Circular RNA Circ＿001422 Promotes Osteosarcoma Proliferation And Metastasis Via The MiR-195-5p/FGF2/PI3K/Akt Axis

Background and objectiveOsteosarcoma（OS）is the most prevalent primary malignant bone neoplasm causing substantial morbidity in adolescents and children.It is derived from mesenchymal cells and characterized by rapid infiltrating growth,early lung metastasis and high recurrence rate.Studies show that the overall 5-year survival rate of patients with localized OS ranges between 65 and 75%,and is only 20%for those with recurrent and metastatic tumors.Despite advances in OS treatment approaches such as adjuvant chemotherapy and surgical resection,the survival rates have plateaued in the last 4 decades,below satisfactory rates.Indeed,there are no specific biomarkers for the diagnostic and prognostic prediction of OS.Consequently,molecular studies aiming to identify promising targets for the treatment of OS are urgently needed.Circular RNAs（circRNAs）are known to regulate various functions of eukaryotic cells.Based on the order of splicing events and different intermediates,two mechanisms exist for the biogenesis of circRNAs:the canonical spliceosome-induced splicing and noncanonical lariat-typed splicing.Accumulating studies have shown that circRNAs modulate diverse physiological and pathophysiological processes via sponging microRNAs（miRNAs）,interacting with RNA-binding proteins,and modulating the epigenetic,transcriptional,or translational alterations of target genes.Abnormal circRNAs expression has been found to correlate with the pathogenesis of various cancers and exert essential regulatory effects on gene expression,cell invasion,cell cycle,migration,apoptosis,and proliferation.Moreover,circRNAs are thought to possess high diagnostic and therapeutic potential given their structural stability,evolutionary conservation,abundance and organ specificity.However,to date,the roles of circRNAs in OS are not clearly known.This study aims to explore the biological function and mechanism underlying the involvement of circ₀01422 in the proliferation and metastasis of OS cells in vitro and in vivo,which may offer new insight into the identification of potential biomarkers or therapeutic targets for OS treatment.Methods1.Identification and characterization of circ₀01422 in OS1.1 Bioinformatics analysis and high-throughput RNA sequencing tools were employed to determine differentially expressed circRNAs between OS and adjacent healthy tissues.The expression levels of circ₀01422 in clinical specimens and cell lines were measured using qRT-PCR.1.2 RNase R digestion and actinomycin D inhibition assays were used to evaluate the stable structures of circ₀01422.1.3 Nuclear-cytoplasmic fractionation and FISH assays were used to uncover the subcellular localization of circ₀01422.1.4 A total of 55 OS patients were recruited to analyze the association of circ₀01422 expression with clinicopathologic features,including clinical stage,tumor size,distant metastasis,primary tumor location and overall survival rate.2.Effect of circ₀01422 on the biological function of OS cells2.1 EdU incorporation assay,colony formation assay,flow cytometry,Transwell assay,and Western blot were performed to detect the impact of circ₀01422 on the proliferation and metastasis of OS cells in vitro.2.2 The subcutaneous xenograft and metastatic lung models were established to explore the role of circ₀01422 in the proliferation and metastasis of OS cells in vivo.3.The molecular mechanism underlying the promotive effect of circ₀01422 on OS proliferation and metastasis3.1 RNA immunoprecipitation,bioinformatics databases,RNA pull-down assay,mRNA sequencing and dual-luciferase reporter assay were conducted to explore the potential downstream target miRNAs and mRNAs regulated by circ₀01422.3.2 Functional rescue experiments were performed to assess whether the malignant phenotypes of OS cells depends on the circ₀01422/miR-195-5p/FGF2 axis.Results1.Circ₀01422 expression was dramatically higher in OS cell lines and tissues relative to normal samples.In addition,circ₀01422 was resistant to RNase R digestion and displayed a longer half-life than linear transcripts.Nuclear-cytoplasmic fractionation and FISH assays further revealed that circ₀01422 was predominantly located in the cytoplasm.2.Overexpression of circ₀01422 significantly correlated with more advanced clinical stage,larger tumor size,more distant metastases and poorer overall survival of OS patients.3.Loss-and gain-of-function experiments were performed to prove that circ₀01422 not only promoted the proliferation,migration and invasion of OS cells,but also inhibited the cell cycle arrest and apoptosis in vitro.Animal models also showed that circ₀01422 increased the volume and weight of subcutaneous xenograft tumors,and accelerated the formation of metastatic pulmonary colonies in nude mice.4.Circ₀01422 exerted its pro-oncogenic effect on OS proliferation and metastasis via targeting the miR-195-5p/FGF2/PI3K/Akt axis.ConclusionCompetitive interactions between circ₀01422 and miR-195-5p upregulated the expression of FGF2 while also initiating the PI3K/Akt signaling pathway,thereby contributing to proliferative and metastatic abilities of OS cells in vitro and in vivo.Our findings reveal that circ₀01422 is a potential therapeutic target against OS.