The Mechanism By Which Hsa?circ?0069382 Inhibits Gastric Cancer Progression By Regulating The MiR-15a-5p/BTG2 Axis

Objective:In this study,a new circRNA molecule was found by analyzing the early miRNA chip and circRNA chip.Then the mechanism of the molecule regulating the biological behavior of gastric cancer was studied in-depth,and a competitive endogenous RNA was found.The pathway provides new biomarkers for the early diagnosis and treatment of gastric cancer.Methods:1.We obtained 31 differentially expressed miRNAs from miRNA chips of 8 pairs of early gastric cancer tissue and adjacent tissue paired samples and detected the expression of miR-15-5p in gastric cancer cells by q RT-PCR and agarose gel electrophoresis.The expression of miR-15a-5p and its clinical features in gastric cancer were analyzed.2.Subcellular localization of miR-15a-5p was performed through fluorescence in situ hybridization(FISH)experiments.miR-15a-5p inhibitor,miR-15a-5p mimics,and miR-15a-5p lentivirus was used to intervene in the expression level of miR-15a-5p in gastric cancer cells and q RT-PCR was used to verify the intervention effect.Gastric cancer cell proliferation,invasion,migration,apoptosis,cell cycle,colony formation,and xenograft models were also detected by CCK8,transwell assay,flow cytometry,wound healing,colony formation assay,and nude mouse subcutaneous tumorigenesis experiment.3.Four databases were performed to predict the target m RNAs of miR-15a-5p,Venny 2.1.0 online tool was used to get intersections,Kmplot online tool was performed for survival analysis,and Cytoscape software was performed to find hub genes.Then we found that BTG2 was the possible target molecular of miR-15a-5p.The luciferase reporter gene assay detected whether miR-15a-5p had an interaction site with BTG2.Next,q RT-PCR was used to detect the expression of BTG2 in gastric cancer cells and 68 paired gastric cancer tissues,and clinical features of BTG2 were analyzed.The TCGA database validated clinical relevance.The expression of BTG2 and FAK in gastric cancer cells was detected by western blotting and the expression of BTG2 in gastric cancer tissues was detected by immunohistochemistry.Mi R-15a-5p inhibitor and miR-15a-5p mimics were used to intervene miR-15a-5p's expression in gastric cancer cells,respectively.Then q RT-PCR was used to detect the level of BTG2 to analyze the interaction between BTG2 and miR-15a-5p in gastric cancer.4.The low-expressed circRNAs in the circRNA microarray were taken intersected with the upstream circRNAs of miR-15a-5p predicted by the circ Bank database.Their expression in gastric cancer cells was detected by q RT-PCR.Rnahybrid 2.2 online tool was used to predict the detailed information of the active sites of significantly low-expressed circRNAs and miR-15a-5p in gastric cancer cells.Then we obtained hsa?circ?0069382 as the most likely upstream regulatory circRNA of miR-15a-5p.The expression of hsa?circ?0069382 in 68 pairs of tissues of gastric cancer was detected through q RT-PCR,and the clinical correlation was analyzed.RNase R digestion experiments,q RT-PCR,and agarose gel electrophoresis were used to verify the circular structure of hsa?circ?0069382.FISH co-localized hsa?circ?0069382 and miR-15a-5p in gastric cancer cells,and the luciferase reporter gene assay confirmed the binding sites of hsa?circ?0069382 and miR-15a-5p.Gastric cancer cells were transfected with hsa?circ?0069382 overexpression plasmid and hsa?circ?0069382 lentivirus,respectively,and the transfection efficiency was confirmed by q RT-PCR.Then we examined the effect of the change expression of hsa?circ?0069382 on the level of miR-15a-5p,BTG2,and FAK and its effect on gastric cancer cell proliferation,invasion,migration and clone formation.Simultaneous overexpression of hsa?circ?0069382 and miR-15a-5p could detect the influence of miR-15a-5p on the overexpression of hsa?circ?0069382.The upstream transcription factors of hsa?circ?0069382 were predicted by the DB toolkit and Animal TFDB database,and the coding potential of hsa?circ?0069382 was predicted by the IRESite database and ORF finder online tool.Results:1.Expression and clinical correlation of miR-15a-5p in gastric cancer: Mi R-15a-5p was up-regulated in miRNA microarrays of early gastric cancer tissues.QRT-PCR and agarose gel electrophoresis showed that the expression of miR-15a-5p was upregulated in various gastric cancer cells.TCGA database showed that miR-15a-5p was a high level in gastric cancer tissues,and its expression was related to the T stage of gastric cancer.2.The affection of miR-15a-5p on the biological behavior of gastric cancer: FISH demonstrated that the localization of miR-15a-5p was in the cytoplasm of gastric cancer cells,and the low expression of miR-15a-5p inhibited the proliferation,invasion,and migration of gastric cancer cells and promoted gastric cancer cell apoptosis.Mi R-15a-5p overexpression had the opposite effect.In addition,overexpression of miR-15a-5p facilitated the growth of gastric cancer cells in nude mice.3.Mi R-15a-5p exerts its biological function by regulating BTG2/FAK: A series of bioinformatics analyses found that BTG2 was a downstream target gene of miR-15a-5p,and luciferase reporter gene experiments confirmed the existence of binding sites for BTG2 and miR-15a-5p.QRT-PCR and western blotting showed that BTG2 was lowly expressed in gastric cancer cells and tissues,and FAK was lowly expressed in gastric cancer cells.Low expression of miR-15a-5p upregulated the level of BTG2 in gastric cancer cells,and its high expression had the opposite effect.4.Hsa?circ?0069382 is an upstream regulatory molecule of miR-15a-5p/BTG2/FAK: Bioinformatics prediction combined with q RT-PCR validation indicated that hsa?circ?0069382 was the most likely upstream regulatory circRNA of miR-15a-5p.The detection of 68 paired gastric cancer tissues by q RT-PCR indicated that hsa?circ?0069382 was lowly expressed in gastric cancer tissues.RNase R digestion experiments,q RT-PCR,and agarose gel electrophoresis confirmed the circular structure of hsa?circ?0069382,and the q RT-PCR results of Oligo d T and Random primers further showed that hsa?circ?0069382 had no poly A-tail.FISH experiments showed that hsa?circ?0069382 and miR-15a-5p were co-localized in the cytoplasm of gastric cancer,and the luciferase reporter gene experiments confirmed that they have sites of action.Overexpression of hsa?circ?0069382 inhibited gastric cancer cells proliferation,invasion,migration,and colony formation by up-regulating the expression of BTG2 and down-regulating the expression of FAK.Overexpression of miR-15a-5p partially inhibited the effects caused by hsa?circ?0069382.Overexpression of hsa?circ?0069382 inhibited the proliferation of gastric cancer cells in nude mice.DB toolkit and Animal TFDB database were performed to predict 58 possible upstream transcription factors of hsa?circ?0069382.The IRESite database and ORF finder online tool found that hsa?circ?0069382 had 19 IRESs and 2 ORFs,suggesting that hsa?circ?0069382 may have coding potential.Conclusions:1.hsa?circ?0069382 is a new circRNA involved in gastric cancer progression.2.The molecular mechanism by which hsa?circ?0069382 regulates the expression of downstream BTG2 and FAK through binding to miR-15a-5p and thus participates in gastric cancer progression is described.