cancer was analyzed by bioinformatic means.Human normal gastric epithelial cells GES-1 were cultured with four human gastric cancer cells:MGC-803,BGC-823,SGC-7901,and AGS.90 gastric cancer tissue specimens and 90 paraneoplastic normal gastric tissue specimens were collected.The expression of OIP5-AS1 in the cells and tissue specimens was detected by qRT-PCR,and the relationship between OIP5-AS1 expression and the clinicopathological characteristics and prognosis of the patients was analyzed.The subcellular localization of OIP5-AS1 was investigated by fluorescence in situ hybridization and nucleoplasmic separation assay.2.Construct OIP5-AS1 stable knockdown and overexpression gastric cancer cell lines.The effect of OIP5-AS1 on the proliferation ability of gastric cancer cells in vitro was observed by CCK-8 assay,EdU assay,and clone formation assay.The effects of OIP5-AS1 on the migration and invasion ability of gastric cancer cells in vitro were observed by cell scratch healing assay and Transwell invasion assay.The effects of OIP5-AS1 on the expression levels of cell proliferation-related proteins and EMT-related proteins in gastric cancer cells were observed by Western Blot assay.The effects of OIP5-AS1 on the glycolysis of gastric cancer cells were observed by the assay of cellular glucose uptake,lactate production,extracellular acidification rate(ECAR),and oxygen consumption rate(OCR).The effect of OIP5-AS1 on the proliferation,invasion,and metastasis of gastric cancer cells in vivo was observed by the mouse PDX model and mouse lung metastasis model.3.To predict the presence of m6A modification sites in OIP5-AS1 and the possible m6A methyl-binding proteins bound to it by bioinformatics.The interaction between OIP5-AS1 and m6A methyl-binding protein IGF2BP3 was examined by RNA Pull-Down and RIP assays.Detection of m6A modifications in OIP5-AS1 by MeRIP assay.The m6A binding site was mutated and RNA Pull-Down assay was performed to verify whether IGF2BP3 protein binds to OIP5-AS1 through the m6A site.Down-regulation of IGF2BP3 and METTL3 protein expression by siRNA transfection.RNA stability assays were performed using actinomycin D intervention to observe the effect of IGF2BP3 protein on OIP5-AS1 stability.The knockdown of IGF2BP3 and overexpression of OIP5-AS1 were combined with a series of cell proliferation,migration,invasion,and glycolysis experiments to verify whether IGF2BP3 protein regulates the proliferation,migration,invasion,and glycolysis of gastric cancer cells through OIP5-AS1 by Rescue.4.To predict whether hnRNPA1 protein is the RNA binding protein of OIP5-AS1 by bioinformatic means.The interaction between OIP5-AS1 and hnRNPA1 protein was observed by RNA Pull-Down and RIP experiments.The specific sequence of OIP5-AS1 binding to hnRNPA1 protein was observed by truncation experiments.The effect of OIP5-AS1 expression levels on hnRNPA1 mRNA and protein expression levels was observed using qRT-PCR and Western Blot assays.Protein stability assays were performed to observe the effect of OIP5-AS1 on hnRNPA1 protein stability using protein synthesis inhibitor cycloheximide intervention.The effect of OIP5-AS1 on the degradation of hnRNPA1 protein via the proteasome pathway was observed using the proteasome inhibitor MG 132 intervention.The effect of OIP5-AS1 on the ubiquitination level of hnRNPA1 protein was observed by protein immunoprecipitation assay(IP)and the assay of ubiquitination level.The effect of OIP5-AS1 on the expression levels of PKM1 and PKM2 proteins was observed by Western Blot assay.Predict whether Trim21 is the E3 ubiquitin ligase of hnRNPA1 protein by bioinformatic means.The expression levels of Trim21 and hnRNPA1 proteins in normal gastric epithelial cells and each gastric cancer cell were observed by Western Blot assay.The expression of hnRNPA1 and Trim21 proteins were up-regulated by plasmid transfection.The effect of Trim21 protein expression level on OIP5-AS1 expression level was observed by qRT-PCR assay.The effect of Trim21 protein expression level on hnRNPA1 protein expression level was observed by Western Blot assay.The mutual binding of hnRNPA1 and Trim21 protein was observed by Co-IP assay,and the effect of the expression level of OIP5-AS1 on their two binding ability was also observed.The knockdown of OIP5-AS1 and overexpression of hnRNPA1 were then combined with a series of cell proliferation,migration,invasion,and glycolysis assays for Rescue verification to observe whether OIP5-AS1 regulates the proliferation,migration,invasion,and glycolysis of gastric cancer cells through hnRNPA1 protein.Results1.Bioinformatics analysis revealed that OIP5-AS1 was aberrantly highly expressed in gastric cancer,testicular cancer,thymic carcinoma,and diffuse large B-cell lymphoma.The qRT-PCR assay revealed that OIP5-AS1 was highly expressed in gastric cancer cells and tissues.the expression level of OIP5-AS1 was strongly correlated with the depth of tumor infiltration,distant metastasis,and TNM stage,but not with the patient’s age,gender,lymph node metastasis,nerve infiltration,vascular invasion,site of the tumor,degree of differentiation and pathological type.Prognostic analysis showed that high expression of OIP5-AS1 was strongly associated with shorter survival time of patients.Fluorescence in situ hybridization and nucleoplasmic separation experiments revealed that OIP5-AS1 was mainly distributed in the cytoplasm of gastric cancer cells.2.After the successful construction of OIP5-AS1 stable knockdown and overexpression gastric cancer cell lines.OIP5-AS1 was found to promote the proliferation ability of gastric cancer cells in vitro by CCK-8 assay,EdU assay,and clone formation assay.OIP5-AS1 was found to promote the migration and invasion ability of gastric cancer cells in vitro by cell scratch healing assay and Transwell invasion assay.By Western Blot assay,OIP5-AS1 was found to increase the expression of pro-cell proliferation-related proteins and pro-EMT-related proteins.OIP5-AS1 was found to promote glycolysis in gastric cancer cells by the assay of cellular glucose uptake,lactate production,ECAR,and OCR.OIP5-AS1 was found to promote the proliferation,invasion,and metastasis of gastric cancer cells in vivo by the mouse PDX model and mouse lung metastasis model.3.Bioinformatic analysis revealed that OIP5-AS1 has m6A modification and binds to m6A methyl-binding protein IGF2BP3.RNA Pull-Down and RIP experiments confirmed that OIP5-AS1 binds to IGF2BP3 protein.RNA Pull-Down experiments after mutation of the m6A binding site confirmed that IGF2BP3 protein binds to OIP5-AS1 through the m6A modification.siRNA transfection successfully down-regulated the expression levels of IGF2BP3 and METTL3 proteins,and subsequent RNA stability assays confirmed that IGF2BP3 protein increased the stability of OIP5-AS1.Combined knockdown of IGF2BP3 and overexpression of OIP5-AS1 were validated by a series of cell proliferation,migration,invasion,and glycolysis assays in Rescue,which confirmed that m6A methyl-binding protein IGF2BP3 increased the stability of OIP5-AS1 in an m6A-dependent manner,thereby promoting the proliferation,migration,invasion,and glycolysis of gastric cancer cells.4.Bioinformatics analysis revealed that hnRNPA1 protein is an RNA-binding protein of OIP5-AS1.RNA Pull-Down and RIP experiments confirmed the mutual binding between OIP5-AS1 and hnRNPA1 protein.qRT-PCR and Western Blot assays showed that the expression level of OIP5-AS1 had no effect on the expression level of hnRNPA1 mRNA expression level,but affected its protein expression level.Further protein stability assays and intervention with the proteasome inhibitor MG 132 confirmed that OIP5-AS1 could increase the stability of hnRNPA1 protein and hinder its degradation via the proteasome pathway.OIP5-AS1 could up-regulate PKM2 protein expression while down-regulating PKM1 protein expression.Bioinformatics analysis revealed that Trim21 was the E3 ubiquitin ligase of hnRNPA1 protein,and Western Blot revealed that Trim21 protein expression was up-regulated and hnRNPA1 protein expression was down-regulated in normal gastric epithelial cells,while Trim21 protein expression was down-regulated and hnRNPA1 protein expression was up-regulated in each gastric cancer cell.Plasmid transfection successfully upregulated the expression levels of hnRNPA1 and Trim21 proteins.qRT-PCR assay showed that Trim21 protein expression level had no effect on OIP5-AS1 expression level.Western blot assay revealed that upregulation of Trim21 protein expression level would lead to downregulation of hnRNPA1 protein expression level.Co-IP assay confirmed that hnRNPA1 and Trim21 protein can bind to each other,and the upregulation of OIP5-AS1 expression level will hinder their binding ability.The combined knockdown of OIP5-AS1 and overexpression of hnRNPA1 was verified by a series of cell proliferation,migration,invasion,and glycolysis assays in Rescue,which confirmed that OIP5-AS1 inhibited the ubiquitination and degradation of hnRNPA1 protein by blocking the binding of ubiquitin ligase Trim21 to hnRNPA1 protein,thus increasing the stability of hnRNPA1 protein stability,and in turn hnRNPA1 protein upregulated PKM2 protein expression,promoting proliferation,migration,invasion,and glycolysis of gastric cancer cells.Conclusion1.OIP5-AS1 is mainly distributed in the cytoplasm,and it is highly expressed in gastric cancer cells and tissues.High expression of OIP5-AS1 is closely associated with poor clinicopathological characteristics and shortened survival time of gastric cancer patients.2.OIP5-AS1 promotes the proliferation,migration,invasion,metastasis and glycolysis of gastric cancer cells.Glycolysis is closely related to the proliferation,migration and invasion of gastric cancer cells.3.m6A methyl-binding protein IGF2BP3 increases the stability of OIP5-AS1 in an m6A-dependent manner and promotes the proliferation,migration,invasion and glycolysis of gastric cancer cells.4.OIP5-AS1 can interact with RNA binding protein hnRNPA1 and inhibit the ubiquitination and degradation of hnRNPA1 protein by blocking the binding of ubiquitin ligase Trim21 to hnRNPA1 protein,thus increasing the stability of hnRNPA1 protein,which in turn upregulates the expression of PKM2 protein and promotes gastric cancer cell proliferation,migration,invasion and glycolysis. |
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