| ObjectiveTo explore the biological effect and possible mechanism of the secretion miR-30a3p,which is differentially expressed by osteoclasts,on osteoblasts in lead-induced osteoporosis,and reveal the communication mechanism of miR-30a-3p between osteoclasts and osteoblasts,so as to further supplement and improve the mechanism of breaking the dynamic balance between fine cells in the process of bone remodeling.Method1.Osteoclast-derived exosomal miR-30a-3p transfers to osteoblast after lead treatmentThe co-culture system of osteoclasts and osteoblasts was constructed.The mouse RAW264.7 cells were selected and induced by 50 ng/mL RANKL and 50 ng/mL MSCF to obtain mature osteoclasts.The success of osteoclast induction was evaluated by TARP staining and TRAP activity determination.Mouse MC3T3-E1 osteoblasts were co-cultured with osteoclasts transfected with fluorescent labeled miR-30a-3p mimic to observe the fluorescence results of lower osteoblasts and detect the expression level of miR-30a-3p.Based on lead and the culture supernatant of osteoclasts in the control group,the existence of miR-30a-3p was verified by adding RNase A,Triton X-100 and exocrine synthesis inhibitor GW4869.2.Effect of lead treated osteoclast secretion miR-30a-3p on osteoblastsThe osteoclasts treated with 5 μmol/L lead acetate and then co-cultured with osteoblasts,and the changes of the viability of the lower layer osteoblasts and related indicators of pyroptosis were detected after 24 hours of co-culture.At the same time,the co-culture group with miR-30a-3p inhibitor was added to observe whether the effect disappeared.In addition,miR-30a-3p mimic was transfected into osteoblasts alone to observe the toxicity of miR-30a-3p on osteoblasts.3.Study on the mechanism by which osteoclast exosomal miR-30a-3p causes osteoblast pyroptosisUse the online target gene prediction database to screen the target gene of miR30a-3p,determine the potential target related to the pyroptotic pathway,randomly detect the expression of this gene in the osteoblast transfected miR-30a-3p mimic group,and detect whether the miR-30a-3p and the gene target bind,at the same time,after the osteoblast transfected siNFKB1,knock down the gene,observe the expression level of pyroptosis related proteins.4.Effect of pyroptotic osteoblasts on osteoclastsThe proliferation activity of osteoclasts after co-culture with pyroptotic osteoblasts(here,osteoblasts after co-culture with lead treated osteoclasts)was detected,and the osteoclasts treated with lead alone were added as a control to eliminate the influence of lead itself on the proliferation ability of osteoclasts.Result1.Osteoclast-derived exosomal miR-30a-3p transfer to osteoblast after lead treatmentThe results showed that osteoclasts and osteoblasts were co-cultured after transfection of fluorescent FAM-labeled miR-30a-3p mimic.Under the fluorescence microscope,it was found that green fluorescence appeared in the field of osteoblasts in the co-culture group of transfected simulants,which could coincide with the results of the nuclear dye DAPI.In addition,the expression level of miR-30a-3p in the co-culture group of transfected simulants increased by 1.5 times(P<0.05).The expression level of miR-30a-3p was not significantly reduced when adding ribonuclease only to the culture supernatant of the lead treatment group,but the expression level of miR-30a-3p was significantly reduced when adding TritonX-100 to destroy the double membrane structure of the exocrine body(P<0.05).In addition,the expression of miR-30a-3p in the supernatant of cell culture decreased by 56%compared with that in the control group after adding the exocrine synthesis inhibitor GW4869(P<0.05).2.Effect of lead treated osteoclast secretion miR-30a-3p on osteoblastsThe results showed that after co-culture with lead treated osteoclasts,the viability of osteoblasts decreased to 86%(P<0.05),the number of PI staining positive cells increased to 21%(P<0.05),the release of LDH increased(P<0.05),the protein levels of caspase-1,IL-1β,IL-18 and GSDMD were significantly higher than those of the cocultured control group,and the number of GSDMD shear increased(P<0.05),pores appear in the cell membrane,the secretion of IL-1β and IL-18 increased(P<0.05).When the lead treated osteoclasts were simultaneously transfected with miR-30a3p inhibitor,the co-culture results showed that compared with the lead treated coculture group,the activity of osteoblasts increased by 12%(P<0.05),the number of PI staining positive cells significantly decreased(P<0.05),the release level of LDH also eased(P<0.05),the addition of miR-30a-3p inhibitor inhibited the increase of pyroptosis related protein levels in lead treated co-culture group(P<0.05),and the secretion of IL-1β and IL-18 decreased(P<0.05).After direct transfection of miR-30a-3p mimic as osteoblasts,compared with the transfected control group,the viability of osteoblasts decreased to 90%(P<0.05),the number of PI staining positive cells increased to 39%(P<0.05),the release of LDH increased by 1.7 times(P<0.05),and the levels of caspase-1,IL-1β,IL-18 and GSDMD increased significantly compared with the transfected control group(P<0.05),GSDMD shear increased(P<0.05),pores appear in the cell membrane,the secretion of IL-1β and IL-18 increased(P<0.05).3.Study on the mechanism of osteoclast exosomal miR-30a-3p causing osteoblast pyroptosisThe online databases were used to predict the miR-30a-3p target genes,and 636 potential target genes were obtained from the intersection.The KEGG enrichment analysis results showed that miR-30a-3p might participate in FOXO,MAPK,NF-kappa B and other signal pathways.Double luciferase reported that the experimental results showed that when NFKB1 3’-UTR and miR-30a-3p were overexpressed at the same time.There was no statistical difference in luciferase activity between the two groups,and there was no difference compared with the mutation group.The expression level of NFKB1 protein in osteoblasts transfected with miR-30a-3p mimic increased(P<0.05).Compared with osteoblasts transfected with miR-30a-3p mimic alone,the expression level of NLRP3 in osteoblasts transfected with miR-30a-3p mimic and NFKB1 siRNA at the same time decreased(P<0.05).The increase of pyroptosis associated proteins also relieved,and the GSDMD shear body decreased(P<0.05).4.Effect of pyroptotic osteoblasts on osteoclastsThe results showed that the proliferative activity of osteoclasts in the lead alone and co-culture group increased compared with that in the control group(P<0.05),while the proliferative activity of osteoclasts in the lead co-culture group was 1.2 times higher than that in the lead alone group(P<0.05).The percentage of G1 phase cells in the lead co-culture osteoclasts decreased compared with that in the lead alone and co-culture control group(P<0.05),while the number of S phase cells increased(P<0.05).Conclusion1.The exosomal miR-30a-3p secreted by lead treated osteoclasts can enter osteoblasts and activate the NLRP3-Caspase-1 classic pyroptotic signal pathway by regulating the activity of NFKB1.At the same time,pyroptotic osteoblasts can promote the proliferation of osteoclasts.2.The exosomal miR-30a-3p can be used as an intercellular messenger to mediate the information exchange between osteoblasts and osteoclasts,and the therapeutic inhibition of miR-30a-3p activity in osteoclasts can be considered as a potential strategy for the treatment of lead induced osteoporosis. |
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