Osteosarcoma Cells Secrete CXCL14 That Activates Integrin α11ß1 on Fibroblasts to Form a Lung Metastatic Niche.

Cooperation between primary malignant cells and stromal cells can mediate the establishment of lung metastatic niches. Here, we characterized the landscape of cell populations in the tumor microenvironment in treatment-naïve osteosarcoma using single-cell RNA sequencing and identified a stem cell-like cluster with tumor cell-initiating properties and prometastatic traits. CXCL14 was specifically enriched in the stem cell-like cluster and was also significantly upregulated in lung metastases compared with primary tumors. CXCL14 induced stromal reprogramming and evoked a malignant phenotype in fibroblasts to form a supportive lung metastatic niche. Binding of CXCL14 to heterodimeric integrin α11ß1 on fibroblasts activated actomyosin contractility and matrix remodeling properties. CXCL14-stimulated fibroblasts produced TGFß and increased osteosarcoma invasion and migration. mAbs targeting the CXCL14-integrin α11ß1 axis inhibited fibroblast TGFß production, enhanced CD8+ T cell-mediated antitumor immunity, and suppressed osteosarcoma lung metastasis. Taken together, these findings identify cross-talk between osteosarcoma cells and fibroblasts that promotes metastasis and demonstrate that targeting the CXCL14-integrin α11ß1 axis is a potential strategy to inhibit osteosarcoma lung metastasis. SIGNIFICANCE: Cooperation between stem-like osteosarcoma cells and fibroblasts mediated by a CXCL14-integrin α11ß1 axis creates a tumor-supportive lung metastatic niche and represents a therapeutic target to suppress osteosarcoma metastasis.

Role of immune cell infiltration and small molecule drugs in adhesive capsulitis: Novel exploration based on bioinformatics analyses.

Background: Adhesive capsulitis (AC) is a type of arthritis that causes shoulder joint pain, stiffness, and limited mobility. The pathogenesis of AC is still controversial. This study aims to explore the role of immune related factors in the occurrence and development of AC. Methods: The AC dataset was downloaded from Gene Expression Omnibus (GEO) data repository. Differentially expressed immune-related genes (DEIRGs) were obtained based on R package "DESeq2" and Immport database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to explore the functional correlation of DEIRGs. MCC method and Least Absolute Shrinkage and Selection Operator (LASSO) regression were conducted to identify the hub genes. The immune cell infiltration in shoulder joint capsule between AC and control was evaluated by CIBERSORTx, and the relationship between hub genes and infiltrating immune cells was analyzed by Spearman's rank correlation. Finally, potential small molecule drugs for AC were screened by the Connectivity Map database (CMap) and further verified by molecular docking. Results: A total of 137 DEIRGs and eight significantly different types of infiltrating immune cells (M0 macrophages, M1 macrophages, regulatory T cells, Tfh cells, monocytes, activated NK cells, memory resting CD4+T cells and resting dendritic cells) were screened between AC and control tissues. MMP9, FOS, SOCS3, and EGF were identified as potential targets for AC. MMP9 was negatively correlated with memory resting CD4+T cells and activated NK cells, but positively correlated with M0 macrophages. SOCS3 was positively correlated with M1 macrophages. FOS was positively correlated with M1 macrophages. EGF was positively correlated with monocytes. Additionally, dactolisib (ranked first) was identified as a potential small-molecule drug for the targeted therapy of AC. Conclusions: This is the first study on immune cell infiltration analysis in AC, and these findings may provide a new idea for the diagnosis and treatment of AC.

Identification of SLC3A2 as a Potential Therapeutic Target of Osteoarthritis Involved in Ferroptosis by Integrating Bioinformatics, Clinical Factors and Experiments.

Osteoarthritis (OA) is a type of arthritis that causes joint pain and limited mobility. In recent years, some studies have shown that the pathological process of OA chondrocytes is related to ferroptosis. Our study aims to identify and validate differentially expressed ferroptosis-related genes (DEFRGs) in OA chondrocytes and to investigate the potential molecular mechanisms. RNA-sequencing and microarray datasets were downloaded from Gene Expression Omnibus (GEO) data repository. Differentially expressed genes (DEGs) were screened by four methods: limma-voom, edgeR, DESeq2, and Wilcoxon rank-sum test. Weighted correlation network analysis (WGCNA), protein-protein interactions (PPI), and cytoHubba of Cytoscape were applied to identify hub genes. Clinical OA cartilage specimens were collected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis, western blotting (WB), histological staining, transmission electron microscopy (TEM), and transfection. Sankey diagram was used to visualize the relationships between the expression level of SLC3A2 in the damaged area and clinical factors. Based on bioinformatics analysis, clinical factors, and experiment validation, SLC3A2 was identified as a hub gene. It was down-regulated in OA cartilage compared to normal cartilage (p < 0.05). Functional enrichment analysis revealed that SLC3A2 was associated with ferroptosis-related functions. Spearman correlation analysis showed that the expression level of SLC3A2 in the OA cartilage-damaged area was closely related to BMI, obesity grade, and Kellgren-Lawrence grade. Furthermore, in vitro experiments validated that SLC3A2 inhibited ferroptosis and suppressed cartilage degeneration in OA. In summary, we demonstrated that SLC3A2 inhibited ferroptosis and suppressed cartilage degeneration in OA. These findings provide a new idea for the study of the pathogenesis of OA, thus providing new means for the clinical diagnosis and targeted therapy of OA.

MiR-654-3p, reduced by the excessive ALKBH5, Alleviated the Inflammation in OA by targeting TNFRSF9, the trigger of the NF-κB pathway.

MicroRNA (miRNA) is one of the most potent therapeutic targets for osteoarthritis (OA). We identified that miR-654-3p protected the phenotype of chondrocytes. We demonstrated that TNF receptor superfamily member 9 (TNFRSF9) was the target of miR-654-3p by binding to its 3'UTR regions, based on a dual-luciferase reporter assay and an RNA binding protein immunoprecipitation (RIP) assay. In addition, further experiments proved that TNFRSF9, as a trigger of the NF-κB pathway, correlated with the inflammation in chondrocytes. MiR-654-3p overexpressed in the knee of mice alleviated the OA in vivo. Moreover, we examined the m6A enzyme level in OA, proving that the abnormal expression of α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) contributed to the miR-654-3p decrease. Our research illustrated the significant role of miR-654-3p in OA, including its maturation and the mechanism in protecting the phenotype of chondrocytes, which could be a new treatment target for OA.

Hyaluronan size alters chondrogenesis of mesenchymal stem cells cultured on tricalcium phosphate-collagen-hyaluronan scaffolds.

Hyaluronan (HA) provides a favorable environment for chondrogenesis of bone marrow mesenchymal stem cells (BMSCs). A previous report from our group indicated that addition of HA increases the chondro-inductive capacity of scaffolds. Therefore, this study aimed to investigate whether the Mw of the HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. Human BMSCs (hBMSCs) and rabbit BMSCs (rBMSCs) were isolated and expanded. TCP-COL scaffolds and TCP-COL-HA scaffolds with two different HA Mws were assessed for their capacity to induce cartilage regeneration from hBMSCs in vitro and in vivo. The results showed that about 96.96% of hBMSCs expressed CD44. Moreover, Hyal-1 and chondrogenic marker genes expressions were increased in hMSCs seeded on TCP-COL-HA scaffolds, and blocking the HA-CD44 interaction with an anti-CD44 antibody reduced the expression levels of Hyal-1 and chondrogenic marker genes. Additionally, TCP-COL-HA scaffolds with 2000 kDa Mw showed greater induction of BMSC chondrogenesis induction compared with those with 80 kDa Mw. Similar results were observed in an ectopic implantation nude mouse model. In a rabbit osteochondral defect repair model, rBMSCs seeded on TCP-COL-HA scaffolds with 2000 kDa Mw showed greater cartilage regeneration than those seeded with 80 kDa Mw. In addition, hBMSC-seeded TCP-COL-HA scaffolds with 2000 kDa Mw showed a significantly higher mechanical strength than those with 80 kDa Mw. Collectively, these results indicate that the Mw of HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. The TCP-COL-HA scaffolds might be used as allogenic off the shelf products in cartilage tissue engineering in future.

Whole-Transcriptome Sequence of Degenerative Meniscus Cells Unveiling Diagnostic Markers and Therapeutic Targets for Osteoarthritis.

Meniscus plays an important role in joint homeostasis. Tear or degeneration of meniscus might facilitate the process of knee osteoarthritis (OA). Hence, to investigate the transcriptome change during meniscus degeneration, we reveal the alterations of messenger RNA (mRNA), microRNA (miRNA), long noncoding RNA (lncRNA), and circular RNA (circRNA) in meniscus during OA by whole-transcriptome sequence. A total of 375 mRNAs, 15 miRNAs, 56 lncRNAs, and 90 circRNAs were significantly altered in the degenerative meniscus treated with interleukin-1ß (IL-1ß). More importantly, highly specific co-expression RNA (ceRNA) networks regulated by lncRNA LOC107986251-miR-212-5p-SESN3 and hsa_circ_0018069-miR-147b-3p-TJP2 were screened out during IL-induced meniscus degeneration, unveiling potential therapeutic targets for meniscus degeneration during the OA process. Furthermore, lipocalin-2 (LCN2) and RAB27B were identified as potential biomarkers in meniscus degeneration by overlapping three previously constructed databases of OA menisci. LCN2 and RAB27B were both upregulated in osteoarthritic menisci and IL-1ß-treated menisci and were highly associated with the severity of OA. This could introduce potential novel molecules into the database of clinical diagnostic biomarkers and possible therapeutic targets for early-stage OA treatment.

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.

MiR-455-3p reduces apoptosis and alleviates degeneration of chondrocyte through regulating PI3K/AKT pathway.

AIMS: This study aimed to explore the functions of miR-455-3p, PTEN, and PI3K/AKT pathway in osteoarthritis. MATERIALS AND METHODS: We used the human bone marrow stem cell (BMSC), healthy chondrocytes, osteoarthritis chondrocytes (OA), and the IL-1ß/TNF-α-treated chondrocyte model to explore the relationship between miR-455-3p and PTEN. Mimic or inhibitor was used to transfect chondrocytes to determine whether miR-455-3p can regulate PTEN and influence COL2A1 and MMP13. Apoptosis was detected by flow cytometry. A luciferase report was applied to verify the targeted binding. KO mice were applied to investigate PTEN and pAKT expression and the effect on chondrocytes in vivo. KEY FINDINGS: MiR-455-3p and PTEN were reverse in chondrogenesis and healthy cartilage versus OA cartilage. Similar trends were noted in IL-1ß model. PTEN and MMP13 decreased and COL2A1 increased after overexpressing miR-455-3p, whereas the inhibition showed opposite results. Flow cytometry showed that miR-455-3p could reduce the apoptosis of chondrocytes. The results of luciferase revealed that miR-455-3p could affect fluorescence activity of PTEN by targeting its 3'-UTR. Finally, we found a marked increased in the expression of PTEN in KO mice relative to WT mice, while pAKT levels decreased. SIGNIFICANCE: It can be supported that miR-455-3p can reduce the apoptosis of chondrocytes and alleviate OA through regulating PI3K/AKT pathway, which may be expected to be a target for the treatment of osteoarthritis.

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.