Comprehensive analysis of the clinical significance and molecular mechanism of T-box transcription factor 3 in osteosarcoma.

Background: T-box transcription factor 3 (TBX3) has been implicated in various malignant tumors, while its exact involvement in osteosarcoma (OS) remains unknown. Methods: Utilizing microarray data and bulk and single-cell RNA-seq data and qRT-PCR, we compared TBX3 mRNA expression levels in different stages of OS. Diagnostic ability testing and prognosis analysis were conducted to better understand the clinical importance of TBX3. Enrichment analysis was performed using gene groups with biological functions similar to TBX3 in different stages of OS to investigate the potential role of TBX3 in OS progression. In addition, we predicted medications targeted at TBX3 and identified downstream target genes to gain a comprehensive understanding of its therapeutic direction and regulatory mechanism. Results: TBX3 expression was highly upregulated in OS and was predominantly expressed in osteoblastic OS cells, with higher expression levels in metastatic tissues. TBX3 expression appeared somewhat suitable for discriminating between OS and normal samples, as well as different stages of OS. We found that TBX3 increased the malignant development of OS by altering cell cycle and cell adhesion molecules; exisulind and tacrolimus, which are targeted small-molecule medicines, were anticipated to counteract this dysregulation. The expression of CCNA2 could potentially be regulated by TBX3, contributing to OS advancement. Conclusion: TBX3 emerges as a potential biomarker for OS. In-depth research into its underlying molecular processes may offer new perspectives on treating OS.

New insights into mesoderm and endoderm development, and the nature of the onychophoran blastopore.

BACKGROUND: Early during onychophoran development and prior to the formation of the germ band, a posterior tissue thickening forms the posterior pit. Anterior to this thickening forms a groove, the embryonic slit, that marks the anterior-posterior orientation of the developing embryo. This slit is by some authors considered the blastopore, and thus the origin of the endoderm, while others argue that the posterior pit represents the blastopore. This controversy is of evolutionary significance because if the slit represents the blastopore, then this would support the amphistomy hypothesis that suggests that a slit-like blastopore in the bilaterian ancestor evolved into protostomy and deuterostomy. RESULTS: In this paper, we summarize our current knowledge about endoderm and mesoderm development in onychophorans and provide additional data on early endoderm- and mesoderm-determining marker genes such as Blimp, Mox, and the T-box genes. CONCLUSION: We come to the conclusion that the endoderm of onychophorans forms prior to the development of the embryonic slit, and thus that the slit is not the primary origin of the endoderm. It is thus unlikely that the embryonic slit represents the blastopore. We suggest instead that the posterior pit indeed represents the lips of the blastopore, and that the embryonic slit (and surrounding tissue) represents a morphologically superficial archenteron-like structure. We conclude further that both endoderm and mesoderm development are under control of conserved gene regulatory networks, and that many of the features found in arthropods including the model Drosophila melanogaster are likely derived.

A novel non-recurrent CNV deletion involving TBX4 and leaving TBX2 intact causes congenital alveolar dysplasia.

Congenital alveolar dysplasia (CAD) belongs to rare lethal lung developmental disorders (LLDDs) in neonates, manifesting with acute respiratory failure and pulmonary arterial hypertension refractory to treatment. The majority of CAD cases have been associated with copy-number variant (CNV) deletions at 17q23.1q23.2 or 5p12. Most CNV deletions at 17q23.1q23.2 were recurrent and encompassed two closely located genes, TBX4 and TBX2. In a few CAD cases, intragenic frameshifting deletions or single-nucleotide variants (SNVs) involved TBX4 but not TBX2. Here, we describe a male neonate who died at 27 days of life from acute respiratory failure caused by lung growth arrest along the spectrum of CAD confirmed by histopathological assessment. Trio-based genome sequencing revealed in the proband a novel non-recurrent ~1.07 Mb heterozygous CNV deletion at 17q23.2, encompassing TBX4 that arose de novo on the paternal chromosome. This is the first report of a larger-sized CNV deletion in a CAD patient involving TBX4 and leaving TBX2 intact. Our results, together with previous reports, indicate that perturbations of TBX4, rather than TBX2, cause severe lung phenotypes in humans.

Diminished TMEM100 Expression in a Newborn With Acinar Dysplasia and a Novel TBX4 Variant: A Case Report.

Acinar dysplasia (AcDys) of the lung is a rare lethal developmental disorder in neonates characterized by severe respiratory failure and pulmonary arterial hypertension refractory to treatment. Recently, abnormalities of TBX4-FGF10-FGFR2-TMEM100 signaling regulating lung development have been reported in patients with AcDys due to heterozygous single-nucleotide variants or copy-number variant deletions involving TBX4, FGF10, or FGFR2. Here, we describe a female neonate who died at 4 hours of life due to severe respiratory distress related to AcDys diagnosed by postmortem histopathologic evaluation. Genomic analyses revealed a novel deleterious heterozygous missense variant c.728A>C (p.Asn243Thr) in TBX4 that arose de novo on paternal chromosome 17. We also identified 6 candidate hypomorphic rare variants in the TBX4 enhancer in trans to TBX4 coding variant. Gene expression analyses of proband's lung tissue showed a significant reduction of TMEM100 expression with near absence of TMEM100 within the endothelium of arteries and capillaries by immunohistochemistry. These results support the pathogenicity of the detected TBX4 variant and provide further evidence that disrupted signaling between TBX4 and TMEM100 may contribute to severe lung phenotypes in humans, including AcDys.

Abnormal activation of the Wnt3a/ß-catenin signaling pathway promotes the expression of T-box transcription factor 3(TBX3) and the epithelial-mesenchymal transition pathway to mediate the occurrence of adenomyosis.

BACKGROUND: T-box transcription factor 3(TBX3) is a transcription factor that can regulate cell proliferation, apoptosis, invasion, and migration in different tumor cells; however, its role in adenomyosis (ADM) has not been previously studied. Some of ADM's pathophysiological characteristics are similar to those of malignant tumors (e.g., abnormal proliferation, migration, and invasion). METHODS AND RESULTS: We hypothesized that TBX3 might have a role in ADM. We used tamoxifen-induced Institute of Cancer research (ICR) mice to establish ADM disease model. The study procedure included western blotting and immunohistochemistry to analyze protein levels; additionally, we used intraperitoneal injection of Wnt/ß-catenin pathway inhibitor XAV-939 to study the relationship between TBX3 and Wnt/ß-catenin pathway as well as Anti-proliferation cell nuclear antigen( PCNA) and TUNEL to detect cell proliferation and apoptosis, respectively. TBX3 overexpression and epithelial-to-mesenchymal transition (EMT) in ADM mice was found to be associated with activation of the Wnt3a/ß-catenin pathway. Treatment with XAV-939 in ADM mice led to the inhibition of both TBX3 and EMT; moreover, abnormal cell proliferation was suppressed, the depth of invasion of endometrium cells was limited. Thus, the use of XAV-939 effectively inhibited further invasion of endometrial cells. CONCLUSION: These findings suggest that TBX3 may play an important role in the development of ADM. The expression of TBX3 in ADM was regulated by the Wnt3a/ß-catenin pathway. The activation of the Wnt3a/ß-catenin pathway in ADM promoted TBX3 expression and induced the occurrence of EMT, thus promoting cell proliferation and inhibiting apoptosis, ultimately accelerating the development of ADM. The study provides a reference for the diagnosis of ADM.

Expression analysis of IL-2, TBX21 and SOCS1 in peripheral blood cells of celiac disease patients reveals the diagnostic potential of IL-2.

BACKGROUND: Celiac disease (CD) is a chronic immune-mediated enteropathy and a cytokine network is involved in its pathogenesis. Interleukin-2 (IL-2) has a key role in the adaptive immune pathogenesis of CD and has been reported to be one of the earliest cytokines to be elicited after gluten exposure by CD patients. This study aimed at investigating the expression level of IL-2 and functionally related genes SOCS1 and TBX21 in active and treated CD patients compared to controls. METHODS AND RESULTS: Peripheral blood (PB) samples were collected from 40 active CD (ACD), 100 treated CD, and 100 healthy subjects. RNA was extracted, cDNA was synthesized and mRNA expression levels of the desired genes were investigated by Real-time PCR. The gene-gene interaction network was also constructed by GeneMANIA. Our results showed a higher PB mRNA expression of IL-2 in ACD patients compared to controls (p = 0.001) and treated CD patients (p˂0.0001). The mRNA expression level of TBX21 was also significantly up-regulated in ACD patients compared to controls (P = 0.03). SOCS1 mRNA level did not differ between active and treated CD patients and controls (p˃0.05) but showed a significant correlation with the patient's aphthous stomatitis symptom (r = 0.37, p = 0.01). ROC curve analysis suggested that the use of IL-2 levels can reach a high specificity and sensitivity in discriminating active CD patients. CONCLUSIONS: The PB level of IL-2 has the potential to be introduced as a diagnostic biomarker for CD. Larger cohort studies, including pediatric patients, are needed to achieve more insights in this regard.

Epigenetic programming of TBX2/CX43 mediates lower sperm quality in male offspring induced by prenatal dexamethasone exposure.

Decreased sperm quality is the main cause of male infertility. Studies have found that prenatal dexamethasone exposure (PDE) decreases sperm quality in male offspring after birth, but the mechanism is unclear. Wistar pregnant rats were subcutaneously injected with 0.1, 0.2 and 0.4 mg/kg.d dexamethasone at gestational day 9-20. The testes and sperm of first-generation (F1) offspring were collected, and F1 offspring were mated with wild-type female rats to obtain F2. Compared with the control group, F1 offspring in PDE group had lower sperm count and motility after birth, and the deformity rate increased. F2 fetal rats' body length and weight decreased, and the intrauterine growth retardation rate increased. Meanwhile, PDE decreased the expression of connexin 43 (CX43) in offspring testes, while T-box transcription factor 2 (TBX2) promoter region histone 3 lysine 9 acetylation (H3K9ac) level and its expression were increased. Traced back to F1 fetus testes, PDE increased the expression of glucocorticoid receptor (GR) and P300, activated GR protein into the nucleus, and made GR act on the TBX2 promoter region. Further, a series of Sertoli cell interventions confirmed that dexamethasone promoted GR to recruit P300, increased the H3K9ac level of TBX2 promoter region and its expression, and inhibited the expression of CX43. This study confirmed that PDE decreased sperm quality of male offspring, which is related to the epigenetic programming of TBX2/CX43 in the Sertoli cells, provided a theoretical and experimental basis for guiding the rational use of drugs during pregnancy.

TBX2 affects proliferation, apoptosis and cholesterol generation by regulating mitochondrial function and autophagy in bovine cumulus cell.

BACKGROUND: T-box transcription factor 2 (TBX2) is a member of T-box gene family whose members are highly conserved in evolution and encoding genes and are involved in the regulation of developmental processes. The encoding genes play an important role in growth and development. Although TBX2 has been widely studied in cancer cell growth and development, its biological functions in bovine cumulus cells remain unclear. OBJECTIVES: This study aimed to investigate the regulatory effects of TBX2 in bovine cumulus cells. METHODS: TBX2 gene was knockdown with siRNA to clarify the function in cellular physiological processes. Cell proliferation and cycle changes were determined by xCELLigence cell function analyzer and flow cytometry. Mitochondrial membrane potential and autophagy were detected by fluorescent dye staining and immunofluorescence techniques. Western blot and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) were used to detect the expression changes of proliferation and autophagy-related proteins. Aadenosine triphosphate (ATP) production, glucose metabolism, and cholesterol synthesis of cumulus cells were measured by optical density and chemiluminescence analysis. RESULTS: After inhibition of TBX2, the cell cycle was disrupted. The levels of apoptosis, ratio of light chain 3 beta II/I, and reactive oxygen species were increased. The proliferation, expansion ability, ATP production, and the amount of cholesterol secreted by cumulus cells were significantly decreased. CONCLUSIONS: TBX2 plays important roles in regulating the cells' proliferation, expansion, apoptosis, and autophagy; maintaining the mitochondrial function and cholesterol generation of bovine cumulus cells.

Dietary polyphenols and their relationship to the modulation of non-communicable chronic diseases and epigenetic mechanisms: A mini-review.

Chronic Non-Communicable Diseases (NCDs) have been considered a global health problem, characterized as diseases of multiple factors, which are developed throughout life, and regardless of genetics as a risk factor of important relevance, the increase in mortality attributed to the disease to environmental factors and the lifestyle one leads. Although the reactive species (ROS/RNS) are necessary for several physiological processes, their overproduction is directly related to the pathogenesis and aggravation of NCDs. In contrast, dietary polyphenols have been widely associated with minimizing oxidative stress and inflammation. In addition to their antioxidant power, polyphenols have also drawn attention for being able to modulate both gene expression and modify epigenetic alterations, suggesting an essential involvement in the prevention and/or development of some pathologies. Therefore, this review briefly explained the mechanisms in the development of some NCDs, followed by a summary of some evidence related to the interaction of polyphenols in oxidative stress, as well as the modulation of epigenetic mechanisms involved in the management of NCDs.

Effects of crocin on T-bet/GATA-3 ratio, and miR-146a and miR-106a expression levels in lung tissue of ovalbumin-sensitized mice.

Objectives: Although various studies have revealed the beneficial effects of crocin (derived from saffron), such as anti-inflammatory, anti-cancer, antioxidant, and immune modulator, however, its exact mechanism is unknown. The present study aimed to investigate the effect of crocin on the expression ratio of T-bet/GATA-3 as an indicator of altered immune responses in the lung tissue of ovalbumin (OVA)-sensitized mice. In addition, the effect of crocin on the expression level of miR-146a and miR-106a in the lung tissue OVA-sensitized mice was investigated. Materials and Methods: Mice were randomly divided into five groups (n=6): Control; OVA, OVA + Crocin 25, OVA + Cro 50, and OVA + Cro100 groups. Crocin was administrated intraperitoneally at doses of 25, 50, and 100 mg/kg for five consecutive days. One day after asthma induction, animals were euthanized, and lungs were sampled for pathological and gene expression analysis. Results: OVA-sensitization led to increased inflammation and histopathological changes in the lung tissue of mice. In addition, GATA-3 expression increased (P<0.001) and T-bet expression decreased (P<0.001) in OVA-sensitized groups. The T-bet/GATA3 ratio was also reduced markedly in asthma groups (P<0.001). Furthermore, increased expression of miR-146a and miR-106a levels was evident in the lung tissue of OVA-sensitized mice (P<0.001 for both). Intervention with high concentrations of crocin (50 and 100 mg/kg) significantly reduced airway inflammation, GATA-3 expression, miR-146a expression, and miR-106a expression and corrected the T-bet/GATA-3 ratio (P<0.05 to P<0.001). Conclusion: Treatment with crocin led to a decrease in the severity of lung inflammation in OVA-sensitized mice, which is probably through the reduction of the T-bet/GATA-3 ratio, and mir-146a and mir-106a expression level.

Functional characteristics of a broad spectrum of TBX6 variants in Mayer-Rokitansky-Küster-Hauser syndrome.

PURPOSE: Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is characterized by congenital absence of the uterus, cervix, and the upper part of the vagina in females. Whole-gene deletion and loss-of-function variants in TBX6 have been identified in association with MRKHS. We aimed to expand the spectrum of TBX6 variants in MRKHS and explore the biological effect of the variant alleles. METHODS: Rare variants in TBX6 were called from a combined multiethnic cohort of 622 probands with MRKHS who underwent exome sequencing or genome sequencing. Multiple in vitro functional experiments were performed, including messenger RNA analysis, western blotting, transcriptional activity assay, and immunofluorescence staining. RESULTS: We identified 16 rare variants in TBX6 from the combined cohort, including 1 protein-truncating variant reported in our previous study and 15 variants with unknown effects. By comparing the prevalence of TBX6 variants in the Chinese MRKHS cohort vs 1038 female controls, we observed a significant mutational burden of TBX6 in affected individuals (P = .0004, odds ratio = 5.25), suggesting a causal role of TBX6 variants in MRKHS. Of the 15 variants with uncertain effects, 7 were shown to induce a loss-of-function effect through various mechanisms. The c.423G>A (p.Leu141=) and c.839+5G>A variants impaired the normal splicing of TBX6 messenger RNA, c.422T>C (p.Leu141Pro) and c.745G>A (p.Val249Met) led to decreased protein expression, c.10C>T (p.Pro4Ser) and c.400G>A (p.Glu134Lys) resulted in perturbed transcriptional activity, and c.356G>A (p.Arg119His) caused protein mislocalization. We observed incomplete penetrance and variable expressivity in families carrying deleterious variants, which indicates a more complex genetic mechanism than classical Mendelian inheritance. CONCLUSION: Our study expands the mutational spectrum of TBX6 in MRKHS and delineates the molecular pathogenesis of TBX6 variants, supporting the association between deleterious variants in TBX6 and MRKHS.

Molecular identification of T-box transcription factor 6 and prognostic assessment in patients with congenital scoliosis: A single-center study.

Background: Congenital scoliosis (CS) is characterized by vertebral malformations. The precise etiology of CS is not fully defined. A compound inheritance of TBX6 was identified in 10% of patients with CS in Han Chinese and formed a distinguishable subtype named TBX6-associated congenital scoliosis (TACS). Methods: To investigate the variants and risk haplotype of TBX6, we recruited 121 patients with CS at Beijing Children's Hospital. We collected the clinical characteristics and surgical treatment options and followed their postoperative prognoses. Results: Eight patients (6.6%) were molecularly diagnosed with TACS and carried the previously defined pathogenic TBX6 compound heterozygous variants. All the eight patients with TACS had the typical TACS clinical feature of hemivertebrae in the lower part of the spine. These patients received posterior hemivertebra resection combined with segmental fusion. Follow-ups revealed satisfactory correction without postoperative complications. Conclusion: We observed a 6.6% prevalence of TACS in our CS cohort. Follow-ups further highlighted that surgical treatment of hemivertebra resection combined with segmental fusion performed well with prognosis for patients with TACS. This could provide valuable information for CS individuals with compound heterozygosity in TBX6.

TBX5 variant with the novel phenotype of mixed­type total anomalous pulmonary venous return in Holt­Oram Syndrome and variable intrafamilial heart defects.

Variants in T­box transcription factor 5 (TBX5) can result in a wide phenotypic spectrum, specifically in the heart and the limbs. TBX5 has been implicated in causing non­syndromic cardiac defects and Holt­Oram syndrome (HOS). The present study investigated the underlying molecular etiology of a family with heterogeneous heart defects. The proband had mixed­type total anomalous pulmonary venous return (mixed­type TAPVR), whereas her mother had an atrial septal defect. Genetic testing through trio­based whole­exome sequencing was used to reveal the molecular etiology. A nonsense variant was identified in TBX5 (c.577G>T; p.Gly193*) initially showing co­segregation with a presumably non­syndromic presentation of congenital heart disease. Subsequent genetic investigations and more complete phenotyping led to the correct diagnosis of HOS, documenting the novel association of mixed­type TAPVR with HOS. Finally, protein modeling of the mutant TBX5 protein that harbored this pathogenic nonsense variant (p.Gly193*) revealed a substantial drop in the quantity of non­covalent bonds. The decrease in the number of non­covalent bonds suggested that the resultant mutant dimer was less stable compared with the wild­type protein, consequently affecting the protein's ability to bind DNA. The present findings extended the phenotypic cardiac defects associated with HOS; to the best of our knowledge, this is the first association of mixed­type TAPVR with TBX5. Prior to the current analysis, the molecular association of TAPVR with HOS had never been documented; hence, this is the first genetic investigation to report the association between TAPVR and HOS. Furthermore, it was demonstrated that the null­variants reported in the T­box domain of TBX5 were associated with a wide range of cardiac and/or skeletal anomalies on both the inter­and intrafamilial levels. In conclusion, genetic testing was highlighted as a potentially powerful approach in the prognostication of the proper diagnosis.

Effect of naturally derived surgical hemostatic materials on the proliferation of A549 human lung adenocarcinoma cells.

Hemostatic materials are generally applied in surgical operations for cancer, but their effects on the growth and recurrence of tumors are unclear. Herein, three commonly used naturally derived hemostatic materials, gelatin sponge, Surgicel (oxidized regenerated cellulose), and biopaper (mixture of sodium hyaluronate and carboxymethyl chitosan), were cocultured with A549 human lung adenocarcinoma cells in vitro. Furthermore, the performance of hemostatic materials and the tumorigenicity of the materials with A549 â€‹cells were observed after subcutaneous implantation into BALB/c mice. The in vitro results showed that biopaper was dissolved quickly, with the highest cell numbers at 2 and 4 days of culture. Gelatin sponges retained their structure and elicited the least cell infiltration during the 2- to 10-day culture. Surgicel partially dissolved and supported cell growth over time. The in vivo results showed that biopaper degraded rapidly and elicited an acute Th1 lymphocyte reaction at 3 days after implantation, which was decreased at 7 days after implantation. The gelatin sponge resisted degradation and evoked a hybrid M1/M2 macrophage reaction at 7-21 days after implantation, and a protumor M2d subset was confirmed. Surgicel resisted early degradation and caused obvious antitumor M2a macrophage reactions. Mice subjected to subcutaneous implantation of A549 â€‹cells and hemostatic materials in the gelatin sponge group had the largest tumor volumes and the shortest overall survival (OS), while the Surgicel and the biopaper group had the smallest volumes and the longest OS. Therefore, although gelatin sponges exhibited cytotoxicity to A549 â€‹cells in vitro, they promoted the growth of A549 â€‹cells in vivo, which was related to chronic M2d macrophage reaction. Surgicel and biopaper inhibited A549 â€‹cell growth in vivo, which is associated with chronic M2a macrophage reaction or acute Th1 lymphocyte reaction.

Patient-Specific TBX5-G125R Variant Induces Profound Transcriptional Deregulation and Atrial Dysfunction.

BACKGROUND: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt-Oram syndrome (also known as hand-heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. METHODS: We analyzed ECGs of an extended family pedigree of Holt-Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome (Tbx5G125R) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). RESULTS: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt-Oram syndrome patients. Tbx5G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. CONCLUSIONS: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

lncRNA Kcnq1ot1 promotes bone formation by inhibiting miR-98-5p/Tbx5 axis in MC3T3-E1 cells.

Long non-coding (lnc)RNA KCNQ1 opposite strand/antisense transcript 1 (Kcnq1ot1) has been shown to regulate multiple biological processes. However, the functional role of Kcnq1ot1 in osteoporosis and the underlying mechanism are still unclear. The present study aimed to investigate the function of lncRNA Kcnq1ot1 in osteogenic differentiation. Alkaline phosphatase (ALP) activity was measured using an ALP assay kit. Western blotting was performed to assess the expression levels of osteogenic differentiation-associated proteins. Reverse transcription-quantitative PCR was performed to detect Kcnq1ot1, microRNA (miR)-98-5p and T-box transcription factor 5 (Tbx5) expression levels. The binding of Kcnq1ot1 with miR-98-5p and that of miR-98-5p with Tbx5 were predicted by starBase and TargetScan databases, respectively, and verified using dual luciferase reporter assays. The mineralization of MC3T3-E1 cells was observed using an Alizarin red S staining assay. The results revealed that expression of Kcnq1ot1 was increased and that of miR-98-5p was decreased during osteogenic differentiation. Additionally, Kcnq1ot1 was shown to target miR-98-5p and inhibit its expression. Inhibiting miR-98-5p reversed the inhibitory effect of Kcnq1ot1 knockdown on osteogenic differentiation and mineralization of MC3T3-E1 cells. Furthermore, Kcnq1ot1 regulated Tbx5 expression via miR-98-5p. Overexpressing miR-98-5p or downregulating Tbx5 expression reversed the promotive effect of Kcnq1ot1 overexpression on osteogenic differentiation and mineralization of MC3T3-E1 cells. In conclusion, these findings suggested that Kcnq1ot1 may promote bone formation by inhibiting miR-98-5p and upregulating Tbx5. Kcnq1ot1, miR-98-5p and Tbx5 may therefore serve as promising targets for the treatment of osteoporosis.

Exacerbation of mild lung disorders to lethal pulmonary hypoplasia by a noncoding hypomorphic SNV in a lung-specific enhancer in trans to the frameshifting TBX4 variant.

Variants involving TBX4 are associated with a wide variety of disorders, including pulmonary arterial hypertension, ischiocoxopodopatellar syndrome (ICPPS)/small patella syndrome (SPS), lethal lung developmental disorders (LLDDs) in neonates, heart defects, and prenatally lethal posterior amelia with pelvic and pulmonary hypoplasia syndrome. The objective of our study was to elucidate the wide variable phenotypic expressivity and incomplete penetrance in a three-generation family with a truncating variant in TBX4. In addition to exome and genome sequencing analyses, a candidate noncoding regulatory single nucleotide variant (SNV) within the lung-specific TBX4 enhancer was functionally tested using an in vitro luciferase reporter assay. A heterozygous frameshift variant c.1112dup (p.Pro372Serfs*14) in TBX4 was identified in patients with mild interstitial lung disease (1), bronchiolitis obliterans (1), recurrent pneumothorax (1), ICPPS/SPS (1), LLDD (2), and in unaffected individuals (4). In two deceased neonates with LLDD, we identified a noncoding SNV rs62069651-C located in trans to the mutated TBX4 allele that reduced the TBX4 promoter activity by 63% in the reporter assay. Our findings provide a functional evidence for the recently reported model of complex compound inheritance in which both TBX4 coding and in trans noncoding hypomorphic variants in the lung-specific enhancer of TBX4 contribute to LLDD.

Role of T-box genes in cancer, epithelial-mesenchymal transition, and cancer stem cells.

Sharing a common DNA binding motif called T-box, transcription factor T-box gene family controls embryonic development and is also involved in cancer progression and metastasis. Cancer metastasis shows therapy resistance and involves complex processes. Among them, epithelial-mesenchymal transition (EMT) triggers cancer cell invasiveness and the acquisition of stemness of cancer cells, called cancer stem cells (CSCs). CSCs are a small fraction of tumor bulk and are capable of self-renewal and tumorsphere formation. Recent progress has highlighted the critical roles of T-box genes in cancer progression, EMT, and CSC function, and such regulatory functions of T-box genes have emerged as potential therapeutic candidates for cancer. Herein we summarize the current understanding of the regulatory mechanisms of T-box genes in cancer, EMT, and CSCs, and discuss the implications of targeting T-box genes as anticancer therapeutics.

Apical periodontitis promotes insulin resistance and alters adaptive immunity markers in rats.

OBJECTIVE: Apical periodontitis (AP) is a chronic or acute inflammatory disease usually developed from endodontic infections, predominantly due to gram-negative anaerobic bacteria invading the dental pulp. This study aimed to evaluate lymphocyte markers to assess the involvement of adaptive immunity in insulin resistance (IR) in a rat model of AP.Design.Forty-five male Wistar albino rats were divided into 3 groups (control, 1AP and 4AP). AP was induced in the upper right first molar (1AP), and in the first and second upper and lower right molars (4AP). The spleen was collected to evaluate the expression of transcription factors involved in lymphocyte polarization, including T-bet (Th1), GATA3 (Th2), and FOXP3 (Treg). Blood samples were assessed for serum cytokine levels transcribed by the respective lymphocyte polarizations, INF-γ (Th1), IL-4 (Th2) and TGF-ß (Treg). In addition, glucose and insulin levels were measured to evaluate IR by the HOMA-IR method. RESULTS: The results showed higher T-bet expression on AP groups, along with lower GATA3 and FOXP3 expression in the 1AP, in addition to increased GATA3 and decreased FOXP3 expression in the 4AP group compared to the CN group. There was no difference in the INF-γ levels, while IL-4 was decreased in the AP groups. Taken together, these results suggest that the adaptive immune system, with a predominance of the Th1 polarization, may be involved in the development of IR in rats with AP. CONCLUSIONS: AP promotes increase in the expression of T-bet (4AP) and decrease of FOXP3 expressions and IL-4 levels (1AP and 4AP). However, depending on the number of lesions (1 or 4 lesions), the expression of GATA3 appears differently. Thus, innate immunity and adaptive immunity may contribute to the IR observed in rats with AP.

Targeting the oncogenic TBX3:nucleolin complex to treat multiple sarcoma subtypes.

Sarcomas are diverse cancers of mesenchymal origin, with compromised clinical management caused by insufficient diagnostic biomarkers and limited treatment options. The transcription factor TBX3 is upregulated in a diverse range of sarcoma subtypes, where it plays a direct oncogenic role, and it may thus represent a novel therapeutic target. To identify versatile ways to target TBX3, we performed affinity purification coupled by mass spectrometry to identify putative TBX3 protein cofactors that regulate its oncogenic activity in sarcomas. Here we identify and validate the multifunctional phosphoprotein nucleolin as a TBX3 cofactor. We show that nucleolin is co-expressed with TBX3 in several sarcoma subtypes and their expression levels positively correlate in sarcoma patients which are associated with poor prognosis. Furthermore, we demonstrate that nucleolin and TBX3 interact in chondrosarcoma, liposarcoma and rhabdomyosarcoma cells where they act together to enhance proliferation and migration and regulate a common set of tumor suppressor genes. Importantly, the nucleolin targeting aptamer, AS1411, exhibits selective anti-cancer activity in these cells and mislocalizes TBX3 and nucleolin to the cytoplasm which correlates with the re-expression of the TBX3/nucleolin target tumor suppressors CDKN1A (p21CIP1) and CDKN2A (p14ARF). Our findings provide the first evidence that TBX3 requires nucleolin to promote features of sarcomagenesis and that disruption of the oncogenic TBX3-nucleolin interaction by AS1411 may be a novel approach for treating sarcomas.