Eun-Jung Jin

PBMC and exosome-derived Hotair is a critical regulator and potent marker for rheumatoid arthritis.

Despite growing importance of long non-coding RNAs (lncRNAs) in normal physiological and disease conditions, our knowledge of RA-related lncRNAs remains limited. Therefore, we aimed to identify lncRNA signatures that have prognostic values in RA. There was a notably high expression level of Hotair in blood mononuclear cells and serum exosome of rheumatoid arthritis (RA) patients, leading the migration of active macrophage. In contrast, markedly lower level of Hotair was detected in differentiated osteoclasts and rheumatoid synoviocytes and enforced expression of Hotair led to significantly decreased levels of MMP-2 and MMP-13. This exploratory study provides novel empirical evidence that Hotair could be one of potential biomarkers for diagnosing RA.

A long non‐coding RNA, GAS5, plays a critical role in the regulation of miR‐21 during osteoarthritis

Growth Arrest‐Specific 5 (GAS5) is known to negatively regulate cell survival and is aberrantly expressed in several cancers. The influence of GAS5 on osteoarthritis (OA) has not been determined. To address this, articular chondrocytes were isolated from relatively normal (Non‐OA) and clear OA regions (OA) of cartilage in total knee replacement (TKR) patients and biopsied normal cartilage. We found that GAS5 was up‐regulated in OA chondrocytes compared with Non‐OA and normal chondrocytes. The over‐expression of GAS5 increased the expression levels of several MMPs, such as MMP‐2, MMP‐3, MMP‐9, MMP‐13, and ADAMTS‐4; stimulated apoptosis; and suppressed autophagic responses. Furthermore, we subsequently identified miR‐21 as a regulator of GAS5 during OA pathogenesis. The expression level of miR‐21 was significantly reduced in OA patients, and the ectopic expression of GAS5 is capable of suppressing miR‐21 induction. Consistent with GAS5 experiments, the introduction of miR‐21 stimulated the apoptosis of chondrocytes and inhibited the expression levels of autophagic complexes, including LC‐3B. In vivo, we found that the introduction of miR‐21 into the cartilage of OA mice significantly stimulated cartilage destruction. Together, these results show that GAS5 contributes to the pathogenesis of OA by acting as a negative regulator of miR‐21 and thereby regulating cell survival.

Titanium oxide nanotube surface topography and microRNA-488 contribute to modulating osteogenesis

Understanding the biocomplexity of cell behavior in relation to the topographical characteristics of implants is essential for successful osseointegration with good longevity and minimum failure. Here, we investigated whether culture on titanium oxide (TiO2) nanotubes of various diameters could affect the behavior and differentiation of MC3T3-E1 cells. Among the tested nanotubes, those of 50 nm in diameter were found to trigger the expression of the osteoblast-specific transcription factors, sp7 and Dlx5, and upregulate the expression of alkaline phosphatase (ALP). Here, we report that miR-488 was significantly induced in osteoblasts cultured on 50 nm nanotubes and continued to increase with the progression of osteoblast differentiation. Furthermore, downregulation of miR-488 suppressed the expression levels of ALP and matrix metalloprotease-2 (MMP-2). This suppression of ALP transcription was overcome by treatment with the MMP-2 activator, bafilomycin A1. Collectively, these results suggest that 50 nm is the optimum TiO2 nanotube diameter for implants, and that modulation of miR-488 can change the differentiation activity of cells on TiO2 nanotubes. This emphasizes that we must fully understand the physicochemical properties of TiO2 nanotubes and the endogenous biomolecules that interact with such surfaces, in order to fully support their clinical application.

MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

BackgroundEven though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488.ResultsHuman articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1β was also suppressed whereas exposure of TGF-β3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation.ConclusionsmiR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.

MicroRNA-181b regulates articular chondrocytes differentiation and cartilage integrity.

MicroRNAs are endogenous gene regulators that have been implicated in various developmental and pathological processes. However, the precise identities and functions of the miRNAs involved in cartilage development are not yet well understood. Here, we report that miR-181b regulates chondrocyte differentiation and maintains cartilage integrity, and is thus a potent therapeutic target. MiR-181b was significantly down-regulated during chondrogenic differentiation of TGF-β3-stimulated limb mesenchymal cells, but it was significantly up-regulated in osteoarthritic chondrocytes isolated from the cartilage of osteoarthritis patients. The use of a mimic or an inhibitor to alter miR-181b levels in chondroblasts and articular chondrocytes showed that attenuation of miR-181b reduced MMP-13 expression while inducing type II collagen expression. Furthermore, over-expression of anti-miR-181b significantly reduced the cartilage destruction caused by DMM surgery in mice. In sum, our data suggest that miR-181b is a negative regulator of cartilage development, and that inhibition of miR-181b could be an effective therapeutic strategy for cartilage-related disease.

MicroRNA-34a Modulates Cytoskeletal Dynamics through Regulating RhoA/Rac1 Cross-talk in Chondroblasts

Background: JNK signaling involved in regulation of chondrogenic differentiation contributes modulation of miR-34a. Results: JNK signaling modulates miR-34a level and regulates stress fiber formation in chondroblasts. Conclusion: miR-34a regulates RhoA/Rac1 cross-talk and negatively modulates the actin cytoskeleton reorganization during chondrogenesis. Significance: This study provides new insights into understanding the regulatory role of miR-34a in the process of chondrogenic differentiation. MicroRNAs (miRNAs) have been implicated in various cellular processes, such as cell fate determination, cell death, and tumorigenesis. In the present study, we investigated the role of miRNA-34a (miR-34a) in the reorganization of the actin cytoskeleton, which is essential for chondrocyte differentiation. miRNA arrays to identify genes that appeared to be up-regulated or down-regulated during chondrogenesis were applied with chondrogenic progenitors treated with JNK inhibitor. PNA-based antisense oligonucleotides and miRNA precursor were used for investigation of the functional roles of miR-34a. We found that, in chick chondroprogenitors treated with JNK inhibitor, which suppresses chondrogenic differentiation, the expression levels of miR-34a and RhoA1 are up-regulated through modulation of Rac1 expression. Blockade of miR-34a via the use of PNA-based antisense oligonucleotides was associated with decreased protein expression of RhoA (a known modulator of stress fiber expression), down-regulation of stress fibers, up-regulation of Rac1, and recovery of protein level of type II collagen. miR-34a regulates RhoA/Rac1 cross-talk and negatively modulates reorganization of the actin cytoskeleton, which is one of the essential processes for establishing chondrocyte-specific morphology.

TGF‐β3 inhibits chondrogenesis of cultured chick leg bud mesenchymal cells via downregulation of connexin 43 and integrin β4

Transforming growth factor β (TGF‐β) is a multifunctional cytokine that regulates a number of biological responses including chemotaxis, cell cycle progression, differentiation, and apoptosis of cells. Even though temporal and spatial expression of TGF‐β3 suggests its role in chick limb development, it is not well characterized how TGF‐β3 regulates chondrogenic differentiation of limb bud mesenchymal cells. In this study, differential display polymerase chain reaction (DD‐PCR) screening and reverse transcription PCR analysis revealed that the mRNA expression of the gap junction protein, connexin 43 (Cx43), was significantly decreased during the first treatment of TGF‐β3 for 24 h in cultured chick leg bud mesenchymal cells. Treatment of these cells with lindane, a general gap junction blocker, or expression of dominant negative Cx43 increased apoptotic cell death and decreased the level of integrin β4 protein, in a manner similar to that observed when these cells were exposed to TGF‐β3. Similarly, exposure of cultured leg chondroblasts to a functional blocking antibody against integrin‐β4 induced an increase in apoptosis. Treatment of cells with TGF‐β3 decreased the membrane translocation of PKC‐α, leading to activation of ERK. The increase in apoptotic cell death triggered by TGF‐β3 and dominant negative Cx43 was blocked by inhibition of ERK but increased by inhibition of PKC. Collectively, these data indicate that, in cultured chick leg bud mesenchyme cells, TGF‐β3 treatment downregulates Cx43 and induces apoptotic cell death via downregulation of integrin β4, activation of ERK and suppression of PKC‐α activation. J. Cell. Physiol. 214: 345–353, 2008.

Two non-coding RNAs, MicroRNA-101 and HOTTIP contribute cartilage integrity by epigenetic and homeotic regulation of integrin-α1

Non-coding RNAs have been less studied in cartilage development and destruction regulated by sophisticated molecular events despite their considerable theranostic potential. In this study, we identified significant down-regulation of mR-101 and up-regulation of lncRNA, HOTTIP in the processes of endochondral ossification and osteoarthritic progression. In wing mesenchymal cells, up-expression of miR-101 by TGF-β3 treatment is targeting DNMT-3B and thereby altered the methylation of integrin-α1 addressed as a positive regulator of endochondral ossification in this study. In like manner, down-regulation of miR-101 also coordinately up-regulated DNMT-3B, down-regulated integrin-α1, and resulted in cartilage destruction. In an OA animal model, introduction of lentiviruses that encoded miR-101 or integrin-α1 successfully reduced cartilage destruction. In like manner, long non-coding RNA (lncRNA), HOTTIP, a known regulator for HoxA genes, was highly up-regulated and concurrent down-regulation of HoxA13 displayed the suppression of integrin-α1 in OA chondrocytes. In conclusion, two non-coding RNAs, miR-101 and HOTTIP regulate cartilage development and destruction by modulating integrin-α1 either epigenetically by DNMT-3B or transcriptionally by HoxA13 and data further suggest that these non-coding RNAs could be a potent predictive biomarker for OA as well as a therapeutic target for preventing cartilage-related diseases.

MicroRNA-222 regulates MMP-13 via targeting HDAC-4 during osteoarthritis pathogenesis

Background Even though increasing evidences on miRNA involvement in human pathological responses, the distinct roles and related mechanisms of miRNAs in the pathology of osteoarthritis (OA) are not yet fully understood. Method RNA levels or protein levels of Apoptotic genes, HDACs, MMP-13, and miRNAs in human chondrocytes isolated from normal biopsy sample and OA cartilages were analyzed by real-time PCR or western blotting. Exogenous modulation of miR-222 level was performed using delivery of its specific precursor or specific inhibitor and target validation assay was applied to identify its potent target. In vivo study using DMM mice model was performed and assessed the degree of cartilage degradation. Results According to miRNA profiling, miR-222 was significantly down-regulated in OA chondrocytes. Over-expression of miR-222 significantly suppressed apoptotic death by down-regulating HDAC-4 and MMP-13 level. Moreover, 3′-UTR reporter assays showed that HDAC-4 is a direct target of miR-222. The treatment of chondrocytes with the HDAC inhibitor, trichostatin A (TSA), suppressed MMP-13 protein level and apoptosis, whereas the over-expression of HDAC-4 displayed opposite effects. The introduction of miR-222 into the cartilage of medial meniscus destabilized mice significantly reduced cartilage destruction and MMP-13 level. Conclusion Taken together, our data suggest that miR-222 may be involved in cartilage destruction by targeting HDAC-4 and regulating MMP-13 level.

MicroRNA-142-3p regulates TGF-β3-mediated region-dependent chondrogenesis by regulating ADAM9

Position-dependent chondrogenesis is regulated by processes that are both common to and differ among all limb types and limb skeletal elements. Despite intrinsic differences between wing and leg bud mesenchyme, the exact regulatory molecules and mechanisms involved in these processes have not been elucidated. Here, we show the limb type-specific role of TGF-β3 during chondrogenic differentiation of chick limb mesenchymal cells. Exposure of wing cells to TGF-β3 stimulated chondrogenic differentiation, whereas in leg bud mesenchymal cells, TGF-β3 induced apoptotic cell death via G2M arrest. Consistent with a limb type-specific effect of TGF-β3 on chondrogenic differentiation, we found different levels of miR-142-3p induction. Inhibition of miR-142-3p via PNA-based antisense oligonucleotides (ASOs) markedly promoted cell migration and precartilage condensation, while exogenous induction of miR-142-3p reduced cell survival and increased cell death. Overexpression of ADAM9 significantly reduced chondrogenic differentiation via downregulation of cell migration and cell survival and upregulation of apoptotic cell death. Limb type-specific expression levels of ADAM9 induced by TGF-β3 were observed. Collectively, this study demonstrates that differential induction of miR-142-3p is involved in the limb type-specific effect of TGF-β3 on wing vs. leg mesenchymal cells through direct modulation of ADAM9 transcription.