Identification of anterior cruciate ligament fibroblasts and their contribution to knee osteoarthritis progression using single-cell analyses.

The mechanical properties of the anterior cruciate ligament (ACL) in the knee have been highlighted, but its role in the regulation of the joint microenvironment remains unclear, especially in the progression of Knee Osteoarthritis (KOA). Here, single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) data were integrated to reveal the transcriptional and epigenomic landscape of ACL in normal and OA states. We identified a novel subpopulation of fibroblasts in ACL, which provides new insights into the role of the ACL in knee homeostasis and disease. Degeneration of the ACL during OA mechanically alters the knee joint homeostasis and influences the microenvironment by regulating inflammatory- and osteogenic-related factors, thereby contributing to the progression of KOA. Additionally, the specific mechanism by which these Inflammation-associated Fibroblasts (IAFs) regulate KOA progression was uncovered, providing new foundation for the development of targeted treatments for KOA.

Exercise combined with administration of adipose-derived stem cells ameliorates neuropathic pain after spinal cord injury.

Experimental studies have shown that exercise and human adipose-derived stem cells (ADSCs) play positive roles in spinal cord injury (SCI). However, whether ADSCs and/or exercise have a positive effect on SCI-induced neuropathic pain is still unclear. Thus, there is a need to explore the effects of exercise combined with administration of ADSCs on neuropathic pain after SCI. In this study, a thoracic 11 (T11) SCI contusion model was established in adult C57BL/6 mice. Exercise was initiated from 7 days post-injury and continued to 28 days post-injury, and approximately 1 × 105 ADSCs were transplanted into the T11 spinal cord lesion site immediately after SCI. Motor function and neuropathic pain-related behaviors were assessed weekly using the Basso Mouse Scale, von Frey filament test, Hargreaves method, and cold plate test. Histological studies (Eriochrome cyanine staining and immunohistochemistry) were performed at the end of the experiment (28 days post-injury). Exercise combined with administration of ADSCs partially improved early motor function (7, 14, and 21 days post-injury), mechanical allodynia, mechanical hypoalgesia, thermal hyperalgesia, and thermal hypoalgesia. Administration of ADSCs reduced white and gray matter loss at the lesion site. In addition, fewer microglia and astrocytes (as identified by expression of ionized calcium-binding adapter molecule 1 and glial fibrillary acidic protein, respectively) were present in the lumbar dorsal horn in the SCI + ADSCs and SCI + exercise + ADSCs groups compared with the sham group. Our findings suggest that exercise combined with administration of ADSCs is beneficial for the early recovery of motor function and could partially ameliorate SCI-induced neuropathic pain.

Circular RNA CREBBP modulates cartilage degradation by activating the Smad1/5 pathway through the TGFß2/ALK1 axis.

Osteoarthritis, characterized by articular cartilage degradation, is the leading cause of chronic disability in older adults. Studies have indicated that circular RNAs are crucial regulators of chondrocyte development and are involved in the progression of osteoarthritis. In this study, we investigated the function and mechanism of a circular RNA and its potential for osteoarthritis therapy. The expression levels of circCREBBP, screened by circular RNA sequencing during chondrogenic differentiation in adipose tissue-derived stem cells, and TGFß2 were significantly increased in the cartilage of patients with osteoarthritis and IL-1ß-induced chondrocytes. circCREBBP knockdown increased anabolism in the extracellular matrix and inhibited chondrocyte degeneration, whereas circCREBBP overexpression led to the opposite effects. Luciferase reporter assays, rescue experiments, RNA immunoprecipitation, and RNA pulldown assays confirmed that circCREBBP upregulated TGFß2 expression by sponging miR-1208, resulting in significantly enhanced phosphorylation of Smad1/5 in chondrocytes. Moreover, intra-articular injection of adeno-associated virus-sh-circCrebbp alleviated osteoarthritis in a mouse model of destabilization of the medial meniscus. Our findings reveal a critical role for circCREBBP in the progression of osteoarthritis and provide a potential target for osteoarthritis therapy.

tRNA-Derived Fragment tRF-5009A Regulates Autophagy and Degeneration of Cartilage in Osteoarthritis via Targeting mTOR.

tRNA-derived fragments (tRFs) have been reported to have critical regulatory roles in osteoarthritis (OA). Recent studies have suggested that autophagy promotes the homeostasis of the extracellular matrix of chondrocytes in OA. However, the role of tRFs in posttranscriptional gene regulation during autophagy in OA is unknown. Therefore, we explored the role of tRF-5009A in the posttranscriptional gene regulation of autophagy and cartilage degeneration in OA. Using RNA sequencing, we identified tRF-5009A, the tRNAValCAC-derived fragment, in OA tissues and explored its expression by quantitative reverse transcription PCR and fluorescence in situ hybridization. We further investigated the relationship between the expression of tRF-5009A and clinical factors in OA. Chondrocytes were transfected with a tRF-5009A inhibitor or mimic to determine their functions, including in relation to autophagy and the cartilage phenotype. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3'-untranslated region (UTR) of mTOR contains a tRF-5009A-binding site. tRF-5009A was downregulated in the cartilage of OA knees, especially in damaged areas. mTOR was highly expressed in damaged cartilage and negatively correlated with the expression of tRF-5009A; transfection with a tRF-5009A inhibitor promoted the expression of mTOR and suppressed autophagy, whereas transfection with a tRF-5009A mimic had the opposite effect. A dual-luciferase reporter assay showed that tRF-5009A silenced the expression of mTOR by binding to its 3'-UTR. Thus, tRF-5009A regulates autophagy and cartilage degeneration in OA by targeting mTOR. In summary, these findings provide an additional tool for the clinical diagnosis and novel targeted therapy of OA.

CircNFIX regulates chondrogenesis and cartilage homeostasis by targeting the miR758-3p/KDM6A axis.

OBJECTIVES: Osteoarthritis (OA) is a degenerative disease causing the progressive destruction of articular cartilage; however, the aetiology has not yet been elucidated. Circular RNAs (circRNAs) are reportedly involved in cartilage degeneration and OA development. In the present study, we identified that circNFIX regulates chondrogenesis and cartilage homeostasis. MATERIALS AND METHODS: Microarray analysis was performed to explore circRNA expression during the chondrogenic differentiation of human adipose-drived stem cells (hADSCs). CircNFIX expression was determined using quantitative reverse transcription-polymerase chain reaction and in situ hybridization. Gain- and loss-of-function assays were performed to validate the role of circNFIX in cartilage homeostasis. RNA pull-down, Argonaute2-RNA immunoprecipitation and luciferase reporter assays were performed to evaluate the interactions among circNFIX, miR758-3p and KDM6A. RESULTS: CircNFIX expression was upregulated in the early and middle stages, whereas downregulated in the late stage of hADSC chondrogenesis. CircNFIX inhibition attenuated hADSC chondrogenesis. CircNFIX was remarkably downregulated in OA samples, circNFIX overexpression protected against chondrocyte degradation and alleviated OA progression in the destabilization of the medial meniscus OA model. Mechanistically, circNFIX acted as a sponge of miR758-3p and played a role in the chondrogenesis and chondrocyte degeneration by targeting the miR-758-3p/KDM6A axis. CONCLUSIONS: Our results revealed a key role of circNFIX in chondrogenesis and cartilage homeostasis, which may provide a potential therapeutic strategy for OA treatment.

Mitochonic Acid-5 Inhibits Reactive Oxygen Species Production and Improves Human Chondrocyte Survival by Upregulating SIRT3-Mediated, Parkin-dependent Mitophagy.

Mitochondrial dysfunction is related to the pathogenesis of osteoarthritis (OA); however, there are no effective drugs to treat OA for maintaining mitochondrial homeostasis. Studies have shown that mitochonic acid-5 (MA-5) has a protective effect against mitochondrial damage and plays a role in mitophagy. However, it is not clear whether MA-5 has a beneficial effect on inflammatory articular cartilage. Here, human OA cartilage was obtained from patients undergoing total joint replacement. Interleukin-1ß (IL-1ß) was used to stimulate chondrocytes and induce inflammatory injury. Cell Counting Kit-8, TUNEL, and flow cytometry assays were used to assess apoptosis. Gene expression was examined using quantitative reverse transcription-polymerase chain reaction. Mitochondrial function was evaluated using immunoblotting, mitochondrial membrane potential assay, JC-1 staining, and immunofluorescence analysis. Mitophagy was detected using immunoblotting and immunofluorescence. 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP), a specific inhibitor of Sirtuin 3 (SIRT3), was used to block the SIRT3/Parkin pathway. Mitophagy in the cartilage sections was evaluated via immunohistochemistry. IL-1ß was found to induce chondrocyte apoptosis by inhibiting SIRT3 expression and mitophagy. In addition, inflammatory damage reduced the mitochondrial membrane potential and promoted the production of intracellular reactive oxygen species (ROS), leading to increased mitochondrial division, mitochondrial fusion inhibition, and the consequent mitochondrial damage. In contrast, the MA-5 treatment inhibited excessive ROS production by upregulating mitophagy, maintaining the mitochondrial membrane potential, and reducing mitochondrial apoptosis. After chemically blocking SIRT3 with 3-TYP, Parkin-related mitophagy was also inhibited, an effect that was prevented by pretreatment of the chondrocytes with MA-5, thereby suggesting that SIRT3 is upstream of Parkin. Overall, MA-5 was found to enhance the activity of SIRT3, promote Parkin-dependent mitophagy, eliminate depolarized/damaged mitochondria in chondrocytes, and protect cartilage cells. In conclusion, MA-5 inhibits IL-1ß-induced oxidative stress and protects chondrocytes by upregulating the SIRT3/Parkin-related autophagy signaling pathway.

tRNA-derived fragment TRF365 regulates the metabolism of anterior cruciate ligament cells by targeting IKBKB.

tRNA-derived fragments (tRFs) are new noncoding RNAs, and recent studies have shown that tRNAs and tRFs have important functions in cell metabolism via posttranscriptional regulation of gene expression. However, whether tRFs regulate cellular metabolism of the anterior cruciate ligament (ACL) remains elusive. The aim of this study was to investigate the role and action mechanism of tRFs in ACL cell metabolism. A tRF array was used to determine tRF expression profiles in different human ACL cells, and quantitative real-time polymerase chain reaction and fluorescence in situ hybridisation were used to determine TRF365 expression. ACL cells were transfected with a TRF365 mimic or a TRF365 inhibitor to determine whether TRF365 regulates IKBKB expression. A rescue experiment and dual-luciferase reporter assay were conducted to determine whether the 3'-untranslated region (UTR) of IKBKB has a TRF365-binding site. TRF365 was weakly expressed in osteoarthritis (OA) ACL and interleukin-1ß-treated ACL cells. IKBKB was highly expressed in OA ACL and interleukin-1ß-treated ACL cells; transfection with the TRF365 mimic suppressed IKBKB expression, whereas transfection with the TRF365 inhibitor had the opposite effect. A dual-luciferase reporter assay showed that TRF365 silenced the expression of IKBKB by binding to its 3'-UTR. Thus, TRF365 regulates the metabolism of ACL cells by targeting IKBKB. In summary, TRF365 may provide a new direction for the study of ACL degeneration and on the pathophysiological process of OA.

Long Non-coding RNAs LOC100126784 and POM121L9P Derived From Bone Marrow Mesenchymal Stem Cells Enhance Osteogenic Differentiation via the miR-503-5p/SORBS1 Axis.

Long non-coding RNAs (lncRNAs) play pivotal roles in mesenchymal stem cell differentiation. However, the mechanisms by which non-coding RNA (ncRNA) networks regulate osteogenic differentiation remain unclear. Therefore, our aim was to identify RNA-associated gene and transcript expression profiles during osteogenesis in bone marrow mesenchymal stem cells (BMSCs). Using transcriptome sequencing for differentially expressed ncRNAs and mRNAs between days 0 and 21 of osteogenic differentiation of BMSCs, we found that the microRNA (miRNA) miR-503-5p was significantly downregulated. However, the putative miR-503-5p target, sorbin and SH3 domain containing 1 (SORBS1), was significantly upregulated in osteogenesis. Moreover, through lncRNA-miRNA-mRNA interaction analyses and loss- and gain-of-function experiments, we discovered that the lncRNAs LOC100126784 and POM121L9P were abundant in the cytoplasm and enhanced BMSC osteogenesis by promoting SORBS1 expression. In contrast, miR-503-5p reversed this effect. Ago2 RNA-binding protein immunoprecipitation and dual-luciferase reporter assays further validated the direct binding of miR-503-5p to LOC100126784 and POM121L9P. Furthermore, SORBS1 knockdown suppressed early osteogenic differentiation in BMSCs, and co-transfection with SORBS1 small interfering RNAs counteracted the BMSCs' osteogenic capacity promoted by LOC100126784- and POM121L9P-overexpressing lentivirus plasmids. Thus, the present study demonstrated that the lncRNAs LOC100126784 and POM121L9P facilitate the osteogenic differentiation of BMSCs via the miR-503-5p/SORBS1 axis, providing potential therapeutic targets for treating osteoporosis and bone defects.

Exosome-transported circRNA_0001236 enhances chondrogenesis and suppress cartilage degradation via the miR-3677-3p/Sox9 axis.

OBJECTIVES: Aberrations in exosomal circular RNA (circRNA) expression have been identified in various human diseases. In this study, we investigated whether exosomal circRNAs could act as competing endogenous RNAs (ceRNAs) to regulate the pathological process of osteoarthritis (OA). This study aimed to elucidate the specific MSC-derived exosomal circRNAs responsible for MSC-mediated chondrogenic differentiation using human bone marrow-derived MSCs (hMSCs) and a destabilization of the medial meniscus (DMM) mouse model of OA. METHODS: Exosomal circRNA deep sequencing was performed to evaluate the expression of circRNAs in human bone marrow-derived MSCs (hMSCs) induced to undergo chondrogenesis from day 0 to day 21. The regulatory and functional roles of exosomal circRNA_0001236 were examined on day 21 after inducing chondrogenesis in hMSCs and were validated in vitro and in vivo. The downstream target of circRNA_0001236 was also explored in vitro and in vivo using bioinformatics analyses. A luciferase reporter assay was used to evaluate the interaction between circRNA_0001236 and miR-3677-3p as well as the target gene sex-determining region Y-box 9 (Sox9). The function and mechanism of exosomal circRNA_0001236 in OA were explored in the DMM mouse model. RESULTS: Upregulation of exosomal circRNA_0001236 enhanced the expression of Col2a1 and Sox9 but inhibited that of MMP13 in hMSCs induced to undergo chondrogenesis. Moreover, circRNA_0001236 acted as an miR-3677-3p sponge and functioned in human chondrocytes via targeting miR-3677-3p and Sox9. Intra-articular injection of exosomal circRNA_0001236 attenuated OA in the DMM mouse model. CONCLUSIONS: Our results reveal an important role for a novel exosomal circRNA_0001236 in chondrogenic differentiation. Overexpression of exosomal circRNA_0001236 promoted cartilage-specific gene and protein expression through the miR-3677-3p/Sox9 axis. Thus, circRNA_0001236-overexpressing exosomes may alleviate cartilage degradation, suppressing OA progression and enhancing cartilage repair. Our findings provide a potentially effective therapeutic strategy for treating OA.

Characterization of exosomal long non-coding RNAs in chondrogenic differentiation of human adipose-derived stem cells.

The exosomes derived from chondrogenic stem cells and long non-coding RNAs (lncRNAs) play a key role in cartilage regeneration. Here, we investigated the expression profile of exosomal lncRNAs in chondrogenesis of human adipose derived stem cells (hADSCs). hADSCs were induced to differentiate into chondrocytes in vitro. Exosomes from undifferentiated hADSCs and chondrogenic hADSCs were isolated. LncRNA and mRNA expression profiles in the isolated exosomes were analyzed by RNA sequencing. The resultant data were subjected to gene ontology (GO) terms and KEGG pathway analysis to identify differentially expressed lncRNAs. We identified 23 upregulated and 163 downregulated lncRNAs in exosomes derived from chondrogenic hADSCs compared to that in exosomes from undifferentiated hADSCs. In addition, analysis of mRNA expression data revealed 968 upregulated genes and 572 downregulated genes in exosomes of chondrogenic hADSCs. Lncrna and mRNA expression levels were further validated by qRT-PCR. Differentially expressed lncRNAs and mRNAs were utilized to construct a coding-non-coding gene co-expression network (CNC network). GO terms and KEGG pathway enrichment analysis revealed several significant processes differentially regulated between undifferentiated hADSCs and chondrogenic hADSCs. Taken together, this study revealed the differential expression of exosomal lncRNAs of chondrogenic hADSCs and provided a foundation for future study on the cartilage recovery mechanism of exosomes derived from chondrogenic stem cells.

Inhibition of miR-490-5p Promotes Human Adipose-Derived Stem Cells Chondrogenesis and Protects Chondrocytes via the PITPNM1/PI3K/AKT Axis.

MicroRNAs (miRNAs) play a pivotal role in cartilage development and homeostasis in osteoarthritis (OA). While the fundamental roles of miRNAs in cartilage degeneration have been extensively studied, their effects on chondrogenic differentiation induced by human adipose-derived stem cells (hADSCs) and the underlying mechanisms remain largely elusive. Here, we investigated the roles and mechanisms of miRNAs in hADSC chondrogenic differentiation and chondrocyte homeostasis. Using microarray analysis, we screened miRNAs expressed in the chondrogenic differentiated hADSCs and identified miR-490-5p as the most significantly down-regulated miRNA. We analyzed its expression patterns during chondrogenesis in vivo and in vitro. Our study showed that miR-490-5p overexpression promoted the transition of hADSCs from chondrogenesis to osteogenesis. In addition, based on miRNA-mRNA prediction analysis and dual-luciferase reporter assay, we proposed and proved that miR-490-5p targeted PITPNM1 by binding to its 3'-UTR and inhibiting its translation. Moreover, loss- and gain-of-function experiments identified the involvement of the PI3K/AKT signaling pathway, and a rescue experiment determined the effect and specific mechanism of the miR-490-5p/PITPNM1/PI3K/AKT axis in hADSC chondrogenic differentiation and chondrocyte homeostasis. Inhibition of miR-490-5p alleviated cartilage injury in vivo as demonstrated using the destabilization of the medial meniscus (DMM) OA model. We identified miR-490-5p as a novel modulator of hADSC-mediated chondrogenesis and chondrocyte phenotype. This study highlighted that miR-490-5p attenuated hADSC chondrogenesis and accelerated cartilage degradation through activation of the PI3K/AKT signaling pathway by targeting PITPNM1. Inhibition of miR-490-5p facilitated hADSC chondrogenic differentiation and protected chondrocyte phenotype via the PITPNM1/PI3K/AKT axis, thus providing a novel stem cell potential therapeutic target for OA treatment.

Long non-coding RNA MEG3 is involved in osteogenic differentiation and bone diseases (Review).

Osteogenic differentiation originating from mesenchymal stem cells (MSCs) requires tight co-ordination of transcriptional factors, signaling pathways and biomechanical cues. Dysregulation of such reciprocal networks may influence the proliferation and apoptosis of MSCs and osteoblasts, thereby impairing bone metabolism and homeostasis. An increasing number of studies have shown that long non-coding (lnc)RNAs are involved in osteogenic differentiation and thus serve an important role in the initiation, development, and progression of bone diseases such as tumors, osteoarthritis and osteoporosis. It has been reported that the lncRNA, maternally expressed gene 3 (MEG3), regulates osteogenic differentiation of multiple MSCs and also acts as a critical mediator in the development of bone formation and associated diseases. In the present review, the proposed mechanisms underlying the roles of MEG3 in osteogenic differentiation and its potential effects on bone diseases are discussed. These discussions may help elucidate the roles of MEG3 in osteogenic differentiation and highlight potential biomarkers and therapeutic targets for the treatment of bone diseases.

Single-cell RNA-seq analysis identifies meniscus progenitors and reveals the progression of meniscus degeneration.

OBJECTIVES: The heterogeneity of meniscus cells and the mechanism of meniscus degeneration is not well understood. Here, single-cell RNA sequencing (scRNA-seq) was used to identify various meniscus cell subsets and investigate the mechanism of meniscus degeneration. METHODS: scRNA-seq was used to identify cell subsets and their gene signatures in healthy human and degenerated meniscus cells to determine their differentiation relationships and characterise the diversity within specific cell types. Colony-forming, multi-differentiation assays and a mice meniscus injury model were used to identify meniscus progenitor cells. We investigated the role of degenerated meniscus progenitor (DegP) cell clusters during meniscus degeneration using computational analysis and experimental verification. RESULTS: We identified seven clusters in healthy human meniscus, including five empirically defined populations and two novel populations. Pseudotime analysis showed endothelial cells and fibrochondrocyte progenitors (FCP) existed at the pseudospace trajectory start. Melanoma cell adhesion molecule ((MCAM)/CD146) was highly expressed in two clusters. CD146+ meniscus cells differentiated into osteoblasts and adipocytes and formed colonies. We identified changes in the proportions of degenerated meniscus cell clusters and found a cluster specific to degenerative meniscus with progenitor cell characteristics. The reconstruction of four progenitor cell clusters indicated that FCP differentiation into DegP was an aberrant process. Interleukin 1ß stimulation in healthy human meniscus cells increased CD318+ cells, while TGFß1 attenuated the increase in CD318+ cells in degenerated meniscus cells. CONCLUSIONS: The identification of meniscus progenitor cells provided new insights into cell-based meniscus tissue engineering, demonstrating a novel mechanism of meniscus degeneration, which contributes to the development of a novel therapeutic strategy.

Long non-coding RNA MALAT1 as a valuable biomarker for prognosis in osteosarcoma: A systematic review and meta-analysis.

BACKGROUND: Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA, MALAT1) has been found to be aberrantly expressed in osteosarcoma, while high MALAT1 expression is correlated with both metastasis and prognosis. This meta-analysis set out to investigate the prognostic value of lncRNA MALAT1 in patients living with osteosarcoma. METHODS: We conducted a systematical search of available databases from inception to May 2019. Odds ratios (OR) of clinical parameters, as well as hazard ratio (HR) of overall survival (OS), were calculated in order to evaluate the relationship between MALAT1 expression and the prognosis of patients living with osteosarcoma. RESULTS: Nine eligible studies which included a total of 599 osteosarcoma patients were enrolled in the present study. Pooled results found that high MALAT1 expression was associated with clinical stage and distant metastasis, but not age, gender, tumor anatomical location or tumor size. When compared to patients with low MALAT1 expression, patients with high MALAT1 expression were markedly correlated with a worse OS. Moreover, MALAT1 may be an independent predictive factor for OS in patients living with osteosarcoma. CONCLUSIONS: This meta-analysis suggests that high MALAT1 expression is associated with advanced clinicopathological features as well as unfavorable prognosis. LncRNA MALAT1 has the potential to serve as a moderate prognostic biomarker for osteosarcoma.

MicroRNA-455-3p promotes TGF-ß signaling and inhibits osteoarthritis development by directly targeting PAK2.

MicroRNAs (miRNAs, miR) play a key role in the pathogenesis of osteoarthritis (OA). Few studies have examined the regulatory role of P21-activated kinases (PAKs), a family of serine/threonine kinases, in OA. The aim of this study was to determine whether miR-455-3p can regulate cartilage degeneration in OA by targeting PAK2. MiR-455-3p knockout mice showed significant degeneration of the knee cartilage. MiR-455-3p expression increased and PAK2 expression decreased in the late stage of human adipose-derived stem cell (hADSC) chondrogenesis and in chondrocytes affected by OA. Furthermore, in both miR-455-3p-overexpressing chondrocytes and PAK2-suppressing chondrocytes, cartilage-specific genes were upregulated, and hypertrophy-related genes were downregulated. A luciferase reporter assay confirmed that miR-455-3p regulates PAK2 expression by directly targeting the 3'-untranslated regions (3'UTRs) of PAK2 mRNA. IPA-3, a PAK inhibitor, inhibited cartilage degeneration due to OA. Moreover, suppressing PAK2 promoted R-Smad activation in the TGF/Smad signaling pathway in chondrocytes. Altogether, our results suggest that miR-455-3p promotes TGF-ß/Smad signaling in chondrocytes and inhibits cartilage degeneration by directly suppressing PAK2. These results thus indicate that miR-455-3p and PAK2 are novel potential therapeutic agents and targets, respectively, for the treatment of OA.

Long Non-coding RNA HOTTIP Promotes CCL3 Expression and Induces Cartilage Degradation by Sponging miR-455-3p.

Long non-coding RNAs (lncRNAs) play pivotal roles in diseases such as osteoarthritis (OA). However, knowledge of the biological roles of lncRNAs is limited in OA. We aimed to explore the biological function and molecular mechanism of HOTTIP in chondrogenesis and cartilage degradation. We used the human mesenchymal stem cell (hMSC) model of chondrogenesis, in parallel with, tissue biopsies from normal and OA cartilage to detect HOTTIP, CCL3, and miR-455-3p expression in vitro. Biological interactions between HOTTIP and miR-455-3p were determined by RNA silencing and overexpression in vitro. We evaluated the effect of HOTTIP on chondrogenesis and degeneration, and its regulation of miR-455-3p via competing endogenous RNA (ceRNA). Our in vitro ceRNA findings were further confirmed within animal models in vivo. Mechanisms of ceRNAs were determined by bioinformatic analysis, a luciferase reporter system, RNA pull-down, and RNA immunoprecipitation (RIP) assays. We found reduced miR-455-3p expression and significantly upregulated lncRNA HOTTIP and CCL3 expression in OA cartilage tissues and chondrocytes. The expression of HOTTIP and CCL3 was increased in chondrocytes treated with interleukin-1ß (IL-1ß) in vitro. Knockdown of HOTTIP promoted cartilage-specific gene expression and suppressed CCL3. Conversely, HOTTIP overexpression reduced cartilage-specific genes and increased CCL3. Notably, HOTTIP negatively regulated miR-455-3p and increased CCL3 levels in human primary chondrocytes. Mechanistic investigations indicated that HOTTIP functioned as ceRNA for miR-455-3p enhanced CCL3 expression. Taken together, the ceRNA regulatory network of HOTTIP/miR-455-3p/CCL3 plays a critical role in OA pathogenesis and suggests HOTTIP is a potential target in OA therapy.

Effects of adductor canal block versus femoral nerve block in patients with anterior cruciate ligament reconstruction: A protocol for a systematic review and meta-analysis.

OBJECTIVE: It is reported that both adductor canal block (ACB) and femoral nerve block (FNB) are commonly used methods for postoperative analgesia in anterior cruciate ligament (ACL) reconstruction. Currently, no record has compared the efficacy of postoperative pain relief and the influence to quadriceps strength between them. This study aims to provide a protocol to compare the efficacy and safety between ACB and FNB for the postoperative analgesia of ACL reconstruction. METHODS: This study will be performed in accordance with the guideline of the Preferred Reporting Items for Systematic Review and Meta-analysis Protocols. Online databases including PubMed, Embase, Web of Science, Cochrane Library, Wanfang database, and the Chinese National Knowledge Infrastructure database will be systematically searched from their inception up May 31, 2019. All randomized controlled trials will be included in present meta-analysis. The quality of enrolled literatures will be evaluated by using the Cochrane Collaboration Risk of bias Tool. Statistical analysis will be calculated by the Review Manager 5.3. RESULTS: This review will investigate the efficacy and safety of ACB compared with FNB in patients undergoing ACL reconstruction. The primary outcomes are visual analog scale, cumulative opioid consumption during 24 hours after surgery, numerical rating scale, and the time to first straight-leg raise. The secondary outcomes include maximal voluntary isometric contraction, stretching torque at 3, 6 months' follow-up, and adverse effects. CONCLUSION: Findings of this systematic review and meta-analysis will summarize the current evidence in postoperative analgesia for ACL reconstruction and also provide implications for clinical practice.

MicroRNA-320c inhibits development of osteoarthritis through downregulation of canonical Wnt signaling pathway.

AIMS: Osteoarthritis (OA) is a leading cause of deformity in aging people. Emerging evidence suggests that microRNAs and Wnt signaling pathway are associated with its pathogenesis. We aimed to determine whether microRNA-320c inhibits the development of osteoarthritis by suppressing Wnt signaling pathway. MATERIALS AND METHODS: MiR-320c and ß-catenin expression was assessed in human adipose derived stem cells (hADSCs) model of chondrogenesis and in normal and OA primary human chondrocytes. OA chondrocytes were transfected with miR-320c or its antisense inhibitor and ß-catenin siRNA respectively. Direct interaction between miR-320c and ß-catenin mRNA as well as activity of ß-catenin/TCF complex were confirmed by luciferase reporter assay. Mmu-miR-320-3p agomir was intra-articularly injected in collagenase-induced OA mouse model. OA progression was evaluated histologically and immunohistochemically. KEY FINDINGS: MiR-320c was decreased and ß-catenin was increased in OA chondrocytes and late stage of hADSCs chondrogenesis. Overexpression of miR-320c and knockdown of ß-catenin had similar effects that the cartilage-specific genes were elevated and hypertrophy-related genes were down-regulated in OA chondrocytes. Luciferase reporter assay confirm that miR-320c regulated the expression of ß-catenin by directly targeting 3'UTR of ß-catenin mRNA and decreased the relative transcriptional activity of the ß-catenin/TCF complex. Injection of mmu-miR-320-3p attenuated OA progression in the OA mouse model. SIGNIFICANCE: Our results supports that miR-320c can inhibits the degeneration of osteoarthritis chondrocytes via suppressing the canonical Wnt signaling pathway and indicates the potential of miR-320c as a novel therapeutic agent for osteoarthritis treatment.

miR-193b-5p regulates chondrocytes metabolism by directly targeting histone deacetylase 7 in interleukin-1ß-induced osteoarthritis.

There is increasing evidence regarding the pivotal roles of microRNAs (miRNAs) and histone deacetylases (HDACs) in the development of osteoarthritis (OA). This study aimed to determine whether miR-193b-5p regulates HDAC7 expression directly to affect cartilage degeneration. Expression levels of miR-193b-5p, HDAC7, matrix metalloproteinase 3 (MMP3), and MMP13 were determined in normal and OA cartilage and primary human chondrocytes (PHCs) stimulated with interleukin-1ß (IL-1ß). PHCs were transfected with a miR-193b-5p mimic or inhibitor to verify whether miR-193b-5p influences the expression of HDAC7 and MMPs. A luciferase reporter assay was performed to demonstrate the binding between miR-193b-5p and the 3'-untranslated region (UTR) of HDAC7. Expression of miR-193b-5p was reduced in IL-1ß-stimulated PHCs and in OA cartilage compared to that in normal cartilage. Luciferase reporter assay exhibited the repressed activity of the reporter construct containing the 3'UTR of HDAC7. Both miR-193b-5p overexpression and HDAC7 inhibition decreased the expression of MMP3 and MMP13, whereas the inhibition of miR-193b-5p enhanced HDAC7, MMP3, and MMP13 expression. miR-193b-5p downregulates HDAC7 directly and, as a result, inhibits MMP3 and MMP13 expression, which suggests that miR-193b-5p has a protective role in OA.