SOX9 promotes stemness in the CAL27 cell line of tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is a prevalent form of oral malignancy, with increasing incidence. Unfortunately, the 5-year survival rate for patients has not exceeded 50%. Studies have shown that sex-determining region Y box 9 (SOX9) correlates with malignancy and tumor stemness in a variety of tumors. To investigate the role of SOX9 in TSCC stemness, we analyzed its influence on various aspects of tumor biology, including cell proliferation, migration, invasion, sphere and clone formation, and drug resistance in TSCC. Our data suggest a close association between SOX9 expression and both the stemness phenotype and drug resistance in TSCC. Immunohistochemical experiments revealed a progressive increase of SOX9 expression in normal oral mucosa, paracancerous tissues, and tongue squamous carcinoma tissues. Furthermore, the expression of SOX9 was closely linked to the TNM stage, but not to lymph node metastasis or tumor diameter. SOX9 is a crucial gene in TSCC responsible for promoting the stemness function of cancer stem cells. Developing drugs that target SOX9 is extremely important in clinical settings.

Transcriptional variations at perilesional site in vitiligo patients: probably a fight for cell-survival.

Exploration of gene expression variations is a potential source to unravel biological pathways involved in pathological changes in body and understand the mechanism underneath. Vitiligo patients were explored for gene expression changes transcriptionally at perilesional site in comparison to normal site of same patients for melanogenesis pathway (TYR, DCT & TYRP1) cell adhesion (MMPs & TIMP1), cell survival (BCL2 & BAX1) as well as proliferation, migration & development (SOX9, SOX10 & MITF) regulatory system, using skin biopsy samples. Results were also compared with changes in gene expression for melanocytes under stress after hydrogen peroxide treatment in-vitro. Gene amplification was carried out via real time PCR. We found increased expression of proliferation, migration & development regulatory genes as well as melanogenesis pathway genes at perilesional site of patients. In-vitro study also supports induced MITF expression and disturbed melanogenesis in melanocytes under stress. Expression level ratio of cell survival regulatory genes' (BCL2/BAX1) as well as cell adhesion regulatory genes (MMPs/TIMP1) was observed upregulated at patient's perilesional site however downregulated in hydrogen peroxide treated melanocytes in-vitro. Observed upregulated gene expression at perilesional site of patients may be via positive feedback loop in response to stress to increase cell tolerance power to survive against adverse conditions. Gene expression analysis suggests better cell survival and proliferation potential at perilesional site in vitiligo patients. It seems in-vivo conditions/growth factors supports cells to fight for survival to accommodate stressed conditions.

SRY-Box transcription factor 9 triggers YAP nuclear entry via direct interaction in tumors.

The translocation of YAP from the cytoplasm to the nucleus is critical for its activation and plays a key role in tumor progression. However, the precise molecular mechanisms governing the nuclear import of YAP are not fully understood. In this study, we have uncovered a crucial role of SOX9 in the activation of YAP. SOX9 promotes the nuclear translocation of YAP by direct interaction. Importantly, we have identified that the binding between Asp-125 of SOX9 and Arg-124 of YAP is essential for SOX9-YAP interaction and subsequent nuclear entry of YAP. Additionally, we have discovered a novel asymmetrical dimethylation of YAP at Arg-124 (YAP-R124me2a) catalyzed by PRMT1. YAP-R124me2a enhances the interaction between YAP and SOX9 and is associated with poor prognosis in multiple cancers. Furthermore, we disrupted the interaction between SOX9 and YAP using a competitive peptide, S-A1, which mimics an α-helix of SOX9 containing Asp-125. S-A1 significantly inhibits YAP nuclear translocation and effectively suppresses tumor growth. This study provides the first evidence of SOX9 as a pivotal regulator driving YAP nuclear translocation and presents a potential therapeutic strategy for YAP-driven human cancers by targeting SOX9-YAP interaction.

SOX9 depletion attenuates retinal ganglion cell ferroptosis through blocking ERK/p38 signaling.

BACKGROUND: Retinal ischemia-refusion (I/R) is a leading cause of irreversible blindness worldwide. This study aims to explore the regulatory role of SOX9 in retinal I/R injury, and attempts to elucidate its potential regulatory mechanism. METHODS: Retinal I/R injury model was established in vivo, and the histological changes was examined by hematoxylin and eosin (H&E) staining and immunofluorescent assay was performed to examine SOX9 expression. Oxygenation-glucose deprivation/reoxygenation (OGD/R)-induced retinal ischemia/reperfusion (I/R) injury in 661 W cells was constructed as an in vitro cellular model of glaucoma. The production of cytokines, lactate dehydrogenase (LDH) and the antioxidant enzymes were assessed by their commercial kits. Cellular reactive oxygen species (ROS) and lipid ROS was detected using DCFH-DA and C11-BODIPY 581/591 staining, respectively. Lipid peroxidation and Fe2+ level were detected to assess the ferroptosis level. Protein expression was examined by western blot. LM22B-10, the agonist of ERK signaling, was used to pretreat 661 W cells for mechanism investigation. RESULTS: SOX9 was aberrantly upregulated following retinal I/R injury both in vivo and in vitro. SOX9 knockdown exerted a protective role against OGD/R-triggered oxidative stress, inflammatory response and ferroptosis in 661 W cells. Further, ERK/p38 signaling was activated in 661 W cells following OGD/R induction, which was repressed by SOX9 knockdown, and the ERK signaling agonist partially counteracted the protective role of SOX9 knockdown against oxidative stress, inflammatory response and ferroptosis in OGD/R-induced 661 W cells. CONCLUSION: Collectively, inhibiting SOX9 to block oxidative stress, inflammation and ferroptosis by inactivating ERK/p38 signaling might be effective to prevent retinal I/R injury, thereby alleviating glaucoma.

Fibroblast-like synoviocytes-derived exosomal circFTO deteriorates rheumatoid arthritis by enhancing N6-methyladenosine modification of SOX9 in chondrocytes.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes disability worldwide. Exosomes released by fibroblast-like synoviocytes in RA (RA-FLSs-Exos) play a role in the development of RA, and circular RNAs (circRNAs) are important for RA progression. This study aimed to investigate the molecular mechanisms underlying the effects of RA-FLSs-Exos in RA and identify the potential pathway responsible for these effects. METHODS: We initially conducted microarray analysis to identify dysregulated circRNAs in exosomes associated with RA. We then co-cultured isolated RA-FLSs-Exos with chondrocytes to examine their role in RA. In vivo experiments were performed using collagen-induced arthritis mouse models, and circFTO knockdown was achieved through intra-articular injection of AAV5 vectors. RESULTS: Our findings revealed increased expression of circFTO in both RA-FLSs-Exos and synovial tissues from patients with RA. Exosomal circFTO hindered chondrocyte proliferation, migration, and anabolism while promoting apoptosis and catabolism. Mechanistically, we discovered that circFTO facilitates the formation of methyltransferases complex to suppress SRY-related high-mobility group box 9 (SOX9) expression with assistance from YTH domain family 2 (YTHDF2) through an m6A-dependent mechanism. Furthermore, inhibition of circFTO improved symptoms of RA in vivo. CONCLUSION: Taken together, our study demonstrates that exosomal circFTO derived from FLSs contributes to the progression of RA by targeting SOX9. These findings highlight a promising target for treating RA.

Skeletal growth is enhanced by a shared role for SOX8 and SOX9 in promoting reserve chondrocyte commitment to columnar proliferation.

SOX8 was linked in a genome-wide association study to human height heritability, but roles in chondrocytes for this close relative of the master chondrogenic transcription factor SOX9 remain unknown. We undertook here to fill this knowledge gap. High-throughput assays demonstrate expression of human SOX8 and mouse Sox8 in growth plate cartilage. In situ assays show that Sox8 is expressed at a similar level as Sox9 in reserve and early columnar chondrocytes and turned off when Sox9 expression peaks in late columnar and prehypertrophic chondrocytes. Sox8-/- mice and Sox8fl/flPrx1Cre and Sox9fl/+Prx1Cre mice (inactivation in limb skeletal cells) have a normal or near normal skeletal size. In contrast, juvenile and adult Sox8fl/flSox9fl/+Prx1Cre compound mutants exhibit a 15 to 20% shortening of long bones. Their growth plate reserve chondrocytes progress slowly toward the columnar stage, as witnessed by a delay in down-regulating Pthlh expression, in packing in columns and in elevating their proliferation rate. SOX8 or SOX9 overexpression in chondrocytes reveals not only that SOX8 can promote growth plate cell proliferation and differentiation, even upon inactivation of endogenous Sox9, but also that it is more efficient than SOX9, possibly due to greater protein stability. Altogether, these findings uncover a major role for SOX8 and SOX9 in promoting skeletal growth by stimulating commitment of growth plate reserve chondrocytes to actively proliferating columnar cells. Further, by showing that SOX8 is more chondrogenic than SOX9, they suggest that SOX8 could be preferred over SOX9 in therapies to promote cartilage formation or regeneration in developmental and degenerative cartilage diseases.

SOX9 Induces Orbital Fibroblast Activation in Thyroid Eye Disease Via MAPK/ERK1/2 Pathway.

Purpose: To evaluate the expression of sry-box transcription factor 9 (SOX9) in orbital fibroblasts (OFs) of thyroid eye disease (TED) and to find its potential role and underlying mechanism in orbital fibrosis. Methods: OFs were cultured from orbital connective tissues obtained from patients with TED (n = 10) and healthy controls (n = 6). SOX9 was depleted by small interfering RNA or overexpressed through lentivirus transduction in OFs. Fibroblast contractile activity was measured by collagen gel contraction assay and proliferation was examined by EdU assay. Transcriptomic changes were assessed by RNA sequencing. Results: The mRNA and protein levels of SOX9 were significantly higher in OFs cultured from patients with TED than those from healthy controls. Extracellular matrix-related genes were down-regulated by SOX9 knockdown and up-regulated by SOX9 overexpression in TED-OFs. SOX9 knockdown significantly decrease the contraction and the antiapoptotic ability of OFs, whereas the overexpression of SOX9 increased the ability of transformation, migration, and proliferation of OFs. SOX9 knockdown suppressed the expression of phosphorylated ERK1/2, whereas its overexpression showed the opposite effect. Epidermal growth factor receptor (EGFR) is one of the notably down-regulated genes screened out by RNA sequencing. Chromatin immunoprecipitation-qPCR demonstrated SOX9 binding to the EGFR promoter. Conclusions: A high expression of SOX9 was found in TED-OFs. SOX9 can activate OFs via MAPK/ERK1/2 signaling pathway, which in turn promotes proliferation and differentiation of OFs. EGFR was a downstream target gene of SOX9. SOX9/EGFR can be considered as therapeutic targets for the treatment of orbital fibrosis in TED.

Hypoxia promotes non-small cell lung cancer cell stemness, migration, and invasion via promoting glycolysis by lactylation of SOX9.

BACKGROUND: Lung cancer is the deadliest form of malignancy and the most common subtype is non-small cell lung cancer (NSCLC). Hypoxia is a typical feature of solid tumor microenvironment. In the current study, we clarified the effects of hypoxia on stemness and metastasis and the molecular mechanism. METHODS: The biological functions were assessed using the sphere formation assay, Transwell assay, and XF96 extracellular flux analyzer. The protein levels were detected by western blot. The lactylation modification was assessed by western blot and immunoprecipitation. The role of SOX9 in vivo was explored using a xenografted tumor model. RESULTS: We observed that hypoxia promoted sphere formation, migration, invasion, glucose consumption, lactate production, glycolysis, and global lactylation. Inhibition of glycolysis suppressed cell stemness, migration, invasion, and lactylation. Moreover, hypoxia increased the levels of SOX9 and lactylation of SOX9, whereas inhibition of glycolysis reversed the increase. Additionally, knockdown of SOX9 abrogated the promotion of cell stemness, migration, and invasion. In tumor-bearing mice, overexpression of SOX9 promoted tumor growth, and inhibition of glycolysis suppressed tumor growth. CONCLUSION: Hypoxia induced the lactylation of SOX9 to promote stemness, migration, and invasion via promoting glycolysis. The findings suggested that targeting hypoxia may be an effective way for NSCLC treatment and reveal a new mechanism of hypoxia in NSCLC.

LncRNA SOX9-AS1 triggers a transcriptional program involved in lipid metabolic reprogramming, cell migration and invasion in triple-negative breast cancer.

At the molecular level, triple-negative breast cancer (TNBC) is frequently categorized as PAM50 basal-like subtype, but despite the advances in molecular analyses, the clinical outcome for these subtypes is uncertain. Long non-coding RNAs (lncRNAs) are master regulators of genes involved in hallmarks of cancer, which makes them suitable biomarkers for breast cancer (BRCA) diagnosis and prognosis. Here, we evaluated the regulatory role of lncRNA SOX9-AS1 in these subtypes. Using the BRCA-TCGA cohort, we observed that SOX9-AS1 was significantly overexpressed in basal-like and TNBC in comparison with other BRCA subtypes. Survival analyzes showed that SOX9-AS1 overexpression was associated with a favorable prognosis in TNBC and basal-like patients. To study the functions of SOX9-AS1, we determined the expression levels in a panel of nine BRCA cell lines finding increased levels in MDA-MB-468 and HCC1187 TNBC. Using subcellular fractionation in these cell lines, we ascertained that SOX9-AS1 was located in the cytoplasmic compartment. In addition, we performed SOX9-AS1 gene silencing using two short-harping constructs, which were transfected in both cell models and performed a genome-wide RNA-seq analysis. Data showed that 351 lncRNAs and 740 mRNAs were differentially expressed in MDA-MB-468 while 56 lncRNAs and 100 mRNAs were modulated in HCC1187 cells (Log2FC < - 1.5 and > 1.5, p.adj value < 0.05). Pathway analysis revealed that the protein-encoding genes potentially regulate lipid metabolic reprogramming, and epithelial-mesenchymal transition (EMT). Expression of lipid metabolic-related genes LIPE, REEP6, GABRE, FBP1, SCD1, UGT2B11, APOC1 was confirmed by RT-qPCR. Functional analysis demonstrated that the knockdown of SOX9-AS1 increases the triglyceride synthesis, cell migration and invasion in both two TNBC cell lines. In conclusion, high SOX9-AS1 expression predicts an improved clinical course in patients, while the loss of SOX9-AS1 expression enhances the aggressiveness of TNBC cells.

Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

Peptide-mediated targeted delivery of SOX9 nanoparticles into astrocytes ameliorates ischemic brain injury.

Astrocytes are highly activated following brain injuries, and their activation influences neuronal survival. Additionally, SOX9 expression is known to increase in reactive astrocytes. However, the role of SOX9 in activated astrocytes following ischemic brain damage has not been clearly elucidated yet. Therefore, in the present study, we investigated the role of SOX9 in reactive astrocytes using a poly-lactic-co-glycolic acid (PLGA) nanoparticle plasmid delivery system in a photothrombotic stroke animal model. We designed PLGA nanoparticles to exclusively enhance SOX9 gene expression in glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes. Our observations indicate that PLGA nanoparticles encapsulated with GFAP:SOX9:tdTOM reduce ischemia-induced neurological deficits and infarct volume through the prostaglandin D2 pathway. Thus, the astrocyte-targeting PLGA nanoparticle plasmid delivery system provides a potential opportunity for stroke treatment. Since the only effective treatment currently available is reinstating the blood supply, cell-specific gene therapy using PLGA nanoparticles will open a new therapeutic paradigm for brain injury patients in the future.

SOX9 promotes the invasion and migration of lung adenocarcinoma cells by activating the RAP1 signaling pathway.

OBJECTIVE: SOX9 has been shown to be related to the metastasis of various cancers. Recently, it has been reported that SOX9 plays a regulatory role in lung adenocarcinoma (LUAD) cell metastasis, but the specific mechanism remains to be explored. Therefore, the objective of this study was to observe the effect and mechanism of SOX9 on the invasion and migration of LUAD cells. METHODS: RT-qPCR was applied to observe the expression of SOX9 and RAP1 in tumor tissues and corresponding normal lung tissues collected from LUAD patients. Co-immunoprecipitation and Pearson correlation to analyze the expression correlation of SOX9 with RAP1. To observe the role of SOX9, the invasion and migration levels of LUAD A549 cells in each group were observed by Transwell invasion assay and Scratch migration assay after knocking down or overexpressing SOX9. Besides, the expression levels of RAP1 pathway-related proteins (RAP1, RAP1GAP and RasGRP33) were observed by RT-qCPR or western blot. Subsequently, RAP1 was overexpressed and SOX9 was knocked down in A549 cells, and then the cell invasion/migration level and RAP1 pathway activity were assessed. RESULTS: The expression levels of SOX9 and RAP1 in tumor tissues and A549 cells of LUAD patients were significantly increased and positively correlated. Overexpression of SOX9 or RAP1 alone in A549 cells enhanced the invasion and migration ability of cells, as well as up-regulated the expression levels of RAP1, RAP1GAP and RasGRP33. However, knocking down SOX9 decreased cell invasion and migration levels and weakened the activity of RAP1 pathway. Notably, overexpressing RAP1 while knocking down SOX9 significantly activated RAP1 pathway and promoted cell invasion and migration. CONCLUSION: Overexpression of SOX9 in LUAD can significantly activate the RAP1 signaling pathway and promote cell invasion and migration.

Role of SOX9 and Hif-1α expression in placentas of patients with HELLP.

PURPOSE: In this study, we investigated the immunohistochemical staining of SRY-box transcription factor 9 (SOX9) and Hif-1α expression in placentas of pregnant woman with hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome. METHODS: Placentas of 20 normotensive and 20 women with HELLP syndrome were processed for routine histological tissue processing. The biochemical and clinical parameters of patients were recorded. Placentas were stained with hematoxylin-eosin and SOX9 and Hif-1α immunostaining. RESULTS: Normotensive placentas showed normal histology of placenta, however placentas of HELLP syndrome showed intense thrombosis, thinning of the villi membrane and vascular dilatation. In placentas of normotensive patients, SOX9 reaction was immunohistochemically negative, however placentas of HELLP group showed SOX9 expression in decidual cells, and syncytial regions of floating villi and inflammatory cells. In placentas of normotensive patients, Hif-1α reaction was mainly negative in vessels and connective tissue cells. Placentas of HELLP group showed increased Hif-1α expression in decidual cell and especially inflammatory cells in the maternal region. CONCLUSIONS: Hif-1α and SOX9 proteins can be used as a marker to show severity of preeclampsia and regulation of cell proliferation and angiogenesis during placental development.

DMRT1 is a testis-determining gene in rabbits and is also essential for female fertility.

DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.

Animals that reproduce sexually have organs called gonads, the ovaries and testes, which produce eggs and sperm. These organs, which are different in males and females, originate from the same cells during the development of the embryo. As a general rule, the chromosomal sex of an embryo, which gets determined at fertilization, leads to the activation and repression of specific genes. This in turn, controls whether the cells that will form the gonads will differentiate to develop testes or ovaries. Disruption of the key genes involved in the differentiation of the gonads can lead to fertility problems, and in some cases, it can cause the gonads to develop in the 'opposite' direction, resulting in a sex reversal. Identifying these genes is therefore essential to know how to maintain or restore fertility. DMRT1 is a gene that drives the differentiation of gonadal cells into the testicular pathway in several species of animals with backbones, including species of fish, frogs and birds. However, its role in mammals ­ where testis differentiation is driven by a different gene called SRY ­ is not well understood. Indeed, when DMRT1 is disrupted in male humans it leads to disorders of sex development, while disrupting this gene in male mice causes infertility. To obtain more information about the roles of DMRT1 in mammalian species, Dujardin et al. disrupted the gene in a third species of mammal: the rabbit. Dujardin et al. observed that chromosomally-male rabbits lacking DMRT1 developed ovaries instead of testes, showing that in rabbits, both SRY and DMRT1 are both required to produce testes. Additionally, this effect is similar to what is seen in humans, suggesting that rabbits may be a better model for human gonadal differentiation than mice are. Additionally, Dujardin et al. were also able to show that in female rabbits, lack of DMRT1 led to infertility, an effect that had not been previously described in other species. The results of Dujardin et al. may lead to better models for gonadal development in humans, involving DMRT1 in the differentiation of testes. Interestingly, they also suggest the possibility that mutations in this gene may be responsible for some cases of infertility in women. Overall, these findings indicate that DMRT1 is a key fertility gene.

A Case of Biphenotypic Adnexal Carcinoma With Bowenoid and Basaloid Features: Focus on the Expression of SOX9 and Wnt Signaling Pathway Molecules, Including CDX2.

ABSTRACT: An 87-year-old woman presented with a pedunculated nodule of 1.2 × 1.2 × 0.6 cm on her left cheek. Microscopic examination of the lesion revealed bowenoid and rosette-like basaloid components, resembling Bowen disease and neuroendocrine carcinoma, respectively. Immunohistochemically, both components were positive for Wnt signaling pathway molecules-nuclear/cytoplasmic beta-catenin, lymphoid enhancer binding factor 1 (LEF1), and caudal type homeobox 2 (CDX2)-and the adnexal marker SRY-box transcription factor 9 (SOX9). Unlike neuroendocrine tumors and basal cell carcinomas, the basaloid component in the present case was negative for chromogranin A, INSM1, synaptophysin, and p40. Previously reported cases of similar CDX2-positive lesions were diagnosed as squamous cell carcinoma with enteric adenocarcinomatous differentiation and basaloid cutaneous carcinoma with a primitive cytomorphology. However, the lesion in the present case was simultaneously positive for SOX9, indicating adnexal differentiation. In particular, the expression of multiple Wnt signaling pathway molecules indicates follicular differentiation despite the absence of morphological follicular features, such as shadow cells. Moreover, shared immunopositivity for SOX9, CDX2, nuclear/cytoplasmic beta-catenin, and LEF1 by both bowenoid and basaloid components indicated that the bowenoid component did not represent Bowen disease but a part of the adnexal tumor, and that the basaloid component was not a tumor-to-tumor metastasis. After complete excision, no recurrence has been observed for 5 months. The findings of the present case expand the histological spectrum of cutaneous adnexal tumors with follicular immunophenotypic differentiation.

EGR1 Promotes Ovarian Hyperstimulation Syndrome Through Upregulation of SOX9 Expression.

Angiogenesis is strongly associated with ovarian hyperstimulation syndrome (OHSS) progression. Early growth response protein 1 (EGR1) plays an important role in angiogenesis. This study aimed to investigate the function and mechanism of EGR1 involved in OHSS progression. RNA-sequencing was used to identify differentially expressed genes. In vitro OHSS cell model was induced by treating KGN cells with human chorionic gonadotropin (hCG). In vivo OHSS model was established in mice. The expression levels of EGR1, SOX1, and VEGF were determined by Quantitative Real-Time polymerase chain reaction (qRT-PCR), Western blot, immunofluorescence staining, and immunochemistry assay. The content of VEGF in the culture medium of human granulosa-like tumor cell line (KGN) cells was accessed by the ELISA assay. The regulatory effect of EGR1 on SRY-box transcription factor 9 (SOX9) was addressed by luciferase reporter assay and chromatin immunoprecipitation. The ERG1 and SOX9 levels were significantly upregulated in granulosa cells from OHSS patients and there was a positive association between EGR1 and SOX9 expression. In the ovarian tissues of OHSS mice, the levels of EGR1 and SOX9 were also remarkedly increased. Treatment with hCG elevated the levels of vascular endothelial growth factor (VEGF), EGR1, and SOX9 in KGN cells. Silencing of EGR1 reversed the promoting effect of hCG on VEGF and SOX9 expression in KGN cells. EGR1 transcriptionally regulated SOX9 expression through binding to its promoter. In addition, administration of dopamine decreased hCG-induced VEGF in KGN cells and ameliorated the progression of OHSS in mice, which were companied with decreased EGR1 and SOX9 expression. EGR1 has a promoting effect on OHSS progression and dopamine protects against OHSS through suppression of EGR1/SOX9 cascade. Our findings may provide new targets for the treatment of OHSS.

Engineering Critical Residues of SOX9 Discovers a Variant With Potent Capacity to Induce Chondrocytes.

Articular cartilage plays vital roles as a friction minimizer and shock absorber during joint movement but has a poor capacity to self-repair when damaged through trauma or disease. Cartilage tissue engineering is an innovative technique for cartilage regeneration, yet its therapeutic application requires chondrocytes in large numbers. Direct reprogramming of somatic cells to chondrocytes by expressing SOX9, KLF4, and c-MYC offers a promising option to generate chondrocytes in sufficient numbers; however, the low efficiency of the reprogramming system warrants further improvement. Here we referred to structural and functional features of SOX9 and performed alanine-scanning mutagenesis of functionally critical residues in the HMG box and at putative posttranslational modification (PTM) sites. We discovered that a SOX9 variant H131A/K398A, doubly mutated in the HMG box (H131) and at a PTM site (K398), significantly upregulated expression of chondrogenic genes and potently induced chondrocytes from mouse embryonic fibroblasts. The H131A/K398A variant remained unsumoylated in cells and exhibited a stronger DNA-binding activity than wild-type SOX9, especially when complexed with other proteins. Our results show that the novel SOX9 variant may be useful for efficient induction of chondrocytes and illuminate the strategic feasibility of mutating a transcription factor at functionally critical residues to expedite discovery of an optimized reprogramming factor.

Promotion of colorectal cancer progression by immune-related lnc-SOX9-4 via suppression of YBX1 poly-ubiquitination and degradation.

BACKGROUND: Recent research has highlighted the versatile functions of long non-coding RNAs (lncRNAs) in the onset and progression of various malignancies. Still, insufficient knowledge is available on how lnc-SOX9-4 functions in colorectal cancer (CRC) progression. METHODS: Bioinformatics analysis was used to identify a novel lncRNA (lnc-SOX9-4), and the expression pattern of the RNA in CRC was verified using qRT-PCR. Gene ontology (GO) term analysis and Gene set enrichment analysis (GSEA) were implemented for the identification of the related mechanisms and roles of lnc-SOX9-4. Immune infiltration analysis was conducted for assessment of how lnc-SOX9-4 is linked to tumor immune cell infiltration level. Both in vitro and in vivo phenotype analyses were conducted for scrutinizing how lnc-SOX9-4 impacts the proliferation and metastasis of CRC. RNA pulldown, mass spectrometry analysis, fluorescent in situ hybridization (FISH), western blotting, and RIP assay aided in verifying lnc-SOX9-4 mechanisms linked to CRC progression. RESULTS: An upregulation of lnc-SOX9-4 was observed in the sample CRC cells and tissues. Elevated lnc-SOX9-4 levels showed a positive association with poor clinical prognosis. Lnc-SOX9-4 was closely correlated to several types of immune infiltrating cells. Functionally, the knockdown of lnc-SOX9-4 significantly inhibited CRC cell proliferation, migration, and invasion abilities. Mechanistically, YBX1 was identified as lnc-SOX9-4, specifically interacting protein in the nucleus. Lnc-SOX9-4 could stabilize YBX1 protein levels by inhibiting poly-ubiquitination and degradation of YBX1. Furthermore, phenotype rescue experiments reveal that lnc-SOX9-4 enhanced the CRC cellular potential to proliferate and metastasize by regulating YBX1 levels. CONCLUSIONS: Lnc-SOX9-4 promoted CRC progression by suppressing cytoplasmic translocation and promoting protein levels of YBX1 can serve as novel treatment targets for diagnosing and treating CRC.

Influence of dexamethasone on the interaction between glucocorticoid receptor and SOX9: A molecular dynamics study.

The glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating the transcription of specific genes. GR transcriptional activity is modulated by a series of ligands and coenzymes, where a ligand can act as an agonist or antagonist. GR agonists, such as the glucocorticoids dexamethasone (DEX) and prednisolone, are widely prescribed to patients with inflammatory and autoimmune diseases. DEX is also used to induce osteogenic differentiation in vitro. Recently, it has been highlighted that DEX induces changes in the osteogenic differentiation of human mesenchymal stromal cells by downregulating the transcription factor SRY-box transcription factor 9 (SOX9) and upregulating the peroxisome proliferator-activated receptor γ (PPARG). SOX9 is fundamental in the control of chondrogenesis, but also in osteogenesis by acting as a dominant-negative of RUNX2. Many processes remain to be clarified during cell fate determination, such as the interplay between the key transcription factors. The main objective pursued by this work is to shed light on the interaction between GR and SOX9 in the presence and absence of DEX at an atomic level of resolution using molecular dynamics simulations. The outcome of this research could help the understanding of possible molecular interactions between GR and SOX9 and their role in the determination of cell fate. The results highlight the key residues at the interface between GR and SOX9 involved in the complexation process and shed light on the mechanism through which DEX modulates GR-SOX9 binding and exerts its biological activity.

ERK1/2-SOX9/FOXL2 axis regulates ovarian steroidogenesis and favors the follicular-luteal transition.

Estradiol and progesterone are the primary sex steroids produced by the ovary. Upon luteinizing hormone surge, estradiol-producing granulosa cells convert into progesterone-producing cells and eventually become large luteal cells of the corpus luteum. Signaling pathways and transcription factors involved in the cessation of estradiol and simultaneous stimulation of progesterone production in granulosa cells are not clearly understood. Here, we decipher that phosphorylated ERK1/2 regulates granulosa cell steroidogenesis by inhibiting estradiol and inducing progesterone production. Down-regulation of transcription factor FOXL2 and up-regulation of SOX9 by ERK underpin its differential steroidogenic function. Interestingly, the incidence of SOX9 is largely uncovered in ovarian cells and is found to regulate FOXL2 along with CYP19A1 and STAR genes, encoding rate-limiting enzymes of steroidogenesis, in cultured granulosa cells. We propose that the novel ERK1/2-SOX9/FOXL2 axis in granulosa cells is a critical regulator of ovarian steroidogenesis and may be considered when addressing pathophysiologies associated with inappropriate steroid production and infertility in humans and animals.