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. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1628–1635, 2014.

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. 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. 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. Taken together, our data suggest that miR-222 may be involved in cartilage destruction by targeting HDAC-4 and regulating MMP-13 level.

Here, we investigated the possible anti-cancer properties of bromelain in Kras mutant human colorectal carcinoma cell lines and a mouse model harboring a Kras mutation. Cell growth and proliferation were significantly reduced in the Kras mutant colorectal carcinoma cell lines following treatment with 50 μg/mL bromelain as assessed by crystal violet staining and a proliferation assay. To identify the molecules responsible for this action, the expression levels of genes involved in signaling pathways and miRNAs were analyzed by real-time PCR. Among the genes tested, down-regulation of ACSL-4 and up-regulation of miRNAs targeting ASCL-4 were observed in Caco₂ cells. Compared to the Kras wild-type colorectal carcinoma cell lines, Kras mutant colorectal carcinoma cell lines exhibited a remarkably up-regulated expression of ACSL-4, which is responsible for ferroptosis sensitivity. Moreover, the knockdown of ACSL-4 by a specific shRNA inhibited erastin-induced ferroptosis in Kras mutant DLD-1 cells as assessed by propidium iodide staining and lipid reactive oxygen species measurement. Our findings indicate that bromelain effectively exerts cytotoxic effects in Kras mutant colorectal cancer cells compared to in Kras wild-type colorectal cancer cells. Differential expression of ACSL-4 is responsible for the differential action of bromelain in regulating ferroptotic cell death.

Background: Even 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. Results: Human 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. Conclusions: miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.

Here, in Ppara^−/− mice, we found that an increased DNL stimulated the cartilage degradation and identified ACOT12 as a key regulatory factor. Suppressed level of ACOT12 was observed in cartilages of OA patient and OA-induced animal. To determine the role and association of ACOT12 in the OA pathogenesis, we generated Acot12 knockout (KO) (Acot12^−/−) mice using RNA-guided endonuclease. Acot12^−/− mice displayed the severe cartilage degradation with the stimulation of matrix MMPs and chondrocyte apoptosis through the accumulation of acetyl CoA. Delivery of acetyl CoA-conjugated chitosan complex into cartilage stimulated DNL and cartilage degradation. Moreover, restoration of ACOT12 into human OA chondrocytes and OA-induced mouse cartilage effectively rescued the pathophysiological features of OA by regulating DNL. Taken together, our study suggested ACOT12 as a novel regulatory factor in maintaining cartilage homeostasis and targeting ACOT12 could contribute to developing a new therapeutic strategy for OA.