Study On Exosomal MiRNA744-5P In Osteosarcoma Metastasis

Objective Osteosarcoma is one of the most common malignant bone tumors.Its prognosis is poor due to its metastasis.Recent studies have shown that overexpression or inhibition of miRNAs in exosome could regulate tumor cell metastasis.Thus,the main purpose of this study is to explore a mechanism of miRNA744-5P in osteosarcoma cell metastasis.We also aim to explore a correlation between miRNA744-5P and tumor metastasis.Methods Part ?: Four osteosarcoma cell lines(high metastatic U20 S,SAOS2,low metastatic MG63,HOS)were cultured in vitro.High and low-metastatic osteosarcoma cell lines were cultured for 48 hours,and then supernatant has been collected.Thereafter,total exosomes were separated from supernatants by ultrahigh-speed centrifugation.Purified exosomes were sent out for high-throughput sequencing and real-time quantitative analysis.Part ?: Tumor cell lines were transfected by miRNA744-5P inhibitor.Then in comparison to miRNA744-5P inhibitor NC highly metastatic potential has been tested.After that Transwell migration and wound healing were investigated to evaluate the miRNA744-5P effect on the metastatic activity.Part ?: We have collected serum samples from 6 patients who were diagnosed with osteosarcoma and 8 healthy volunteers.Total Exosome Isolation kit was used to extract exosomes from the serum of patients with osteosarcoma,and then RNA exogenously extracted by Trizol method.Total exosomal RNA was used to quantitatively assess exosomal miRNA744-5P expression in the serum of osteosarcoma patients and healthy volunteers.Result Part ?: According to high-throughput sequencing results and relative gene expression,miRNA744-5P expression in the different metastatic osteosarcoma cell lines showed significant variations.The expression of miRNA744-5P in high metastatic cell line was significantly higher than low metastatic cells.Part ?: The inhibition of miRNA744-5P in the metastatic cell line SAOS2 and U2 OS showed a significant association with cell number.We also found that transfected U2 OS and SAOS2 cells have a slower rate of scratch healing after miRNA744-5P inhibitor transfection.Part ?: According to real-time quantitative comparison of miRNA744-5P in serum exosomes of osteosarcoma patients and healthy volunteers,miRNA744-5P in serum of patients with osteosarcoma was higher than that of healthy volunteers.Conclusion : miRNA744-5P has significant expression differences between different metastatic osteosarcoma cells.Decreasing the expression of miRNA744-5P in osteosarcoma cells significantly inhibits the migration of osteosarcoma cells.Preliminary results showed that miRNA744-5P is significantly higher in the serum of patients with osteosarcoma than in healthy.