SOX9-mediated UGT8 expression promotes glycolysis and maintains the malignancy of non-small cell lung cancer.

UDP-glycosyltransferases (UGTs) catalyze the covalent addition of sugars to small lipophilic chemicals and are associated with a wide range of diseases including cancer. The human genome contains 22 UGT genes which could be classified into four families: UGT1, UGT2, UGT3, and UGT8. The UGT8 family contains only one member which utilizes UDP galactose to galactosidate ceramide. Although higher UGT8 mRNA was observed in some types of cancer, its pathological significances remain elusive. Here, by integrating the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and the Genotype-Tissue Expression (GTEx) databases, we showed that UGT8 was selectively highly expressed in non-small cell lung cancer (NSCLC) and associated with worse prognosis. The transcription factor SOX9 promoted UGT8 expression in NSCLC by recognizing two putative response elements localized on the promoter region of UGT8. Silencing UGT8 impaired glycolysis and reduced the malignancy of NSCLC cells both in vitro and in vivo. On the contrary, inhibition of glycolysis by 2-deoxy-d-glucose (2-DG) significantly impaired the pro-proliferation function of UGT8 in NSCLC cells. In conclusion, our results suggest that UGT8 maintains the malignancy of NSCLC mainly via enhanced glycolysis and provides a promising therapeutic target for NSCLC.

Dexmedetomidine upregulates microRNA-185 to suppress ovarian cancer growth via inhibiting the SOX9/Wnt/ß-catenin signaling pathway.

Dexmedetomidine (DEX) could serve as an adjuvant analgesic during cancer therapies. Abnormal expression of microRNAs (miRNAs) could lead to cancer development. This study was aimed to explore the roles of DEX in ovarian cancer (OC) development. OC cell lines SKOV3 and HO-8910 were treated with DEX, after which OC development and the miR-185, SOX9, and Wnt/ß-catenin pathway were measured. DEX-treated HO-8910 cells were transfected with miR-185 mimic, miR-185 antisense or miR-185 antisense + silenced SOX9 to further measure the OC cell growth. The target relation between miR-185 and SOX9 was identified, and SOX9 and Wnt/ß-catenin pathway were protein levels detected after miR-185 transfection. The role of miR-185 in OC in vivo was also measured. Our study found DEX had a dose-dependent inhibition on OC growth, and DEX promoted miR-185 but suppressed SOX9 expression in OC cells. miR-185 targeted SOX9. After interfering with miR-185 expression, HO-8910 cell proliferation, invasion, migration, and apoptosis were affected. SOX9 knockdown repressed OC development and Wnt/ß-catenin pathway. The volume, weight, positive rate of Ki67, CyclinD1, p53 and the degree of tumor necrosis were affected by miR-185 expression. This study demonstrated that DEX could inhibit OC development via upregulating miR-185 expression and inactivating the SOX9/Wnt/ß-catenin signaling pathway.

Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis.

Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P < 0.01) and KrasG12D/+; p53R172H/+; Pdx-1 Cre (KPC) (P < 0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P < 0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P < 0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P < 0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P < 0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan-EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of pan-EGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.

Cbx2, a PcG Family Gene, Plays a Regulatory Role in Medaka Gonadal Development.

Chromobox homolog 2 (CBX2), a key member of the polycomb group (PcG) family, is essential for gonadal development in mammals. A functional deficiency or genetic mutation in cbx2 can lead to sex reversal in mice and humans. However, little is known about the function of cbx2 in gonadal development in fish. In this study, the cbx2 gene was identified in medaka, which is a model species for the study of gonadal development in fish. Transcription of cbx2 was abundant in the gonads, with testicular levels relatively higher than ovarian levels. In situ hybridization (ISH) revealed that cbx2 mRNA was predominately localized in spermatogonia and spermatocytes, and was also observed in oocytes at stages I, II, and III. Furthermore, cbx2 and vasa (a marker gene) were co-localized in germ cells by fluorescent in situ hybridization (FISH). After cbx2 knockdown in the gonads by RNA interference (RNAi), the sex-related genes, including sox9 and foxl2, were influenced. These results suggest that cbx2 not only plays a positive role in spermatogenesis and oogenesis but is also involved in gonadal differentiation through regulating the expression levels of sex-related genes in fish.

A novel natural antisense transcript at human SOX9 locus is down-regulated in cancer and stem cells.

OBJECTIVE: SOX9 is a key transcription factor with important roles in regulating proliferation and differentiation of various cell types. Dysregulation of SOX9 expression has been involved with pathogenesis of different developmental, degenerative, and neoplastic disorders. Natural antisense transcripts (NATs) are long non-coding RNAs with increasing significance in regulation of gene expression. However, the presence of a NAT at SOX9 locus has been so far unclear. RESULT: We detected a natural antisense transcript at SOX9 locus (SOX9-NAT) through strand-specific RT-PCR. In contrast to SOX9 sense RNA (mRNA), SOX9-NAT was down-regulated in cancer tissues and cell lines compared with their normal counterparts. In addition, reciprocal to SOX9 mRNA, SOX9-NAT was also down-regulated in human embryonic stem cells in comparison with human fibroblasts in vitro. CONCLUSION: The negative correlation between SOX9 mRNA and SOX9-NAT was confirmed by analyzing qPCR data, as well as RNA-Seq datasets of several human cancers. Our data suggest a functional role for SOX9-NAT in the regulation of SOX9 mRNA as a potential target in cancer treatment and regenerative medicine.

MicroRNA-215-3p suppresses the growth and metastasis of cervical cancer cell via targeting SOX9.

OBJECTIVE: The aim of the current study was to investigate the potential roles of miR-215-3p in the progression of cervical cancer. PATIENTS AND METHODS: The levels of miR-215-3p in both cervical cancer tissues and cell lines were detected using quantitative Real-time polymerase chain reaction (qRT-PCR) assay. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), colony formation, migration and invasion assays were applied to investigate the role of miR-215-3p on the growth and aggressiveness of cervical carcinoma SiHa cell. The expression of SRY-Box 9 (SOX9) was assessed by Western blotting assay. The Xenograft model and lung metastasis model were applied to reveal the impact of miR-215-3p on the growth and distant metastasis of cervical carcinoma cell in vivo. Moreover, miR-215-3p and a SOX9 siRNA were co-transfected into the SiHa cell to investigate the underlying mechanism of miR-215-3p-SOX9 on cervical cancer tumorigenesis. RESULTS: We used genome-wide gene expression analysis using clinical cervical cancer samples to identify that miR-215-3p was down-regulated in cervical cancer. We then collected 31 pairs of cervical cancer and the corresponding non-cancerous tissues to determine miR-215-3p level and indicated that miR-215-3p was significantly down-expressed in cervical cancer. Furthermore, the functional analysis suggested that over-expression of miR-215-3p suppressed the aggressiveness of SiHa cell, whereas down-regulation led to the opposite results. We identified SOX9 as a direct target of miR-215-3p, and its level was negatively related to the level of miR-215-3p in cervical carcinoma tissue. Up-regulation of SOX9 reversed the suppressive impact of miR-215-3p on cervical carcinoma cell, and down-regulation of SOX9 reversed the promote effects of miR-215-3p CONCLUSIONS: These findings showed the important role of the miR-215-3p/SOX9 axis in the progression of cervical carcinoma.

Up-regulation of miRNA-105 inhibits the progression of gastric carcinoma by directly targeting SOX9.

OBJECTIVE: MicroRNAs (miRNAs) are involved in the tumorigenesis and progression of multiple tumor types and function as either tumor suppressor genes or oncogenes. This study was designed to investigate the functional behaviors and regulatory mechanisms of miR-105 in the progression of gastric carcinoma. PATIENTS AND METHODS: 24 pairs of patients with gastric carcinoma were enrolled in this study. The levels of miR-105 in gastric carcinoma tissues and cells were determined using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assay. The biological functions of miR-105 in gastric carcinoma cell were detected by colony formation, transwell invasion and wound-healing assay. Luciferase activity assay and immunoblotting assay were applied to validate the direct target of miR-105. The expression of SRY-Box 9 (SOX9) was detected using immunofluorescence staining assay. Furthermore, the role of miR-105 on the growth of gastric carcinoma cell was examined in the established xenograft model. The role of miR-105 in the metastasis of gastric carcinoma cell in vivo, an experimental metastasis assay was performed. RESULTS: Herein, we proved that miR-105 was down-regulated in gastric carcinoma specimens as well as gastric cancer cells. Up-regulation of miR-105 suppressed the colony formation and aggressiveness traits of gastric carcinoma cell lines BGC823 and SGC7901 in vitro. Furthermore, over-expression of miR-105 inhibited the tumor growth as well as lung metastasis of gastric carcinoma cell in vivo. Further investigation identified SOX9 was the target gene of miR-105 in gastric cancer and its expression was negatively associated with the expression of miR-105 in gastric carcinoma tissues. Finally, overexpression of SOX9 partially reversed the influence of miR-105 on the growth and aggressiveness of gastric carcinoma cell. CONCLUSIONS: These results revealed the crucial role of miR-105 in the progression and metastasis of gastric carcinoma, which indicated the potential application of miR-105 in the treatment of gastric carcinoma.

Overexpression of HIF-2α-Dependent NEAT1 Promotes the Progression of Non-Small Cell Lung Cancer through miR-101-3p/SOX9/Wnt/ß-Catenin Signal Pathway.

BACKGROUND/AIMS: The present study aimed to explore the function of NEAT1 on non-small cell lung cancer (NSCLC), as well as its underlying mechanisms. METHODS: Quantitative realtime PCR (qRT-PCR) was used to measure NEAT1 expression in NSCLC tissues and cells. MTT assay and transwell assay were performed to detect cell proliferation, migration and invasion. Potential target genes were identified via luciferase reporter assay. Protein analysis was performed through western blotting. RESULTS: The expressions of NEAT1 were significantly higher in both of NSCLC tissues and cells than in normal controls. High expression of NEAT1 was significantly associated with TNM stage (P=0.000) and metastasis (P=0.000). NEAT1 knockdown inhibited the proliferation, migration and invasion of NSCLC cells. Hypoxia induction mediated by HIF-2α promoted EMT and NEAT1 expressions. Moreover, miR-101-3p was a target of NEAT1. We also found that SOX9 was a target of miR-101-3p. Oncogenic function of NEAT1 on NSCLC progression was mediated by miR-101-3p/SOX9/Wnt/ß-catenin signaling pathway. CONCLUSION: NEAT1 up-regulation induced by HIF-2α over-expression could promote the progression of NSCLC under hypoxic condition. Moreover, NEAT1 also takes part in NSCLC progression via miR-101-3p/SOX9/Wnt/ß-catenin axis.

FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells.

Merkel cells are innervated mechanosensory cells responsible for light-touch sensations. In murine dorsal skin, Merkel cells are located in touch domes and found in the epidermis around primary hairs. While it has been shown that Merkel cells are skin epithelial cells, the progenitor cell population that gives rise to these cells is unknown. Here, we show that during embryogenesis, SOX9-positive (+) cells inside hair follicles, which were previously known to give rise to hair follicle stem cells (HFSCs) and cells of the hair follicle lineage, can also give rise to Merkel Cells. Interestingly, while SOX9 is critical for HFSC specification, it is dispensable for Merkel cell formation. Conversely, FGFR2 is required for Merkel cell formation but is dispensable for HFSCs. Together, our studies uncover SOX9(+) cells as precursors of Merkel cells and show the requirement for FGFR2-mediated epithelial signalling in Merkel cell specification.

Increase of SOX9 promotes hepatic ischemia/reperfusion (IR) injury by activating TGF-ß1.

Ischemia/reperfusion (IR) injury causes damage in aerobically metabolizing organs or tissues, which is an essential injury mechanism in various clinical settings. SRY-related high mobility group-Box gene 9 (SOX9) is a transcription factor of the SRY family, modulating various cellular processes, including fibrosis formation and tumor growth. However, the effects of SOX9 on hepatic IR injury have not been explored. In the present study, a hepatic IR injury model was established, supported by a significant histological alteration with high Suzuki scores, and a remarkable up-regulation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Importantly, we found that SOX9 was over-expressed in liver of mice after IR operation. Suppressing SOX9 markedly reduced inflammatory response, as evidenced by the reduced mRNA expressions of tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1ß and inactivation of inhibitor of κBα (IκBα)/nuclear factor (NF)-κB pathway. In addition, SOX9 suppression alleviated apoptosis in liver of mice after IR injury, as supported by the reduced number of terminal deoxyribonucleotidyl transferse (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL)-staining cells and decreased expression of Caspase-3 in liver tissue sections. The role of SOX9 in accelerating hepatic IR injury was further confirmed in primary hepatocytes under hypoxiaand reoxygenation (HR) treatment by enhancing inflammatory response and apoptosis. Of note, we found that transforming growth factor (TGF)-ß1 was highly induced in liver of mice after IR injury. HR treatment also stimulated TGF-ß1 expressions in vitro. Significantly, SOX9 over-expression-induced inflammation and apoptosis were obviously reduced by pirfenidone (Pirf), TGF-ß1 inhibitor. In contrast, TGF-ß1 exposure to cells further enhanced inflammation and apoptosis in HR-operated cells either with SOX9 knockdown or over-expression. Therefore, we identified a novel SOX9-dependent pathway that contributed to hepatic IR injury through enhancing inflammation and apoptosis by activating TGF-ß1.

Association between SOX9 and CA9 in glioma, and its effects on chemosensitivity to TMZ.

Temozolomide (TMZ) is a standard chemotherapeutic drug used in the treatment of glioblastoma multiforme (GBM); however, resistance to this drug is common. SRY-Box 9 (SOX9) expression is associated with a poor prognosis of patients with GBM and with resistance to TMZ. Therefore, the aim of this study was to examine the effects of SOX9 inhibition on the sensitivity of glioma cells to TMZ treatment. We knocked down the expression of SOX9 (SOX9KD) via lentiviral infection in two glioblastoma (U87 and U251) cell lines, and the cells were then subjected to gene microarray, Gene Ontology and KEGG analysis pathway, all of which revealed a close association between SOX9 and the carbonic anhydrase 9 (CA9) gene. The TMZ-mediated apoptosis of glioma cells was significantly increased in the cells in the SOX9KD group. The potential underlying mechanism involved the downregulation of SOX9 and CA9 expression, which in turn decreased Akt phosphorylation, downregulated BCL­2 expression, and upregulated BAX expression, as assessed by western blot analysis and RT-qPCR. The effects were found to be substantially enhanced in the cells in the SOX9KD group treated with TMZ. Subsequently, considering the association between SOX9 and CA9, the effects of CA9 inhibition, using a CA9 inhibitor (U­104), on the chemosensitivity of glioma cells to TMZ were assessed. The results revealed that the use of U­104 + TMZ effectively induced glioma cell death, compared to treatment with TMZ alone. The underlying mechanisms were similar to those observed with the silencing of SOX9 in the TMZ-treated glioma cells. On the whole, the findings of this study establish the SOX9/CA9-mediated oncogenic pathway in glioma, the inhibition of which enhances the sensitivity of glioma cells to TMZ treatment, and thus highlights the value of developing small molecules or antibodies against the SOX9/CA9 pathway, for combination therapy with TMZ, in the more efficient management of glioma.

Inhibition of SOX9 Promotes Inflammatory and Immune Responses of Dental Pulp.

INTRODUCTION: The process of pulpitis is characterized by extracellular matrix imbalance and inflammatory cell infiltration. As an essential transcription factor, sex-determining region Y-box 9 (SOX9) is significantly inhibited by tumor necrosis factor alpha in inflammatory joint diseases. The aim of this study was to explore the role of SOX9 in extracellular matrix balance, cytokine expression, and the immune response in dental pulp. METHODS: The expression of SOX9 in normal and inflamed pulp tissue/human dental pulp cells (HDPCs) was detected by immunohistochemistry, Western blot, and quantitative polymerase chain reaction (qPCR). SOX9 small interfering RNA was used to knock down SOX9 expression of dental cells in vitro; extracellular matrix imbalance was analyzed by qPCR, Western blot, and gelatin/collagen zymography, and the secretion of cytokines was scanned by antibody arrays. The immune response of THP-1 was investigated by cell migration assay, cell attachment assay, phagocytosis assay, and enzyme-linked immunosorbent assay. The interaction of SOX9 with target genes was explored by chromatin immunoprecipitation (ChIP). RESULTS: SOX9 was strongly expressed in normal dental pulp tissue and HDPCs and reduced in inflamed pulp. SOX9 knockdown could inhibit the production of type I collagen, stimulate the enzymatic activities of MMP2 and MMP13, and regulate the production of interleukin (IL) 8 of HDPCs. SOX9 knockdown also effectively suppressed the differentiation and functional activities of THP-1. ChIP showed that the binding of the SOX9 protein with matrix metalloproteinase (MMP)-1, MMP-13, and IL-8 gene promoters was reduced after being treated with recombinant human tumor necrosis factor alpha. CONCLUSIONS: SOX9 was inhibited in inflamed dental pulp and may participate in the regulation of extracellular matrix balance, the inflammatory process, and the immune response.

SOX9/miR-130a/CTR1 axis modulates DDP-resistance of cervical cancer cell.

Cisplatin (DDP) -based chemotherapy is a standard strategy for cervical cancer, while chemoresistance remains a huge challenge. Copper transporter protein 1 (CTR1), a copper influx transporter required for high affinity copper (probably reduced Cu I) transport into the cell, reportedly promotes a significant fraction of DDP internalization in tumor cells. In the present study, we evaluated the function of CTR1 in the cell proliferation of cervical cancer upon DDP treatment. MicroRNAs (miRNAs) have been regarded as essential regulators of cell proliferation, apoptosis, migration, as well as chemoresistance. By using online tools, we screened for candidate miRNAs potentially regulate CTR1, among which miR-130a has been proved to promote cervical cancer cell proliferation through targeting PTEN in our previous study. In the present study, we investigated the role of miR-130a in cervical cancer chemoresistance to DDP, and confirmed the binding of miR-130a to CTR1. SOX9 also reportedly act on cancer chemoresistance. In the present study, we revealed that SOX9 inversely regulated miR-130a through direct targeting the promoter of miR-130a. Consistent with previous studies, SOX9 could affect cervical cancer chemoresistance to DDP. Taken together, we demonstrated a SOX9/miR-130a/CTR1 axis which modulated the chemoresistance of cervical cancer cell to DDP, and provided promising targets for dealing with the chemoresistance of cervical cancer.

Expression and Therapeutic Potential of SOX9 in Chordoma.

Purpose: Conventional chemotherapeutic agents are ineffective in the treatment of chordoma. We investigated the functional roles and therapeutic relevance of the sex-determining region Y (SRY)-box 9 (SOX9) in chordoma.Experimental Design: SOX9 expression was examined by immunohistochemistry (IHC) using 50 chordoma tissue samples. SOX9 expression in chordoma cell lines was examined by Western blot and immunofluorescent assays. We used synthetic human SOX9 siRNA to inhibit the expression of SOX9. Cell proliferation ability and cytotoxicity of inhibiting SOX9 were assessed by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) and clonogenic assays. The effect of SOX9 knockdown on chordoma cell motility was evaluated by a wound-healing assay and a Transwell invasion chamber assay. Knockdown of SOX9 induced apoptosis, cell-cycle arrest, as well as decreased expression of cancer stem cell markers were determined by Western blot and flow cytometric assays. The effect of the combination of SOX9 siRNA and the chemotherapeutic drug doxorubicin/cisplatin on chordoma cells was assessed by an MTT assay.Results: Tissue microarray and IHC analysis showed that SOX9 is broadly expressed in chordomas and that higher expression levels of SOX9 correlated with a poor prognosis. RNA interference (RNAi)-mediated knockdown of SOX9 inhibited chordoma cell growth, decreased cell motility, and induced apoptosis as well as cell-cycle arrest. Moreover, the combination of SOX9 inhibition and chemotherapeutic drugs had an enhanced anti-cancer effect on chordoma cells.Conclusions: Our results demonstrate that SOX9 plays a crucial role in chordoma. Targeting SOX9 provides a new rationale for treatment of chordoma. Clin Cancer Res; 23(17); 5176-86. ©2017 AACR.

Fgf10 and Sox9 are essential for the establishment of distal progenitor cells during mouse salivary gland development.

Salivary glands are formed by branching morphogenesis with epithelial progenitors forming a network of ducts and acini (secretory cells). During this process, epithelial progenitors specialise into distal (tips of the gland) and proximal (the stalk region) identities that produce the acini and higher order ducts, respectively. Little is known about the factors that regulate progenitor expansion and specialisation in the different parts of the gland. Here, we show that Sox9 is involved in establishing the identity of the distal compartment before the initiation of branching morphogenesis. Sox9 is expressed throughout the gland at the initiation stage before becoming restricted to the distal epithelium from the bud stage and throughout branching morphogenesis. Deletion of Sox9 in the epithelium results in loss of the distal epithelial progenitors, a reduction in proliferation and a subsequent failure in branching. We demonstrate that Sox9 is positively regulated by mesenchymal Fgf10, a process that requires active Erk signalling. These results provide new insights into the factors required for the expansion of salivary gland epithelial progenitors, which can be useful for organ regeneration therapy.

cFOS-SOX9 Axis Reprograms Bone Marrow-Derived Mesenchymal Stem Cells into Chondroblastic Osteosarcoma.

Bone marrow-derived mesenchymal stem cells (BMSCs) are proposed as the cells of origin of several subtypes of osteosarcoma (OS). However, signals that direct BMSCs to form different subtypes of OS are unclear. Here we show that the default tumor type from spontaneously transformed p53 knockout (p53_KO) BMSCs is osteoblastic OS. The development of this default tumor type caused by p53 loss can be overridden by various oncogenic signals: RAS reprograms p53_KO BMSCs into undifferentiated sarcoma, AKT enhances osteoblastic OS, while cFOS promotes chondroblastic OS formation. We focus on studying the mechanism of cFOS-induced chondroblastic OS formation. Integrated genome-wide studies reveal a regulatory mechanism whereby cFOS binds to the promoter of a key chondroblastic transcription factor, Sox9, and induces its transcription in BMSCs. Importantly, SOX9 mediates cFOS-induced cartilage formation in chondroblastic OS. In summary, oncogenes determine tumor types derived from BMSCs, and the cFOS-SOX9 axis is critical for chondroblastic OS formation.

ZNF606 interacts with Sox9 to regulate chondrocyte differentiation.

SRY-related high-mobility-group box 9 (Sox9) gene is a transcription factor that plays an essential role in chondrocyte differentiation and cartilage formation. In this study, we identified the transcriptional factor ZNF606 as an interacting partner for Sox9 in cells. We further demonstrated that overexpression of ZNF606 inhibited the transcriptional activity of Sox9, while knockdown of ZNF606 increased Sox9-mediated transcription. Chromatin immunoprecipitation analysis revealed that ZNF606 prevents Sox9 binding to the enhancers of its target gene col2a1. Importantly, the interaction between ZNF606 and Sox9 was decreased during chondrocyte differentiation. Consistent with these findings, ZNF606 inhibited chondrocyte differentiation. Thus, our results demonstrate that ZNF606 acts as a novel Sox9 co-regulator that inhibits Sox9-mediated chondrocyte differentiation.

MicroRNA-455-3p modulates cartilage development and degeneration through modification of histone H3 acetylation.

Histone acetylation regulated by class I histone deacetylases (HDACs) plays a pivotal role in matrix-specific gene transcription and cartilage development. While we previously demonstrated that microRNA (miR)-455-3p is upregulated during chondrogenesis and can enhance early chondrogenesis, the mechanism underlying this process remains largely unclear. In this study, we characterized the effect of miR-455-3p on histone H3 acetylation and its role during cartilage development and degeneration. We observed that miR-455-3p was highly expressed in proliferating and pre-hypertrophic chondrocytes, while HDAC2 and HDAC8 were primarily expressed in hypertrophic chondrocytes. Meanwhile, miR-455-3p suppressed the activity of reporter constructs containing the 3'-untranslated regions of HDAC2/8, inhibited HDAC2/8 expression and promoted histone H3 acetylation at the collagen 2 (COL2A1) promoter in human SW1353 chondrocyte-like cells. Treatment with the HDAC inhibitor trichostatin A (TSA) resulted in increased expression of cartilage-specific genes and promoted glycosaminoglycan deposition. Moreover, TSA inhibited matrix metalloproteinase 13 (Mmp13) expression and promoted nuclear translocation of SOX9 in interleukin-1-treated primary mouse chondrocytes. Lastly, knockdown of HDAC2/3/8 increased SRY (sex-determining region Y)-box 9 (SOX9) and decreased Runt-related transcription factor 2 (RUNX2) expression. Taken together, these findings suggest that miR-455-3p plays a critical role during chondrogenesis by directly targeting HDAC2/8 and promoting histone H3 acetylation, which raises possibilities of using miR-455-3p to influence chondrogenesis and cartilage degeneration.

PPARγ suppressed Wnt/ß-catenin signaling pathway and its downstream effector SOX9 expression in gastric cancer cells.

Wnt signaling pathway activation plays a critical role in biological processes of tumor progression. SOX9 belongs to the sry-related high-mobility group box (SOX) family and is a key transcription factor in the development and differentiation of multiple cell lineages. The purpose of this study was to investigate whether suppression of Wnt signaling pathway by PPARγ gene affects target SOX9 gene expression. The pEGFP-N1-PPARγ overexpression recombinant plasmid was structured by molecular biology technology. The overexpression plasmid and empty vector pEGFP-N1 were transfected into three types of human gastric cancer cell lines, with different levels of differentiation, MKN-28, SGC-7901 and BGC-823. The PPARγ, ß-catenin and SOX9 mRNA levels and proteins were examined by real-time PCR and Western blot analysis. The pEGFP-N1-PPARγ recombinant plasmid was constructed and transfected into MKN-28, SGC-7901 and BGC-823 successfully. High expression of PPARγ (p < 0.05) for transfection recombinant plasmid group induced obviously decreased expression of ß-catenin (p < 0.05), whereas SOX9 expression decreased significantly (p < 0.05) compared with the transfection empty vector group and normal comparison group. PPARγ can suppress ß-catenin expression in Wnt signaling pathway and its downstream effector SOX9 expression in gastric cancer cells.

Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes via repression of Sox9.

The terminal differentiation of hypertrophic chondrocytes is a tightly regulated process that plays a pivotal role in endochondral ossification. As a negative regulator, Sox9 is essentially downregulated in terminally differentiated hypertrophic chondrocytes. However, the underlying mechanism of Sox9 silencing is undefined. Here we show that the zinc finger protein Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes by repressing Sox9. In the developing skeleton of the mouse, Zbtb20 protein is highly expressed by hypertrophic chondrocytes from late embryonic stages. To determine its physiological role in endochondral ossification, we have generated chondrocyte-specific Zbtb20 knockout mice and demonstrate that disruption of Zbtb20 in chondrocytes results in delayed endochondral ossification and postnatal growth retardation. Zbtb20 deficiency caused a delay in cartilage vascularization and an expansion of the hypertrophic zone owing to reduced expression of Vegfa in the hypertrophic zone. Interestingly, Sox9, a direct suppressor of Vegfa expression, was ectopically upregulated at both mRNA and protein levels in the late Zbtb20-deficient hypertrophic zone. Furthermore, knockdown of Sox9 greatly increased Vegfa expression in Zbtb20-deficient hypertrophic chondrocytes. Our findings point to Zbtb20 as a crucial regulator governing the terminal differentiation of hypertrophic chondrocytes at least partially through repression of Sox9.