M2 macrophage-derived exosomal miR-26b-5p regulates macrophage polarization and chondrocyte hypertrophy by targeting TLR3 and COL10A1 to alleviate osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent chronic musculoskeletal diseases among the elderly population. In this study, macrophage-derived exosomes were isolated and identified. Exosomes were subjected to microRNA (miRNA) sequencing and bioinformatic analysis, and differentially expressed miRNAs were verified. miR-26b-5p target genes were confirmed through target-site mutation combined with a dual-luciferase reporter assay. The effects of miR-26b-5p on macrophage polarization and chondrocyte hypertrophy were assessed in vitro. miR-26b-5p agomir was applied to mice with OA induced by anterior cruciate ligament transection (ACLT). The therapeutic effects of miR-26b-5p were evaluated via pain behavior experiments and histological observations. In vitro, miR-26b-5p repolarized M1 macrophages to an anti-inflammatory M2 type by targeting the TLR3 signaling pathway. miR-26b-5p could target COL10A1, further inhibiting chondrocyte hypertrophy induced by M1 macrophage-conditioned medium (M1-CM). In vivo, miR-26b-5p agomir ameliorated gait abnormalities and mechanical allodynia in OA mice. miR-26b-5p treatment attenuated synovitis and cartilage degeneration, thereby delaying OA progression. In conclusion, M2 macrophage-derived exosomal miR-26b-5p could protect articular cartilage and ameliorate gait abnormalities in OA mice by targeting TLR3 and COL10A1. miR-26b-5p further affected macrophage polarization and chondrocyte hypertrophy. Thus, this exosomal miR-26b-5p-based strategy might be a potential method for OA treatment.

Experimental validation and pan-cancer analysis identified COL10A1 as a novel oncogene and potential therapeutic target in prostate cancer.

BACKGROUND: Type X collagen (COL10) is a homologous trimeric non-fibrillar collagen found in the extracellular matrix of human tissues, and it exhibits a distinctive white appearance. Type X collagen α1 chain (COL10A1) is a specific cleaved fragment of type X collagen. However, the expression, prognostic significance, clinicopathological attributes and immune-related associations of COL10A1 in prostate cancer as well as in pan-cancer contexts remain poorly understood. METHODS: Using bioinformatic analysis of data from the most recent databases (TCGA, GTEx and GEO databases), we have extensively elucidated the role played by COL10A1 in terms of its expression patterns, prognostic implications, and immune efficacy across a pan-cancer spectrum. Subsequently, the biological functions of COL10A1 in prostate cancer were elucidated by experimental validation. RESULTS: Our findings have confirmed that COL10A1 was highly expressed in most cancers and was associated with poorer prognosis in cancer patients. Immune correlation analysis of COL10A1 in various cancers showed its significant correlation with Tumor mutational burden (TMB), microsatellite instability (MSI) and immune cell infiltration. In addition, knockdown of COL10A1 in prostate cancer resulted in a substantial reduction in the proliferation, migration, and invasive potential of prostate cancer cells. CONCLUSION: Our pan-cancer analysis of COL10A1 gene provided novel insights into its pivotal role in cancer initiation, progression, and therapeutic implications, underscoring its potential significance in prognosis and immunotherapeutic interventions for cancer, particularly prostate cancer.

GuiLu-ErXian Glue extract promotes mesenchymal stem cells (MSC)-Induced chondrogenesis via exosomes release and delays aging in the MSC senescence process.

ETHNOPHARMACOLOGICAL RELEVANCE: The treatment of osteoarthritis (OA) patients is a challenging problem. Mesenchymal stem cells (MSCs) are multipotent cells and play key roles in regenerative medicine for cartilage degeneration. GuiLu-ErXian Glue (GLEXG) is an herbal remedy widely used in traditional Chinese medicine to treat joint pain and disability in elderly OA patients. However, the mechanisms of how GLEXG affects MSCs-induced chondrogensis remains to be elucidated. AIM OF THE STUDY: The aim of this study was to investigate the effects of GLEXG on MSC-derived chondrogenesis, both in vitro and in vivo and its potential mechanisms. METHODS: Using human MSC (hMSCs) as in vitro model, the effects of HPLC-profiled GLEXG water extract on chondrogenic differentiation were investigated by 3D spheroid cultures under chondrogenesis-inducing medium (CIM) condition. The chondrogenesis process was evaluated by measuring the sphere sizes, chondrogenesis-related genes expression by reverse transcription real-time PCR that targeted type II/X collagens, SOX9, aggrecan, and protein expression by immunostaining. Anti-TGF-ß1 neutralization antibody was used for mechanistic study. Mono-iodoacetate (MIA) induced OA joint was used to evaluate the effects of GLEXG on in vivo model. MSCs-derived exosomes were purified for proteomics study and senescence process was evaluated by cumulative population doublings and senescence-associated ß-Galactosidase staining. RESULTS: The results showed that GLEXG enhanced hMSCs chondrogenesis and upregulated RNA expression of type II/X collagen, SOX9 and aggrecan at 0.1 µg/mL, 0.3 µg/mL in vitro. In vivo, GLEXG at the dose of 0.3 µg intraarticular (i.a.) injection rescued the MIA-induced cartilage defect. Proteomics and ingenuity pathway analysis obtained from MSCs-released exosomes suggested that senescence pathway was less activated in GLEXG group than in vehicle group. Besides, GLEXG was able to increase cumulative population doubling and delayed hMSCs senescence process after four passages in cultures. CONCLUSION: we conclude that GLEXG promotes in vitro MSC-induced chondrogenesis possibly via exosomes release and delays aging in the MSC senescence process and that treatment with GLEXG (0.3 µg, i.a.) rescued cartilage defects in rat OA knee model.

Cancer-Associated Fibroblasts Hinder Lung Squamous Cell Carcinoma Oxidative Stress-Induced Apoptosis via METTL3 Mediated m6A Methylation of COL10A1.

Background: Cancer-associated fibroblasts (CAFs) within the tumor microenvironment are key players in tumorigenesis and tumor development. Nevertheless, the regulatory mechanisms of CAFs on lung squamous cell carcinoma- (LUSC-) associated remain poorly elucidated. Methods: The microarray dataset GSE22874, containing 30 specimens of primary culture of normal fibroblasts (NFs) and 8 specimens of cancer-associated fibroblasts (CAFs) samples derived from LUSC, was retrieved from the Gene Expression Omnibus (GEO) database and then calculated by using the R language (limma package) to identify differentially expressed genes (DEGs). CAF-conditioned medium (CAF-CM) was collected and used to culture LUSC cells, followed by assessment of cell proliferation, apoptosis, and oxidative stress levels by using CCK-8, annexin V-FITC/PI double staining and ELISA assays. Subsequently, COL10A1 was knocked down in CAFs to assess the role of COL10A1 in CAF regulation of LUSC behavior. Bioinformatics online analysis and MeRIP were applied to predict and test the m6A modification of COL10A1 mRNA and the regulatory relationship with METTL3. Rescue experiments were next performed to explore the effects of METTL3 and COL10A1 in CAFs on LUSC cell proliferation, apoptosis, and oxidative stress. LUSC tumor cells with or without (COL10A1-silenced) CAFs were subcutaneously inoculated in nude mice to evaluate the effect of COL10A1 in CAFs on LUSC tumor growth. Results: Elevated expression of COL10A1 was found in LUSC-derived CAFs by GSE22874 dataset analysis. We discovered that COL10A1 and METTL3 was expressed in both LUSC cells and matched CAFs, while COL10A1 expression was prominently higher in CAFs than in LUSC cells. CAF-CM memorably encouraged LUSC cell proliferation and suppressed apoptosis-induced oxidative stress, which was reversed by interfering with COL10A1 expression in CAFs, suggesting that COL10A1 might be secreted by CAFs into the culture medium to exert its effects inside LUSC cells. Global m6A modification was decreased in METTL3 knocked down CAFs. M6A modification, expression levels, and stability of COL10A1 mRNA were impaired upon METTL3 knockdown in CAFs. Overexpression of COL10A1 in CAFs partially reversed the effect of METTL3 knockdown on the malignant behavior of LUSC cells. In vivo studies confirmed that CAFs accelerated LUSC tumor growth, and this effect was counteracted by COL10A1 silencing. Conclusions: COL10A1 secreted by CAFs could facilitate LUSC cell proliferation and repress apoptosis-induced oxidative stress, and the mechanism was due to elevated expression mediated by METTL3 promoting its mRNA m6A modification, thereby accelerating tumor growth.

Downregulation of miR-30b-5p Facilitates Chondrocyte Hypertrophy and Apoptosis via Targeting Runx2 in Steroid-Induced Osteonecrosis of the Femoral Head.

As a widely used steroid hormone medicine, glucocorticoids have the potential to cause steroid-induced osteonecrosis of the femoral head (SONFH) due to mass or long-term use. The non-coding RNA hypothesis posits that they may contribute to the destruction and dysfunction of cartilages as a possible etiology of SONFH. MiR-30b-5p was identified as a regulatory factor in cartilage degeneration caused by methylprednisolone (MPS) exposure in our study through cell transfection. The luciferase reporter assay confirmed that miR-30b-5p was downregulated and runt-related transcription factor 2 (Runx2) was mediated by miR-30b-5p. The nobly increased expression of matrix metallopeptidase 13 (MMP13) and type X collagen (Col10a1) as Runx2 downstream genes contributed to the hypertrophic differentiation of chondrocytes, and the efficiently upregulated level of matrix metallopeptidase 9 (MMP9) may trigger chondrocyte apoptosis with MPS treatments. The cell transfection experiment revealed that miR-30b-5p inhibited chondrocyte hypertrophy and suppressed MPS-induced apoptosis. As a result, our findings showed that miR-30b-5p modulated Runx2, MMP9, MMP13, and Col10a1 expression, thereby mediating chondrocyte hypertrophic differentiation and apoptosis during the SONFH process. These findings revealed the mechanistic relationship between non-coding RNA and SONFH, providing a comprehensive understanding of SONFH and other bone diseases.

Bioinformatics-Based Analysis: Noncoding RNA-Mediated COL10A1 Is Associated with Poor Prognosis and Immune Cell Infiltration in Pancreatic Cancer.

Background: Collagen type X alpha 1 (COL10A1) is a structural component of the extracellular matrix that is aberrantly expressed in a variety of cancer tissues. However, its role in pancreatic cancer progression is not well understood. Methods: The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Gene Expression Profiling Interaction Analysis (GEPIA) data were employed to explore the expression of COL10A1 in normal and tumor tissues and its prognostic value in pancreatic adenocarcinoma. The clinical data of pancreatic cancer in TCGA were used to explore the relationship between COL10A1 and clinical features. Genes coexpressed with COL10A1 were explored using multiple databases and analyzed for functional enrichment. In addition, the lncRNA/miRNA/COL10A1 axis that may be involved in COL10A1 regulation in pancreatic cancer was explored by constructing a competitive endogenous RNA (ceRNA) regulatory axis. Finally, COL10A1 was analyzed for correlation with immune cell infiltration and various immune checkpoint molecules in pancreatic cancer. Results: It was found that the expression of COL10A1 was significantly increased in pancreatic cancer tissues. High expression of COL10A1 was related to the clinicopathological characteristics and the worse prognosis of pancreatic cancer patients. The TUG1/miR-144-3p/COL10A1 axis was identified as the most likely upstream noncoding RNA pathway for COL10A1 in pancreatic cancer. Besides, in pancreatic adenocarcinoma, the expression level of COL10A1 showed a significant positive correlation with tumor immune cell infiltration, biomarkers of immune cells, and expression of immune checkpoint molecules. Conclusion: COL10A1 is an early diagnostic marker, and its high expression correlates with immune infiltration in pancreatic cancer. The TUG1/miR-144-3p/COL10A1 axis was identified as the most likely upstream noncoding RNA pathway for COL10A1 in pancreatic cancer.

High COL10A1 expression potentially contributes to poor outcomes in gastric cancer with the help of LEF1 and Wnt2.

BACKGROUND: COL10A1 is a secreted, short-chain collagen found in several types of cancer. Studies have shown that COL10A1 aberrant expression is considered an oncogenic factor. However, its underlying mechanisms and regulation of gastric cancer remain undefined. METHODS: The data on the expression of COL10A1, clinicopathological characteristics, and outcome of patients with GC were obtained from The Cancer Genome Atlas. The ALGGEN-PROMO database defined the related transcription factors. Quantitative real-time reverse transcription-polymerase chain reaction and western blotting analysis were used to identify the differential expression levels of COL10A1 and related transcription factors. RESULTS: We found that high COL10A1 expression is an independent risk factor for gastric cancer. Upregulation of LEF1 and Wnt2 was also observed in gastric cancer, suggesting a potential correlation between LEF1/COL10A1 regulation in the Wnt2 signaling pathway. CONCLUSION: High COL10A1 expression may contribute to poor outcomes via upregulation of LEF1 and Wnt2 in gastric cancer.

Collagen X Marker Levels are Decreased in Individuals with Achondroplasia.

Collagen X marker (CXM) is a degradation fragment of collagen type X. It is a real-time biomarker of height velocity with established norms. Plasma C-type natriuretic peptide (CNP) and NTproCNP levels have also been found to correlate with growth velocity in the general population and are elevated in individuals with achondroplasia compared with age- and sex-matched controls. Collagen X marker levels in people with fibroblast growth factor receptor 3 (FGFR3)-opathies have never been systematically measured. The objective of this study was to measure CXM in a population of dwarfism caused by FGFR3-opathies. Using the same cohort in which CNP and NTproCNP levels were previously measured, archived serum aliquots from 63 children with achondroplasia, six with hypochondroplasia, and two with thanatophoric dysplasia had CXM concentrations measured. Results were plotted against age- and sex-specific norms, and standard deviation scores were plotted for comparison between clinical diagnoses. CXM levels were significantly decreased (p < 0.0001) in children with achondroplasia compared with age- and sex-matched controls. Temporal patterns of change in CXM levels were sex-dependent. As the FGFR3 pathway was more constitutively active, CXM levels decreased. New tools are emerging to study impact of skeletal dysplasia on growth plate regulation and function.

VSNL1 Promotes Cell Proliferation, Migration, and Invasion in Colorectal Cancer by Binding with COL10A1.

OBJECTIVE: The study aimed to explore the role of VSNL1/COL10A1 axis in colorectal cancer. METHODS: The differential-expressed mRNA in colorectal cancer tissues and adjacent tissues were analyzed through GEO database and GEPIA database. The target genes of mRNA were predicted through the Starbase database, and the targeting relationship of mRNA was verified by co-IP assay. The expressions of VSNL1 and COL10A1 were detected by RT-PCR and immunohistochemistry. Cell viability and proliferation were detected by CCK8 assay and EdU assay, respectively. Cell migration and invasion were detected by transwell assay. The expression of related proteins was detected by western blot. RESULTS: VSNL1 was significantly overexpressed in colorectal cancer tissues compared with adjacent tissues. In addition, downregulation of VSNL1 could inhibit the proliferation, migration, and invasion of colorectal cancer cells. The co-IP experiment indicated that VSNL1 could bind with COL10A1. Further studies demonstrated that upregulation of COL10A1 could promote colorectal cells proliferation, migration, invasion, and reverse the effect of sh-VSNL1 on colorectal cancer cells. CONCLUSION: VSNL1 could promote the proliferation, migration, and invasion of colorectal cancer by targeting COL10A1. VSNL1 might be a potential target for colorectal cancer treatment.

Downregulation of miR-1-3p expression inhibits the hypertrophy and mineralization of chondrocytes in DDH.

BACKGROUND: Developmental dysplasia of the hip (DDH) is a highly prevalent hip disease among children. However, its pathogenesis remains unclear. MicroRNAs (miRNA) are important regulators of cartilage development. In a previous study, high-throughput miRNA sequencing of tissue samples from an animal model of DDH showed a low level of miR-1-3p in the cartilage of the acetabular roof (ARC), but its role in DDH pathogenesis was not addressed. Therefore, our aim here was to investigate the effects of miR-1-3p in the ARC. METHODS: The diagnosis of acetabular dysplasia was confirmed with X-ray examination, while imaging and HE staining were conducted to further evaluate the ARC thickness in each animal model. FISH was employed to verify miR-1-3p expression in the ARC and chondrocytes. The miR-1-3p target genes were predicted by a bioinformatics database. A dual-luciferase reporter assay was used to confirm the targeting relationship between miR-1-3p and SOX9. The gene expression of miR-1-3p, SOX9, RUNX2 and collagen type X was evaluated by qPCR analysis. The protein expression of SOX9, RUNX2 and collagen type X was detected by western blot analysis. The levels of SOX9, RUNX2, and collagen type X in the ARC were further assessed via immunohistochemistry analysis. Finally, Alizarin Red S staining was used to observe the mineralized nodules produced by the chondrocytes. RESULTS: We observed low expression of miR-1-3p in the ARC of animals with DDH. SOX9 is a miR-1-3p target gene. Using miR-1-3p silencing technology in vitro, we demonstrated significantly reduced chondrocyte-generated mineralized nodules compared to those of the control. We also confirmed that with miR-1-3p silencing, SOX9 expression was upregulated, whereas the expression of genes associated with endochondral osteogenesis such as RUNX2 and collagen type X was downregulated. To confirm the involvement of miR-1-3p silencing in abnormal ossification through SOX9, we also performed a rescue experiment in which SOX9 silencing restored the low expression of RUNX2 and collagen type X produced by downregulated miR-1-3p expression. Finally, the elevated SOX9 levels and reduced RUNX2 and collagen type X levels in the ARC of rabbits with DDH were also verified using immunohistochemistry, RT-PCR, and western blots. CONCLUSION: The relatively low expression of miR-1-3p in the ARC may be the cause of abnormal endochondral ossification in the acetabular roof of animals with DDH.

Identification of a novel COL10A1: c.1952 G>T variant in a family with Schmid metaphyseal chondrodysplasia and development of a noninvasive prenatal testing method.

BACKGROUND: The collagen alpha-1(X) chain gene (COL10A1) is a known causative gene for Schmid metaphyseal chondrodysplasia (SMCD). This study clinically examined a Chinese family (n = 42) for SMCD and inheritance pattern. Fifteen individuals were diagnosed with SMCD based on characteristic skeletal phenotypes with autosomal dominant inheritance mode. METHODS: Four clinically diagnosed patients and three healthy relatives were selected for subsequent genetic tests. Trio-whole exome sequencing (Trio-WES) followed by Sanger sequencing and familial co-segregation analysis were performed to identify SMCD-associated variants. RESULTS: COL10A1 (NM_000493.4):c.1952 G>T(p.Trp651Leu) variant was detected only in the four patients and not in the three healthy relatives. The variant was evaluated as "likely pathogenic" according to the American College of Medical Genetics and Genomics variation classification guidelines with evidence of PM2, PM5, PP1, and PP3. To test the presence of the target variant in proband's fetal offspring, we developed a noninvasive prenatal testing method by extracting cell-free fetal DNA in maternal plasma followed by high-depth sequencing. The variant was also detected in the fetus and later confirmed by amniocentesis. CONCLUSION: We identified a new disease-causing variant in COL10A1. Cell-free fetal DNA in maternal peripheral blood can be used as the rapid and noninvasive prenatal diagnostic method to detect the pathogenic/or likely pathogenic variant.

Sodium hyaluronate supplemented culture medium combined with joint-simulating mechanical loading improves chondrogenic differentiation of human mesenchymal stem cells.

In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-ß1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.

Characterization of a novel COL10A1 variant associated with Schmid-type metaphyseal chondrodysplasia and a literature review.

BACKGROUND: Schmid-type metaphyseal chondrodysplasia (SMCD) is a rare autosomal dominant skeletal dysplasia caused by heterozygous mutations in COL10A1, the gene which encodes collagen type X alpha 1 chain. However, its genotype-phenotype relationship has not been fully determined. Subjects and Methods The proband is a 2-year-old boy, born of non-consanguineous Chinese parents. We conducted a systematic analysis of the clinical and radiological characteristics and a follow-up study of the proband. Whole-exome sequencing was applied for the genetic analysis, together with bioinformatic analysis of predicted consequences of the identified variant. A homotrimer model was built to visualize the affected region and predict possible outcomes of this variant. Furthermore, a literature review and genotype-phenotype analysis were performed by online searching all cases with SMCD. RESULTS: A novel heterozygous variant (NM_000493.4: c.1863_1866delAATG, NP_000484.2: p.(Met622 Thrfs*54)) was identified in COL10A1 gene in the affected child. And it was predicted to be pathogenic by in silico analysis. Protein modeling revealed that the variant was located in the NC1 domain, which was predicted to produce truncated collagen and impair the trimerization of collagen type X alpha 1 chain and combination with molecules in the matrix. Moreover, genotype-phenotype correlation analysis demonstrated that patients with truncating variants or variants in NC1 domain often presented earlier onset and severer symptoms compared with those with non-truncating or variants in non-NC1 domains. CONCLUSION: The NC1 domain of COL10A1 was proved to be the hotspot region underlying SMCD, patients with variants in NC1 domain were more likely to present severer manifestations at an earlier age.

Collagen Type X Alpha 1 (COL10A1) Contributes to Cell Proliferation, Migration, and Invasion by Targeting Prolyl 4-Hydroxylase Beta Polypeptide (P4HB) in Breast Cancer.

BACKGROUND Breast cancer, a common malignant tumor, has been considered as the leading cause of cancer-related death in women. Collagen type X alpha 1 (COL10A1) is overexpressed in breast cancer. The current study was designed to determine the functional involvement and regulatory mechanism of COL10A1 on the growth and metastasis of breast cancer. MATERIAL AND METHODS COL10A1 and Prolyl 4-hydroxylase beta polypeptide (P4HB) expressions in normal tissues and tumor tissues of breast cancer patients were obtained from the GEPIA dataset. COL10A1 and P4HB levels in breast cancer cell lines were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Furthermore, the interaction between COL10A1 and P4HB was confirmed by co-immunoprecipitation (Co-IP) assay. Cell Counting Kit-8 (CCK-8) and colony formation assay were applied to evaluate cell proliferation and clone-forming abilities of breast cancer cells. In addition, wound healing assay and transwell assay were performed to measure cell migration and invasion capabilities, respectively, in breast cancer. RESULTS The GEPIA dataset presented overexpressed COL10A1 and P4HB in tumor tissues of breast cancer patients. COL10A1 and P4HB expression levels were greatly upregulated in breast cancer cell lines. In addition, COL10A1 could directly interact with P4HB. Functionally, overexpressed COL10A1 boosted the proliferation and metastasis of breast cancer cells and silenced COL10A1 impeded the progression of breast cancer. More importantly, knockdown of P4HB weakened the promoting effects of overexpressed COL10A1 on cell proliferation, migration, and invasion in breast cancer. CONCLUSIONS COL10A1 promotes the malignant progression of breast cancer by upregulating P4HB expression, indicating that COL10A1 functions as an oncogene in breast cancer.

Differentiation of Hypertrophic Chondrocytes from Human iPSCs for the In Vitro Modeling of Chondrodysplasias.

Chondrodysplasias are hereditary diseases caused by mutations in the components of growth cartilage. Although the unfolded protein response (UPR) has been identified as a key disease mechanism in mouse models, no suitable in vitro system has been reported to analyze the pathology in humans. Here, we developed a three-dimensional culture protocol to differentiate hypertrophic chondrocytes from induced pluripotent stem cells (iPSCs) and examine the phenotype caused by MATN3 and COL10A1 mutations. Intracellular MATN3 or COL10 retention resulted in increased ER stress markers and ER size in most mutants, but activation of the UPR was dependent on the mutation. Transcriptome analysis confirmed a UPR with wide-ranging changes in bone homeostasis, extracellular matrix composition, and lipid metabolism in the MATN3 T120M mutant, which further showed altered cellular morphology in iPSC-derived growth-plate-like structures in vivo. We then applied our in vitro model to drug testing, whereby trimethylamine N-oxide led to a reduction of ER stress and intracellular MATN3.

Linc-ROR promotes mesenchymal stem cells chondrogenesis and cartilage formation via regulating SOX9 expression.

OBJECTIVE: The present study is to characterize the role of long intergenic non-coding RNA, regulator of reprogramming (linc-ROR) in bone marrow mesenchymal stem cell (BMSCs) chondrogenesis, cartilage formation and OA development. METHODS: Linc-ROR expression pattern in articular cartilage tissue sample from OA patients were studied by real-time PCR. Linc-ROR lentivirus mediated BMSCs were constructed. In vitro micromass cultured BMSCs chondrogenesis or in vivo MeHA hydrogel encapsulated BMSCs cartilage formation activity were studied. Linc-ROR associating miRNAs which repressed SOX9 expression were characterized by luciferase assay, real-time PCR and Western blot. Linc-ROR was co-transfected with miRNAs into BMSCs to study its rescue effect on SOX9 expression and chondrogenesis activity. RESULTS: Linc-ROR was down-regulated in articular cartilage tissue from OA patients and was positively correlated with the expression level of SOX9 (R2 = 0.43). Linc-ROR expression was upregulated during BMSCs chondrogenesis. Linc-ROR ectopic expression significantly promoted in vitro BMSCs chondrogenesis and in vivo cartilage formation activities as revealed by safranin O, alcian blue and COL II staining. The mRNA expression level of chondrogenesis markers including COL II, SOX9 and ACAN were increased, and the hypertrophy markers MMP13 and COL X were decreased upon linc-ROR overexpression in BMSCs. Linc-ROR functioned as a miRNA sponge for miR-138 and miR-145. Both miR-138 and miR-145 suppressed BMSCs chondrogenesis activity and SOX9 expression, while co-expression of linc-ROR displayed a rescuing effect. CONCLUSIONS: Taken together, linc-ROR modulated BMSCs chondrogenesis differentiation and cartilage formation by acting as a competing endogenous RNA for miR-138 and miR-145 and activating SOX9 expression. Linc-ROR could be considered as a new diagnostic and therapeutic target for OA treatment.

Analysis of Collagen type X alpha 1 (COL10A1) expression and prognostic significance in gastric cancer based on bioinformatics.

Collagen type X alpha 1 (COL10A1) is a member of the collagen family and the main matrix component. However, COL10A1 expression and prognosis relationship remains unclear in gastric cancer (GC). Through the analysis of database of Oncomine, the Cancer Genome Atlas (TCGA) as well as the Gene Expression Omnibus (GEO), in contrast to the tissue of normal gastric, COL10A1 in gastric cancer, had been upregulated. The high expression of COL10A1 was obviously related to T stage (P = 0.025) and lymph node metastasis (P = 0.025). It has been illustrated by the analysis of logistic regression that COL10A1's heightened expression in gastric cancer had been essentially linked with pathological stage, tumor differentiation, and T classification. The Kaplan-Meier curve in the Kaplan-Meier plotter database (P = 0.0371) and GSE84437 (P = 0.002) indicate that patients with high COL10A1 expression possess poor prognosis, specifically GC patients with lymph node metastasis have it. TCGA's Multivariate analysis (P = 0.025) and GSE84437 dataset (P = 0.034) show that high expression COL10A1 is a key independent predictor of poor overall survival. Searching KEGG pathway enrichment by GSEA, the results suggested that 29 pathways were enriched. qRT-PCR technique was used for verification of the COL10A1's high expression in gastric cancer in contrast to the normal gastric tissues. In conclusion, COL10A1 is of great importance in predicting the survival rate of GC patients.

Investigating the role of culture conditions on hypertrophic differentiation in human cartilage endplate cells.

Cartilage endplate degeneration/calcification has been linked to the onset and progression of intervertebral disc degeneration and there is a critical need to understand mechanisms, such as hypertrophic differentiation, of cartilage endplate degeneration/calcification to inform treatment strategies for discogenic back pain. In vitro cell culture conditions capable of inducing hypertrophic differentiation are used to study pathophysiological mechanisms in articular chondrocytes, but culture conditions capable of inducing a hypertrophic cartilage endplate cell phenotype have yet to be explored. The goal of this study was to investigate the role of culture conditions capable of inducing hypertrophic differentiation in articular chondrocytes on hypertrophic differentiation in human cartilage endplate cells. Isolated human cartilage endplate cells were cultured as pellets for 21 days at either 5% O2 (physiologic for cartilage) or 20.7% O2 (hyperoxic) and treated with 10% fetal bovine serum or Wnt agonist, two stimuli used to induce hypertrophic differentiation in articular chondrocytes. Cartilage endplate cells did not exhibit a hypertrophic cell morphology in response to fetal bovine serum or Wnt agonist but did display other hallmarks of chondrocyte hypertrophy and degeneration such as hypertrophic gene and protein expression, and a decrease in healthy proteoglycans and an increase in fibrous collagen accumulation. These findings demonstrate that cartilage endplate cells take on a degenerative phenotype in response to hypertrophic stimuli in vitro, but do not undergo classical changes in morphology associated with hypertrophic differentiation regardless of oxygen levels, highlighting potential differences in the response of cartilage endplate cells versus articular chondrocytes to the same stimuli.

Histone deacetylase 4 deletion results in abnormal chondrocyte hypertrophy and premature ossification from collagen type 2α1­expressing cells.

Histone deacetylase 4 (HDAC4) plays a vital role in chondrocyte hypertrophy and bone formation. To investigate the function of HDAC4 in postnatal skeletal development, the present study developed lineage­specific HDAC4­knockout mice [collagen type 2α1 (Col2α1)­Cre, HDAC4d/d mice] by crossing transgenic mice expressing Cre recombinase. Thus, a specific ablation of HDAC4 was performed in Col2α1­expressing mice cells. The knee joints of HDAC4fl/fl and Col2α1­Cre, HDAC4d/d mice were analyzed at postnatal day (P)2­P21 using an in vivo bromodeoxyuridine (BrdU) assay, and Safranin O, Von Kossa and whole­body staining were used to evaluate the developmental growth plate, hypertrophic differentiation, mineralization and skeletal mineralization patterns. The trabecular bone was analyzed using microcomputed tomography. The expressions of BrdU, proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)­13, runt­related transcription factor (Runx)­2, osteoprotegerin (OPG), CD34, type X collagen (ColX), osteocalcin and Wnt5a were determined using immunohistochemistry, in situ hybridization (ISH) and reverse transcription­quantitative (RT­q)PCR. The results demonstrated that HDAC4­null mice (HDAC4d/d mice) were severely runted; these mice had a shortened hypertrophic zone (histopathological evaluation), accelerated vascular invasion and articular mineralization (Von Kossa staining), elevated expressions of MMP­13, Runx2, OPG and CD34 (RT­qPCR and immunohistochemistry), downregulated expression of the proliferative marker BrdU and PCNA (immunohistochemistry), increased expression of ColX and decreased expression of Wnt5a (ISH). In conclusion, chondrocyte­derived HDAC4 was responsible for regulating chondrocyte proliferation and differentiation as well as endochondral bone formation.

Once-daily, subcutaneous vosoritide therapy in children with achondroplasia: a randomised, double-blind, phase 3, placebo-controlled, multicentre trial.

BACKGROUND: There are no effective therapies for achondroplasia. An open-label study suggested that vosoritide administration might increase growth velocity in children with achondroplasia. This phase 3 trial was designed to further assess these preliminary findings. METHODS: This randomised, double-blind, phase 3, placebo-controlled, multicentre trial compared once-daily subcutaneous administration of vosoritide with placebo in children with achondroplasia. The trial was done in hospitals at 24 sites in seven countries (Australia, Germany, Japan, Spain, Turkey, the USA, and the UK). Eligible patients had a clinical diagnosis of achondroplasia, were ambulatory, had participated for 6 months in a baseline growth study and were aged 5 to less than 18 years at enrolment. Randomisation was done by means of a voice or web-response system, stratified according to sex and Tanner stage. Participants, investigators, and trial sponsor were masked to group assignment. Participants received either vosoritide 15·0 µg/kg or placebo, as allocated, for the duration of the 52-week treatment period administered by daily subcutaneous injections in their homes by trained caregivers. The primary endpoint was change from baseline in mean annualised growth velocity at 52 weeks in treated patients as compared with controls. All randomly assigned patients were included in the efficacy analyses (n=121). All patients who received one dose of vosoritide or placebo (n=121) were included in the safety analyses. The trial is complete and is registered, with EudraCT, number, 2015-003836-11. FINDINGS: All participants were recruited from Dec 12, 2016, to Nov 7, 2018, with 60 assigned to receive vosoritide and 61 to receive placebo. Of 124 patients screened for eligibility, 121 patients were randomly assigned, and 119 patients completed the 52-week trial. The adjusted mean difference in annualised growth velocity between patients in the vosoritide group and placebo group was 1·57 cm/year in favour of vosoritide (95% CI [1·22-1·93]; two-sided p<0·0001). A total of 119 patients had at least one adverse event; vosoritide group, 59 (98%), and placebo group, 60 (98%). None of the serious adverse events were considered to be treatment related and no deaths occurred. INTERPRETATION: Vosoritide is an effective treatment to increase growth in children with achondroplasia. It is not known whether final adult height will be increased, or what the harms of long-term therapy might be. FUNDING: BioMarin Pharmaceutical.