Circ-REOS Inhibits Osteosarcoma Via Suppressing HuR-mediated MYC Activation

Objective: Osteosarcoma is highly invasive,and the complications associated with metastasis have a significant impact on patients’ quality of life while also posing a significant burden on the economy and society.As a result,a thorough analysis of the molecular mechanisms of osteosarcoma invasion and metastasis is critical for improving the therapeutic effect of osteosarcoma and patient prognosis.Circular RNA is associated to the occurrence and development of tumors and plays an important role in the malignant process.However,we still know very little about the mechanism of circRNA regulation of osteosarcoma invasion and metastasis,which requires further investigation.The purpose of this study is to investigate the expression of circRNA derived from MYC gene in osteosarcoma,and to detect its effect on the expression of MYC and the invasion and metastasis activity of osteosarcoma,so as to further explore the mechanism of its regulation of MYC expression and the proliferation and invasion of osteosarcoma.Method:1.R2 online analysis platform(http://r2.amc.nl,data source: GSE42352,88 osteosarcoma samples)was used to analyze the relationship between MYC gene expression and survival time and metastasis of osteosarcoma.2.Circ RNA generated by MYC gene was analyzed through circ Base and Circ Interactome databases;The expression of the above circRNA in osteosarcoma cells was verified by reverse transcription PCR and Sanger sequencing,and the expression difference of the above circRNA in osteoblasts and osteosarcoma cells was analyzed by real-time fluorescent quantitative PCR to screen out circ-REOS;The circular structure of circRNA was analyzed by RNase R assay,and the expression position of circRNA in osteosarcoma cells was detected by fluorescence in situ hybridization.3.Using lentivirus to transfect 143 B and MG63 osteosarcoma cell lines,construct circ-REOS over-expression vector.The changes of MYC mRNA and protein in osteosarcoma cells were detected by q RT-PCR and Western blot;MTT and matrix gel invasion test were used to detect the effect of circ-REOS on the proliferation and invasion activity of osteosarcoma cells MG-63 and 143 B.4.The proteins binding with circ-REOS were analyzed by circRNA online database;The interaction between HuR and circ-REOS was verified by RNA binding protein immunoprecipitation test;Construction of circ-REOS over-expression vector,complement HuR,observe the changes of MYC mRNA and protein expression level by q RT-PCR and Western blot;Osteosarcoma cells were treated with actinomycin D,and the abundance of MYC mRNA at different time points was detected by q RT-PCR.5.Construct a stable cell line overexpressing circ-REOS and HuR;The effects of circ-REOS combined with HuR on the proliferation and invasion of osteosarcoma cells were observed by cell cloning and matrix gel invasion experiments.Result:1.The expression of MYC was significantly correlated with the survival and prognosis of sarcoma patients.The mortality and malignant metastasis rate of patients with high expression of MYC were higher(* P<0.05).2.Database analysis showed that MYC gene could transcribe three circRNAs,among which hsa＿circ＿0085533 and hsa＿circ＿0085535 expression could be detected in osteosarcoma cells MG63 and 143 B.hsa＿circ＿0085535 expression was significantly reduced in osteosarcoma cells compared to normal osteoblasts(*P<0.05).The expression of hsa＿circ＿0085535(circ-REOS)was significantly lower in osteosarcoma cells compared with normal osteoblasts(*P<0.05).RNase R assay confirmed the presence of circ-REOS in the nucleus,while RNA-FISH assay suggested that circ-REOS was widely expressed in the nucleus.3.Overexpression of circ-REOS significantly decreased the mRNA and protein expression levels of MYC in osteosarcoma cells(*P<0.05),and the proliferation and invasive activity of osteosarcoma cells were significantly reduced(*P<0.05).4.Circ-REOS can combine with HuR.After overexpression of circ-REOS,supplementing HuR can restore the mRNA and protein expression level of MYC in osteosarcoma cells(* P<0.05).The half-life of MYC mRNA was shortened after overexpression of circ-REOS,and the inhibition was counteracted by supplementation of HuR(* P<0.05).5.Overexpression of circ-REOS and supplementation of HuR and MYC can save the proliferation and invasion of osteosarcoma cells.Conclusion:1.circRNA hsa＿circ＿0085533 and hsa＿circ＿0085535 can be produced by MYC,of which hsa＿circ＿0085535(circ-REOS)is lowly expressed in osteosarcoma cells.2.circ-REOS regulates the expression of parental gene MYC by binding to HuR.3.circ-REOS competitively binds to HuR,decreases the regulatory effect of HuR on MYC mRNA stability,reduces the expression of osteosarcoma MYC gene,and thus inhibits osteosarcoma invasion and metastasis.4.circ-REOS can inhibit the proliferation and invasive ability of osteosarcoma cells,which is expected to provide a new way for the treatment of osteosarcoma.