| Objective: Osteoporosis(OP)is a common disease of bone metabolism in the whole body,which is characterized by osteoporosis,low bone mineral density and easy to fracture.In recent years,many studies have shown that miRNAs can target genes and signaling pathways related to osteogenic differentiation and participate in the process of osteogenic differentiation.Therefore,this study was designed to investigate the role of miR-148a-3p in the regulation of osteoblastic differentiation and bone remodeling in MC3T3-E1 cells,and further clarify the role of miR-148a-3p/p300/Nrf2,a potential regulatory axis,in the process of osteoblastic differentiation of MC3T3-E1 cells,providing a new potential target for the prevention and treatment of osteoporosis.Methods: In this study,we investigated the effect of miR-148a-3p on the osteogenic differentiation and bone remodeling of MC3T3-E1 cells by regulating the expression of p300.I Cell Experiments1.When MC3T3-E1 cells were induced with osteogenic medium,the expression levels of miR-148a-3p,p300,Nrf2,Runx2,osteocalcin and Col1a1 were detected by q RT-PCR and Western blot.2.The expression levels of miR-148a-3p,Runx2,osteocalcin and Col1a1 in MC3T3-E1 cells were detected by q RT-PCR after transfection with miR-148a-3p inhibitor;The cell proliferation was detected by CCK-8,the ALP activity,the number of calcified nodules and the number of mineralized nodules were detected by ALP staining,ALP activity and ARS staining,the m RNA and protein expressions of Runx2,Osteocalcin and Col1a1 were detected by Western blot.3.The relationship between miR-148a-3p and p300 was detected by dual luciferase reporter gene.When miR-148a-3p was overexpressed or inhibited in MC3T3-E1 cells,the expression level of p300 was detected by q RT-PCR and Western blot.When p300 was overexpressed,the expression level of p300 was detected by q RT-PCR and Western blot.In MC3T3-E1 cells,the expression of miR-148a-3p was upregulated alone or simultaneously with the expression of miR-148a-3p with p300,the expression levels of miR-148a-3p,p300,Runx2,Osteocalcin and Col1a1 were detected by q RT-PCR.The cell proliferation was detected by CCK-8.The activity of ALP was detected by ALP staining,and the activity of ALP was detected by ARS staining,the m RNA and protein expression levels of Runx2,Osteocalcin and Col1a1 were detected by Western blot.4.In MC3T3-E1 cells,dual luciferase reporter gene assay was used to detect the association and regulation of p300 with NRF2 gene.Chip assay was used to detect the enrichment of p300 in the promoter region of NRF2 gene,overexpression of miR-148a-3p alone or both miR-148a-3p and p300 was detected by Western blot.5.In MC3T3-E1 cells transfected with Si-Nrf2 plasmid,the expression of Nrf2,Runx2,osteocalcin and Col1a1 were detected by q RT-PCR,the protein expressions of Runx2,osteocalcin and Col1a1 were detected by Western blot.II Animal ExperimentsThe osteoporosis model of OVX(ovariectomy)mice was established:Thirty-two12-week-old female C57 BL / 6J mice were randomly divided into 4 groups with 8 in each group: Sham group(sham operation),OVX group(bilateral ovariectomy to establish osteoporosis model),OVX antagomir-NC group(antagomir-NC lentivirus vector was injected in vitro after bilateral ovary resection),OVX antagomiR-148a-3p group(antagomiR-148a-3p lentivirus vector was injected in vitro after bilateral ovary resection),all mice were raised for 6 weeks after modeling.Bone mineral density(BMD),bone volume fraction(BV/TV),trabecular number(Tb.N),trabecular thickness(Tb.Th)and trabecular spacing(Tb.Sp)were measured by Micro-CT.The trabecular number(Tb.N)and trabecular thickness(Tb.Th)of bone tissue were measured by HE staining.The expression levels of miR-148a-3p and p300 in bone tissue were detected by q RT-PCR,and the expression levels of p300,Runx2,Osteocalcin and Col1a1 in bone tissue were detected by Western blot.Results:1.The expression of miR-148a-3p gradually decreased during osteoblast differentiation,while the m RNA and protein expressions of p300,Nrf2,Runx2,Osteocalcin and Col1a1 gradually increased.Inhibition of miR-148a-3p could increase the expression of Runx2,OC and Col1a1.The results of CCK-8,ALP staining,ALP activity and ARS staining suggested that the osteogenic differentiation of cells was enhanced.2.The miRNA target gene prediction site shows that miR-148a-3p is able to combine with p300.Dual luciferase reporter results showed that p300 was a downstream gene of miR-148a-3p.Overexpression of miR-148a-3p inhibited the expression of p300,and the expression of p300 increased significantly when the expression of miR-148a-3p was low.Overexpression of p300 could break the inhibitory effect of miR-148a-3p.When miR-148a-3p was overexpressed alone,the m RNA and protein expressions of Runx2,Osteocalcin and Col1a1 decreased significantly.The results of CCK-8 detection,ALP staining,ALP activity detection and ARS staining suggested that the osteoblast differentiation of cells was decreased,while miR-148a-3p and p300 were overexpressed,the m RNA and protein expressions of Runx2,Osteocalcin and Col1a1 were significantly increased.The results of CCK-8,ALP staining,ALP activity and ARS staining indicated that the osteogenic differentiation of the cells was enhanced.3.Dual luciferase reporter gene results showed that p300 bound to Nrf2 promoter.Ch IP results further confirmed that p300 could directly bind to Nrf2 and affect its transcriptional activity.Western blot showed that overexpression of miR-148a-3p inhibited the expression of Nrf2 and P65 proteins,while overexpression of p300 reversed the inhibition of Nrf2 and p65 by miR-148a-3p.The expression of Runx2,Osteocalcin and Col1a1 were decreased by down-regulation of Nrf2.The results of ALP staining,ALP activity and ARS staining indicated that the osteogenic differentiation ability of the cells was decreased.4.Compared with the sham-operated mice,the results of Micro-CT showed that BMD,BV/TV and Tb.N of mouse femur decreased,Tb.Th thinned,Tb.Sp decreased,the number and thickness of trabecular bone decreased in OVX mice.The expression of miR-148a-3p was significantly increased and the expression of p300 was significantly decreased by q RT-PCR,the expressions of Runx2,Osteocalcin and Col1a1 in bone tissue were significantly decreased by Western blot.Further inhibit the expression of miR-148a-3p in OVX mice.The results of Micro-CT showed that BMD,BV/TV and Tb.N increased significantly,Tb.Th thickened significantly,Tb.Sp decreased significantly,and the number and thickness of trabecular bone increased significantly.The results of q RT-PCR showed that the expression of miR-148a-3p was significantly decreased and the expression of p300 was significantly increased in bone tissue.The protein expression of Runx2,Osteocalcin and Col1a1 was significantly increased by Western blot.Conclusions:1.During osteogenic differentiation,the expression of miR-148a-3p in MC3T3-E1 cells gradually decreased,and the expression of p300 and Nrf2 gradually increased.Downregulation of miR-148a-3p expression could promote the osteogenic differentiation of MC3T3-E1 cells.2.MiR-148a-3p regulates p300.Overexpression of miR-148a-3p can inhibit the expression of p300 and inhibit the osteogenic differentiation of MC3T3-E1 cells.Overexpression of p300 can reverse the inhibitory effect of miR-148a-3p on the osteogenic differentiation of MC3T3-E1 cells,these results suggest that miR-148a-3p influences the osteogenic differentiation of MC3T3-E1 cells by regulating the expression of p300.3.p300 could bind to Nrf2 specifically and affect its transcriptional activity.Overexpression of miR-148a-3p could inhibit the expression of Nrf2 in MC3T3-E1 cells,while overexpression of p300 could reverse the inhibitory effect of miR-148a-3p on Nrf2 and P65,downregulation of NRF2 expression could inhibit osteoblastic differentiation of MC3T3-E1 cells,these results suggest that miR-148a-3p can affect the activation of Nrf2 pathway by regulating p300,thus affecting cell osteoblastic differentiation.4.Inhibiting the expression of miR-148a-3p in OVX mice could significantly increase the expression of p300 and promote bone remodeling in osteoporotic mice. |
PDF Full Text Request