SOX9+ enthesis cells are associated with spinal ankylosis in ankylosing spondylitis.

OBJECTIVE: Although cartilage degeneration and invasion of the subchondral bone plate in entheseal lesion has been considered to consequently lead bony ankylosis in ankylosing spondylitis (AS), no evident mechanisms are known. DESIGN: To identify histopathological and physiological changes in enthesitis-related ankylosis in AS, we performed molecular characterization of transcription factors and surface markers, and transcriptome analysis with human tissues. Entheseal tissue containing subchondral bone was obtained from the facet joints of 9 patients with AS and 10 disease controls, and assessed by using differential staining techniques. Enthesis cells were isolated, characterized, stimulated with TNF and/or IL-17A, and analysed by cell-based experimental tools. RESULTS: We found diffusely distributed granular tissue and cartilage in the subchondral bone in AS. Co-expression of SOX9, a specific transcription factor in cartilage, and matrix metalloproteinase 13 (MMP13) was found in the granular tissues within the subchondral bone from AS patients. Intriguingly, SOX9 expression was significantly higher in AS enthesis cells than controls and correlated with TNFR1 and IL-17RA expressions, which is important for high reactivity to TNF and IL-17A cytokines. Co-stimulation by TNF and IL-17A resulted in accelerated mineralization/calcification features, and increased OCN expression in AS enthesis cells. Furthermore, SOX9 overexpression in enthesis leads to promoting mineralization feature by TNF and IL-17A stimuli. Finally, OCN expression is elevated in the destructive enthesis of advanced AS. CONCLUSION: These findings provide insight into the links between inflammation and the mineralization of entheseal tissue as the initiation of spinal ankylosis, emphasizing the importance of SOX9+ enthesis cells.

Temporospatial regulation of intraflagellar transport is required for the endochondral ossification in mice.

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, the unique functions and roles of each IFT during endochondral ossification remain unclear. Here, we show that IFT20 is required for endochondral ossification in mice. Utilizing osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre and Col2-Cre), we deleted Ift20 to examine its function. Although chondrocyte-specific Ift20 deletion with Col2-Cre mice did not cause any overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) mice resulted in shortened limb outgrowth. Primary cilia were absent on chondrocytes of Ift20:Prx1-Cre mice, and ciliary-mediated Hedgehog signaling was attenuated in Ift20:Prx1-Cre mice. Interestingly, loss of Ift20 also increased Fgf18 expression in the perichondrium that sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate that IFT20 is a critical regulator of temporospatial FGF signaling that is required for endochondral ossification.

Differential Expression Levels of Sox9 in Early Neocortical Radial Glial Cells Regulate the Decision between Stem Cell Maintenance and Differentiation.

Radial glial progenitor cells (RGCs) in the dorsal telencephalon directly or indirectly produce excitatory projection neurons and macroglia of the neocortex. Recent evidence shows that the pool of RGCs is more heterogeneous than originally thought and that progenitor subpopulations can generate particular neuronal cell types. Using single-cell RNA sequencing, we have studied gene expression patterns of RGCs with different neurogenic behavior at early stages of cortical development. At this early age, some RGCs rapidly produce postmitotic neurons, whereas others self-renew and undergo neurogenic divisions at a later age. We have identified candidate genes that are differentially expressed among these early RGC subpopulations, including the transcription factor Sox9. Using in utero electroporation in embryonic mice of either sex, we demonstrate that elevated Sox9 expression in progenitors affects RGC cell cycle duration and leads to the generation of upper layer cortical neurons. Our data thus reveal molecular differences between progenitor cells with different neurogenic behavior at early stages of corticogenesis and indicates that Sox9 is critical for the maintenance of RGCs to regulate the generation of upper layer neurons.SIGNIFICANCE STATEMENT The existence of heterogeneity in the pool of RGCs and its relationship with the generation of cellular diversity in the cerebral cortex has been an interesting topic of debate for many years. Here we describe the existence of RGCs with reduced neurogenic behavior at early embryonic ages presenting a particular molecular signature. This molecular signature consists of differential expression of some genes including the transcription factor Sox9, which has been found to be a specific regulator of this subpopulation of progenitor cells. Functional experiments perturbing expression levels of Sox9 reveal its instructive role in the regulation of the neurogenic behavior of RGCs and its relationship with the generation of upper layer projection neurons at later ages.

An in vivo evaluation of induced chondrogenesis by decellularized extracellular matrix particles.

Bioengineered scaffolds composed of synthetic materials and extracellular matrix (ECM) components can offer a tissue-specific microenvironment capable of regulating cells to regenerate the structure and function of the native cartilage. Here, given the potential preservation of biomechanical and biochemical cues found in the native cartilage, particulate decellularized ECM (DC-ECM) was utilized for immobilization on the surface of nanofibrous scaffolds. Afterward, the chondro-inductive potential and ectopic cartilage formation after subcutaneous implantation of bioengineered DC-ECM scaffolds were investigated in mice model. Eight weeks post-implantation, no growth of considerable inflammatory response and neovascularization was observed in histological images of bioengineered DC-ECM scaffolds. Pre-seeded bioengineered scaffolds with human adipose-derived stem cells exhibited high levels of chondro-induction capability, indicated with immunohistochemical and gene expression results. In both interval times, we also observed chondrogenesis and tissue formation after implanting unseeded bioengineered scaffolds, which denote that the presence of DC-ECM particles can even enhance attachment and migration of the host cells and induce chondrogenesis to them. To sum up, the incorporation of DC-ECM materials to tissue engineered constructs is a promising avenue to mimic the native tissue environment for regulation of cartilage regeneration in both in vivo and in vitro settings.

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. METHODS: The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and ß-Catenin were quantified by Western blot. RESULTS: Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho ß-Catenin, and BMP4. CONCLUSIONS: These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation.

Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP-based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell-free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system.

Combined therapies with exercise, ozone and mesenchymal stem cells improve the expression of HIF1 and SOX9 in the cartilage tissue of rats with knee osteoarthritis.

PURPOSE: Knee osteoarthritis (OA) is a common type of degenerative joint disease which decreases the quality of life. Sex-determining region Y box 9 (SOX9) and hypoxia-inducible factor-1 (HIF1) are considered as the key regulators of OA. We investigated the effect of combined therapies with mesenchymal stem cells (MSCs), ozone (O3) and exercise training on SOX9 and HIF1 expression in the cartilage of rats with knee OA. METHODS: Knee OA was induced by surgical method. OA rats were divided into model, MSCs, ozone, exercise, MSCs + ozone, MSCs + exercise, ozone + exercise and MSCs + ozone + exercise groups. Rats in the MSCs group received intraarticular injection of 1 × 106 cells/kg. Rats in the ozone group received O3 at the concentration of 20 µg/mL, once weekly for 3 weeks. Rats in the exercise group were trained on rodent treadmill three times per week. 48 hours after the programs, cartilage tissues were isolated and the expression of SOX9 and HIF1 was determined using Real-Time PCR. RESULTS: Significant differences were found in the expression of SOX9 and HIF1 between groups (P < 0.0001). Although combined therapies with exercise, MSCs and O3 significantly increased the expression of SOX9 and HIF1 in the cartilage tissue of rats with knee OA, combination of exercise with O3 was significantly more effective compared to the other combined therapies (P < 0.001). CONCLUSIONS: Combined therapy with exercise, MSCs and O3 significantly increased the expression of SOX9 and HIF1 genes in the cartilage of rats with knee OA; however, exercise + O3 was significantly more effective.

Temporal and spatial expression of Sox9, Pax1, TGF-ß1 and type I and II collagen in human intervertebral disc development.

PURPOSE: An accurate understanding of cellular biochemical changes in human intervertebral disc (IVD)s and the corresponding mechanisms during the developmental process still remain unknown and important for investigating the function of critical factors in normal IVD development as well as ascertaining the therapeutic targets for the IVD degeneration. METHODS: Under ethical conditions, human fetal cervical IVDs at 4, 5, and 6 months of pregnancy were collected at abortion surgery. Normal adult human C3-C7 cervical IVDs were taken from cadaveric donors. Sox9, Pax1, TGF-ß1 and type I/II collagen protein and RNA were detected. The number of positive cells was counted to calculate the optical density value for each factor. RESULTS: Sox9, Pax1, and TGF-ß1 expression in the IVD was remarkably reduced with the developmental stage. The location of high expression of Sox9, Pax1, and TGF-ß1 changed with the developmental stage, and migrated from the nucleus pulposus to the annulus fibrosus and endplate. Higher Sox9, Pax1, and TGF-ß1 expression was finally observed around the sclerotome of the vertebral body. The anabolism of type I/II collagens is significantly increased in the IVD in the mid-trimester fetus. CONCLUSIONS: Sox9, Pax1 and TGF-ß1 participate in the developmental process of the human IVD and vertebral body. However, these factors show a separate expression of mRNA and protein, suggesting that they are expressed in the strict time and spatial order.

Reduced hypertrophy in vitro after chondrogenic differentiation of adult human mesenchymal stem cells following adenoviral SOX9 gene delivery.

BACKGROUND: Mesenchymal stem cell (MSC) based-treatments of cartilage injury are promising but impaired by high levels of hypertrophy after chondrogenic induction with several bone morphogenetic protein superfamily members (BMPs). As an alternative, this study investigates the chondrogenic induction of MSCs via adenoviral gene-delivery of the transcription factor SOX9 alone or in combination with other inducers, and comparatively explores the levels of hypertrophy and end stage differentiation in a pellet culture system in vitro. METHODS: First generation adenoviral vectors encoding SOX9, TGFB1 or IGF1 were used alone or in combination to transduce human bone marrow-derived MSCs at 5 × 102 infectious particles/cell. Thereafter cells were placed in aggregates and maintained for three weeks in chondrogenic medium. Transgene expression was determined at the protein level (ELISA/Western blot), and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. RESULTS: SOX9 cDNA was superior to that encoding TGFB1, the typical gold standard, as an inducer of chondrogenesis in primary MSCs as evidenced by improved lacuna formation, proteoglycan and collagen type II staining, increased levels of GAG synthesis, and expression of mRNAs associated with chondrogenesis. Moreover, SOX9 modified aggregates showed a markedly lower tendency to progress towards hypertrophy, as judged by expression of the hypertrophy markers alkaline phosphatase, and collagen type X at the mRNA and protein levels. CONCLUSION: Adenoviral SOX9 gene transfer induces chondrogenic differentiation of human primary MSCs in pellet culture more effectively than TGFB1 gene transfer with lower levels of chondrocyte hypertrophy after 3 weeks of in vitro culture. Such technology might enable the formation of more stable hyaline cartilage repair tissues in vivo.

Dual functional nanoparticles containing SOX duo and ANGPT4 shRNA for osteoarthritis treatment.

In our previous studies, we found that adult stem cells transfected with sex-determining region Y-box (SOX)-9, -6 and -5 genes (SOX trio) enhanced chondrogenesis and suppressed the progression of osteoarthritis (OA). The inhibition of angiopoietin-like 4 (ANGPT4) is known to reduce levels of cartilage damaging enzymes, such as, matrix metalloproteinases (MMPs). In this study, we designed nanoparticles comprising dexamethasone-conjugated polyethylenimine (DEX PEI) complexed with minicircle plasmid (MC) harboring SOX duo (SOX-9, -6) and ANGPTL4 small hairpin RNA (shANG) [MC SOX9/6/shANG] in the expectation that transfection of these nanoparticles would enhance chondrogenesis of stem cells and suppress inflammation in OA. Adipose-derived stem cells (ADSCs) transfected with MC SOX9/6/shANG (MC SOX9/6/shANG-tADSCs) showed significantly higher expressions of COL2 gene and protein than MC SOX9/6-transfected ADSCs (MC SOX9/6-tADSCs) during in vitro chondrogenesis while both enhanced chondrogenesis in the absence of growth factor addition as compared with negative controls. Furthermore, the expressions of MMP13 and MMP3 genes were significantly more diminished in MC SOX9/6/shANG-tADSCs than in MC SOX9/6-tADSCs. In vivo experiments using surgically-induced OA rats showed MC SOX9/6/shANG-tADSC-treated rats had significantly lower levels of cyclooxygenase (COX-2) and MMP13 in synovial fluids than MC SOX9/6-tADSC-treated rats, but no significant difference was observed between them in histological appearances. Both groups showed significantly less joint destruction than control groups did. These results demonstrate that dual functional nanoparticles containing SOX duo and ANGPT4 shRNA enhance chondrogenesis of ADSCs and suppress inflammation in OA. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:234-242, 2020.

Blastula stage specification of avian neural crest.

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of ß-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.

PPARδ Interacts with the Hippo Coactivator YAP1 to Promote SOX9 Expression and Gastric Cancer Progression.

Despite established functions of PPARδ in lipid metabolism and tumorigenesis, the mechanisms underlying its role in gastric cancer are undefined. Here, we demonstrate that SOX9 was dramatically induced by stably expressing PPARδ and by its agonist GW501516 in human gastric cancer cell lines. PPARδ knockdown in patient-derived gastric cancer cells dramatically reduced SOX9 expression and transcriptional activity, with corresponding decreases in invasion and tumor sphere formation. Mechanistically, PPARδ induced SOX9 transcription through direct interaction with and activation of the Hippo coactivator YAP1. PPARδ-YAP1 interaction occurred via the C-terminal domain of YAP1, and both TEAD- and PPARE-binding sites were required for SOX9 induction. Notably, CRISPR/Cas9-mediated genetic ablation of YAP1 or SOX9 abolished PPARδ-mediated oncogenic functions. Finally, expression of PPARδ, YAP1, and SOX9 were significantly correlated with each other and with poor survival in a large cohort of human gastric cancer tissues. Thus, these findings elucidate a novel mechanism by which PPARδ promotes gastric tumorigenesis through interaction with YAP1 and highlights the PPARδ/YAP1/SOX9 axis as a novel therapeutic target in human gastric cancer. IMPLICATIONS: Our discovery of a new model supports a distinct paradigm for PPARδ and a crucial oncogenic function of PPARδ in gastric cancer through convergence on YAP1/TEAD signaling. Therefore, PPARδ/YAP1/SOX9 axis could be a novel therapeutic target that can be translated into clinics.

Signaling Events Downstream of AHR Activation That Contribute to Toxic Responses: The Functional Role of an AHR-Dependent Long Noncoding RNA (slincR) Using the Zebrafish Model.

BACKGROUND: A structurally diverse group of chemicals, including dioxins [e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)] and polycyclic aromatic hydrocarbons (PAHs), can xenobiotically activate the aryl hydrocarbon receptor (AHR) and contribute to adverse health effects in humans and wildlife. In the zebrafish model, repression of sox9b has a causal role in several AHR-mediated toxic responses, including craniofacial cartilage malformations; however, the mechanism of sox9b repression remains unknown. We previously identified a long noncoding RNA, sox9b long intergenic noncoding RNA (slincR), which is increased (in an AHR-dependent manner) by multiple AHR ligands and is required for the AHR-activated repression of sox9b. OBJECTIVE: Using the zebrafish model, we aimed to enhance our understanding of the signaling events downstream of AHR activation that contribute to toxic responses by identifying: a) whether slincR is enriched on the sox9b locus, b) slincR's functional contributions to TCDD-induced toxicity, c) PAHs that increase slincR expression, and d) mammalian orthologs of slincR. METHODS: We used capture hybridization analysis of RNA targets (CHART), qRT-PCR, RNA sequencing, morphometric analysis of cartilage structures, and hemorrhaging screens. RESULTS: The slincR transcript was enriched at the 5' untranslated region (UTR) of the sox9b locus. Transcriptome profiling and human ortholog analyses identified processes related to skeletal and cartilage development unique to TCDD-exposed controls, and angiogenesis and vasculature development unique to TCDD-exposed zebrafish that were injected with a splice-blocking morpholino targeting slincR. In comparison to TCDD exposed control morphants, slincR morphants exposed to TCDD resulted in abnormal cartilage structures and a smaller percentage of animals displaying the hemorrhaging phenotype. In addition, slincR expression was significantly increased in six out of the sixteen PAHs we screened. CONCLUSION: Our study establishes that in zebrafish, slincR is recruited to the sox9b 5' UTR to repress transcription, can regulate cartilage development, has a causal role in the TCDD-induced hemorrhaging phenotype, and is up-regulated by multiple environmentally relevant PAHs. These findings have important implications for understanding the ligand-specific mechanisms of AHR-mediated toxicity. https://doi.org/10.1289/EHP3281.

Inhibition of microRNA-30d attenuates the apoptosis and extracellular matrix degradation of degenerative human nucleus pulposus cells by up-regulating SOX9.

Accumulating evidence has suggested that microRNAs are critical regulators of intervertebral disc degeneration (IDD). The excessive apoptosis and extracellular matrix degradation of nucleus pulposus (NP) cells contribute to the initiation of IDD. However, the precise regulatory role of miRNAs in NP cell apoptosis and extracellular matrix degradation remains largely unknown. MicroRNA-30d (miR-30d) has been reported to be involved in regulating apoptosis and bone homeostasis. In this study, we aimed to investigate the role of miR-30d in regulating apoptosis and the extracellular matrix degradation of NP cells, along with the potential underlying molecular mechanism. Herein, our results showed that miR-30d was significantly increased in degenerative NP tissues compared with normal controls. Functional experiments showed that the inhibition of miR-30d promoted the viability and reduced the apoptosis of NP cells in vitro. Moreover, miR-30d inhibition increased the expression of type II collagen and aggrecan and inhibited the expression of matrix metalloproteinase. In contrast, the overexpression of miR-30d showed the opposite effects. Bioinformatics analysis, the dual-luciferase reporter assay, real-time quantitative PCR and western blot analysis showed that miR-30d directly targeted the 3'-untranslated region of SRY-related high mobility group box 9 (SOX9) and negatively regulated SOX9 expression. Correlation analysis showed that miR-30d expression was inversely correlated with SOX9 expression in degenerative NP tissues. Moreover, siRNA-mediated silencing of SOX9 expression significantly blocked the protective effects of miR-30d inhibition against NP cell apoptosis and extracellular matrix degradation. Overall, these results demonstrate that the inhibition of miR-30d attenuates the apoptosis and extracellular matrix degradation of degenerative human NP cells by up-regulating SOX9, suggesting a potential therapeutic target for IDD.

Inhibition of microRNA-384-5p alleviates osteoarthritis through its effects on inhibiting apoptosis of cartilage cells via the NF-κB signaling pathway by targeting SOX9.

Osteoarthritis (OA), a major cause of pain and disability, is a serious public issue worldwide. Some microRNAs (miRNAs) and SOX9 have been found to be expressed in OA. Therefore, the aim of this study is to investigate effects of microRNA-384-5p (miR-384-5p) on cartilage cell proliferation and apoptosis in mice with OA by targeting SOX9 through the NF-κB signaling pathway. First, bioinformatics was used to predict the SOX9-mediated miRNA (miR-384-5p), and dual luciferase reporter gene assay was conducted to further verify the relationship between miR-384-5p and SOX9. Then, the expression of miR-384-5p, SOX9, and NF-kB in mice modeled with OA was detected. To investigate the specific mechanism of miR-384-5p in OA, mimic and inhibitor of miR-384-5p and siRNA against SOX9 were used to transfect cartilage cells. Finally, proliferation, cell cycle, and cell apoptosis were detected using MTT assay and flow cytometry, respectively. Our results indicated that OA mice exhibited decreased expression of SOX9 and NF-kB but higher miR-384-5p expression. In addition, over-expressed miR-384-5p or silenced SOX9 could inhibit cell proliferation, and block cell cycle entry and induces apoptosis. SOX9 was a target gene of miR-384-5p. The NF-kB signaling pathway was inactivated after overexpression of miR-384-5p. Furthermore, we also observed that the effect of miR-384-5p inhibition was rescued when SOX9 was knocked down. The results support the view that inhibition of miR-384-5p could impede apoptosis and promote proliferation of cartilage cells through activating the NF-κB signaling pathway by promoting SOX9, thereby preventing the development of OA.

Linc-ROR promotes esophageal squamous cell carcinoma progression through the derepression of SOX9.

BACKGROUND: Novel therapies tailored to the molecular composition of esophageal squamous cell carcinoma (ESCC) are needed to improve patient survival. We investigated the regulatory network of long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) and sex-determining region Y-box 9 (SOX9), and their therapeutic relevance in ESCC. METHODS: Linc-ROR and SOX9 expression were examined in ESCC specimens, cell lines, and cultured tumorspheres. We investigated the effects of linc-ROR on SOX9 expression and malignant phenotypes by CCK8, colony formation, Transwell, and sphere-forming assay. The linc-ROR/SOX9 interaction mediated by multiple microRNAs (miRNAs) was confirmed by bioinformatic analysis, luciferase assay, and RNA-binding protein immunoprecipitation, transient overexpression or antagonizing endogenous candidate miRNAs. The effect of linc-ROR depletion on tumor growth was assessed by xenograft assay. RESULTS: A positive correlation between linc-ROR and SOX9 expression was found in clinical ESCC specimens (r = 0.562, P = 0.036), cell lines, and tumorspheres. Silencing of linc-ROR significantly inhibited cell proliferation, motility, chemoresistance, and self-renewal capacity. Mechanistically, linc-ROR modulating the derepression of SOX9 by directly sponging multiple miRNAs including miR-15b, miR-33a, miR-129, miR-145, and miR-206. Antagonizing these miRNAs counteracted with linc-ROR silencing, whereas the repression of SOX9 abrogated malignant phenotypes induced by the cocktail of miRNA inhibitors. Moreover, linc-ROR disruption was sufficient to attenuate tumor growth and cancer stem cell marker expression in vivo. CONCLUSIONS: Our results demonstrate that the linc-ROR-miRNA-SOX9 regulatory network may represent a novel therapeutic target for ESCC.

The long non-coding RNA ROCR contributes to SOX9 expression and chondrogenic differentiation of human mesenchymal stem cells.

Long non-coding RNAs (lncRNAs) are expressed in a highly tissue-specific manner and function in various aspects of cell biology, often as key regulators of gene expression. In this study, we established a role for lncRNAs in chondrocyte differentiation. Using RNA sequencing we identified a human articular chondrocyte repertoire of lncRNAs from normal hip cartilage donated by neck of femur fracture patients. Of particular interest are lncRNAs upstream of the master chondrocyte transcription factor SOX9 locus. SOX9 is an HMG-box transcription factor that plays an essential role in chondrocyte development by directing the expression of chondrocyte-specific genes. Two of these lncRNAs are upregulated during chondrogenic differentiation of mesenchymal stem cells (MSCs). Depletion of one of these lncRNAs, LOC102723505, which we termed ROCR (regulator of chondrogenesis RNA), by RNA interference disrupted MSC chondrogenesis, concomitant with reduced cartilage-specific gene expression and incomplete matrix component production, indicating an important role in chondrocyte biology. Specifically, SOX9 induction was significantly ablated in the absence of ROCR, and overexpression of SOX9 rescued the differentiation of MSCs into chondrocytes. Our work sheds further light on chondrocyte-specific SOX9 expression and highlights a novel method of chondrocyte gene regulation involving a lncRNA.

Tomo-Seq Identifies SOX9 as a Key Regulator of Cardiac Fibrosis During Ischemic Injury.

BACKGROUND: Cardiac ischemic injury induces a pathological remodeling response, which can ultimately lead to heart failure. Detailed mechanistic insights into molecular signaling pathways relevant for different aspects of cardiac remodeling will support the identification of novel therapeutic targets. METHODS: Although genome-wide transcriptome analysis on diseased tissues has greatly advanced our understanding of the regulatory networks that drive pathological changes in the heart, this approach has been disadvantaged by the fact that the signals are derived from tissue homogenates. Here we used tomo-seq to obtain a genome-wide gene expression signature with high spatial resolution spanning from the infarcted area to the remote to identify new regulators of cardiac remodeling. Cardiac tissue samples from patients suffering from ischemic heart disease were used to validate our findings. RESULTS: Tracing transcriptional differences with a high spatial resolution across the infarcted heart enabled us to identify gene clusters that share a comparable expression profile. The spatial distribution patterns indicated a separation of expressional changes for genes involved in specific aspects of cardiac remodeling, such as fibrosis, cardiomyocyte hypertrophy, and calcium handling (Col1a2, Nppa, and Serca2). Subsequent correlation analysis allowed for the identification of novel factors that share a comparable transcriptional regulation pattern across the infarcted tissue. The strong correlation between the expression levels of these known marker genes and the expression of the coregulated genes could be confirmed in human ischemic cardiac tissue samples. Follow-up analysis identified SOX9 as common transcriptional regulator of a large portion of the fibrosis-related genes that become activated under conditions of ischemic injury. Lineage-tracing experiments indicated that the majority of COL1-positive fibroblasts stem from a pool of SOX9-expressing cells, and in vivo loss of Sox9 blunted the cardiac fibrotic response on ischemic injury. The colocalization between SOX9 and COL1 could also be confirmed in patients suffering from ischemic heart disease. CONCLUSIONS: Based on the exact local expression cues, tomo-seq can serve to reveal novel genes and key transcription factors involved in specific aspects of cardiac remodeling. Using tomo-seq, we were able to unveil the unknown relevance of SOX9 as a key regulator of cardiac fibrosis, pointing to SOX9 as a potential therapeutic target for cardiac fibrosis.

In Vivo Characterization of an AHR-Dependent Long Noncoding RNA Required for Proper Sox9b Expression.

Xenobiotic activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) prevents the proper formation of craniofacial cartilage and the heart in developing zebrafish. Downstream molecular targets responsible for AHR-dependent adverse effects remain largely unknown; however, in zebrafish sox9b has been identified as one of the most-reduced transcripts in several target organs and is hypothesized to have a causal role in TCDD-induced toxicity. The reduction of sox9b expression in TCDD-exposed zebrafish embryos has been shown to contribute to heart and jaw malformation phenotypes. The mechanisms by which AHR2 (functional ortholog of mammalian AHR) activation leads to reduced sox9b expression levels and subsequent target organ toxicity are unknown. We have identified a novel long noncoding RNA (slincR) that is upregulated by strong AHR ligands and is located adjacent to the sox9b gene. We hypothesize that slincR is regulated by AHR2 and transcriptionally represses sox9b. The slincR transcript functions as an RNA macromolecule, and slincR expression is AHR2 dependent. Antisense knockdown of slincR results in an increase in sox9b expression during both normal development and AHR2 activation, which suggests relief in repression. During development, slincR was expressed in tissues with sox9 essential functions, including the jaw/snout region, otic vesicle, eye, and brain. Reducing the levels of slincR resulted in altered neurologic and/or locomotor behavioral responses. Our results place slincR as an intermediate between AHR2 activation and the reduction of sox9b mRNA in the AHR2 signaling pathway.