PTCH1 Expression Research Articles

CPLANE protein INTU regulates growth and patterning of the mouse lungs through cilia-dependent Hh signaling

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.

Shh Gene Regulates the Proliferation and Apoptosis of Dermal Papilla Cells to Affect Its Differential Expression in Secondary Hair Follicle Growth Cycle of Cashmere Goats.

Sonic hedgehog (Shh) is a component of the Hedgehog signaling pathway, playing an important role in regulating cell proliferation, differentiation, apoptosis, and the repair of damaged organisms. To further clarify the expression pattern of Shh gene in the secondary hair follicle growth cycle of cashmere goats and its mechanism of action on secondary hair follicle papilla cells, and improve cashmere quality, in this study, we took Inner Mongolia Albas white cashmere goats as the research objects and collected skin samples at different growth stages to obtain secondary hair follicles, detected Shh and its gene expression by RT-qPCR, Western blot, immunohistochemistry, and other techniques, while we also cultured DPCs in vitro. Shh gene overexpression and interference vectors were constructed, and the effects of Shh gene on the proliferation and apoptosis of DPCs were studied through cell transfection technology. The results showed that there are significant differences in Shh and its gene expression in the secondary hair follicle growth cycle skins of cashmere goats, with the highest expression level in anagen, followed by catagen, and the lowest expression level in telogen. Shh was mainly expressed in the inner root sheath, outer root sheath, and secondary hair follicle papilla. After the overexpression of Shh gene, the proliferation and vitality of the hair papilla cells were enhanced compared to the interference group. After Shh gene interference, the apoptosis rate of the cells increased, indicating that Shh gene can regulate downstream Ptch, Smo, and Gli2 gene expression to promote the proliferation of DPCs, and thus form its expression pattern in the secondary hair follicle growth cycle of cashmere goats.

Implicating clinical utility of altered expression of PTCH1 & SMO in oral squamous cell carcinoma.

Oral cancer poses a significant burden on public health in India, with higher incidence and mortality rates. Despite advancements in treatment modalities, prognosis remains poor due to factors such as localized recurrence and lymph node metastasis, potentially influenced by cancer stem cells. Among signaling pathways implicated in CSC regulation, the Hedgehog pathway plays a crucial role in oral squamous cell carcinoma (OSCC). 97 OSCC patients' tissue samples were collected and subjected to RNA isolation, cDNA synthesis and quantitative real-time PCR to analyze PTCH1 and SMO expression. Protein expression was assessed through immunohistochemistry. Clinicopathological parameters were correlated with gene and protein expression. Statistical analysis included Pearson chi-square tests, co-relation co-efficient tests, Kaplan-Meier survival analysis and ROC curve analysis. PTCH1 expression correlated with lymphatic permeation (p = 0.002) and tumor stage (p = 0.002), while SMO expression correlated with lymph node status (p = 0.034) and tumor stage (p = 0.021). PTCH1 gene expression correlated with lymph node status (p = 0.024). High PTCH1 gene expression was associated with shorter survival in tongue cancer patients. ROC curve analysis indicated diagnostic potential for PTCH1 and SMO gene and cytoplasmic SMO expression in distinguishing malignant tissues from adjacent normal tissues. PTCH1 and SMO play a crucial role in oral cancer progression, correlating with tumor stages and metastatic potential. Despite not directly influencing overall survival, PTCH1 expression at specific anatomical sites hints at its prognostic implications. PTCH1 and SMO exhibit diagnostic potential, suggesting their utility as molecular markers in oral cancer management and therapeutic strategies.

TRIM37 interacts with EZH2 to epigenetically suppress PTCH1 and regulate stemness in glioma stem cells through sonic hedgehog pathway.

Glioma stem cells (GSCs), which are known for their therapy resistance, play a substantial role in treatment inefficacy for glioblastoma multiforme (GBM). TRIM37, a member of the tripartite motif (TRIM) protein family initially linked to a rare growth disorder, has been recognized for its oncogenic role. However, the mechanism by which TRIM37 regulates tumor growth in glioma and GSCs is unclear. For the in vitro experiments, gene expression was measured by western blotting, RT-qPCR, and immunofluorescence. Cell viability was detected by CCK-8, and cell apoptosis was detected by flow cytometry. The interaction between Enhancer of Zeste Homolog 2 (EZH2) and TRIM37 was verified by co-immunoprecipitation (Co-IP). The interaction between EZH2 and the PTCH1 promoter was verified using dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP). For the in vivo experiments, an orthotopically implanted glioma mouse model was used to validate tumor growth. The expression of TRIM37 is higher in GSCs compared with matched non-GSCs. TRIM37 knockdown promotes apoptosis, decreased stemness in GSCs, and reduces tumor growth in GSCs xenografts of nude mice. TRIM37 and EZH2 co-localize in the nucleus and interact with each other. TRIM37 knockdown or EZH2 inhibition downregulates the protein expressions associated with the Sonic Hedgehog (SHH) pathway. EZH2 epigenetically downregulates PTCH1 to activate SHH pathway in GSCs. TRIM37 maintains the cell growth and stemness in GSCs through the interaction with EZH2. EZH2 activates SHH stem cell signaling pathway by downregulating the expression of SHH pathway suppressor PTCH1. Our findings suggest that TRIM37 may be a potential therapeutic target for GBM.

Impaired Sonic Hedgehog Responsiveness of Induced Pluripotent Stem Cell-Derived Floor Plate Cells Carrying the LRRK2-I1371V Mutation Contributes to the Ontogenic Origin of Lower Dopaminergic Neuron Yield.

Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

MiRNA-210 is involved in cigarette smoke extract-induced apoptosis of MLE-12 via the Shh signaling pathway.

The aim of the study is the regulatory effect of MicroRNA-210 (MiR-210) on cigarette smoke extract (CSE)-induced mouse lung epithelial type II cells (MLE-12) apoptosis and determine whether the MiR-210 is involved in cigarette smoke extract-induced apoptosis of MLE-12 via Shh signaling pathway. Expression of MiR-210 in CSE-induced MLE-12 was assessed by qRT-PCR. The emphysema mouse model and MiR-210 knockdown mice were each established by inhaling cigarette smoke or intratracheal lentiviral vector instillation. The Sonic hedgehog (Shh), Ptch1, Gli1, B-cell lymphoma-2 (Bcl-2), and Caspase 3 protein expressions were detected by Western blotting. mRNA expressions of MiR-210, Shh, Ptch1, and Gli1 were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Apoptotic ratios in mice and CSE-induced HPVEC were assessed using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays and flow cytometry. Our results showed that MiR-210 mRNA levels were significantly down-regulated in the CSE-induced MLE 12. MLE 12 apoptosis with down-regulated Shh, Ptch1, Gli1, and Bcl-2 expression, increased Caspase 3 expression in the emphysema mouse model and CSE-induced MLE 12. Knockdown MiR-210 can facilitate cell apoptosis and emphysema via the Shh signaling pathway in mice. In vitro, MiR-210 can attenuate the apoptosis of CSE-exposed MLE 12. Moreover, MiR-210 regulated the Shh pathway and promoted its expression. MiRNA-210 is involved in cigarette smoke extract-induced apoptosis of MLE-12 via the Shh signaling pathway. The present study reveals that MiRNA-210 may be a key regulator of cellular apoptosis and could be explored as a potential therapeutic target in the future.

ZMYND8 Is a Regulator of Sonic Hedgehog Signaling in ATRA-Mediated Differentiation of Neuroblastoma Cells.

Zinc Finger MYND (Myeloid, Nervy, and DEAF-1) type containing 8 (ZMYND8) is a crucial epigenetic regulator that plays a multifaceted role in governing a spectrum of vital cellular processes, encompassing proliferation, apoptosis, migration, tumor suppression, and differentiation. It has emerged as a key player in neuronal differentiation by orchestrating the expression of neuronal lineage-committed genes. The present study uncovers the role of ZMYND8 in regulating the Sonic Hedgehog (SHH) signaling axis, which is crucial for neuronal differentiation. Genetic deletion of ZMYND8 leads to a significant reduction in SHH pathway genes, GLI1, and PTCH1 expression during all-trans-retinoic acid (ATRA)-induced differentiation. ZMYND8 and RNA pol II S5P are found to co-occupy the GLI1 and PTCH1 gene promoters, positively impacting their gene transcription upon ATRA treatment. Interestingly, ZMYND8 is found to counteract the inhibitory effects of Cyclopamine that block the upstream SHH pathway protein SMO, resulting in enhanced neurite formation in neuroblastoma cells following their treatment with ATRA. These results indicate that ZMYND8 is an epigenetic regulator of the SHH signaling pathway and has tremendous therapeutic potential in ATRA-mediated differentiation of neuroblastoma.

Laser-assisted topical delivery of vismodegib reduces hedgehog gene expression in human basal cell carcinomas in vivo.

Systemically delivered hedgehog inhibitors including vismodegib and sonidegib are widely used to treat basal cell carcinomas (BCCs). Ablative fractional laser (AFL)-assisted topical delivery of vismodegib has been demonstrated in preclinical studies. The aim of this explorative clinical study was to evaluate intratumoral vismodegib concentrations and effect on hedgehog pathway gene expression following AFL-assisted topical vismodegib delivery to BCCs. In an open-label clinical trial, 16 nodular BCCs (in n = 9 patients) received one application of CO2 -AFL (40 mJ/microbeam, 10% density) followed by topical vismodegib emulsion. After 3-4 days, vismodegib concentrations in tumor biopsies (n = 15) and plasma were analyzed and compared with samples from patients receiving oral treatment (n = 3). GLI1, GLI2, PTCH1, and PTCH2 expression was determined by quantitative polymerase chain reaction (n = 7) and GLI1 additionally by in situ hybridization (n = 3). Following AFL-assisted topical administration, vismodegib was detected in 14/15 BCCs and reached a median concentration of 6.2 µmol/L, which compared to concentrations in BCC tissue from patients receiving oral vismodegib (9.5 µmol/L, n = 3, p = 0.8588). Topical vismodegib reduced intratumoral GLI1 expression by 51%, GLI2 by 55%, PTCH1 and PTCH2 each by 73% (p ≤ 0.0304) regardless of vismodegib concentrations (p ≥ 0.3164). In situ hybridization demonstrated that GLI1 expression was restricted to tumor tissue and downregulated in response to vismodegib exposure. A single AFL-assisted topical application of vismodegib resulted in clinically relevant intratumoral drug concentrations and significant reductions in hedgehog pathway gene expressions.

Molecular mechanism of angiogenesis for cerebral infarction rats by acupuncture intervention based on sonic hedgehog signaling pathway

BackgroundTo study the factors of the Sonic Hedgehog (Shh) signaling pathway after permanent cerebral ischemic and the effects by acupuncture. MethodsMale Wistar rats were divided into Electro-acupuncture (EA) group, Model Control (MC) group, and blank control (Control) group. EA and MC were divided into 9 phases, namely 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 3 d, 7 d, and 12 d after the operation. The neurological deficits and permanent cerebral ischemic volume were observed. The immunofluorescence method was used to examine the angiogenesis. (Polymerase Chain Reaction) PCR and (Immunohistochemistry) IHC were used to test the changes in Shh, Ptch, Smo, and Gli2 mRNA and proteins. ResultsThe neurological severity scores (NSS) of the Control was 0, the score of the EA group was less than that of the MC. The cerebral permanent ischemic volume of the Control was 0 %, and the EA group's was smaller than that of the MC. The expression of copositive cells in the EA group was higher than the MC's from 12 h to 12 d, and the EA group had more peripheral blood vessels. The rat brain expressions of Shh, Ptch, Smo and Gli2 mRNA and proteins in the MC was higher than that of the Control, the rat brain expression of the EA group was higher than that of the MC. ConclusionsEA can upregulate the expression of the Shh signaling pathway factors, thereby promoting angiogenesis.

Selegiline induced differentiation of rat bone marrow mesenchymal stem cells to dopaminergic neurons in vitro

Today, the use of mesenchymal stem cells (MSCs) for treating human diseases has attracted wide attention. The aim of this study is the expression of dopaminergic genes such as Nestin, patched Tumor Suppressor (PTCH), Sonic Hedgehog (SHH), Tyrosine Hydroxylase (TH) and Nuclear receptor-related factor 1 (NURR1) in MSCs after induction with selegiline. Rat bone marrow mesenchymal stem cells (rBMSCs) were extracted from femur and tibia bones and incubated with alpha Minimum Essential Medium (α-MEM) and 10% Fetal bovine serum (FBS). The stemness of cells at passage 4 was determined by the positive response to CD71 and CD90 markers and their differentiation into adipocytes and osteoblasts. The expression of SHH, PTCH, TH, NURR1 and Nestin genes in the cells after induction by 10-8 M selegiline for 48 hours was investigated by Reverse transcription polymerase chain reaction (RT-PCR) and Real Time-PCR methods. Isolated rBMSCs expressed CD71 and CD90 markers in culture conditions and could differentiate into adipocytes and osteoblasts. Induced cells showed neuronal morphology, positive response to Nestin and TH immunostaining. There was a significant increase of dopaminergic genes TH and NURR1 compared to the untreated cells. The results showed that selegiline with a dose of 10-8 M for 48 hours can lead to dopaminergic differentiation in rBMSCs.

Evaluation of Expression of WNT1 and PTCH Genes in Peripheral Blood of Patients with Odontogenic Cysts and Tumours of the Jaws by Quantitative RT-PCR: A Pilot Study.

Odontogenic lesions of the maxillofacial region constitute a complex group of lesions with diverse histopathologic types and clinical behaviour. Early diagnosis is important to minimize the need for radical surgery and to improve quality of life of the patients. Tumour markers play an essential role in the molecular level understanding of Odontogenic lesions and also used for early diagnosis and target therapies which improves the quality of life of the patients. Patched, a tumour suppressor gene encodes the transmembrane protein PTCH and is a receptor for the morphogen Sonic Hedgehog. It is evident that PTCH gene mutations occur in odontogenic keratocysts and the Hedgehog signalling pathway has an important role during tooth formation. WNT 1 is a key signal molecule that controls cell growth and proliferation. WNT pathway abnormalities are reported to induce tumour occurrence. Hence, my study was to determine the presence of WNT1 and PTCH in peripheral blood of patients with Odontogenic lesions using quantitative RT-PCR. In this cross-sectional study, two groups were included: Group 1-blood samples from 8 individuals with odontogenic cysts and tumours, and Group 2-blood samples of 8 individuals without Odontogenic lesions. 2ml of blood sample was collected from radial veins into PAX gene tubes containing RNA stabilizing agent and stored at a temperature of 2 to 4 degrees and transported to Enable Biolabs India Pvt Ltd., Chennai. PAX gene tubes were subjected to centrifugation at 8000rpm to separate plasma fraction. Reverse transcription of mRNA was performed using miScript II RT Kit (Cat#218161, Qiagen, Germany) to synthesize cDNA. GAPDH house-keeping gene used as control. The study group had 3 males and 5 females (n = 8) with a mean age group of 32.6years and the control group had 2 males and 6 females (n = 8) with mean age of 35.2years. Group I (study group) showed 37.5% positive expression of WNT1 gene with a p value of 0.055 (p > 0.05) and 50% positive expression of PTCH with a p value of 0.021 (p < 0.05) (Figs. 3 and 4) which was statistically significant when compared with control group. Group II (control group) showed 100% negative expression for WNT1 and PTCH genes. WNT1 and PTCH genes were expressed in peripheral blood of patients with odontogenic lesions. WNT1 and PTCH genes may be potential predictors in individuals who would develop odontogenic lesions. Further studies on expression of WNT1 and PTCH genes with larger number of samples might give a future scope for target therapy in odontogenic lesions.

Itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of hemangioma endothelial cells by downregulating the hedgehog signaling pathway

Infantile hemangioma (IH) is among the most prevalent benign vascular tumours in infants. The pathogenesis of IH mainly involves abnormal proliferation of vascular endothelial cells and the formation of new vessels. Itraconazole was shown to be effective in treating IH; however, the mechanism underlying its action is still unclear. The purpose of this study was to examine the effects of itraconazole on the proliferation, apoptosis, and angiogenesis of hemangioma endothelial cells (HemECs); human umbilical vein endothelial cells served as the control group. The expression of genes involved in the hedgehog (HH) signaling pathway (SHH, PTCH1, SMO, and GLI1) was determined using real-time quantitative polymerase chain reaction. Western blotting was used to determine the expression of related proteins. In this study, itraconazole significantly dose- and time-dependently inhibited the viability of HemECs. Itraconazole suppressed the expression of PCNA, Ki67, and vascular endothelial growth factor (VEGF), demonstrating that this treatment inhibited cell proliferation and angiogenesis. Moreover, itraconazole induced apoptosis of HemECs by activating the expression of BAX and inhibiting the expression of BCL2. Itraconazole inhibited SHH, PTCH1, SMO, and GLI1 expression. Activation of the HH pathway by recombinant human sonic hedgehog (rhSHH) protein attenuated the effect of itraconazole on HemECs. In conclusion, itraconazole inhibits proliferation, induces apoptosis, and reduces angiogenesis of HemECs via the downregulation of the HH signaling pathway. Therefore, itraconazole may be an alternative choice for the treatment of IH.

LAMA5: A new pathogenic gene for non-syndromic cleft lip with or without cleft palate

BackgroundThis study aimed to investigate the effect of LAMA5 on palatal development in mice. MethodsThe palatine process of C57BL/6 J fetal mice on the embryonic day 13.5 (E13.5) was cultured in vitro via the rotating culture method. The LAMA5-shRNA adenovirus vector was constructed, then transfected into the palatal process of E13.5 for 48 h in vitro. A fluorescence microscope was used to visualize the fusion of palates. The expression of LAMA5 was also detected. The expression of ki67, cyclin D1, caspase 3, E-cadherin, vimentin and SHH signaling pathway-related signaling factors in the blank control group, the negative control group, and the LAMA5 interference group were detected after virus transfection. ResultsThe bilateral palates in the LAMA5 interference group were not fused after virus transfection. PCR and WB showed that the mRNA and protein expressions of LAMA5 were decreased in the LAMA5 interference group. Furthermore, the mRNA and protein expressions of ki67, cyclin D1 and gli1 were decreased in the LAMA5 interference group, while the mRNA and protein expressions of caspase 3 were increased. However, the mRNA and protein expression of E-cadherin, vimentin, Shh and ptch1 did not significantly change in the LAMA5 interference group. ConclusionsLAMA5 silencing causes cleft palate by inhibiting the proliferation of mouse palatal cells and promoting apoptosis, which may not be involved in EMT. LAMA5 silencing can also cause cleft palate by interfering with the SHH signaling pathway.

Total flavonoid extract from Dracocephalum moldavica L. improves pulmonary fibrosis by reducing inflammation and inhibiting the hedgehog signaling pathway.

Dracocephalum Moldavica L. is a traditional herb for improving pharynx and relieving cough. However, the effect on pulmonary fibrosis is not clear. In this study, we explored the impact and molecular mechanism of total flavonoid extract from Dracocephalum moldavica L. (TFDM) on bleomycin-induced pulmonary fibrosis mouse model. Lung function testing, lung inflammation and fibrosis, and the related factors were detected by the lung function analysis system, HE and Masson staining, ELISA, respectively. The expression of proteins was studied through Western Blot, immunohistochemistry, and immunofluorescence while the expression of genes was analyzed by RT-PCR. The results showed that TFDM significantly improved lung function in mice, reduced the content of inflammatory factors, thereby reducing the inflammation. It was found that expression of collagen type I, fibronectin, and α-smooth muscle actin was significantly decreased by TFDM. The results further showed that TFDM interferes with hedgehog signaling pathway by decreasing the expression of Shh, Ptch1, and SMO proteins and thereby inhibiting the generation of downstream target gene Gli1 and thus improving pulmonary fibrosis. Conclusively, these findings suggest that TFDM improve pulmonary fibrosis by reducing inflammation and inhibition of the hedgehog signaling pathway.

Carvacrol instigates intrinsic and extrinsic apoptosis with abrogation of cell cycle progression in cervical cancer cells: Inhibition of Hedgehog/GLI signaling cascade.

Recent times have seen a strong surge in therapeutically targeting the hedgehog (HH)/GLI signaling pathway in cervical cancer. HH signaling pathway is reported to be a crucial modulator of carcinogenesis in cervical cancer and is also associated with recurrence and development of chemoresistance. Moreover, our previous reports have established that carvacrol (CAR) inhibited the proliferation of prostate cancer cells via inhibiting the Notch signaling pathway and thus, it was rational to explore its antiproliferative effects in cervical cancer cell lines. Herein, the present study aimed to investigate the anticancer and apoptotic potential of CAR on C33A cervical cancer cells and further explore the underlying mechanisms. We found that CAR significantly suppressed the growth of C33Acells, induced cell cycle arrest, and enhanced programmed cell death along with augmentation in the level of ROS, dissipated mitochondrial membrane potential, activation of caspase cascade, and eventually inhibited the HH signaling cascade. In addition, CAR treatment increased the expression of pro-apoptotic proteins (Bax, Bad, Fas-L, TRAIL, FADDR, cytochrome c) and concomitantly reduced the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL) in C33Acells. CAR mediates the activation of caspase-9 and -3 (intrinsic pathway) and caspase-8 (extrinsic pathway) accompanied by the cleavage of PARP in cervical cancer cells. Thus, CAR induced apoptosis by both the intrinsic and extrinsic apoptotic pathways. CAR efficiently inhibited the growth of cervical cancer cells via arresting the cell cycle at G0/G1 phase and modulated the gene expression of related proteins (p21, p27, cyclin D1 and CDK4). Moreover, CAR inhibited the HH/GLI signaling pathway by down regulating the expression of SMO, PTCH and GLI1 proteins in cervical carcinoma cells. With evidence of the above results, our data revealed that CAR treatment suppressed the growth of HPV-C33A cervical cancer cells and further elucidated the mechanistic insights into the functioning of CAR.

Leonurine Regulates Hippocampal Nerve Regeneration in Rats with Chronic and Unpredictable Mild Stress by Activating SHH/GLI Signaling Pathway and Restoring Gut Microbiota and Microbial Metabolic Homeostasis.

Depression is a highly prevalent and heterogeneous disorder that requires new strategies to overcome depression. In this study, we aimed to investigate whether leonurine modulated hippocampal nerve regeneration in chronic and unpredictable mild stress (CUMS) rats through the SHH/GLI signaling pathway and restoring gut microbiota and microbial metabolic homeostasis. The CUMS rat model was constructed and treated with leonurine. The body weight of rats was recorded, and a series of tests were performed. Western blot was utilized to measure the expression of BDNF and 5-HT in the hippocampus. Then the expression of SHH, GLI, PTCH, and SMO were measured by qRT-PCR and western blot. The colocalization of BrdU+DCX and BrdU+NeuN was evaluated by IF. 16S rDNA high-throughput sequencing was applied to detect the composition and distribution of gut microbiota. The differential metabolites were analyzed by untargeted metabolomics. The correlation between gut microbiota and microbial metabolites was analyzed by Pearson correlation coefficient. After CUMS modeling, the body weight of rats was decreased, and the expression of BDNF and 5-HT were decreased, while the body weight was recovered, and the expression of BDNF and 5-HT were increased after leonurine treatment. Leonurine reversed the reduction in the colocalization of BrdU+DCX and BrdU+NeuN and the reduction in the levels of SHH, GLI, PTCH, and SMO induced by CUMS modeling. Leonurine also restored gut microbiota and microbial metabolites homeostasis in CUMS rats. Furthermore, Prevotellaceae_Ga6A1_group was negatively correlated with 3-Oxocholic acid, nutriacholic acid, and cholic acid. Collectively, leonurine regulated hippocampal nerve regeneration in CUMS rats by activating the SHH/GLI signaling pathway and restoring gut microbiota and microbial metabolic homeostasis.

Sequential application of small molecule therapy enhances chondrogenesis and angiogenesis in murine segmental defect bone repair.

The increasing incidence of physiologic/pathologic conditions that impair the otherwise routine healing of endochondral bone fractures and the occurrence of severe bone injuries necessitate novel approaches to enhance clinically challenging bone fracture repair. To promote the healing of nonunion fractures, we tested an approach that used two small molecules to sequentially enhance cartilage development and conversion to the bone in the callus of a murine femoral segmental defect nonunion model of bone injury. Systemic injections of smoothened agonist 21k(SAG21k) were used to stimulate chondrogenesis through the activation of the sonic hedgehog (SHH) pathway early in bone repair, while injections of the prolyl hydroxylase domain (PHD)2 inhibitor, IOX2, were used to stimulate hypoxia signaling-mediated endochondral bone formation. The expression of SHH pathway genes and Phd2 target genes was increased in chondrocyte cell lines in response to SAG21k and IOX2 treatment, respectively. The segmental defect responded to sequential systemic administration of these small molecules with increased chondrocyte expression of PTCH1, GLI1, and SOX9 in response to SAG and increased expression of hypoxia-induced factor-1α and vascular endothelial growth factor-A in the defect tissues in response to IOX2. At 6 weeks postsurgery, the combined SAG-IOX2 therapy produced increased bone formation in the defect with the bony union over the injury.Clinical significance: This therapeutic approach was successful in promoting cartilage and bone formation within a critical-size segmental defect and established the utility of a sequential small molecule therapy for the enhancement of fracture callus development in clinically challenging bone injuries.

Long Non-coding RNA SNHG16 Facilitates Esophageal Cancer Cell Proliferation and Self-renewal through the microRNA-802/PTCH1 Axis.

This research sought to explore the effect and mechanism of long non-coding RNA SNHG16 on esophageal cancer (EC) cell proliferation and self-renewal. SNHG16 expression was measured in EC9706 and KYSE150 cells. EC9706 and KYSE150 cells were transfected with Lenti-SNHG16, sh-SNHG16, Lenti-protein patched homolog 1 (PTCH1), miR-802 mimic, or miR-802 inhibitor. Flow cytometry was used to sort cancer stem cells (CSCs) in EC9706 and KYSE150 cells. Cell proliferation in EC cells was measured, in addition to colony and tumorsphere numbers. The possible interactions among SNHG16, PTCH1, and miR-802 were identified by dual luciferase reporter and RNA pull-down assays. The expression of the genes in the Hedgehog pathway was detected. Nude mice were injected with SNHG16-silenced EC9706 cells to observe the tumorigenicity of EC9706 cells. Upregulated SNHG16 expression was found in CSCs, whose expression was decreased during the differentiation of CSCs. SNHG16 or PTCH1 overexpression or miR-802 inhibition promoted the proliferation, colony formation, and tumorsphere formation of EC9706 and KYSE150 cells as well as SOX2, OCT4, Bmi-1, and PTCH1 expression. Consistently, SNHG16 knockdown or miR-802 overexpression inhibited EC progression. Moreover, SNHG16 and PTCH1 were competitively bound to miR-802, and SNHG16 orchestrated the miR-802/PTCH1 axis to activate the Hedgehog pathway. SNHG16 silencing repressed the tumorigenicity of EC9706 in nude mice. Conclusively, SNHG16 acts as a sponge of miR-802 to upregulate PTCH1 and activate the Hedgehog pathway, thus promoting EC cell proliferation and selfrenewal.

Expression of Hedgehog Signaling Pathway Proteins in Basal Cell Carcinoma: Clinicopathologic Study.

Basal cell carcinoma (BCC) is the most common type of cancer with a growing incidence rate over recent decades. The increasing economic burden and incidence of BCC have generated major interest in developing targeted therapies for this disease. The critical role of the Hedgehog (Hh) pathway in the pathogenesis of BCC has become evidently demonstrated. The purpose of this study was to observe the expression of PTCH1 and Gli1 in BCC and further evaluate their relationship with clinicopathological features. This retrospective study included 84 patients with BCC. Information of 84 patients with pathologically diagnosed BCC (including location, sex, tumor size, pathological type, and depth of invasion) were collected, and tissue paraffin blocks were collected for immunohistochemical staining. Western blot analysis for PTCH1 and Gli1 were also performed. The staining intensity and percentage of stained cells were expressed as a histochemical score (HSCORE). PTCH1 and Gli1 were overexpressed in BCC compared with adjacent normal epidermis. Our study found that the expression of PTCH1 and Gli1 in BCC in exposed sites was significantly higher than in non-exposed sites. Moreover, no significant difference was observed in sex, Breslow thickness, tumor size or pathological type (P>0.05). PTCH1 and Gli1 were overexpressed in BCC. Higher PTCH1 and Gli1 expression were in exposed sites lesions. Our study suggests that UV radiation may be associated with aberrant activation of the Hh-PTCH1-Gli1 intercellular signaling pathway in BCC. The molecular mechanism of UV-related PTCH1 and Gli1 differential expression deserves more rigorous research in the future.

Activation of the hedgehog signaling pathway is associated with the promotion of cell proliferation and epithelial-mesenchymal transition in chronic rhinosinusitis with nasal polyps.

To investigate the pathogenesis of the hedgehog (Hh) signaling pathway activated by inflammation in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). The 82 people including CRSwNP patients (case group) and nasal septal deviation patients (control group) were recruited. The samples in the case group were collected and classified into two groups: mucosal tissue of nasal polyps (NP group) and mucosal tissue adjacent to nasal polyps (NM group), the samples were collected from the control group as CM group. Clinical characteristics were assessed. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were performed to detect eosinophils (EOS), the expression of the key genes of the pathway and epithelial-mesenchymal transition (EMT) markers in the samples. There were significant differences in the nasal obstruction visual analog scale (VAS) score, rhinorrhea VAS score, percentage of blood EOS, blood EOS absolute counts and tissue EOS counts in the case group compared with the control group (P < 0.05). The EOS level and expression levels of PTCH1, SMO, Gli1, Gli2, Ki67 and vimentin were higher in NP group than in the other two groups (P < 0.05). E-cadherin expression was decreased in NP group (P < 0.05). A positive correlation between PTCH1 expression and CRSwNP Lund-Mackay score in NP group. Our results indicated that the activation of Hh signaling pathway might promote cell proliferation and EMT occurrence, ultimately leading to the development of CRSwNP, which might provide a new target for treatment.