Sox9 mRNA Research Articles

MiR-155 promotes m6A modification of SOX2 mRNA through targeted regulation of HIF-1α and delays wound healing in diabetic foot ulcer in vitro models.

Diabetic foot ulcers (DFU) are one of the most destructive complications of diabetes mellitus. The aim of this study was to link miR-155 and SOX2 with DFU to explore the regulation of wound healing by DFU and its potential mechanism. Human keratinocytes (HaCaT) were induced with advanced glycation end products (AGEs) to construct DFU models in vitro. AGE-induced HaCaTcells were subjected to CCK-8 assays, flow cytometry, and wound healing assays to evaluate cell proliferation, apoptosis, and migration capacity, respectively. RT-qPCR and Western blotting were used to determine gene and protein expression levels, respectively. N6-methyladenosine (M6A) levels in total RNA were assessed using an M6A methylation quantification kit. Our results suggested that the inhibition of miR-155 promoted wound healing in an in vitro DFU model, while the knockdown of HIF-1α reversed this process, and that HIF-1α was a target protein of miR-155. In addition, knockdown of HIF-1α promoted the m6A level of SOX2 mRNA, inhibited the expression of SOX2, and inhibited the activation of the EGFR/MEK/ERK signaling pathway, thus inhibiting the proliferation and migration of HaCaTcells and promoting the apoptosis of HaCaTcells, while overexpression of SOX2 reversed this effect. We also found that METTL3 knockdown had the opposite effect of HIF-1α knockdown. Inhibition of miR-155 promoted the expression of HIF-1α and attenuated the m6A modification of SOX2 mRNA, thereby promoting the expression of SOX2 and activating the downstream EGFR/MEK/ERK signaling pathway to promote wound healing in an in vitro DFU model.

The essential role of the miRNA-208a/TARBP2/Sox6 axis in myosin expression in the end stage human heart failure

Abstract Despite diverse aetiologies, failing human hearts (HF) of various origins (coronary artery disease (CAD), dilated cardiomyopathy (DCM), restrictive cardiomyopathy, and hypertrophic cardiomyopathy (HCM)) exhibit impaired contractility and share alterations in MYH6, MYH7, and MYH7B gene expression. Here, we explore the miRNA-208a/TARBP2/Sox6 axis in regulating myosin expression within left ventricles of explanted human hearts. Methods Samples of left ventricles were obtained from explanted hearts of patients with terminal HF (NYHA III-IV) and reduced left ventricle ejection fraction (LVEF <25%) due to CAD (n=10), DCM (n=10), HCM (n=6), and controls (n=5). We analysed the expression of cardiac myosin heavy chains (MYH6, MYH7, MYH7B), cardiac damage markers (ANP, BNP), and heart-related MyoMiRs using quantitative real-time PCR. Results Pre-transplant data for the 31 subjects (25 male, 3 female) included: age 54.4±8.2y, BP 112±18/67±11 mmHg, HR 79±16 min-1, LVEF 27±14%, LVEDD 8.8±15.8mm, RVEDD 32.8±5.1mm, QRS 0.140±0.05s, QT 0.42±0.04s, NT-proBNP 5551±4259 ng/l. In all diseased groups, MYH6 mRNA was downregulated, while MYH7 and MHRT ncRNA were upregulated. We observed strong positive correlations between MYH7, MYH7B, and MHRT transcription, suggesting coordinated expression. The miRNA-208a/miRNA-208b ratio shifted heavily towards miRNA-208b in controls, with a similar effect for miRNA-499 and MYH7B. A strong negative correlation existed between miRNA-499 and MYH7B expression across HF samples. TARBP2 expression decreased slightly in all pathological groups, while Sox6 mRNA increased. Conclusion Elevated Sox6 expression may contribute to MYH6 downregulation and MYH7/MYH7B upregulation. Altered TARBP2 expression could promote cardiomyopathy via Sox6-mediated repression of cardiac slow-twitch myofiber proteins. Changes in miRNA-208a processing leading to Sox6 upregulation may inhibit Sox6-dependent myosin transcript expression.

Feedback loop LINC00511-YTHDF2-SOX2 regulatory network drives cholangiocarcinoma progression and stemness.

Cholangiocarcinoma (CCA) was identified as a malignant tumor with rising incidence and mortality rates, and the roles of long noncoding RNA (lncRNA) in CCA remained not entirely clear. In this study, LINC00511 had high expression in CCA, which was closely related to poor prognosis. Knockdown of LINC00511 significantly inhibited cell malignant biological behaviors. It also affected the stemness of CCA, evidenced by decreased SOX2 protein expression. Moreover, the study revealed the interaction of LINC00511, YTHDF2, and SOX2 in CCA. Specifically, LINC00511 facilitated the formation of a complex with YTHDF2 on SOX2 mRNA, which uniquely enhances the mRNA's stability through m6A methylation sites. This stabilization appears crucial for maintaining malignant behaviors in CCA cells. Additionally, LINC00511 modulated SOX2 expression via the PI3K/AKT signaling pathway. Meanwhile, SOX2 can also promote LINC00511 expression as an upstream transcription factor, thereby confirming a positive feedback loop formed by LINC00511, YTHDF2, and SOX2, which plays a significant role in the occurrence and development of CCA. Finally, the study successfully constructed two patient-derived xenograft models, revealing the vital role of LINC00511 in CCA development. In summary, this research provides a comprehensive understanding of the role of LINC00511 in the pathogenesis of CCA.

MIAT promotes myofibroblastic activities and transformation in oral submucous fibrosis through sponging the miR-342-3p/SOX6 axis.

Oral submucous fibrosis (OSF) is an oral potentially malignant disorder that is closely related to the habit of areca nut chewing. Long non-coding RNA (lncRNA) myocardial infarction-associated transcript (MIAT) has been identified as an essential regulator in the fibrosis progression. However, the role of MIAT in the development of OSF remains unknown. The transcriptomic profile showed that MIAT is significantly overexpressed in the OSF cohort, with a positive correlation to fibrotic markers. The silencing of MIAT expression in primary buccal mucosal fibroblasts (BMFs) markedly inhibited arecoline-induced myofibroblast transformation. Mechanistically, MIAT functioned as a miR-342-3p sponge and suppressed the inhibitory effect of miR-342-3p on SOX6 mRNA, thereby reinstating SOX6 expression. Subsequent RNA expression rescue experiments confirmed that MIAT enhanced resistance to apoptosis and facilitated myofibroblastic properties such as cell mobility and collagen gel contraction by regulating the miR-342-3p/SOX6 axis. Taken together, these results suggest that the abnormal upregulation of MIAT is important in contributing persistent activation of myofibroblasts in fibrotic tissue, which may result from prolonged exposure to the constituents of areca nut. Furthermore, our findings demonstrated that therapeutic avenues that target the MIAT/miR-342-3p/SOX6 axis may be a promising approach for OSF treatments.

Repair of Mechanical Cartilage Damage Using Exosomes Derived from Deer Antler Stem Cells.

Articular cartilage has limited self-repair capacity, and current clinical treatment options for cartilage defects are inadequate. However, deer antler cartilage possesses unique regenerative properties, with the ability to rapidly repair itself. This rapid self-repair process is closely linked to the paracrine factors released by deer antler stem cells. These findings present potential for the development of cell-free therapies for cartilage defects in clinical settings. The aim of this study was to investigate a novel method for repairing cartilage. A rat model with articular cartilage defects was established through surgery. Hydrogels loaded with exosomes (Exos) derived from antler stem cells (ASC-Exos) were implanted into the rat cartilage defects. The extent of cartilage damage repair was assessed using histological methods. The effects of ASC-Exos on chondrocytes and rat bone marrow mesenchymal stem cells (BMSCs) were evaluated using cell viability assays, proliferation assays, and scratch assays. Additionally, the maintenance of the chondrocyte phenotype by ASC-Exos was assessed using real-time fluorescence quantitative PCR (qPCR) and western blot analysis. The protein components contained of the Exos were identified using data-independent acquisition (DIA) mass spectrometry. ASC-Exos significantly promoted the repair of cartilage tissue damage. The level of cartilage repair in the experimental group (ASC-Exos) was higher than that in the positive control (human adipose-derived stem cells, hADSC-Exos) and negative control (dulbecco's modified eagle medium) groups (p < 0.05). In vitro experiments demonstrated that ASC-Exos significantly enhanced the proliferation abilities of chondrocytes and the proliferation abilities and the migration abilities of BMSCs (p < 0.05). ASC-Exos up-regulated the expression levels of Aggrecan, Collagen II (COLII), and Sox9 mRNA and proteins in chondrocytes. Analysis of ASC-Exos protein components revealed the presence of active components such as Serotransferrin (TF), S100A4, and Insulin-like growth factor-binding protein 1 (IGF1). ASC-Exos have a significant effect on cartilage damage repair, which may be attributed to their promotion of chondrocyte and BMSCs proliferation and migration, as well as the maintenance of chondrocyte phenotype. This effect may be mediated by the presence of TF, S100A4, and IGF1.

Oxygenator assisted dynamic microphysiological culture elucidates the impact of hypoxia on valvular interstitial cell calcification

IntroductionMicrophysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation.MethodsHuman valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts.ResultsHypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h.ConclusionIn hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Elastin-derived peptides (EDPs) affect gene and protein expression in human mesenchymal stem cells (hMSCs) – preliminary study

During the aging process, elastin is degraded and the level of elastin-derived peptides (EDPs) successively increases. The main peptide released from elastin during its degradation is a peptide with the VGVAPG sequence. To date, several papers have described that EDPs or elastin-like peptides (ELPs) affect human mesenchymal stem cells (hMSCs) derived from different tissues. Unfortunately, despite the described effect of EDPs or ELPs on the hMSC differentiation process, the mechanism of action of these peptides has not been elucidated. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG and VVGPGA peptides on the hMSC stemness marker and elucidation of the mechanism of action of these peptides. Our data show that both studied peptides (VGVAPG and VVGPGA) act with the involvement of ERK1/2 and c-SRC kinases. However, their mechanism of activation is probably different in hMSCs derived from adipose tissue. Both studied peptides increase the KI67 protein level in hMSCs, but this is not accompanied with cell proliferation. Moreover, the changes in the NANOG and c-MYC protein expression and in the SOX2 and POU5F1 mRNA expression suggest that EDPs reduced the hMSC stemness properties and could initiate cell differentiation. The initiation of differentiation was evidenced by changes in the expression of AhR and PPARγ protein as well as specific genes (ACTB, TUBB3) and proteins (β-actin, RhoA) involved in cytoskeleton remodeling. Our data suggest that the presence of EDPs in tissue can initiate hMSC differentiation into more tissue-specific cells.

Interaction between microRNA-195 and HuR regulates Paneth cell function in the intestinal epithelium by altering SOX9 translation.

Paneth cells at the bottom of small intestinal crypts secrete antimicrobial peptides, enzymes, and growth factors and contribute to pathogen clearance and maintenance of the stem cell niche. Loss of Paneth cells and their dysfunction occur commonly in various pathologies, but the mechanism underlying the control of Paneth cell function remains largely unknown. Here, we identified microRNA-195 (miR-195) as a repressor of Paneth cell development and activity by altering SOX9 translation via interaction with RNA-binding protein HuR. Tissue-specific transgenic expression of miR-195 (miR195-Tg) in the intestinal epithelium decreased the levels of mucosal SOX9 and reduced the numbers of lysozyme-positive (Paneth) cells in mice. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restored Paneth cell development ex vivo. miR-195 did not bind to Sox9 mRNA but it directly interacted with HuR and prevented HuR binding to Sox9 mRNA, thus inhibiting SOX9 translation. Intestinal mucosa from mice that harbored both Sox9 transgene and ablation of the HuR locus exhibited lower levels of SOX9 protein and Paneth cell numbers than those observed in miR-195-Tg mice. Inhibition of miR-195 activity by its specific antagomir improved Paneth cell function in HuR-deficient intestinal organoids. These results indicate that interaction of miR-195 with HuR regulates Paneth cell function by altering SOX9 translation in the small intestinal epithelium.NEW & NOTEWORTHY Our results indicate that intestinal epithelial tissue-specific transgenic miR-195 expression decreases the levels of SOX9 expression, along with reduced numbers of Paneth cells. Ectopically expressed SOX9 in the intestinal organoids derived from miR-195-Tg mice restores Paneth cell development ex vivo. miR-195 inhibits SOX9 translation by preventing binding of HuR to Sox9 mRNA. These findings suggest that interaction between miR-195 and HuR controls Paneth cell function via SOX9 in the intestinal epithelium.

SOX9 regulates epithelial‐mesenchymal transformation by mediating the Wnt/β‐catenin signaling pathway in hypospadias

AbstractThe transcription factor SOX9 is crucial in the development and differentiation of various tissues and cells. However, the roles of SOX9‐dependent genes and pathways in normal urethral development and the mechanism of hypospadias are unclear. This study collected 15 foreskin tissue specimens from patients who underwent hypospadias repair surgery and compared them to normal foreskin tissue specimens obtained during circumcision. The expression levels of SOX9, WNT signaling pathway markers, and epithelial‐mesenchymal transition (EMT) markers were analyzed in both groups. It was found that mRNA and protein levels of SOX9, WNT signaling pathway, and EMT mesenchymal markers were significantly reduced in the hypospadias group compared to the normal foreskin group. In contrast, mRNA and protein levels of epithelial markers were significantly increased in the hypospadias group. Immunofluorescence confirmed the decrease in SOX9 expression. Experiments using siRNA to inhibit SOX9 expression in foreskin fibroblasts yielded similar results to the hypospadias group. The findings suggest that down‐regulation of SOX9 expression may contribute to the development of hypospadias by down‐regulating the WNT pathway and inhibiting EMT. These findings provide new insights into the embryonic development of the urethra.

METTL14-Mediate the Biological Effects of EMT in Bladder Cancer Cells by Methylating SOX4 mRNA with m6A

METTL14-Mediate the Biological Effects of EMT in Bladder Cancer Cells by Methylating SOX4 mRNA with m6A

Isorhapontigenin inhibition of basal muscle-invasive bladder cancer attributed to its downregulation of SNHG1 and DNMT3b

BackgroundBladder cancer (BC) is among the most prevalent malignant urothelial tumors globally, yet the prognosis for patients with muscle-invasive bladder cancer (MIBC) remains dismal, with a very poor 5-year survival rate. Consequently, identifying more effective and less toxic chemotherapeutic alternatives is critical for enhancing clinical outcomes for BC patients. Isorhapontigenin (ISO), a novel stilbene isolated from a Gnetum found in certain provinces of China, has shown potential as an anticancer agent due to its diverse anticancer activities. Despite its promising profile, the specific anticancer effects of ISO on BC and the underlying mechanisms are still largely unexplored.MethodsThe anchorage-independent growth, migration and invasion of BC cells were assessed by soft agar and transwell invasion assays, respectively. The RNA levels of SOX2, miR-129 and SNHG1 were quantified by qRT-PCR, while the protein expression levels were validated through Western blotting. Furthermore, methylation-specific PCR was employed to assess the methylation status of the miR-129 promoter. Functional assays utilized siRNA knockdown, plasmid-mediated overexpression, and chemical inhibition approaches.ResultsOur study demonstrated that ISO treatment significantly reduced SNHG1 expression in a dose- and time-dependent manner in BC cells, leading to the inhibition of anchorage-independent growth and invasion in human basal MIBC cells. This effect was accompanied by the downregulation of MMP-2 and MMP-9 and the upregulation of the tumor suppressor PTEN. Further mechanistic investigations revealed that SOX2, a key upstream regulator of SNHG1, played a crucial role in mediating the ISO-induced transcriptional suppression of SNHG1. Additionally, we found that ISO treatment led to a decrease in DNMT3b protein levels, which in turn mediated the hypomethylation of the miR-129 promoter and the subsequent suppression of SOX2 mRNA 3’-UTR activity, highlighting a novel pathway through which ISO exerts its anticancer effects.ConclusionsCollectively, our study highlights the critical role of SNHG1 downregulation as well as its upstream DNMT3b/miR-129/SOX2 axis in mediating ISO anticancer activity. These findings not only elucidate the mechanism of action of ISO but also suggest novel targets for BC therapy.

FLRT2 mediates chondrogenesis of nasal septal cartilage and mandibular condyle cartilage.

Nasal septal cartilages (NSCs) and mandibular condyle cartilages (MCCs) are two important cartilages for craniomaxillofacial development. However, the role of FLRT2 in the formation of NSCs and MCCs remains undiscovered. NSCs and MCCs were used for immunocytochemistry staining of collagen II, toluidine blue staining, and alcian blue staining. Quantitative reverse transcription‑PCR and western blot were used to detect mRNA and protein expressions of FLRT2, N-cadherin, collagen II, aggrecan, and SOX9. Cell proliferation of MCCs and NSCs was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell counting kit‑8 assay. Cell migration of MCCs and NSCs was examined by wound healing assay and Transwell. Chondrogenesis of MCCs and NSCs were similar in morphological characteristics, while different in cell proliferation, migration, and extracellular matrix. FLRT2 promotes the proliferation and migration of NSCs. There were up-regulation of N-cadherin and down-regulation of collagen II, aggrecan, and SOX9 in NSC with knock down FLRT2. The current study, as demonstrated by Xie et al., reveals that FLRT2 overexpression in Sprague-Dawley neonatal rats promotes the proliferation and migration of NSCs and MCCs, decreases N-cadherin while increases collagen II, aggrecan, and SOX9 in NSC and MCCs. Altogether, FLRT2 mediates chondrogenesis of NSCs and MCCs.

Evaluation of the Effects of Opium on the Expression of SOX2 and OCT4 in Wistar Rat Bladder.

Bladder cancer is a malignancy greatly affected by behavioral habits. The aim of this study was to examine the effect of opium on changes in the expression of OCT4 and SOX2 in the bladder tissue of rats. Thirty six rats were divided into six groups: 24 rats in the addicted group received morphine and opium for 4 months with 12 rats in the control group. Blood testing was done for the evaluation of CBC, MDA, and TAC. The bladder tissue was removed and checked by histopathological examination. All total RNA was extracted, then cDNAs were synthesized and the OCT4 and SOX2 gene expressions were evaluated by Real-time PCR. The OCT4 mRNA expression level in the opium group of rats was significantly increased compared to the control group (13.5 and 6.8 fold in males and females respectively). Also, in the morphine group, similar augmentation was detected (3.8 and 6.7 fold in males and females respectively). The SOX2 mRNA over-expression level was seen in the morphine group of both genders as compared to the control group (3.7 and 4.2 fold in male and female respectively) but in the opium group, enhancement of mRNA level was seen only in males (6.6 fold). Opium increases both OCT4 and SOX2 expression more than morphine in male rats, but in female rats, SOX2 is increased more by morphine. Over expression of OCT4 and SOX2 was observed in rats treated with opium and morphine. Increased OCT4 and SOX2 expression was seen in opium-treated male rats, but in female rats, SOX2 was increased more by morphine.

Effect of valproic acid on the formation and migration of cranial neural crest cells at the early developmental stages in rat embryos.

Cranial neural crest cells (NCCs) are critical for craniofacial development. The administration of valproic acid (VPA) to pregnant females causes craniofacial malformations in offspring. However, the in vivo influence of VPA on mammalian cranial NCCs remains unclear. In this study, we aimed to elucidate the developmental stage-specific effect of VPA on cranial NCCs through the administration of a single dose of VPA to pregnant rat females immediately prior to the formation of the cranial neural crest (NC). We performed whole-mount immunohistochemistry or in situ hybridization to examine localization changes of gene transcripts associated with the epithelial-mesenchymal transition of the cranial NC (i.e., cranial NCC formation) and cranial NCC migration. The results showed that Hoxa2 mRNA was abnormally detected and Sox9 mRNA expression was decreased in the midbrain-rhombomere (R) 1/2 NC, which forms cranial NCCs that migrate to the frontonasal mass (FNM) and branchial arch (BA) 1, through VPA administration, thus reducing the formation of SNAI2-positive NCCs. Hoxa2-positive NCCs were detected normally in BA2 and abnormally in FNM and BA1, which are normally Hox-free, implying VPA-induced abnormal cranial NCC migration. In vitro verification experiments using the whole embryo culture system revealed that midbrain-R4 NCC migration was abnormal. These results indicate that VPA reduces the formation/delamination of the midbrain-R1/2 NCCs in a developmental stage-specific manner and subsequently causes the abnormal migration of R4 NCCs, which suggests that the abnormal formation and migration of cranial NCCs contribute to the inhibition of axonal elongation in the trigeminal nerve and a reduction in head size.

Human Umbilical Cord Mesenchymal Stem Cells Alleviate Rat Knee Osteoarthritis via Activating Wnt/ β-catenin Signaling Pathway.

Osteoarthritis (OA) is a chronic disease characterized by joint cartilage degeneration, destruction, and osteogenic hyperplasia. Human umbilical cord mesenchymal stem cells (hUCMSCs) have attracted increasing research interest due to their high clonogenic, proliferative, and migratory potential, as well as their improved secretion of relevant chondrogenic factors. This study evaluated the therapeutic potential and underlying mechanism of hUC-MSCs in alleviating pathological symptoms of OA. For the in vivo study, OA rats were established by the Hulth method to observe the therapeutic effect of intra-articular injection of hUC-MSCs. X-ray tests, gross observations, and histological and immunohistochemical assessments were conducted in rats. Levels of interleukin-1 beta (IL-1β), IL-6, matrix metalloproteinase-13 (MMP-13), and tissue inhibitor matrix metalloproteinase-1 in rats' synovial fluid were measured using enzyme-linked immunosorbent assay kits. For the in vitro study, hUC-MSCs and chondrocytes were cultured to explore the effect and underlying mechanisms of hUC-MSCs on OA. Apoptosis, proliferation, and glycosaminoglycan (GAG) were measured in the chondrocytes. The relative expression of aggrecan, COL-2, and SOX-9 mRNA was quantified by real-time polymerase chain reaction. Expressions of Wnt/β-catenin signaling molecules were measured by Western blot. We found that intra-articular injection of hUC-MSCs reduced the combined score, increased the expression of collagen II, and decreased the expression of MMP-13, IL-1β, and IL-6 in rat knee joints. Additionally, hUC-MSCs increased the content of GAGs, inhibited chondrocyte apoptosis, and promoted chondrocyte proliferation. The expression of aggrecan, COL-2, and SOX-9 mRNA in chondrocytes was promoted by hUC-MSCs via activation of the Wnt/β-catenin signaling pathway. Overall, this study demonstrated that hUC-MSCs induce the secretion of some cytokines via the paracrine function to activate the Wnt/β-catenin signaling pathway to reduce the pathological condition of OA and maintain the proper expression of cytokines and extracellular matrix proteins.

Propranolol inhibits EMT and metastasis in breast cancer through miR-499-5p-mediated Sox6

PurposeThis study will focus on 4T1 cells, a murine mammary adenocarcinoma cell line, as the primary research subject. We aim to investigate the inhibitory effects and mechanisms of propranolol on epithelial–mesenchymal transition (EMT) in breast cancer cells, aiming to elucidate this phenomenon at the miRNA level.MethodsIn this study, the EMT inhibitory effect of propranolol was observed through in vitro and animal experiments. For the screening of potential target miRNAs and downstream target genes, second-generation sequencing (SGS) and bioinformatics analysis were conducted. Following the screening process, the identified target miRNAs and their respective target genes were confirmed using various experimental methods. To confirm the target miRNAs and target genes, Western Blot (WB), reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence experiments were performed.ResultsIn this study, we found that propranolol significantly reduced lung metastasis in 4T1 murine breast cancer cells (p < 0.05). In vitro and in vivo experiments demonstrated that propranolol inhibited the epithelial–mesenchymal transition (EMT) as evidenced by Western Blot analysis (p < 0.05). Through next-generation sequencing (SGS), subsequent bioinformatics analysis, and PCR validation, we identified a marked downregulation of miR-499-5p (p < 0.05), suggesting its potential involvement in mediating the suppressive effects of propranolol on EMT. Overexpression of miR-499-5p promoted EMT, migration, and invasion of 4T1 cells, and these effects were not reversed or attenuated by propranolol (Validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). Sox6 was identified as a functional target of miR-499-5p, with its downregulation correlating with the observed EMT changes (p < 0.05). Silencing Sox6 or overexpressing miR-499-5p inhibited Sox6 expression, further promoting the processes of EMT, invasion, and migration in 4T1 cells. Notably, these effects were not alleviated by propranolol (validated via Western Blot, wound healing assay, transwell migration, and invasion assays, p < 0.05). The direct interaction between miR-499-5p and Sox6 mRNA was confirmed by dual-luciferase reporter gene assay.ConclusionThese results suggest that propranolol may have potential as a therapeutic agent for breast cancer treatment by targeting EMT and its regulatory mechanisms.

Morphogenesis of fungiform papillae in developing miniature pigs

ObjectiveFungiform papillae contain taste buds and play a critical role in mastication and the gustatory system. In this study, we report a series of sequential observations of organogenesis of fungiform papillae in miniature pigs, as well as changes in the expression of BMP2, BMP4, Wnt5a, Sox2, and Notch1 signaling pathway components. DesignIn this study, we investigated the spatiotemporal expression patterns of BMP, Wnt, Sox2 and Notch in the fungiform papillae of miniature pigs at the bud stage (E40), cap stage (E50) and bell stage (E60). Pregnant miniature pigs were obtained, and the samples were processed for histological staining. Immunohistochemistry and real-time PCR were used to detect the mRNA and protein expression levels of BMP2, BMP4, Wnt5a, Sox2, and Notch1. ResultsAt E40, fungiform papillae were present on the anterior two-thirds of the tongue in a specific array and pattern. The fungiform papillae were enlarged and basically developed at E50 and were largest at the earlier stage (E60). Most of the BMP2 was concentrated in the epithelial layer and the connective tissue core of the fungal papilloma and gradually accumulated from E40-E60. BMP-4 was weakly expressed in the fungiform papillae epithelia, but BMP-4-positive cells were also observed in the developing tongue muscle at E50 and E60. Wnt5a-positive cells were observed in the fungiform papillae epithelia and developing tongue muscle at all three time points. Sox2-positive cells were observed only in fungiform papillae epithelial cells, and Notch1-positive cells could not be detected. ConclusionsThis study provides primary data regarding the morphogenesis and expression of developmental signals in the fungiform papillae of miniature pigs, establishing a foundation for further research in both this model and humans.

SOX4/HDAC2 Axis Enhances Cell Survivability and Reduces Apoptosis by Activating AKT/MAPK Signaling in Colorectal Cancer.

Increased SOX4 (SRY-related HMG-box) activity aids cellular transformation and metastasis. However, its specific functions and downstream targets remain to be completely elusive in colorectal cancer (CRC). To investigate the role of SOX4 in CRC progression and the underlying mechanism. In the current study, online available datasets of CRC patients were explored to check the expression status of SOX4. To investigate the further functions, SOX4 was overexpressed and knocked down in CRC cells. Colony formation assay, flowcytometry analysis, and MTT assay were used to check for proliferation and apoptosis. Acridine orange staining was done to check the role of SOX4 in autophagy induction. Furthermore, western blot, qRT-PCR, and bioinformatic analysis was done to elucidate the downstream molecular mechanism of SOX4. GEPIA database showed enhanced expression of SOX4 mRNA in CRC tumor, and the human protein atlas (HPA) showed strong staining of SOX4 protein in tumor when compared to the normal tissue. Ectopic expression of SOX4 enhanced colony formation ability as well as rescued cells from apoptosis. SOX4 overexpressed cells showed the formation of acidic vesicular organelles (AVOs) which indicated autophagy. Further results revealed the activation of p-AKT/MAPK molecules upon overexpression of SOX4. SOX4 expression was found to be positively correlated with histone deacetylase 2 (HDAC2). Knockdown of SOX4 or HDAC2 inhibition induced apoptosis, revealed by decrease in BCL2 and increase in BAX expression, and inactivated the p-AKT/MAPK signaling. The study uncovers that SOX4/HDAC2 axis improves cell survivability and reduces apoptosis via activation of the p-AKT/MAPK pathway.

Establishment and characterization of cisplatin-resistant cell lines from canine mammary gland tumors

The development of cisplatin resistance is one of the major causes of mammary cancer treatment failure, and is associated with changes in Sox4 gene expression. To investigate the characteristic changes that occur in canine mammary gland tumor (CMGT) cells following the development of acquired cisplatin resistance, along with the relationship between these changes and the Sox4 gene. We constructed cisplatin-resistant cell line, CHMpCIS, from the cell line CHMp, which was isolated from the primary lesion of a malignant CMGT. The biological characteristics of these cells were examined by Western blot analysis, Transwell assays, and mammosphere formation assays. Compared to CHMp cells, CHMpCIS cells exhibited elevated cisplatin resistance, apoptotic escape ability, enhanced epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) features, in addition to over-activation of the Wnt/β-catenin signaling pathway and increased Sox4 protein. In CMGT cases, CMGT tissues (CMGTT) expressed higher levels of Sox4 protein and mRNA compared to adjacent tissues (CAMGTT). We found that these changes were inhibited by silencing of Sox4 expression in CHMpCIS cells. Furthermore, activation of the Wnt/β-catenin signaling pathway increased Sox4 expression levels through a positive feedback loop. These results suggested that CHMpCIS cells circumvented the damage caused by cisplatin through altering the expression of the Sox4 gene and activating the Wnt/β-catenin pathway, thereby changing the cellular biological characteristics.

HuR (ELAVL1) Stabilizes SOX9 mRNA and Promotes Migration and Invasion in Breast Cancer Cells.

RNA-binding proteins play diverse roles in cancer, influencing various facets of the disease, including proliferation, apoptosis, angiogenesis, senescence, invasion, epithelial-mesenchymal transition (EMT), and metastasis. HuR, a known RBP, is recognized for stabilizing mRNAs containing AU-rich elements (AREs), although its complete repertoire of mRNA targets remains undefined. Through a bioinformatics analysis of the gene expression profile of the Hs578T basal-like triple-negative breast cancer cell line with silenced HuR, we have identified SOX9 as a potential HuR-regulated target. SOX9 is a transcription factor involved in promoting EMT, metastasis, survival, and the maintenance of cancer stem cells (CSCs) in triple-negative breast cancer. Ribonucleoprotein immunoprecipitation assays confirm a direct interaction between HuR and SOX9 mRNA. The half-life of SOX9 mRNA and the levels of SOX9 protein decreased in cells lacking HuR. Cells silenced for HuR exhibit reduced migration and invasion compared to control cells, a phenotype similar to that described for SOX9-silenced cells.