Circular RNA CircEMB Promotes Malignant Behavior And Methotrexate Resistance Of Osteosarcoma Through MiR-3184-5p/EGFR Axis

Osteosarcoma（OSA）is the most common malignant bone tumor,which often occurs in the metaphyses of the extremities,such as the distal femur and proximal tibia.Due to its high degree of invasiveness,approximately 10%to 15%of newly diagnosed metastatic diseases appear,which has reduced the five-year survival rate from 60%to 20%,greatly affecting the clinical prognosis of patients.At present,the treatment methods include surgery and neoadjuvant chemotherapy,but the survival rate of metastatic patients and chemotherapy resistance is still relatively low.Many recent articles report that the genesis and prognosis of OSA are closely associated with gene variations.As a result,the understanding of the molecular mechanisms related to the genesis and development of OSA,as well as identification of the specific targets to create management strategies,is of great importance.circular RNAs（circRNAs）,as a new type of endogenous non-coding RNA,form a continuous covalent closed loop structure through reverse splicing of precursor mRNA（pre-mRNA）transcripts,without 5’-cap structure and 3’-poly A tail.circRNA is highly stable and conserved because it is not affected by exonucases.Therefore,circRNA has the potential to be used as a new biomarker for disease.Recent studies indicate that circRNA is involved in the occurrence and development of OSA by regulating the proliferation,apoptosis,metabolism,invasion,metastasis and drug resistance of tumor cells.Therefore,interpreting the function of circRNA is particularly important for understanding the molecular mechanism of OSA and addressing chemotherapy resistance of OSA.In this study,we identified a novel circRNA named circEMB（circBase ID:hsa_circ₀01310）through high-throughput sequencing analysis of OSA patient tissues.circEMB is formed by splicing from the second exon to the eighth exon of the parent gene EMB.Our study found that the expression of circEMB in circEMB was highly expressed in OSA patient tissues compared with normal tissues.More importantly,the high expression of circEMB is related to the proliferation,apoptosis,invasion and metastasis of OSA cells.Mechanistically,circEMB can affect the expression of miR-3184-5p on EGFR by regulating sponge adsorption,thus achieving the effect of influencing tumor occurrence and development.More importantly,we found that the circEMB/miR-3184-5p/EGFR axis regulates the resistance of methotrexate.Methods1.Biological characteristics and clinical expression characteristics of circEMB were identified;（1）The target circRNA were screened by tissue sequencing analysis and tissue qRT-PCR analysis and the expression level of circrnas in tissues was detected;（2）Identification of biological characteristics of circEMB;（3）Fluorescence in situ hybridization and nucleo-cytoplasmic separation were used to identify the cell localization of circEMB.（4）To identify the relationship between circEMB and clinical characteristics of cancer patients;（5）CCK8 cell proliferation assay,plate cloning assay,flow cytometry（apoptosis and cell cycle）,Transwell（migration and invasion）and plate scratch assay were used to detect the biological functions of circEMB.2.CircEMB can play a role through mir-3184-5p（1）RIP,RNA poll down,luciferase binding assay and bioinformatics analysis proved that circEMB could bind Mir-3184-5p.（2）The relationship between the expression of mir-3184-5p and the clinical characteristics of patients with osteosarcoma;（3）CCK8 cell proliferation assay,plate cloning assay,flow cytometry（apoptosis and cell cycle）and Transwell（migration and invasion）detection of biological functions of circEMB/mir-3184-5p axis3.CircEMB/mir-3184-5p/EGFR axis can influence the malignant behavior of osteosarcoma and chemotherapy resistance of methotrexate（1）Transcriptome sequencing analysis,qRT-PCR and double luciferase gene reporting experiments confirmed the existence of circEMB/mir-3184-5p/EGFR axis;（2）A series of in vitro and in vivo rescue experiments demonstrated that circEMB could affect the malignant behavior of osteosarcoma.Results1.CircEMB as an oncogene of osteosarcoma is associated with the malignant behavior of osteosarcoma;2.CircEMB affects the proliferation,invasion and metastasis of osteosarcoma cells through mir-3184-5p;3.CircEMB/mir-3184-5p/EGFR axis plays a role in promoting tumor development.