LncRNA NEAT1 promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by inhibiting cell pyroptosis
Objective To investigate the effect of long non-coding RNA(lncRNA)nuclear paraspeckle assembly transcript 1(NEAT1)in regulating osteogenic differentiation of human bone marrow-derived mesenchymal stem cells(hBMSCs)through regulating cell pyroptosis.Methods hBMSCs were cultured for 7 days to induce osteogenic differentiation,and then divided into the control group(common culture),the osteogenic differentiation group(osteogenic differentiation induction),the pcD-NEAT1 group(transfected with NEAT1 overexpression plasmid),the pcD-null group(transfected with negative control of NEAT1 overexpression plasmid),the osteogenic differentiation+CML group[treated with osteogenic differentiation induction and NOD-like receptor family protein 3(NLRP3)inflammasome activator of carboxy methyl lysine(CML)],and the osteogenic differentiation+CML+pcD-NEAT1 group(treated with osteogenic differentiation and pcD-NEAT1 as well as CML).The cell mineralization was detected using alizarin red staining,the alkaline phosphatase(ALP)activity was determined by ALP activity assay,the cell survival rate was determined by CCK-8,and the morphological change was observed under a high-power microscope.The cell apoptosis was determined using the TUNEL assay.The expression of NEAT1 was detected using qRT-PCR.The protein expression of IL-1β,IL-18,NLRP3,cleaved-caspase 1(cleaved-CASP1),gasdermin D,Runt-related transcription factor 2(RUNX2),ALP,and osteopontin(OPN)were detected using Western blot.Results Compared with the control group,the degree of cell mineralization in the osteogenic differentiation group was increased,the ALP activity was elevated(P<0.05),and the expression of NEAT1 and the protein expression of RUNX2,ALP,and OPN were upregulated(P<0.05).Compared with the pcD-null group,the degree of cell mineralization in the pcD-NEAT1 group was increased,the ALP activity was elevated(P<0.05),and the expression of NEAT1 and the protein expression of RUNX2,ALP,and OPN were upregulated(P<0.05).Compared with the osteogenic differentiation group,the degree of cell mineralization in the osteogenic differentiation+CML group was reduced,the ALP activity was decreased(P<0.05),the cell survival rate was reduced(P<0.05),the cell apoptosis was increased(P<0.05),cell membrane rupture occurred,cells showed enlarged deformation,the expression of NLRP3 and the protein expression of IL-1β,IL-18,cleaved-CASP1,and gasdermin D were significantly upregulated(P<0.05),while the protein expression of RUNX2,ALP and OPN were significantly downregulated(P<0.05).Compared with the osteogenic differentiation+CML group,the degree of cell mineralization in the osteogenic differentiation+CML+pcD-NEAT1 group was increased,the ALP activity was increased(P<0.05),the protein expression of RUNX2,ALP and OPN were significantly upregulated(P<0.05),the cell survival rate was increased(P<0.05),the cell apoptosis rate was decreased(P<0.05),the cell membrane was intact with the normal cell shape,the expression of NLRP3 and the protein expression of IL-1β,IL-18,cleaved-CASP1 and gasdermin D were significantly downregulated(P<0.05).Conclusion The overexpression of NEAT1 promotes the osteogenic differentiation of hBMSCs by inhibiting NLRP3 inflammasome-mediated cell pyroptosis.
lncRNA NEAT1cell pyroptosisNLRP3 inflammasomehuman bone marrow-derived mesenchymal stem cellsosteo-genic differentiation
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