BACKGROUND:Casein kinase 2 binding protein 1 (CKIP-1) is an important negative control gene in bone formation.After the deletion of this gene,the overall bone of mice was significantly enhanced,and bone formation and bone density were significantly increased.microRNA (miRNA) as the early found small molecular regulator has a regulatory effect on most of the coding genes and plays an important role in osteogenic differentiation.OBJECTIVE:To investigate the effect and molecular mechanism of miRNA/CKIP-1 axis on osteogenic differentiation of bone marrow mesenchymal stem cells from osteoporosis patients.METHODS:The miRNA-Seq technology was used to detect the changes of miRNA in bone marrow mesenchymal stem cells in 32 patients with osteoporosis treated in the Department of Orthopedics,Kaifeng Central Hospital from March to June 2022 and healthy people in the physical examination center during the same period.The Targetscan website was used to predict the miRNA targeted to regulate CKIP-1.Luciferase reporter gene assay was used to detect the binding of miRNA and CKIP-1 promoter DNA.miR-192-5p analogs (miR-192-5p mimics)/negative control (NC mimics) or miR-192-5p inhibitors (miR-192-5p inhibitor)/negative control (NC inhibitor) were transfected in bone marrow mesenchymal stem cells.On days 7 and 14 after osteogenic induction,the expression levels of Runt-related transcription factor 2 (Runx2),osteocalcin,anti-osteopontin,bone sialoprotein,and CKIP-1,and the differentiation of bone marrow mesenchymal stem cells into osteoblasts were detected by real-time fluorescence quantitative PCR and alizarin red staining.The regulatory effect of miR-192-5p/CKIP-1/axis on osteogenic differentiation of cells was detected by western blot assay and alizarin red staining.RESULTS AND CONCLUSION:Compared with the healthy group,the expression levels of 16 miRNAs were significantly up-regulated and those of 53 miRNAs were significantly down-regulated in the osteoporosis group (P<0.05).Using Targetscan website and verified by luciferase reporter gene experiment,it was found that miR-192-5p and CKIP-1 had complementary nucleotide sequences (P<0.05).Overexpression of miR-192-5p significantly increased the expression levels of Runx2,osteocalcin,osteopontin,and bone sialoprotein (P<0.05),and inhibition of miR-192-5p significantly decreased the expression levels of Runx2,osteocalcin,osteopontin,and bone sialoprotein (P<0.05).After silencing the expression of CKIP-1,the protein levels of Runx2,osteocalcin,and osteopontin increased (P<0.05);the inhibitory effect of knockdown of miR-192-5p on osteogenic differentiation of cells was reversed.Above results confirm that the expression of miR-192-5p is decreased in osteoporosis.miR-192-5p promotes osteogenic differentiation of bone marrow mesenchymal stem cells by targeting the inhibition of CKIP-1 expression.

BACKGROUND:Casein kinase 2 binding protein 1 (CKIP-1) is an important negative control gene in bone formation.After the deletion of this gene,the overall bone of mice was significantly enhanced,and bone formation and bone density were significantly increased.microRNA (miRNA) as the early found small molecular regulator has a regulatory effect on most of the coding genes and plays an important role in osteogenic differentiation.OBJECTIVE:To investigate the effect and molecular mechanism of miRNA/CKIP-1 axis on osteogenic differentiation of bone marrow mesenchymal stem cells from osteoporosis patients.METHODS:The miRNA-Seq technology was used to detect the changes of miRNA in bone marrow mesenchymal stem cells in 32 patients with osteoporosis treated in the Department of Orthopedics,Kaifeng Central Hospital from March to June 2022 and healthy people in the physical examination center during the same period.The Targetscan website was used to predict the miRNA targeted to regulate CKIP-1.Luciferase reporter gene assay was used to detect the binding of miRNA and CKIP-1 promoter DNA.miR-192-5p analogs (miR-192-5p mimics)/negative control (NC mimics) or miR-192-5p inhibitors (miR-192-5p inhibitor)/negative control (NC inhibitor) were transfected in bone marrow mesenchymal stem cells.On days 7 and 14 after osteogenic induction,the expression levels of Runt-related transcription factor 2 (Runx2),osteocalcin,anti-osteopontin,bone sialoprotein,and CKIP-1,and the differentiation of bone marrow mesenchymal stem cells into osteoblasts were detected by real-time fluorescence quantitative PCR and alizarin red staining.The regulatory effect of miR-192-5p/CKIP-1/axis on osteogenic differentiation of cells was detected by western blot assay and alizarin red staining.RESULTS AND CONCLUSION:Compared with the healthy group,the expression levels of 16 miRNAs were significantly up-regulated and those of 53 miRNAs were significantly down-regulated in the osteoporosis group (P<0.05).Using Targetscan website and verified by luciferase reporter gene experiment,it was found that miR-192-5p and CKIP-1 had complementary nucleotide sequences (P<0.05).Overexpression of miR-192-5p significantly increased the expression levels of Runx2,osteocalcin,osteopontin,and bone sialoprotein (P<0.05),and inhibition of miR-192-5p significantly decreased the expression levels of Runx2,osteocalcin,osteopontin,and bone sialoprotein (P<0.05).After silencing the expression of CKIP-1,the protein levels of Runx2,osteocalcin,and osteopontin increased (P<0.05);the inhibitory effect of knockdown of miR-192-5p on osteogenic differentiation of cells was reversed.Above results confirm that the expression of miR-192-5p is decreased in osteoporosis.miR-192-5p promotes osteogenic differentiation of bone marrow mesenchymal stem cells by targeting the inhibition of CKIP-1 expression.