Xiaoyi Zhao

MiR-455-3p regulates early chondrogenic differentiation via inhibiting Runx2.

The expression of miR‐455‐3p has been shown to be up‐regulated in chondrogenesis of mesenchymal stem cell, but its role in different stages during chondrogenesis remains unknown. Here, we show that miR‐455‐3p is increased in ATDC5 cells from 0 d to 21 d, but rapidly decreases at 28 d, and a similar expression kinetic is detected in the development of mouse embryos. We show that miR‐455‐3p functions as an activator for early chondrogenic differentiation, most likely by inhibiting the expression of Runt‐related transcription factor 2 (Runx2) as indicated by luciferase reporter assays. In conclusion, miR‐455‐3p may activate early chondrogenesis by directly targeting Runx2.

Expression patterns of transcription factor PPARγ and C/EBP family members during in vitro adipogenesis of human bone marrow mesenchymal stem cells

In the past decades increasing lines of evidence have demonstrated that adipose tissue, as an endocrine organ plays a central role in metabolic homeostasis and its related maladies. CCAAT/enhancer‐binding protein (C/EBP) family members and the nuclear receptor peroxisome proliferator‐activated receptor gamma (PPARγ) were known to be the vital transcription factors in the regulation of adipogenesis. However, the exact mechanism for increased marrow fat in patients with bone metabolic diseases, such as osteoporosis, is still poorly understood. Herein, we studied the expression pattern of PPARγ and C/EBPs in human bone marrow mesenchymal stem cell (hBMSC) adipogenesis and evaluated the effects of individual components of an adipogenic cocktail on the differentiation and transcription factor expression. We furthermore examined whether the ERK signaling pathway was involved in mediating these effects. These findings showed that C/EBPβ and C/EBPδ were detected in undifferentiated hBMSC and maintained during the whole process of adipogenesis, and could initiate the expression of PPARγ1 under the treatment of dexamethasone and IBMX. Subsequently, the activation of PPARγ1 by indomethacin, its exogenous ligand, activated C/EBPα, which, together with IBMX, up‐regulated PPARγ2 expression and therefore the fullest adipogenesis. Insulin and its downstream signal pathway extracellular signal‐regulated kinases (ERK), however, were found not necessary for hBMSC adipogenesis. Our results revealed some unique characteristics of human adipocyte formation, which may help to understand the molecular mechanisms of bone marrow adipogenesis and give insights into the treatment of osteoporosis.

CCL3 serves as a potential plasma biomarker in knee degeneration (osteoarthritis)

OBJECTIVE To explore the ability of chemokines in plasma to detect the presence of pre-X-rays defined knee degeneration and the extent (burden). METHODS A total of 181 subjects (75 control subjects, 47 pre-X-KD patients and 50 X-KOA patients) were included and subdivided into three subgroups. Articular cartilage loss in pre-X-KD patients were scored on the basis of the ICRS classification during the arthroscopy or documented on MRI with chondral WORMS. The severity of X-KOA was graded using the Kellgren-Lawrence classification through the posterior-anterior knee X-rays. The concentrations of the inflammatory cytokines and chemokines in plasma were quantified using Luminex microbead-based suspension array (SA) and were cross-validated by enzyme-linked immunosorbent assay (ELISA). RESULTS CCL3 in plasma showed the highest ability to discriminate pre-X-KD patients from the controls with an AUC of 0.799. At a cutoff value of 0.168 pg/ml, the sensitivity was 70.21%, the specificity was 96.00%, the positive predictive value was 91.67% and the negative predictive value was 83.72%. As to define disease burden, the plasma levels of resistin, IL6, IL8, CCL3 and CCL4 showed significant association with the severity of X-rays defined knee OA, with regard to the KL classification. Moreover, significant elevation of IL6, IL8, CCL3 and CCL4 levels in plasma were observed in severe knee OA patients (KL grade IV) compared with those with pre-X-KD (KL grade 0-I). CONCLUSION We firstly showed that the plasma CCL3 could be potential serum biomarker for knee OA with the capacity to detect pre-X-rays defined changes and stage the severity of damage in knee.

Presence and function of microRNA-92a in chondrogenic ATDC5 and adipose-derived mesenchymal stem cells

The aim of the present study was to investigate the presence and biological function of microRNA-92a (miR-92a) in chondrogenesis and cartilage degeneration. Human adipose-derived mesenchymal stem cells (hADSCs) in micromass and chondrocyte-like ATDC5 cells were induced to chondrogenesis, and primary human/mouse chondrocytes (PHCs/PMCs) and chondrogenic ATDC5 cells were stimulated with interleukin-1β (IL-1β). An miR-92a mimic/inhibitor was transfected into the ATDC5 cells using lipofectamine 2000. Gene expression was analyzed using reverse transcription-quantitative polymerase chain reaction. Alcian blue was used to stain the cartilage nodules and chondrogenic micromass. The potential target genes, signaling pathways and functions of miR-92a were examined using miRanda, miRDB, CLIP-Seq, TargetScan and Kyoto Encyclopedia of Genes and Genomes. The expression of miR-92a was elevated in the chondrogenic ATDC5 cells and hADSCs, and also in the IL-1β-induced ATDC5 cells, PMCs and PHCs. Forced expression of miR-92a enhanced the expression levels of col9a2 and aggrecan. A total of 279 genes were predicted as potential target genes of miR-92a. The phosphoinositide 3-kinase/PI3K)-Akt, ErbB and focal adhesion kinase pathways, extracellular matrix (ECM)-receptor interaction and the mammalian target of rapamycin (mTOR) signaling pathway were suggested to mediate the effects of miR-92a on chondrogenesis and cartilage degeneration. These results demonstrated that miR-92a was involved in chondrogenesis and the chondrocyte response induced by IL-1β. miR-92a positively contributed to the expression of col9a2 and of aggrecan.

MicroRNA-193b-3p regulates chondrogenesis and chondrocyte metabolism by targeting HDAC3

Histone deacetylase 3 (HDAC3) plays a pivotal role in the repression of cartilage-specific gene expression in human chondrocytes. The aim of this study was to determine whether microRNA-193b-3p (miR-193b-3p) regulates the expression of HDAC3 during chondrogenesis and chondrocyte metabolism. Methods: miR-193b-3p expression was assessed in a human mesenchymal stem cell (hMSC) model of chondrogenesis, in interleukin-1β (IL-1β)-treated primary human chondrocytes (PHCs), and in non-degraded and degraded cartilage. hMSCs and PHCs were transfected with miR-193b-3p or its antisense inhibitor. A direct interaction between miR-193b-3p and its putative binding site in the 3′-untranslated region (3′-UTR) of HDAC3 mRNA was confirmed by performing luciferase reporter assays. Chondrocytes were transfected with miR-193b-3p before performing a chromatin immunoprecipitation assay with an anti-acetylated histone H3 antibody. To investigate miR-193b-3p-transfected PHCs in vivo, they were seeded in tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffolds, which were then implanted in nude mice. In addition, plasma exosomal miR-193b-3p in samples from normal controls and patients with osteoarthritis (OA) were measured. Results: miR-193b-3p expression was elevated in chondrogenic and hypertrophic hMSCs, while expression was significantly reduced in degraded cartilage compared to non-degraded cartilage. In addition, miR-193b-3p suppressed the activity of reporter constructs containing the 3′-UTR of HDAC3, inhibited HDAC3 expression, and promoted histone H3 acetylation in the COL2A1, AGGRECAN, COMP, and SOX9 promoters. Treatment with the HDAC inhibitor trichostatin A (TSA) increased cartilage-specific gene expression and enhanced hMSCs chondrogenesis. TSA also increased AGGRECAN expression and decreased MMP13 expression in IL-1β-treated PHCs. Further, 8 weeks after implanting PHC-seeded TCP-COL-HA scaffolds subcutaneously in nude mice, we found that miR-193b overexpression strongly enhanced in vivo cartilage formation compared to that found under control conditions. We also found that patients with OA had lower plasma exosomal miR-193b levels than control subjects. Conclusions: These findings indicate that miR-193b-3p directly targets HDAC3, promotes H3 acetylation, and regulates hMSC chondrogenesis and metabolism in PHCs.

Exosomal miR-95-5p regulates chondrogenesis and cartilage degradation via histone deacetylase 2/8

Abstract MicroRNAs play critical roles in the pathogenesis of osteoarthritis, the most common chronic degenerative joint disease. Exosomes derived from miR‐95‐5p‐overexpressing primary chondrocytes (AC‐miR‐95‐5p) may be effective in treating osteoarthritis. Increased expression of HDAC2/8 occurs in the tissues and chondrocyte‐secreted exosomes of patients with osteoarthritis and mediates cartilage‐specific gene expression in chondrocytes. We have been suggested that exosomes derived from AC‐miR‐95‐5p (AC‐miR‐95‐5p‐Exos) would enhance chondrogenesis and prevent the development of osteoarthritis by directly targeting HDAC2/8. Our in vitro experiments showed that miR‐95‐5p expression was significantly lower in osteoarthritic chondrocyte‐secreted exosomes than in normal cartilage. Treatment with AC‐miR‐95‐5p‐Exos promoted cartilage development and cartilage matrix expression in mesenchymal stem cells induced to undergo chondrogenesis and chondrocytes, respectively. In contrast, co‐culture with exosomes derived from chondrocytes transfected with an antisense inhibitor of miR‐95‐5p (AC‐anti‐miR‐95‐5p‐Exos) prevented chondrogenic differentiation and reduced cartilage matrix synthesis by enhancing the expression of HDAC2/8. MiR‐95‐5p suppressed the activity of reporter constructs containing the 3ʹ‐untranslated region of HDAC2/8, inhibited HDAC2/8 expression and promoted cartilage matrix expression. Our results suggest that AC‐miR‐95‐5p‐Exos regulate cartilage development and homoeostasis by directly targeting HDAC2/8. Thus, AC‐miR‐95‐5p‐Exos may act as an HDAC2/8 inhibitor and exhibit potential as a disease‐modifying osteoarthritis drug.

MiR-455-3p inhibits the degenerate process of chondrogenic differentiation through modification of DNA methylation

The aim of this work was to determine whether miR-455-3p regulates DNA methylation during chondrogenic differentiation of hMSCs. The expression of miR-455-3p and de novo methyltransferase DNMT3A was assessed in micromass culture of hBMSCs, which induced chondrogenic differentiation in vitro, and in E16.5 mice in vivo. A luciferase reporter assay was used to confirm whether miR-455-3p directly targets DNMT3A by interaction with the 3′-UTR. Using an Illumina Infinium Methylation EPIC microarray, genome-wide DNA methylation of hBMSCs with or without overexpressed miR-455-3p was examined for 28 days during induced chondrogenic differentiation. Here, we showed that miR-455-3p was more expressed during the middle stage of hBMSC chondrogenic differentiation, and less expressed in the late stage. DNMT3A was less expressed in the middle stage and more expressed in the late stage, and was also more expressed in the palms of miR-455-3p deletion mice compared to those of wild-type mice. The luciferase reporter assay demonstrated that miR-455-3p directly targets DNMT3A 3′-UTR. miR-455-3p overexpression inhibits the degenerate process during chondrogenic differentiation, while deletion of miR-455-3p in mice accelerated cartilage degeneration. Genome-wide DNA methylation analysis showed miR-455-3p overexpression regulates DNA methylation of cartilage-specific genes. GO analysis revealed PI3K-Akt signaling pathway was most hypomethylated. Our data show that miR-455-3p can regulate hMSC chondrogenic differentiation by affecting DNA methylation. Overexpression of miR-455-3p and DNA methylation inhibitors can thus potentially be utilized to optimize chondrogenic differentiation.

Radiographic Measurement of Femoral Lateral Bowing and Distal Femoral Condyle Resection Thickness: Variances and Effects on Total Knee Arthroplasty Planning

Background: Accurate evaluation of the plain radiography of lower limb is critical for preoperative planning of total knee arthroplasty (TKA). We aimed to investigate the effect of femoral lateral bowing and rotation on the radiographic measurements of distal femoral condyle resection thickness (DRT) and the distal femoral resection valgus angle (FVA). Methods: We analyzed 246 three-dimensional femoral models generated from computed tomography images of 123 patients, acquiring projected contours in seven positions – 20° and 10° internal rotation; 0° rotation; 10°, 20°, 30°, and 40° external rotation – for each model. Medial and lateral condyle DRTs, femoral shaft lateral bowing angle (FBA), and distal FVA were determined for each position. Linear mixed effect model was used to determine the effect of degree of femur rotation on repeated measurements of DRT or FVA. Results: FBA significantly affected the FVA and DRT (Pearsons R = 0.767 and −0.408, respectively; P < 0.000). Samples were divided into three groups according to the FBA measured in neutral position: FBA <0°: DRT 3.75 ± 1.30 mm, FVA 4.53° ± 1.27°; FBA >0° but <3°: DRT 3.39 ± 1.31 mm, FVA 5.92° ± 1.31°; FBA >3°: DRT 2.22 ± 1.31 mm, FVA 7.37° ± 1.31°. From simulated 20° internal rotation to 40° external rotation in each femoral model, the average variation ranges of radiographically measured DRT, FVA, and FBA were 0.50 ± 0.28 mm, 2.93° ± 0.96°, and 10.33° ± 1.90°, respectively, with no significant differences among the FBA groups. The degree of femoral rotation significantly affected the FVA (F = 62.148, P < 0.000), whereas there was no effect on condyle resection thickness (F = 0.4705, P = 0.494). Conclusions: Axial femoral rotation has less effect on radiographic measurements of differences in the DRT than on those of the distal FVA.