Exosomal mir-1307 of osteosarcoma and the proliferation and apoptosis of osteosarcoma cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Studies have shown that the occurrence of osteosarcoma is associated with abnormal expression of various microRNAs (miRNAs). Exosomes (Exos) containing miRNAs get much more attentions in intracellular communications. OBJECTIVE: To investigate the effects of exosomal miR-1307 from osteosarcoma on proliferation and apoptosis of osteosarcoma cells and its mechanism. METHODS: Human osteosarcoma cell lines (143B, MG63, U2OS, Saos-2 and SW1353) and human normal osteoblastic cell line (hFOB 1.19) were purchased. The expression level of miR-1307 in five kinds of osteosarcoma cell lines and normal osteoblastic cell line was detected by qRT-PCR. Finally, SW1353 cell line was selected for functional verification of osteosarcoma cells. Osteosarcoma cells and normal osteoblasts were cultured. Osteosarcoma exosomes and normal osteoblastic exosomes were extracted by differential centrifugation. Then miR-1307 inhibitor and miR-NC mimic were transfected into SW1353 osteosarcoma cells and normal osteoblasts, and then SW1353 osteosarcoma cell exosomes and normal osteoblast exosomes were extracted. The above exosomes (25 mg/L) were added to SW1353 osteosarcoma cells to intervene for 24 hours. Cell proliferation assay and flow cytometry were used to observe the proliferation and apoptosis of osteosarcoma cells. Targetscan, miRBase and miRDIP databases were used to predict the target genes of miR-1307. The activity of luciferin was assayed by luciferase reporter gene. mRNA and protein expression levels of AGAP1 were assayed by qRT-PCR and western blot assay in osteosarcoma cells. RESULTS AND CONCLUSION: (1) Compared with hFOB 1.19-exosomes of osteoblasts, SW1353 osteosarcoma cell exosomes significantly promoted the proliferation and inhibited the apoptosis of osteosarcoma cells (P < 0.01). Compared with SW1353 osteosarcoma cell exosomes, the level of miR-1307 in SW1353 osteosarcoma cell exosomes was down-regulated; the proliferation of osteosarcoma cells was significantly decreased but the apoptosis rate was significantly increased (P < 0.01). (2) AGAP1 was verified as a direct target gene of miR-1307. Compared with miR-NC, miR-1307 significantly inhibited the mRNA and protein levels of AGAP1 (P < 0.01). Compared with miR-NC, miR-1307 significantly inhibited the luciferase activity of wild-type PGLO-AGAP1-WT 3'UTR (P < 0.05). (3) The results show that exosomal miR-1307 of osteosarcoma promoted the proliferation and inhibited the apoptosis of osteosarcoma cells via targeting AGAP1.