Gli1 Expression Research Articles

Tumor-Associated Macrophage-Derived TGF-β1 Activates GLI2 via the Smad2/3 Signaling Pathway to Affect Cisplatin Resistance in Lung Adenocarcinoma.

Transforming growth factor-β1 (TGF-β1) is an immunosuppressive cytokine that is highly expressed in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD). TGF-β1 plays important roles in regulating tumor metastasis and chemotherapy resistance. However, the specific molecular mechanisms by which TGF-β1 regulates cisplatin resistance in the TAM of LUAD remain unclear. THP-1 induced macrophages were co-cultured with A549 and H1975 cells, and subsequently transfected with silencing TGF-β1 (siTGF-β1), GLI2 (siGLI2), a GLI2 overexpression plasmid, and their negative controls. Cellular activity was measured by CCK-8 and colony formation assays. Cell apoptosis was evaluated by flow cytometry and TUNEL staining. Transwell assays were performed to assess cell migration and invasion capabilities. The levels of Smad2/3, GLI2, cyclin D, and cyclin E expression were evaluated by qPCR, western blotting, and immunofluorescence methods. TGF-β1 levels were determined by ELISA. Macrophages suppressed the apoptosis and promoted the migration and invasion of LUAD cells. TAM siTGF-β1 downregulated the Smad2/3 signaling pathways and GLI2 expression, deceased cell proliferation, and promoted apoptosis. SiGLI2 increased apoptosis and decreased the proliferation of LUAD cell lines. GLI2 decreased cisplatin resistance in LUAD cells. High expression of TGF-β1 in the TAM positively activates GLI2 expression via the Smad2/3 pathway, which subsequently regulates cyclin D and cyclin E expression, and promotes the cisplatin resistance of LUAD.

Hedgehog signalling is involved in acquired resistance to KRASG12C inhibitors in lung cancer cells

Although KRASG12C inhibitors have shown promising activity in lung adenocarcinomas harbouring KRASG12C, acquired resistance to these therapies eventually occurs in most patients. Re-expression of KRAS is thought to be one of the main causes of acquired resistance. However, the mechanism through which cancer cells re-express KRAS is not fully understood. Here, we report that the Hedgehog signal is induced by KRASG12C inhibitors and mediates KRAS re-expression in cancer cells treated with a KRASG12C inhibitor. Further, KRASG12C inhibitors induced the formation of primary cilia and activated the Hedgehog-GLI-1 pathway. GLI-1 binds to the KRAS promoter region, enhancing KRAS promoter activity and KRAS expression. Inhibition of GLI using siRNA or the smoothened (Smo) inhibitor suppressed re-expression of KRAS in cells treated with a KRASG12C inhibitor. In addition, we demonstrate that KRASG12C inhibitors decreased Aurora kinase A (AURKA) levels in cancer cells, and inhibition of AURKA using siRNA or inhibitors led to increased expression levels of GLI-1 and KRAS even in the absence of KRAS inhibitor. Ectopic expression of AURKA attenuated the effect of KRASG12C inhibitors on the expression of GLI-1 and re-expression of KRAS. Together, these findings demonstrate the important role of AURKA, primary cilia, and Hedgehog signals in the re-expression of KRAS and therefore the induction of acquired resistance to KRASG12C inhibitors, and provide a rationale for targeting Hedgehog signalling to overcome acquired resistance to KRASG12C inhibitors.

Hepatic progenitor cell-originated ductular reaction facilitates liver fibrosis through activation of hedgehog signaling.

Background: It is poorly understood what cellular types participate in ductular reaction (DR) and whether DR facilitates recovery from injury or accelerates hepatic fibrosis. The aim of this study is to gain insights into the role of hepatic progenitor cell (HPC)-originated DR during fibrotic progression. Methods: DR in liver specimens of PBC, chronic HBV infection (CHB) or NAFLD, and four rodent fibrotic models by different pathogenic processes was evaluated. Gli1 expression was inhibited in rodent models or cell culture and organoid models by AAV-shGli1 or treating with GANT61. Results: Severity of liver fibrosis was positively correlated with DR extent in patients with PBC, CHB or NAFLD. HPCs were activated, expanded, differentiated into reactive cholangiocytes and constituted "HPC-originated DR", accompanying with exacerbated fibrosis in rodent models of HPC activation & proliferation (CCl4/2-AAF-treated), Μdr2-/- spontaneous PSC, BDL-cholestatic fibrosis or WD-fed/CCl4-treated NASH-fibrosis. Gli1 expression was significantly increased in enriched pathways in vivo and in vitro. Enhanced Gli1 expression was identified in KRT19+-reactive cholangiocytes. Suppressing Gli1 expression by administration of AAV-shGli1 or GANT61 ameliorated HPC-originated DR and fibrotic extent. KRT19 expression was reduced after GANT61 treatment in sodium butyrate-stimulated WB-F344 cells or organoids or in cells transduced with Gli1 knockdown lentiviral vectors. In contrast, KRT19 expression was elevated after transducing Gli1 overexpression lentiviral vectors in these cells. Conclusions: During various modes of chronic injury, Gli1 acted as an important mediator of HPC activation, expansion, differentiation into reactive cholangiocytes that formed DR, and subsequently provoked hepatic fibrogenesis.

Combined transcriptomics and proteomics studies on the effect of electrical stimulation on spinal cord injury in rats

Electrical stimulation (ES) of the spinal cord is a promising therapy for functional rehabilitation after spinal cord injury (SCI). However, the specific mechanism of action is poorly understood. We designed and applied an implanted ES device in the SCI area in rats and determined the effect of ES on the treatment of motor dysfunction after SCI using behavioral scores. Additionally, we examined the molecular characteristics of the samples using proteomic and transcriptomic sequencing. The differential molecules between groups were identified using statistical analyses. Molecular, network, and pathway-based analyses were used to identify group-specific biological features. ES (0.5 mA, 0.1 ms, 50 Hz) had a positive effect on motor dysfunction and neuronal regeneration in rats after SCI. Six samples (three independent replicates in each group) were used for transcriptome sequencing; we obtained 1026 differential genes, comprising 274 upregulated genes and 752 downregulated genes. A total of 10 samples were obtained: four samples in the ES group and six samples in the SCI group; for the proteome sequencing, 48 differential proteins were identified, including 45 up-regulated and three down-regulated proteins. Combined transcriptomic and proteomic studies have shown that the main enrichment pathway is the hedgehog signaling pathway. Western blot results showed that the expression levels of Sonic hedgehog (SHH) (P < 0.001), Smoothened (SMO) (P = 0.0338), and GLI-1 (P < 0.01) proteins in the ES treatment group were significantly higher than those in the SCI group. The immunofluorescence results showed significantly increased expression of SHH (P = 0.0181), SMO (P = 0.021), and GLI-1 (P = 0.0126) in the ES group compared with that in the SCI group. In conclusion, ES after SCI had a positive effect on motor dysfunction and anti-inflammatory effects in rats. Moreover, transcriptomic and proteomic sequencing also provided unique perspectives on the complex relationships between ES on SCI, where the SHH signaling pathway plays a critical role. Our study provides a significant theoretical foundation for the clinical implementation of ES therapy in patients with SCI.

Umbilical cord mesenchymal stem cells inhibited inflammation of bronchial epithelial cells by regulating Hedgehog pathway.

This study aimed to explore the role and mechanism of umbilical cord mesenchymal stem cells (UCMSCs) inregulating inflammation of bronchial epithelial cells. Transforming growth factor beta-1 (TGF-β1) was used to induce inflammation in human bronchial epithelial cells. Cell proliferation was detected through CCK8 and cellapoptosis was detected by Annexin V and propidium iodide double staining. E-cadherin and α-smooth muscle actin (α-SMA) were detected by immunofluorescence, and tumor necrosis factor alpha (TNF-α) and interleukin6 (IL-6) in culture medium supernatant were detected by ELISA. The expression of E-cadherin, α-SMA, Sonic hedgehog (Shh), Gli1 and Snail was detected by Western blot analysis. Compared with the control group,bronchial epithelial cells treated with TGF-β1 showed significantly decreased proliferation, increased apoptosis, increased secretion of TNF-α and IL-6, increased expression of α-SMA, Shh, Gli1 and Snail and decreased E-cadherin expression. However, co-culture with UCMSCs inhibited TGF-β1-induced changes in humanbronchial epithelial cell proliferation, apoptosis, secretion of TNF-α and IL-6 and activation of the Hedgehog pathway. In conclusion, UCMSCs have protective effects on TGF-β1-induced inflammation in humanbronchial epithelial cells by regulating the Hedgehog pathway.

GLI1+ perivascular, renal, progenitor cells: The likely source of spontaneous neoplasia that created the AGMK1-9T7 cell line

The AGMK1-9T7 cell line has been used to study neoplasia in tissue culture. By passage in cell culture, these cells evolved to become tumorigenic and metastatic in immunodeficient mice at passage 40. Of the 20 x 106 kidney cells originally plated, less than 2% formed the colonies that evolved to create this cell line. These cells could be the progeny of some type of kidney progenitor cells. To characterize these cells, we documented their renal lineage by their expression of PAX-2 and MIOX, detected by indirect immunofluorescence. These cells assessed by flow-cytometry expressed high levels of CD44, CD73, CD105, Sca-1, and GLI1 across all passages tested; these markers have been reported to be expressed by renal progenitor cells. The expression of GLI1 was confirmed by immunofluorescence and western blot analysis. Cells from passages 13 to 23 possessed the ability to differentiate into adipocytes, osteoblasts, and chondrocytes; after passage 23, their ability to form these cell types was lost. These data indicate that the cells that formed the AGMK1-9T7 cell line were GLI1+ perivascular, kidney, progenitor cells.

Tumor suppressor miR-361-3p inhibits prostate cancer progression through Gli1 and AKT/mTOR signaling pathway

BackgroundThe primary challenge in prostate cancer (PCa) is tumor metastasis, which seriously affects the survival time of patients. Growing evidence suggests that microRNAs play a crucial regulatory role in various malignancies and that the tumor suppressor miR-361-3p is responsible for regulating migration, proliferation, and invasion in different cancer types. However, the underlying regulatory mechanism of miR-361-3p in PCa remains unknown. MethodsThe expression of miR-361-3p in PCa cells was analyzed using quantitative real time-polymerase chain reaction. The clinical utility of miR-361-3p in PCa was evaluated using in vitro assays. The mechanism of action of miR-361-3p was investigated using western blotting, luciferase reporter assays, immunofluorescence, and rescue studies. ResultsThe function, invasiveness, migration, and proliferation of PCa cells, as well as epithelial-mesenchymal transition (EMT), were aided by the downregulation of miR-361-3p, whereas its overexpression exerted the opposite effect. Repression of glioma-associated oncogene homolog 1 (Gli1) expression by miR-361-3p led to activation of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) signaling pathway, triggering EMT and promoting PCa metastasis. ConclusionsDownregulation of miR-361-3p along the Gli1 axis promoted tumor malignancy. Collectively, the results of this study imply that miR-361-3p has the potential to be both a biomarker and therapeutic target in PCa.

BushenHuoxue formula promotes osteogenic differentiation via affecting Hedgehog signaling pathway in bone marrow stem cells to improve osteoporosis symptoms.

The BushenHuoxue formula (BSHX) has been previously demonstrated to ameliorate osteoporosis, but the mechanisms underlying this phenomenon are currently unclear. The present study aims at investigating the mechanisms that BSHX induces osteogenesis. We established an osteoporosis model in rats by bilateral ovariectomy and then treated the rats with an osteogenic inducer (dexamethasone, β-sodium glycerophosphate and Vitamin C) and BSHX. After that, bone marrow density and histopathological bone examination were evaluated by using HE staining and immunohistochemistry, respectively. We also assessed the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts by using immunofluorescence staining. ALP, BMP, and COL1A1 levels were determined by ELISA. We identified genes involved in pathogenesis of osteoporosis through Gene Expression Omnibus (GEO) database and subsequently selected Hedgehog signaling-related genes Shh, Ihh, Gli2, and Runx2 for assessment via qRT-PCR and ELISA, Western blotting. Network pharmacology analysis was performed to identify bioactive metabolites of BSHX. BSHX treatment in osteoporosis model rats promoted tightening of the morphological structure of the trabecular bone and increased the bone mineral density (BMD). BSHX also increased levels of osteoblast makers ALP, BMP, and COL1A1. Additionally, bioinformatics analysis of the GEO dataset showed that Hedgehog signaling pathway was involved in pathogenesis of osteoporosis, especially related genes Shh, Ihh, Gli2, and Runx2. Remarkably, BHSX upregulated these genes indispensably involved in the osteogenesis-related Hedgehog signaling pathway in both bone tissue and BMSCs. Importantly, we identified that quercetin was the active compounds that involved in the mechanism of BSHX-improved OP via affecting Hedgehog-related genes. Our results indicate that BSHX promotes osteogenesis by improving BMSC differentiation into osteoblasts via increased expression of Hedgehog signaling-related genes Shh, Ihh, Gli2, and Runx2, and quercetin was the bioactive compound of BSHX.

Long non-coding RNA HIF1A-AS2 promotes carcinogenesis by enhancing Gli1-mediated HIF1α expression in clear cell renal cell carcinoma

BackgroundThe most common urologic tumor in humans with the highest incidence rate is clear cell renal cell carcinoma (ccRCC). Long non-coding RNAs (lncRNAs) act as regulatory factors in several tumors. Here, we studied ccRCC regulated by hypoxia-inducible factor 1α (HIF1α)-antisense RNA 2 (AS2) or HIF1A-AS2. MethodsWe performed wound-healing, transwell, and CCK-8 assays by decreasing or increasing the HIF1A-AS2 expression in RCC cell lines. Western blotting and qRT-PCR were used to identify the expression of downstream genes of the HIF1A-AS2 pathway. Gli1 and HIF1A-AS2 relationship was assessed using RIP and RNA pull-down assays. Lastly, transcriptome sequencing was performed on kidney cancer cells that had been knocked down to find possible regulatory mechanisms. ResultsOur results suggest that high expression of HIF1A-AS2 may promote RCC cell proliferation and Gli1 expression as a downstream factor. Furthermore, they have physical binding sites and together regulate HIF1α to encourage the development of ccRCC. HIF1A-AS2 lncRNA may offer a new molecular target for ccRCC treatment. ConclusionlncRNA HIF1A-AS2 affects ccRCC development by regulating HIF1a expression through Gli1.

RCC2 promotes prostate cancer cell proliferation and migration through Hh/GLI1 signaling pathway and cancer stem-like cells

BackgroundRegulator of chromosome condensation 2 (RCC2) was a telophase disk-binding protein on mitosis, and functions as an oncogene in many human cancers. However, its role on prostate cancer (PCa) was unknown. The goal of this study is to explore the function of RCC 2 on PCa development.MethodsThe expression of RCC2 and its methylation level, its correlation with lymph node metastasis or disease-free survival (DFS) was analyzed using TCGA database. The effect of RCC2 on PCa cell proliferation, migration and invasion were detected using CCK-8, cell colony formation, Transwell and wood healing assays. RNA-seq and GSEA analysis were used to search the downstream genes and pathways of RCC2 in mediated PCa progression. Western blot was used to detect the proteins in PCa cells transfected with indicated siRNAs or plasmids.ResultsRCC2 had high expression and low promoter methylation level in PCa, and its expression was correlated with regional node metastasis and disease-free survival. Cell proliferation, migration, invasion and EMT of PCa cells in vitro were greatly enhanced after RCC2 overexpression, while the RCC2 knockdown suppressed these processes. RNA-seq and GSEA results showed the Hedgehog signaling regulator Gli1 and Gli3 were involved in RCC2 knockdown DU145 cells. Gli1 was also a marker of cancer stem-like cells (CSCs). Mechanistically, RCC2 induced cell growth, EMT, CSCs markers through Gli1; inhibiting Gli1 expression using siGli1 or GLI inhibitor suppressed cell progression in vitro and tumor growth in vivo.ConclusionIn summary, RCC2 promoted PCa development through Hh/Gli1 signaling pathway via regulating EMT and CSCs.

LncRNA MIR31HG fosters stemness malignant features of non-small cell lung cancer via H3K4me1- and H3K27Ace-mediated GLI2 expression

Non-coding RNAs are responsible for oncogenesis and the development of stemness features, including multidrug resistance and metastasis, in various cancers. Expression of lncRNA MIR31HG in lung cancer tissues and peripheral sera of lung cancer patients were remarkably higher than that of healthy individuals and indicated a poor prognosis. Functional analysis showed that MIR31HG fosters stemness-associated malignant features of non-small cell lung cancer cells. Further mechanistic investigation revealed that MIR31HG modulated GLI2 expression via WDR5/MLL3/P300 complex-mediated H3K4me and H3K27Ace modification. In vivo MIR31HG repression with an antisense oligonucleotide attenuated tumor growth and distal organ metastasis, whereas MIR31HG promotion remarkably encouraged cellular invasion in lung and liver tissues. Our data suggested that MIR31HG is a potential diagnostic indicator and druggable therapeutic target to facilitate multiple strategic treatments for lung cancer patients.

Oxidative Stress Induces Skin Pigmentation in Melasma by Inhibiting Hedgehog Signaling.

There is growing evidence that oxidative stress plays a role in melasma and disrupts primary cilia formation. Additionally, primary cilia have been suggested to have an inhibitory role in melanogenesis. This study examined the potential link between oxidative stress, skin hyperpigmentation, and primary cilia. We compared the expression levels of the nuclear factor E2-related factor 2 (NRF2), intraflagellar transport 88 (IFT88), and glioma-associated oncogene homologs (GLIs) in skin samples from patients with melasma, both in affected and unaffected areas. We also explored the roles of NRF2, IFT88, and GLIs in ciliogenesis and pigmentation using cultured adult human keratinocytes, with or without melanocytes. Our findings revealed decreased levels of NRF2, heme oxygenase-1, IFT88, and GLIs in lesional skin from melasma patients. The knockdown of NRF2 resulted in reduced expressions of IFT88 and GLI1, along with fewer ciliated cells. Furthermore, NRF2, IFT88, or GLI1 knockdown led to increased expressions in protease-activated receptor-2 (PAR2), K10, involucrin, tyrosinase, and/or melanin. These effects were reversed by the smoothened agonist 1.1. Calcium also upregulated these proteins, but not NRF2. The upregulation of involucrin and PAR2 after NRF2 knockdown was mitigated with a calcium chelator. In summary, our study suggests that oxidative stress in NRF2-downregulated melasma keratinocytes impedes ciliogenesis and related molecular processes. This inhibition stimulates keratinocyte differentiation, resulting in melanin synthesis and melanosome transfer, ultimately leading to skin hyperpigmentation.

Gli1 marks a sentinel muscle stem cell population for muscle regeneration.

Adult skeletal muscle regeneration is mainly driven by muscle stem cells (MuSCs), which are highly heterogeneous. Although recent studies have started to characterize the heterogeneity of MuSCs, whether a subset of cells with distinct exists within MuSCs remains unanswered. Here, we find that a population of MuSCs, marked by Gli1 expression, is required for muscle regeneration. The Gli1+ MuSC population displays advantages in proliferation and differentiation both in vitro and in vivo. Depletion of this population leads to delayed muscle regeneration, while transplanted Gli1+ MuSCs support muscle regeneration more effectively than Gli1- MuSCs. Further analysis reveals that even in the uninjured muscle, Gli1+ MuSCs have elevated mTOR signaling activity, increased cell size and mitochondrial numbers compared to Gli1- MuSCs, indicating Gli1+ MuSCs are displaying the features of primed MuSCs. Moreover, Gli1+ MuSCs greatly contribute to the formation of GAlert cells after muscle injury. Collectively, our findings demonstrate that Gli1+ MuSCs represents a distinct MuSC population which is more active in the homeostatic muscle and enters the cell cycle shortly after injury. This population functions as the tissue-resident sentinel that rapidly responds to injury and initiates muscle regeneration.

FOXS1 promotes prostate cancer progression through the Hedgehog/Gli1 pathway

BackgroundProstate cancer (PCa) remains the most common malignant tumor in men, and the clinical treatment still faces many challenges. Several molecular biomarkers of PCa progression have been reported, however, whether FOXS1 can serve as a new biomarker in PCa remains unknown. MethodsFOXS1 and Gli1 expression was assessed by RT-qPCR and western blot. The binding and regulation roles between FOXS1 and Gli1 were confirmed by Co-IP and ubiquitination assays. Cell viability, proliferation, apoptosis, migration, invasion and EMT progress were assessed through CCK-8, colony formation, flow cytometry, wound-healing, transwell and western blot assays, respectively. In vivo nude mice tumorigenesis model was also conducted to verify PCa growth. ResultsFOXS1 was upregulated in the PCa TCGA dataset and cells. High FOXS1 level was correlated with PCa patients’ worse tumor stage and shorter survival. FOXS1 knockdown inhibited PCa cell proliferation, invasion, migration, EMT and tumor growth while increased cell apoptosis. Furthermore, FOXS1 knockdown decreased the inactivation of Hedgehog (Hh) pathway. FOXS1 bind to Gli1 and decreased the ubiquitination of Gli1, which resulted in the upregulation of Gli1. Besides, both Gil1 overexpression and Hh signal activation reversed the suppression function of FOXS1 silencing on PCa growth and metastasis. ConclusionFOXS1 bind and stabilized Gli1 by blocking Gli1 ubiquitination, thereby activating Hh signaling to promote PCa cell growth and metastasis.

Towards modular engineering of cell signalling: Topographically-textured microparticles induce osteogenesis via activation of canonical hedgehog signalling

Polymer microparticles possess great potential as functional building blocks for advanced bottom-up engineering of complex tissues. Tailoring the three-dimensional architectural features of culture substrates has been shown to induce osteogenesis in mesenchymal stem cells in vitro, but the molecular mechanisms underpinning this remain unclear. This study proposes a mechanism linking the activation of Hedgehog signalling to the osteoinductive effect of surface-engineered, topographically-textured polymeric microparticles. In this study, mesenchymal progenitor C3H10T1/2 cells were cultured on smooth and dimpled poly(D,l-lactide) microparticles to assess differences in viability, cellular morphology, proliferation, and expression of a range of Hedgehog signalling components and osteogenesis-related genes. Dimpled microparticles induced osteogenesis and activated the Hedgehog signalling pathway relative to smooth microparticles and 2D-cultured controls without the addition of exogenous biochemical factors. We observed upregulation of the osteogenesis markers Runt-related transcription factor2 (Runx2) and bone gamma-carboxyglutamate protein 2 (Bglap2), as well as the Hedgehog signalling components, glioma associated oncogene homolog 1 (Gli1), Patched1 (Ptch1), and Smoothened (Smo). Treatment with the Smo antagonist KAAD-cyclopamine confirmed the involvement of Smo in Gli1 target gene activation, with a significant reduction in the expression of Gli1, Runx2 and Bglap2 (p ≤ 0.001) following KAAD-cyclopamine treatment. Overall, our study demonstrates the role of the topographical microenvironment in the modulation of Hedgehog signalling, highlighting the potential for tailoring substrate topographical design to offer cell-instructive 3D microenvironments. Topographically-textured microparticles allow the modulation of Hedgehog signalling in vitro without adding exogenous biochemical agonists, thereby eliminating potential confounding artefacts in high-throughput drug screening applications.

Sonic hedgehog signaling promotes angiogenesis of endothelial progenitor cells to improve pressure ulcers healing by PI3K/AKT/eNOS signaling.

Pressure ulcer is a severe disease in the paralyzed and aging populations. Endothelial progenitor cells (EPCs) are able to regulate ulcer healing by modulating angiogenesis, but the molecular mechanism is still obscure. Sonic hedgehog (SHH) signaling contributes to angiogenesis in various diseases and has been identified to modulate EPCs function. Here, we aimed to explore the significance of SHH signaling in EPCs function during pressure ulcers. The EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1. Cell proliferation was detected by cell counting kit 8 (CCK-8). The DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 were analyzed by immunofluorescent analysis. The angiogenesis of EPCs was analyzed by tube formation assay. The pressure ulcers rat model was constructed, the wound injury was analyzed by H&E staining and angiogenesis was analyzed by the accumulation of CD31 based on immunofluorescent analysis. The expression of patched-1 and Gli-1 was enhanced by SHH activator SAG but reduced by SHH inhibitor cyclopamine in the EPCsThe PI3K, Akt, eNOS expression and the Akt phosphorylation were induced by SAG, while the treatment of cyclopamine presented a reversed result. The proliferation and migration of EPCs were enhanced by SAG but repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse the effect of SAG. Thus, we found that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. SHH signaling may serve as the potential target for attenuating pressure ulcers.

GANT61, an inhibitor of Gli1, inhibits the proliferation and migration of hepatocellular carcinoma cells.

Constitutive activation of Hedgehog (Hh) signaling has been implicated in many cancers including hepatocellular carcinoma (HCC). Among them, the terminal glioma-associated oncogene homolog 1 (Gli1) regulates the expression of critical genes in the Hh pathway. The current study aims to evaluate the anti-HCC effect of the Gli1 inhibitor, GANT61. In vitro analysis including cell counting kit-8 (CCK-8) assay, flow cytometry, and migration and invasion assay were adopted to evaluate the effect of GANT61 on HCC cell lines. In vivo, xenograft studies were also performed to verify the effect of GANT61 on HCC. By CCK-8 assay, we found that GANT61 could significantly reduce the growth of HCC cell lines Huh7 and hemophagocytic lymphohistiocytosis (HLE), and their IC50 concentrations were 4.481 and 6.734 μM, respectively. Flow cytometry shows that GANT61 induced cell cycle arrest in the G2/M phase and accelerated apoptosis of both HLE and Huh7 cells. While migration and invasion assay shows that GANT61 weakens cells' migration and invasion ability. Besides that, GANT61 inhibits the expression of Gli1, FoxM1, CyclinD1, and Bcl-2, upregulates the level of Bax protein, and also reverses the epithelial-mesenchymal transition program by downregulating the expression of Vimentin and N-Cadherin and upregulating the expression of epithelial E-Cadherin expression. Furthermore, GANT61 inhibits the growth of subcutaneous xenografts of Huh7 cells in nude mice. Overall, this study suggests that Gli1 is a potential target for therapy and GANT61 shows promising therapeutic potential for future treatment in HCC.

Synthesis of Vismodegib Derivatives as Potential Smo Agonist: A Case of Undergraduate Experimental Teaching

The activation of Hh pathway demonstrates therapeutic potential for many diseases. Smo is the main target for the development of Hh pathway modulators. However, compared with Smo antagonist, the development of Smo agonists is lagging behind. Based on our previous work, a series of vismodegib derivatives were designed and synthesized, and their potential to activate the Hh pathway were evaluated via determining the up-regulation of known pathway target genes Gli1, whcih revealed that many target compounds could activate the Hh pathway. XH-16 and XH-17, with the strongest agonistic effect, could upregulate the expression of Gli1 by more than 50% at a concentration of 10 μM. In vitro cytotoxicity against A549 and the MDA-MB-231 cells was evaluated and the results revealed that XH-16 and XH-17 did not exhibit cytotoxicity at a concentration of 50 μM. Molecular docking results confirmed that XH-16 and XH-17 could bind to Smo indicating that their agonistic effect on the Hh pathway may be due to the activation of Smo. XH-16 and XH-17 with novel molecular scaffold could be used as a lead compound for the development of Smo agonists. Moreover, the research process was introduced in a medicinal chemistry experimental course to assist undergraduates in understanding the core of medicinal chemistry and building capabilities for independently carrying out a medicinal chemistry project. And the teaching practice experiences were summed up to provide suggestions for the development of exploratory experimental course.

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

Transcription Factor GLI1 Induces IL-6-Mediated Inflammatory Response and Facilitates the Progression of Adamantinomatous Craniopharyngioma.

Adamantinomatous craniopharyngioma (ACP) is a neuroendocrine tumor whose pathogenesis remains unclear. This study investigated the role of glioma-associated oncogene family zinc finger 1 (GLI1), a transcription factor in the sonic hedgehog (SHH) signaling pathway, in ACP. We discovered that GLI1 regulates the expression of IL-6, thereby triggering inflammatory responses in ACP and influencing the tumor's progression. Analyzing the Gene Expression Omnibus (GEO) database chip GSE68015, we found that GLI1 is overexpressed in ACP, correlating positively with the spite of ACP and inflammation markers. Knockdown of GLI1 significantly inhibited the levels of tumor necrosis factor α, interleukin-6 (IL-6), and IL-1β in ACP cells, as well as cell proliferation and migration. We further identified a binding site between GLI1 and the promoter region of IL-6, demonstrating that GLI1 can enhance the expression of IL-6. These findings were verified in vivo, where activation of the SHH pathway significantly promoted GLI1 and IL-6 expressions in nude mice, inducing inflammation and tumor growth. Conversely, GLI1 knockdown markedly suppressed these processes. Our study uncovers a potential molecular mechanism for the occurrence of inflammatory responses and tumor progression in ACP.