Pathogenesis Of Glucocorticoid-induced Osteonecrosis Of Femoral Head With Syndrome Of Blood Stasis By Histone Demethylase JMJD Family

ObjectiveTo study the expression of histone demethylase JMJD family genes in glucocorticoid-induced osteonecrosis of femoral head (GIONFH) and their role in osteogenic differentiation of human mesenchymal stem cells.Methods1.Four cases of non-GIONFH patients (control group) and six cases of GIONFH patients with syndrome of blood stasis were involved to isolate the bone marrow from proximal femur during surgery. Human bone-marrow derived mesenchymal stem cells (hBMSCs) were isolated by density gradient centrifugation, then purified, cultured and passaged to P4 for the subsequent experiments. Flow cytometry was used to identify the surface markers for hBMSCs including CD105、CD29、CD73、CD44、CD34、CD45 and CDllb/c.ALP staining and activity, alizarin red staining were taken to examine the Osteogenic differentiation capacity. Oil red 0 staining were taken to examine the adipogenic differentiation capacity. Total RNA were extracted to perform real-time PCR assay.2. Normal hBMSCs from Lonza were cultured and passaged to P5 for the subsequent experiments. Cells from 0,3,7,14,21 days of osteogenic differentiation of hBMSCs were collected for the RNA isolation for RNA-seq profile and total protein extraction for western blot experiments. Plasmids for KDM4A knock down and over-expression were constructed separately to transfected with 293T cells for the production of corresponding lentivirus. Effects of KDM4A knock down and over-expression on osteogenic differentiation of hBMSCs were performed by ALP staining and activity, alizarin red staining, RNA-seq and western blot. JIB-04, a pan-selective Jumonji histone demethylase inhibitor were used to examine its effect on osteogenic differentiation of hBMSCs including ALP staining and activity, alizarin red staining, RNA-seq and western blot.Results1.The hBMSCs isolated from patients met the criteria of hBMSCs characteristic including the surface markers, the morphology, the osteogenic differentiation capacity and adipogenic differentiation capacity.2. KMD6A and RUNX2 mRNA expression were significantly lower in ONFH group than in control group(P<0.05). mRNA expression of KDM4A、KDM4B and KDM4D were dynamic down-regulated, while KDM4C、KDM5B、KDM5C、KDM6A、KDM6B、UTY were dynamic up-regulated during osteogenic differentiation of hBMSCs.Protein expression of KDM4A、KDM4B、KDM5A、KDM6B were dynamic up-regulated in 0,3,7,14,21 days of osteogenic differentiation of hBMSCs.3. There were no significant difference among shKDM4A#1,shKDM4A#2 and shNeal (P<0.05) when KDM4A were knock down whether in regular medium or osteogenic differentiation medium, indicating that knock down of KMD4A did not affect proliferation of hBMSCs. ALP staining on 8-1 day osteogenic differentiation was decreased in shKDM4A#1 and shKDM4A#2 group than in shNeal group. ALP activity on 8th day osteogenic differentiation was significantly lower in shKDM4A#1 and shKDM4A#2 group than in shNeal group (P<0.05), indicating that knock down of KMD4A inhibited ALP activity of hBMSCs during osteogenic differentiation. Alizarin red staining on 14th day osteogenic differentiation was decreased in shKDM4A#1 and shKDM4A#2 group than in shNeal group, indicating that knock down of KMD4A inhibited mineralization of hBMSCs during advanced period of osteogenic differentiation. The mRNA level by RNA-seq and protein expression of RUNX2, SPP1, ALP were lower in shKDM4A#1 and shKDM4A#2 group than in shNeal group.When KDM4A were over expression, ALP staining on 8th day osteogenic differentiation was increased in KDM4A (wild type) and KDM4AH188A (mutant type) group than in Vector group (control). ALP activity on 8th day osteogenic differentiation was significantly higher in KDM4A and KDM4AH188A group than in Vector group (P<0.05), indicating that over expression of KMD4A promoted ALP activity of hBMSCs during osteogenic differentiation. Alizarin red staining on 14th day osteogenic differentiation was increased in KDM4A and KDM4AH188A group than in Vector group, indicating that over expression of KMD4A promoted mineralization of hBMSCs during advanced period of osteogenic differentiation. Protein expression of RUNX2 and SPP1 were higher in KDM4A and KDM4AH188A group than in Vector group.4.800nM of JIB-04 significantly inhibited proliferation of hBMSCs compared with control group (DMSO)(P<0.05) in osteogenic differentiation medium, while there were no significant difference in 100nM,200nM and 400nM of JIB-04 compared with control group (P>0.05). ALP staining on 8th day osteogenic differentiation was decreased in 50 nM,100 nM,200 nM,400 nM and 800 nM of JIB-04 group than in control group. ALP activity on 8"’day osteogenic differentiation was significantly lower in 50 nM,100 nM,200 nM, 400 nM and 800nM of JIB-04 group than in control group (P<0.05). Alizarin red staining on 16th,19th and 21th day osteogenic differentiation was decreased in 50 nM,100 nM,200 nM,400 nM and 800nM of JIB-04 group than in control group. The mRNA level by RNA-seq of RUNX2, SPP1 and ALP were lower in 100 nM and 400 nM of JIB-04 than in control group, while protein expression of RUNX2 and SPP1 were lower in 100 nM and 400 nM of JIB-04 than in control group.ConclusionKMD6A may contribute to the pathogenesis of glucocorticoid-induced osteonecrosis of femoral head with syndrome of blood stasis.KMD4A is an important gene regulating osteogenic differentiation of hBMSCs via RUNX2-OPN/ALP signaling pathway. JIB-04 negatively regulates osteogenic differentiation of hBMSCs via RUNX2-OPN signaling pathway, indicating that the integral function of JMJD family genes is closely correlated with osteogenic differentiation of hBMSCs.