| Objectives: The most common bone malignancy in adolescents is osteosarcoma(OS)and the incidence is about 0.0004% per year worldwide.The occurrence of OS is associated with abnormal expression of many micro RNAs(mi RNAs).Exosomal mi RNAs get much more attentions in intracellular communications.Tumor-derived exosomes(Exos)promote the growth and angiogenesis of tumor cells.mi R-1307 has been studied in many cancers,but its effects in OS has not been studied.The purpose of this study is to test the effect of OS derived exosomal mi R-1307 on proliferation,migration,invasion and apoptosis of OS cells.Methods: Purchased human OS cell lines(143B,HOS,U2 OS,Saos-2 and SW1353)and human normal osteoblastic cell line(h FOB 1.19).SW1353 OS cells and h FOB 1.19 normal osteoblasts were cultured,exosomes of osteosarcoma(OS-Exos)and normal osteoblastic Exos(h FOB 1.19-Exos)were extracted by differential centrifugation.TEM and NTA were detected to verify whether the isolated extracellular particles were Exos.CCK-8 was used to observe the cell proliferation of OS cells at different times(0h,24 h,48h and 72h)after Exos were added.Flow Cytometry was used to assay the apoptosis of OS cells.Transwell was used to assay the migration and invasion ability of OS cells.The expression level of mi R-1307 in 5different OS cell lines,h FOB 1.19 normal osteoblastic cell line and OS-Exos were detected by q RT-PCR.Secondly,mi R-1307 in OS-Exos was down-regulated,and the effects of OSExos on proliferation,migration,invasion and apoptosis of OS cells were observed.Used Targetscan,mi RBase and mi RDIP databases to predict the target genes of mi R-1307 might be AGAP1.Used q RT-PCR and Western blot to assay the effects of overexpression of mi R-1307 on m RNA and protein expression levels of AGAP1 and the m RNA and protein expression levels of AGAP1 in OS cells.Luciferase report gene technology was used to further determine whether mi R-1307 was bound the 3'-UTR of AGAP1.Finally,the effects of OS cells transfected with mi R-NC mimics,mi R-1307 mimics or mi R-1307 mimics+AGAP1on proliferation,migration,invasion and apoptosis levels of OS cells were assayed.Results: 1.The shape of extracellular particle was round vesicle and bilayer membrane by Transmission Electron Microscope(TEM).Nanoparticle Tracking Analysis(NTA)suggested that the diameter of extracellular particles,with a size of 30-150 nm.The results indicated that the extracellular particles were exosomes.2.The results of CCK-8 assay showed that OSExos significantly promoted the proliferation of OS cells.Transwell results showed that the OS-Exos significantly promoted the migration and invasion of OS cells compared with the control group.The results of flow cytometry showed that the apoptotic cells were significantly reduced of OS cells treated with OS-Exos compared with the control group.3.The results of q RT-PCR suggested that the expression levels of mi R-1307 in five OS cell lines and OS-Exos were significantly higher than control group.OS-Exos were extracted after OS cells were cultured and transfected with mi R-1307 inhibitor.Compared with control group,the level of mi R-1307 in OS-Exos was significantly decreased after transfection with mi R-1307 inhibitor.After transfection with mi R-1307 inhibitor,the level of migration,invasion and proliferation of OS cells was significantly decreased,however the level of apoptosis was increased.4.Online predictive analysis of Targetscan,mi RDB and mi RDIP databases found that one of the 3 '-UTR sequence of AGAP1 was complementary to mi R-1307,and the combination score was the highest among the predicted targets.5.After transfection with mi R-1307 mimics in OS cells,the m RNA and protein levels of AGAP1 were significantly down-regulated.The m RNA expression level of AGAP1 in five OS cell lines were all significantly lower than h FOB1.19 cell line.The protein level of AGAP1 in SW1353 OS cells was significantly lower than control group,suggested that AGAP1 might be a potential therapeutic target for OS.6.Luciferase assay results showed that mi R-1307 significantly inhibited the activity of wild-type PGLO?AGAP1-WT 3'-UTR.However,mi R-1307 not affacted the luciferase activity in the mutant group of PGLO?AGAP1-MUT 3'-UTR.The above data indicated that mi R-1307 directly bound the 3'-UTR of AGAP1,thereby inhibiting the m RNA and protein levels of AGAP1.7.Compared with normal control,the proliferation,migration and invasion of OS cells were significantly enhanced after transfection with mi R-1307 mimics in OS cells and the level of apoptosis was significantly inhibited,however,the regulation of mi R-1307 on OS cells was significantly inhibited after supplementation with AGAP1.Conclusions: 1.OS-Exos significantly promoted the proliferation,migration,invasion and inhibited the apoptosis of OS cells.2.mi R-1307 was highly expressed in OS cells and OSExos.After mi R-1307 was down-regulated in OS-Exos,the effects of OS-Exos on proliferation,migration,invasion and apoptosis of OS cells were significantly reduced.3.mi R-1307 directly bound the 3'-UTR of AGAP1,thereby inhibiting the m RNA and protein levels of AGAP1.4.AGAP1 was significantly underexpressed in OS cells.5.mi R-1307 could significantly promote the proliferation,migration,invasion and inhibit the apoptosis of OS cells,while overexpression of AGAP1 could inhibit the above regulatory effects of mi R-1307,which could be used as a potential therapeutic target for OS.In summary,OS derived exosomal mi R-1307 promotes the proliferation,migration,invasion and inhibits the apoptosis of OS cells via targeting AGAP1. |
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