RESUMO
Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.
Assuntos
Cílios , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog , Pulmão , Transdução de Sinais , Animais , Camundongos , Padronização Corporal/genética , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Pulmão/embriologia , Pulmão/metabolismo , Morfogênese/genética , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.
Assuntos
Quinases Dyrk , Proteínas Hedgehog , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Transdução de Sinais , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de Zinco , Animais , Proteína Gli3 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Camundongos , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Proliferação de Células , Cílios/metabolismo , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Proteínas Nucleares , Proteínas RepressorasRESUMO
Tangeretin (Tan), a citrus flavonoid, possesses a strong anti-tumor efficacy in various human cancers. However, the precise role of Tan in the development of esophageal squamous cell carcinoma (ESCC) remains unclear. RNA sequencing (RNA-seq) analysis was performed to observe the Tan-related genes in Tan-treated TE-1 cells. The direct relationship between GLI family zinc finger 2 (GLI2) and the promoter of glycoprotein non-metastatic melanoma protein B (GPNMB) was predicted by bioinformatics analysis and validated by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Cell survival after Tan treatment was assessed by CCK8 assay. Gene expression levels were evaluated by a qRT-PCR, western blot, or immunofluorescence method. Cell migration and invasion were detected by wound-healing and transwell assays. The function of Tan in vivo was examined using xenograft studies. Our data indicated anti-migration and anti-invasion functions of Tan in ESCC cells in vitro. Tan also diminished tumor growth in vivo. Mechanistically, Tan diminished the expression and transcriptional activity of GLI2 in ESCC cells. Silencing of GLI2 resulted in decreased expression of GPNMB by inhibiting GPNMB transcription via the binding site at the GPNMB promoter at position +(1539-1550). Moreover, Tan down-regulated GPNMB expression in ESCC cells, and re-expression of GPNMB reversed anti-migration and anti-invasion functions of Tan in ESCC cells. Our findings uncover anti-migration and anti-invasion effects of Tan in ESCC cells by down-regulating GPNMB by suppressing GLI2-mediated GPNMB transcription, providing new evidence that Tan can function as a therapeutic agent against ESCC.
Assuntos
Movimento Celular , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Flavonas , Regulação Neoplásica da Expressão Gênica , Glicoproteínas de Membrana , Proteína Gli2 com Dedos de Zinco , Humanos , Carcinoma de Células Escamosas do Esôfago/tratamento farmacológico , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Animais , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Linhagem Celular Tumoral , Camundongos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Flavonas/farmacologia , Movimento Celular/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Transcrição Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , Proliferação de Células/efeitos dos fármacos , Proteínas do Olho/metabolismo , Proteínas do Olho/genética , Proteínas NuclearesRESUMO
The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.
Assuntos
Proteínas Hedgehog , Transdução de Sinais , Papilas Gustativas , Língua , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Língua/metabolismo , Língua/crescimento & desenvolvimento , Camundongos , Papilas Gustativas/metabolismo , Papilas Gustativas/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Homeostase , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Proteínas do Tecido Nervoso , Proteínas de Ciclo Celular , Proteínas Ligadas por GPIRESUMO
Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.
Assuntos
Carcinoma Ductal Pancreático , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Proteína Gli2 com Dedos de Zinco , Animais , Humanos , Camundongos , Carcinogênese , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Histonas/metabolismo , Histonas/genética , Camundongos Transgênicos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transcrição Gênica , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
Tumors of the head and neck, more specifically the squamous cell carcinoma, often show upregulation of the Hedgehog signaling pathway. However, almost nothing is known about its role in the sinonasal adenocarcinoma, either in intestinal or non-intestinal subtypes. In this work, we have analyzed immunohistochemical staining of six Hedgehog pathway proteins, sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched1 (PTCH1), Gli family zinc finger 1 (GLI1), Gli family zinc finger 2 (GLI2), and Gli family zinc finger 3 (GLI3), on 21 samples of sinonasal adenocarcinoma and compared them with six colon adenocarcinoma and three salivary gland tumors, as well as with matching healthy tissue, where available. We have detected GLI2 and PTCH1 in the majority of samples and also GLI1 in a subset of samples, while GLI3 and the ligands SHH and IHH were generally not detected. PTCH1 pattern of staining shows an interesting pattern, where healthy samples are mostly positive in the stromal compartment, while the signal shifts to the tumor compartment in tumors. This, taken together with a stronger signal of GLI2 in tumors compared to non-tumor tissues, suggests that the Hedgehog pathway is indeed activated in sinonasal adenocarcinoma. As Hedgehog pathway inhibitors are being tested in combination with other therapies for head and neck squamous cell carcinoma, this could provide a therapeutic option for patients with sinonasal adenocarcinoma as well.
Assuntos
Adenocarcinoma , Proteínas Hedgehog , Imuno-Histoquímica , Transdução de Sinais , Proteína Gli2 com Dedos de Zinco , Humanos , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Masculino , Feminino , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Pessoa de Meia-Idade , Projetos Piloto , Idoso , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismo , Proteína Gli3 com Dedos de Zinco/genética , Neoplasias dos Seios Paranasais/metabolismo , Neoplasias dos Seios Paranasais/patologia , Adulto , Regulação Neoplásica da Expressão Gênica , Proteínas do Tecido Nervoso , Proteínas NuclearesRESUMO
Cardiomyocyte (CM) proliferation and maturation are highly linked processes, however, the extent to which these processes are controlled by a single signaling axis is unclear. Here, we show the previously undescribed role of Hedgehog (HH)-GLI2-CKS1B cascade in regulation of the toggle between CM proliferation and maturation. Here we show downregulation of GLI-signaling in adult human CM, adult murine CM, and in late-stage hiPSC-CM leading to their maturation. In early-stage hiPSC-CM, inhibition of HH- or GLI-proteins enhanced CM maturation with increased maturation indices, increased calcium handling, and transcriptome. Mechanistically, we identified CKS1B, as a new effector of GLI2 in CMs. GLI2 binds the CKS1B promoter to regulate its expression. CKS1B overexpression in late-stage hiPSC-CMs led to increased proliferation with loss of maturation in CMs. Next, analysis of datasets of patients with heart disease showed a significant enrichment of GLI2-signaling in patients with ischemic heart failure (HF) or dilated-cardiomyopathy (DCM) disease, indicating operational GLI2-signaling in the stressed heart. Thus, the Hh-GLI2-CKS1B axis regulates the proliferation-maturation transition and provides targets to enhance cardiac tissue engineering and regenerative therapies.
Assuntos
Proliferação de Células , Miócitos Cardíacos , Proteína Gli2 com Dedos de Zinco , Miócitos Cardíacos/metabolismo , Humanos , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Animais , Camundongos , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Células-Tronco Pluripotentes Induzidas/metabolismo , Diferenciação CelularRESUMO
A common cause of deafness in humans is dysregulation of the endocochlear potential generated by the stria vascularis (SV). Thus, proper formation of the SV is critical for hearing. Using single-cell transcriptomics and a series of Shh signaling mutants, we discovered that the Shh receptor Patched1 (Ptch1) is essential for marginal cell (MC) differentiation and SV formation. Single-cell RNA sequencing analyses revealed that the cochlear roof epithelium is already specified into discrete domains with distinctive gene expression profiles at embryonic day 14, with Gsc as a marker gene of the MC lineage. Ptch1 deficiency leads to defective specification of MC precursors along the cochlear basal-apical regions. We demonstrated that elevated Gli2 levels impede MC differentiation through sustaining Otx2 expression and maintaining the progenitor state of MC precursors. Our results uncover an early specification of cochlear non-sensory epithelial cells and establish a crucial role of the Ptch1-Gli2 axis in regulating the development of SV.
Assuntos
Diferenciação Celular , Cóclea , Receptor Patched-1 , Estria Vascular , Receptor Patched-1/metabolismo , Receptor Patched-1/genética , Animais , Camundongos , Estria Vascular/metabolismo , Estria Vascular/citologia , Cóclea/metabolismo , Cóclea/embriologia , Cóclea/citologia , Transdução de Sinais , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genéticaRESUMO
Hedgehog signaling is activated in response to liver injury, and modulates organogenesis. However, the role of non-canonical hedgehog activation via TGF-ß1/SMAD3 in hepatic carcinogenesis is poorly understood. TGF-ß1/SMAD3-mediated non-canonical activation was found in approximately half of GLI2-positive hepatocellular carcinoma (HCC), and two new GLI2 isoforms with transactivating activity were identified. Phospho-SMAD3 interacted with active GLI2 isoforms to transactivate downstream genes in modulation of stemness, epithelial-mesenchymal transition, chemo-resistance and metastasis in poorly-differentiated hepatoma cells. Non-canonical activation of hedgehog signaling was confirmed in a transgenic HBV-associated HCC mouse model. Inhibition of TGF-ß/SMAD3 signaling reduced lung metastasis in a mouse in situ hepatic xenograft model. In another cohort of 55 HCC patients, subjects with high GLI2 expression had a shorter disease-free survival than those with low expression. Moreover, co-positivity of GLI2 with SMAD3 was observed in 87.5% of relapsed HCC patients with high GLI2 expression, indicating an increased risk of post-resection recurrence of HCC. The findings underscore that suppressing the non-canonical hedgehog signaling pathway may confer a potential strategy in the treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/patologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Neoplasias Hepáticas/patologia , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
Missense variants are the most common type of coding genetic variants. Their functional assessment is fundamental for defining any implication in human diseases and may also uncover genes that are essential for human organ development. Here, we apply CRISPR-Cas9 gene editing on human iPSCs to study a heterozygous missense variant in GLI2 identified in two siblings with early-onset and insulin-dependent diabetes of unknown cause. GLI2 is a primary mediator of the Hedgehog pathway, which regulates pancreatic ß-cell development in mice. However, neither mutations in GLI2 nor Hedgehog dysregulation have been reported as cause or predisposition to diabetes. We establish and study a set of isogenic iPSC lines harbouring the missense variant for their ability to differentiate into pancreatic ß-like cells. Interestingly, iPSCs carrying the missense variant show altered GLI2 transcriptional activity and impaired differentiation of pancreatic progenitors into endocrine cells. RNASeq and network analyses unveil a crosstalk between Hedgehog and WNT pathways, with the dysregulation of non-canonical WNT signaling in pancreatic progenitors carrying the GLI2 missense variant. Collectively, our findings underscore an essential role for GLI2 in human endocrine development and identify a gene variant that may lead to diabetes.
Assuntos
Diabetes Mellitus , Ilhotas Pancreáticas , Humanos , Camundongos , Animais , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Mutação de Sentido Incorreto/genética , Ilhotas Pancreáticas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Nucleares/metabolismoRESUMO
Depleted uranium (DU) retains the radiological toxicities, which accumulates preferentially in the kidneys. Hedgehog (Hh) pathway plays a critical role in tissue injury. However, the role of Hh in DU-induced nephrotoxicity was still unclear. This study was carried out to investigate the effect of Gli2, which was an important transcription effector of Hh signaling, on DU induced nephrotoxicity. To clarify it, CK19 positive tubular epithelial cells specific Gli2 conditional knockout (KO) mice model was exposed to DU, and then histopathological damage and Hh signaling pathway activation was analyzed. Moreover, HEK-293 T cells were exposed to DU with Gant61 or Gli2 overexpression, and cytotoxicity of DU as analyzed. Results showed that DU caused nephrotoxicity accompanied by activation of Hh signaling pathway. Meanwhile, genetic KO of Gli2 reduced DU-induced nephrotoxicity by normalizing biochemical indicators and reducing Hh pathway activation. Pharmacologic inhibition of Gli1/2 by Gant61 reduced DU induced cytotoxicity by inhibiting apoptosis, ROS formation and Hh pathway activation. However, overexpression of Gli2 aggravated DU-induced cytotoxicity by increasing the levels of apoptosis and ROS formation. Taken together, these results revealed that Hh signaling negatively regulated DU-inducted nephrotoxicity, and that inhibition of Gli2 might serve as a promising nephroprotective target for DU-induced kidney injury.
Assuntos
Proteínas Hedgehog , Rim , Camundongos Knockout , Transdução de Sinais , Proteína Gli2 com Dedos de Zinco , Animais , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Humanos , Células HEK293 , Transdução de Sinais/efeitos dos fármacos , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Rim/efeitos dos fármacos , Rim/patologia , Rim/metabolismo , Camundongos , Urânio/toxicidade , Apoptose/efeitos dos fármacos , Piridinas/farmacologia , Piridinas/toxicidade , Masculino , Nefropatias/induzido quimicamente , Nefropatias/patologia , Nefropatias/metabolismo , Pirimidinas/farmacologia , Pirimidinas/toxicidade , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Anoikis is a speed-limited procedure to inhibit tumor metastasis during epithelial-mesenchymal transition (EMT). Previous studies have explored anoikis-related genes (ARG) in predicting prognosis and distinguishing tumoral immunity in many types of cancer. However, the role of ARGs in regulating NK cell exhaustion (NKE) and in predicting chemotherapy sensitivity is not clear. Therefore, it is necessary to work on it. METHODS: Gene expression profiles and clinical features are collected from TCGA and GEO, and data analysis is performed in R4.2.0. RESULTS: The ARGs-based no-supervised learning algorithm identifies three ARG subgroups, amongst which the prognosis is different. WCGNA and Artificial intelligence (AI) are applied to construct an NKE-related drug sensitivity stratification and prognosis identification model in digestive system cancer. Pathways association analysis screens out GLI2 is a key gene in regulating NKE by non-classic Hedgehog signaling (GLI2/TGF-ß/IL6). In vitro experiments show that down-regulation of GLI2 enhances the CAPE-mediated cell toxicity and accompanies with down-regulation of PD-L1, tumor-derive IL6, and snial1 whereas the expression of cleaved caspas3, cleaved caspase4, cleaved PARP, and E-cadherin are up-regulated in colorectal cancer. Co-culture experiments show that GLI2- decreased colorectal tumor cells lead to down-regulation of TIM-3 and PD1 in NK cells, which are restored by TGF-bate active protein powder. Besides, the Elisa assay shows that GLI2-decreased colorectal tumor cells lead to up-regulation of IFN-gamma in NK cells.
Assuntos
Anoikis , Neoplasias Colorretais , Proteínas Hedgehog , Proteína Gli2 com Dedos de Zinco , Humanos , Anoikis/genética , Inteligência Artificial , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Interleucina-6 , Proteínas Nucleares/genética , Fator de Crescimento Transformador beta , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
Objective: Congenital hypopituitarism (CH) is a rare disease characterized by one or more hormone deficiencies of the pituitary gland. To date, many genes have been associated with CH. In this study, we identified the allelic variant spectrum of 11 causative genes in Turkish patients with CH. Materials and methods: This study included 47 patients [21 girls (44.6%) and 26 boys (55.4%)] from 45 families. To identify the genetic etiology, we screened 11 candidate genes associated with CH using next-generation sequencing. To confirm and detect the status of the specific familial variant in relatives, Sanger sequencing was also performed. Results: We identified 12 possible pathogenic variants in GHRHR, GH1, GLI2, PROP-1, POU1F1, and LHX4 in 11 patients (23.4%), of which six were novel variants: two in GHRHR, two in POU1F1, one in GLI2, and one in LHX4. In all patients, these variants were most frequently found in GLI2, followed by PROP-1 and GHRHR. Conclusion: Genetic causes were determined in only 23.4% of all patients with CH and 63% of molecularly diagnosed patients (7/11) from consanguineous families. Despite advances in genetics, we were unable to identify the genetic etiology of most patients with CH, suggesting the effect of unknown genes or environmental factors. More genetic studies are necessary to understand the etiology of CH.
Assuntos
Hipopituitarismo , Feminino , Humanos , Masculino , Alelos , Hipopituitarismo/diagnóstico , Hipopituitarismo/genética , Mutação , Proteínas Nucleares/genética , Fator de Transcrição Pit-1/genética , Fatores de Transcrição/genética , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
Increasing evidence has shown that circular RNAs (circRNAs) interact with RNA-binding proteins (RBPs) and promote cancer progression. However, the function and mechanism of the circRNA/RBP complex in esophageal squamous cell carcinoma (ESCC) are still largely unknown. Herein, we first characterized a novel oncogenic circRNA, circ-FIRRE, by RNA sequencing (Ribo-free) profiling of ESCC samples. Furthermore, we observed marked circ-FIRRE overexpression in ESCC patients with high TNM stage and poor overall survival. Mechanistic studies indicated that circ-FIRRE, as a platform, interacts with the heterogeneous nuclear ribonucleoprotein C (HNRNPC) protein to stabilize GLI2 mRNA by directly binding to its 3'-UTR in the cytoplasm, thereby resulting in elevated GLI2 protein expression and subsequent transcription of its target genes MYC, CCNE1, and CCNE2, ultimately contributing to ESCC progression. Moreover, HNRNPC overexpression in circ-FIRRE knockdown cells notably abolished circ-FIRRE knockdown-mediated Hedgehog pathway inhibition and ESCC progression impairment in vitro and in vivo. Clinical specimen results showed that circ-FIRRE and HNRNPC expression was positively correlated with GLI2 expression, which reveals the clear significance of the circ-FIRRE/HNRNPC-GLI2 axis in ESCC. In summary, our results indicate that circ-FIRRE could serve as a valuable biomarker and potential therapeutic target for ESCC and highlight a novel mechanism of the circ-FIRRE/HNRNPC complex in ESCC progression regulation.
Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , MicroRNAs , Humanos , Carcinoma de Células Escamosas do Esôfago/patologia , RNA Circular/genética , RNA Circular/metabolismo , Neoplasias Esofágicas/patologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , RNA Mensageiro/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , MicroRNAs/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Linhagem Celular Tumoral , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteínas Nucleares/genéticaRESUMO
Hedgehog (Hh) signaling is involved in multiple biological events including development and cancers. It is processed through primary cilia, which are assembled from the mother centriole in most mammalian cells. Pancreatic ductal adenocarcinoma (PDAC) cells generally lose their primary cilia; thus, the Hh signaling pathway is postulated to be independent of the organelle in PDAC. We previously reported that the mother centriole-specific protein, centrosomal protein 164 (CEP164), is required for centriolar localization of the GLI2 transcription factor in Hh signaling and for suppressing the expression of Hh-target genes. In this study, we demonstrated the physical interaction between CEP164 and GLI2, and delineated their binding modes at the mother centriole. The ectopically expressed GLI2-binding region of CEP164 reduced the centriolar GLI2 localization and enhanced the expression of Hh-target genes in PDAC cells. Furthermore, similar phenotypes were observed in PDAC cells lacking primary cilia. These results suggest that the CEP164-GLI2 association at the mother centriole is responsible for controlling Hh signaling, independent of primary cilia in PDAC cells.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Cílios/metabolismo , Transdução de Sinais , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Fatores de Transcrição Kruppel-Like/metabolismo , Mamíferos/metabolismo , Neoplasias PancreáticasRESUMO
Culler-Jones syndrome is a rare clinical phenomenon with diverse manifestations and is prone to misdiagnosis. We report one patient who presented with a 10-year history of anosmia and a 1-year history of epididymal pain. Kallmann syndrome was suspected initially. The results of his laboratory tests, imaging, and genetic testing, however, combined to provide a conclusive diagnosis of Culler-Jones syndrome. With the aid of high-throughput sequencing technology, the GLI2 gene c.527A>G (p.Tyr176Cys) heterozygous mutation in the child was identified. No published works have yet described this mutation site. We described Culler-Jones syndrome in a child at length. We recommend that Culler-Jones syndrome be taken into account when considering the spectrum of disorders associated with abnormal growth and development in children. Once diagnosed, individualized hormone replacement treatment is required for each patient.
Assuntos
Síndrome de Kallmann , Criança , Humanos , Síndrome de Kallmann/diagnóstico , Síndrome de Kallmann/genética , Síndrome de Kallmann/complicações , Anosmia/complicações , Mutação , Proteína Gli2 com Dedos de Zinco/genética , Proteínas Nucleares/genéticaRESUMO
OBJECTIVE: Being a prevalent endocrine and metabolic disease, polycystic ovary syndrome (PCOS) severely threatens women's physical and mental health. Glioma-associated oncogene family zinc finger 2 (GLI2) expression is up-regulated in granulosa cells of PCOS patients, but its specific role in PCOS remains unclear. METHODS: Following the treatment of human ovarian granulosa cells (KGN) with dihydrotestosterone (DHT), RT-qPCR and western blot were utilized to check GLI2 expression. After GLI2 expression was silenced, cell activity was detected through CCK8 and apoptosis was examined via TUNEL and western blot. Inflammation and oxidative stress were tested utilizing ELISA and western blot. The binding between GLI2 and neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4L) promoter was predicted by JASPAR database and verified by luciferase reporter and ChIP assay. In addition, RT-qPCR and western blot were applied to check the mRNA and protein expressions of NEDD4L. Following the knockdown of NEDD4L in GLI2-silencing cells, CCK8 assay, TUNEL assay, western blot, ELISA and other methods were performed again. Finally, western blot detected the expressions of Wnt pathway-related proteins. RESULTS: GLI2 was up-regulated in DHT-treated KGN cells. Interference with GLI2 increased the viability, decreased the apoptosis, and inhibited the inflammatory response and oxidative stress of DHT-induced KGN cells. GLI2 could bind to NEDD4L promoter and transcriptionally suppress NEDD4L expression. Further experiments testified that NEDD4L depletion reversed the impacts of GLI2 deficiency on the viability, apoptosis, inflammation, oxidative stress and Wnt signaling pathway in DHT-challenged KGN cells. CONCLUSION: GLI2 activated Wnt signaling to promote androgen-induced granulosa cell damage through transcriptional inhibition of NEDD4L.
Assuntos
Glioma , MicroRNAs , Síndrome do Ovário Policístico , Humanos , Feminino , Via de Sinalização Wnt/genética , Androgênios/metabolismo , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/metabolismo , Células da Granulosa/metabolismo , Apoptose/genética , Proteínas Wnt/metabolismo , Inflamação/metabolismo , Dedos de Zinco , Oncogenes , Proliferação de Células/genética , MicroRNAs/genética , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Proteínas Nucleares/genéticaRESUMO
PURPOSE: Metastatic lung squamous cell carcinoma (LUSC) is one of the most common causes of cancer death worldwide. As yet, however, the molecular mechanism underlying LUSC metastasis remains elusive. In this study, we report a novel mechanism involving signaling interactions between FGF19 and GLI2 that could drive the progression of LUSC. METHODS: The expression of FGF19 in human LUSC samples was assessed by immunohistochemistry. The concentration of FGF19 in serum samples was assessed by ELISA. RNA sequencing, scratch wound-healing, trans-well, GO analysis, GSEA, luciferase reporter, Western blotting, immunofluorescence and immunohistochemistry assays, as well as an animal model were used to investigate the molecular mechanism underlying FGF19 driven LUSC progression. The therapeutic effect of a GLI2 inhibitor was determined using both in vitro cellular and in vivo animal experiments. RESULTS: We found that FGF19, a member of the fibroblast growth factor family, plays a crucial role in the invasion and metastasis of LUSC, and identified GLI2 as an important downstream effector of FGF19 involved in metastasis. Surprisingly, we found that FGF19 and GLI2 could reciprocally induce the expression of each other, and form a positive feedback loop to promote LUSC cell invasion and metastasis. These findings were corroborated by an association between a poor prognosis of LUSC patients and FGF19/GLI2 co-expression. In addition, we found that the GLI inhibitor GANT61 could effectively reduce FGF19-mediated LUSC invasion and metastasis. CONCLUSION: Our data suggest that FGF19 may serve as a novel biomarker for predicting metastatic LUSC. Intervening with the FGF19-GLI2 feedback loop may be a strategy for the treatment of FGF19-driven LUSC metastasis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Transição Epitelial-Mesenquimal , Fatores de Crescimento de Fibroblastos , Neoplasias Pulmonares , Proteína Gli2 com Dedos de Zinco , Animais , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Pulmão/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Proteína Gli2 com Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/metabolismoRESUMO
OBJECTIVE: To explore the genetic basis for a child featuring short stature and postaxial polydactyly. METHODS: A child who presented at Ningbo Women & Children's Hospital in May 2021 due to the"discovery of growth retardation for more than two years" was selected as the subject. Peripheral blood samples of the child and his parents were collected for the extraction of genomic DNA. Whole exome sequencing was carried out for the child, and candidate variant was verified by Sanger sequencing of his family members. RESULTS: The child was found to harbor a heterozygous c.3670C>T (p.Q1224) variant of the GLI2 gene, which may lead to premature termination of protein translation. The variant was not detected in either parent. CONCLUSION: The child was diagnosed with Culler-Jones syndrome. The c.3670C>T (p.Q1224*) variant of the GLI2 gene probably underlay the disease in this child.