The Role And Mechanism Of Circ＿0001852 In Gastric Cancer

Background and Objective:Gastric cancer(GC)is one of the most common and deadly malignancies worldwide and is the fourth leading cause of cancer-related deaths across the globe.Despite advances in the diagnosis and systemic treatment of gastric cancer over the past several decades,there have been no breakthroughs and the overall prognosis for patients with gastric cancer is poor,with five-year survival rates remaining low,primarily due to the high incidence of metastasis and recurrence of gastric cancer.However,our current understanding of the molecular mechanisms associated with gastric cancer metastasis remains minimal.Circular RNAs(circ RNAs)are closed-loop RNA molecules present in eukaryotic organisms,the vast majority of which do not encode proteins,and can affect various aspects of cellular homeostasis,including proliferation,migration,invasiveness or genomic stability.Increasingly,studies have shown that circRNAs may play a role in oncogene or oncogene stability in malignant tumors,and circRNAs may be potential biomarkers and targets for early diagnosis of tumors,assessment of prognosis,treatment and reduction of chemotherapy resistance.In this study,we screened the differential gene circ＿0001852 based on high-throughput sequencing results,and focused on the expression of circ＿0001852 in gastric cancer and its correlation with survival,prognosis and clinical staging of gastric cancer patients,as well as the biological functions and regulatory mechanisms of the involved networks,to explore the potential therapeutic targets and biological markers and provide theoretical basis for gastric cancer treatment.Methods:1.In 3 pairs of gastric and paracancerous tissues,high-throughput sequencing and differential gene screening were performed,and qRT-PCR was used to verify the expression of circ RNA in gastric cancer samples and cells;divergent primers and convergent primers were designed for amplification of circ RNA cyclization sites in gDNA PCR amplification was performed in gDNA and cDNA,and the amplified products were separated by gel electrophoresis and sent to sanger for sequencing to verify the presence of circ＿0001852;the gastric cancer cell lines were treated with RNase R and putrescine D to analyze the stability and half-life of circ＿0001852;fluorescence in situ hybridization(FISH)and nucleoplasmic separation were used to demonstrate the specificity of circ＿0001852 in the cells.The specific localization of circ＿0001852 in cells was demonstrated by fluorescence in situ hybridization(FISH)and nucleoplasmic separation.2.After stable interference with circ＿0001852 in HGC-27 cells and overexpression in MKN-45 and AGS cells,the interference and overexpression efficiencies were examined by qRT-PCR;cell proliferation capacity was examined using CCK-8,Edu and clonal colony formation assays;changes in the ability of cells to invade and migrate were examined using Transwell assays,and the Western blot assay was used to investigate how the epithelial-mesenchymal transition(EMT)process was affected.In an in vivo experiment,a subcutaneous transplantation tumour model was established in nude mice to assess the effect of interfering with circ＿0001852 expression on the growth and malignancy of gastric cancer cell tumours and the target genes.3Using online bioinformatics analysis tools and RNA-seq high-throughput sequencing results to predict potential miRNAs downstream of circ＿0001852;qRT-PCR was used to further investigate the expression of miR-139-5p and the correlation between circ＿0001852 and miRNAs in clinical gastric cancer tissues.The interaction between circ＿0001852 and miR-139-5p was verified by RNA-pulldown,RIP and FISH assays and luciferase reporter assay;miR-139-5p mimics/NC were cotransfected in AGS cells overexpressing circ＿0001852 and in HGC-27 cells interfering with circ＿0001852.HGC27 cells were co-transfected with miR-139-5p inhibitors/inhibitor NC,and the ability of circ＿0001852/miR-139-5p axis to regulate the proliferation,migration and invasion of gastric cancer cells was verified by CCK-8,plate cloning,EdU assay and transwell assay.4.To predict the expression of target genes in gastric cancer and adjacent normal tissues through the TCGA database,and also to assess their clinical relevance(?)and prognostic potential in gastric cancer patients and their validity as gastric cancer biomarkers.The qRT-PCR method,Western blot method and immunohistochemistry were used to explore the expression of the target genes in clinical gastric cancer tissues and gastric cancer cell lines.The interaction with the target genes was verified by dual luciferase reporter gene assay,qRT-PCR method and/or Western blot method,respectively.Co-transfection of si-CCT5/NC in AGS cells overexpressing circ＿0001852 and cotransfection of oe-CCT5/NC in HGC-27 cells interfering with circ＿0001852 was verified by CCK-8,plate cloning,EdU assay and transwell assay.circ＿0001852/miR-139-5p axis on the regulation of proliferation,migration and invasion of gastric cancer cells.5HGC-27 cell line with stable silencing of circ＿0001852 was selected for high-throughput transcriptome sequencing,and the enrichment of signaling pathways associated with circ＿0001852 expression was analyzed using bioinformatics methods,and the activation level of related signaling pathway proteins was detected by Western blot methodResults:1.The increased expression of circ＿0001852 in gastric cancer tissues and gastric cancer cell lines correlated with the pathological grading and staging of tumours,patient prognosis and survival rates.This suggests that circ＿0001852 may be involved in regulating the proliferation and invasive migration of gastric cancer as an oncogenic factor,and suggests that circ＿0001852 may serve as a clinical and prognostic biomarker for gastric cancer,which may have significant clinical and prognostic implications.2.functional cellular results showed that silencing circ＿0001852 expression in gastric cancer cells inhibited cell proliferation and clone formation while suppressing cell invasion and migration as well as the epithelial-mesenchymal transition(EMT)process.In in vivo experiments,silencing circ＿0001852 expression inhibited the growth and,at the same time,reduced the malignancy of subcutaneous transplanted tumours in nude mice..3.Bioinformatics analysis,RNA-pulldown,RIP and dual luciferase reporter gene confirmed the binding between circ＿0001852 and miR-139-5p.qRT-PCR showed that miR-139-5p was lowly expressed in gastric cancer tissues and gastric cancer cells.circ＿0001852 was significantly correlated with miR-139-5p.The FISH assay also analyzed the cytoplasmic co-localization of miR-139-5p and circ＿0001852;the rescue function assay confirmed that circ＿0001852 could reply to the inhibitory effect of miR-13 9-5p on gastric cancer cell function.4.Bioinformatics and dual luciferase reporter gene assays showed that CCT5 could be a target gene regulating miR-139-5p.qRT-PCR and Western blot confirmed the high expression of CCT5 in gastric cancer tissues and the positive correlation with circ＿0001852;meanwhile,qRT-PCR and Western blot assay showed that miR-1395p might target CCT5 in gastric cancer.rescue assay CCT5 could reverse the procarcinogenic effect of circ＿0001852.5.In the HGC-27 cell line with stable silencing of circ＿0001852 expression,KEGG analysis showed that the differential gene was mainly enriched in the Wnt//β-catenin signaling pathway.western blot analysis showed that circ＿0001852/miR-139-5p could affect CCT5 expression and thus regulate the Wnt//β-catenin pathway.Conclusions:Circ＿0001852 is stably highly expressed in gastric cancer tissues and cell lines,and is mainly localized in the cytoplasm,with a role in promoting the malignant progression of gastric cancer;Circ＿0001852 is an independent risk factor for the prognosis of gastric cancer patients;in vivo and in vitro experiments confirmed that interference or overexpression of circ＿0001852 can affect gastric cancer cell proliferation,migration,invasion and EMT changes.In specific mechanistic studies,it was noted that circ＿0001852 could sponge-like adsorption of miR-139-5p to increase CCT5 expression and promote the progression of gastric cancer malignancy;the above findings point to a new research direction for the exploration of gastric cancer.