| Objective: Osteosarcoma(OS)is a rare mesenchymal tumor and the most common primary bone tumor.It mainly occurs in the metaphysis of the long shaft,especially in the distal femur,proximal tibia and humerus.OS is highly complex and heterogeneous among different tissue types,which makes it very difficult to understand its molecular pathogenesis and identify specific therapeutic molecules.The number of cancer deaths caused by bone tumors accounts for 8.9% of all cancer deaths in children and adolescents.Although the multi-drug chemotherapy strategy has significantly improved the prognosis and survival of patients with non-metastatic osteosarcoma in the past few years,about20% of patients have clinical metastasis at the time of presentation,and the 5-year survival rate is less than 30%.Therefore,it is of great clinical significance to search for good molecular markers for early diagnosis and prognosis evaluation of osteosarcoma and to search for targeted treatment sites.This study mainly discussed the potential role of miR-941 as a new biomarker for diagnosis,prognosis and clinical monitoring of OS patients.Methods: 1.We used the GEO database to query the differentially expressed genes in osteosarcoma and normal paracancerous tissues,so as to screen the target genes studied.2.Fresh osteosarcoma and adjacent normal tissues from 18 patients were collected and the expression of miR-941 was measured by q RT-PCR.3.The relationship between miR-941 and the survival period of patients was determined by clinicopathological factor analysis.4.MTT,EDU and colony formation test were used to detect the effect of miR-941 on the proliferation of osteosarcoma cells,and Transwell and scratch healing test were used to detect the effect of miR-941 on the invasion and metastasis of osteosarcoma cells.5.The effect of miR-941 on the proliferation of osteosarcoma cells was verified by nude mice tumorigenesis experiment in vivo,and the effect of miR-941 on the metastasis of osteosarcoma cells was verified by tail vein injection.6.Use the bioinformatics to predict the genes that miR-941 may regulate,find the possible binding protein CROT and determine the binding site with CROT.We used the double luciferase reporter gene experiment to verify this interaction.7.Fresh osteosarcoma and adjacent normal tissues from 18 patients were collected and the expression of CROT was determined by Western Blot test.8.Collect osteosarcoma cell lines to detect the expression of CROT in osteosarcoma cells.9.We used MTT,EDU,colony formation,Transwell,and scratch healing experiments to detect the biological behavior of CROT after overexpression or silencing.10.GSEA Bioinformatics analysis The mechanism regulated by the biological function of CROT is significantly enriched in NF-κB channel.11.Western Blot experiment verification NF-κB Activation of B pathway.12.Western Blot experiment verification NF-κ B The crosstalk relationship between channel B and m TOR is clear that AKT-m TOR is NF-κB Upstream of access road B.13.Use NF-κB Bay117082 and Rapamycin,inhibitors of B and m TOR pathway,were used for recovery experiment.Results: 1.The expression of miR-941 in osteosarcoma tissue was significantly higher than that in adjacent tissues.2.Clinicopathological factor analysis data showed that the survival period of patients in the high expression group was significantly shorter than that in the low expression group,and its expression was highly correlated with advanced osteosarcoma,but there was no significant correlation with other characteristics such as sex,age or family history.3.miR-941 can promote the proliferation,invasion and metastasis of osteosarcoma cells.4.miR-941 combines with CROT to play biological functions.5.Compared with normal bone tissue,the protein level of CROT is low expressed in osteosarcoma tissue,and it is also low expressed in osteosarcoma cells compared with normal osteoblasts.6.CROT can inhibit the proliferation,invasion and metastasis of osteosarcoma cells.7.miR-941 can regulate the activation of NFKB pathway through CROT.8.The results show that miR-941 regulates the crosstalk relationship between NF-κB and m TOR pathways,and AKT-m TOR is NF-κB Upstream of access road B.9.miR-941 promotes the drug resistance of osteosarcoma cells.Conclusion:MiR-941 activates Akt/NF by down-regulating CROT expression-κB-axis promotes osteosarcoma progression and mediates doxorubicin resistance... |
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