GLI1 facilitates collagen-induced arthritis in mice by collaborative regulation of DNA methyltransferases.

Rheumatoid arthritis (RA) is characterized by joint synovitis and bone destruction, the etiology of which remains to be explored. Many types of cells are involved in the progression of RA joint inflammation, among which the overactivation of M1 macrophages and osteoclasts has been thought to be an essential cause of joint inflammation and bone destruction. Glioma-associated oncogene homolog 1 (GLI1) has been revealed to be closely linked to bone metabolism. In this study, GLI1 expression in the synovial tissue of RA patients was positively correlated with RA-related scores and was highly expressed in collagen-induced arthritis (CIA) mouse articular macrophage-like cells. The decreased expression and inhibition of nuclear transfer of GLI1 downregulated macrophage M1 polarization and osteoclast activation, the effect of which was achieved by modulation of DNA methyltransferases (DNMTs) via transcriptional regulation and protein interactions. By pharmacological inhibition of GLI1, the proportion of proinflammatory macrophages and the number of osteoclasts were significantly reduced, and the joint inflammatory response and bone destruction in CIA mice were alleviated. This study clarified the mechanism of GLI1 in macrophage phenotypic changes and activation of osteoclasts, suggesting potential applications of GLI1 inhibitors in the clinical treatment of RA.

["Huayu Tongluo" moxibustion improves learning-memory ability and promotes myelin regeneration by regulating Sonic Hedgehog signaling pathway in vascular dementia rats].

OBJECTIVE: To investigate the effect of "Huayu Tongluo" (resolving blood stagnation to dredge meridian-collaterals) moxibustion on remyelination and Sonic Hedgehog (Shh) signaling pathway in the corpus callosum of vascular dementia (VD) rats, so as to explore its mechanisms underlying improvement of VD. METHODS: Male Wistar rats were randomized into sham-operation, model, medication and moxibustion groups, with 12 rats in each group.The VD model was established by bilateral common carotid artery occlusion. Moxibustion was applied to "Shenting"(GV24), "Baihui"(GV20) and "Dazhui"(GV14) for 20 min once a day, 7 d as a treatment course, for 3 courses, with one day's rest between every two courses. Rats of the medication group were treated by gavage of 10 mg/kg of chloromastine solution once a day, and the course of treatment was the same as that of the moxibustion group. The rat's learning-memory ability was assessed by Morris water maze test (escape latency). The neurological deficits were evaluated by using Longa's scale.The mRNA and protein expressions of Shh and Gli1 in the corpus callosum were measured by quantitative real-time fluorescence PCR and Western blot, separately. The ultrastructure of the myelin sheath and myelinated axons was observed under transmission electron microscopy (TCM). RESULTS: Compared with the sham-operation group, the neurologic score and escape latency were significantly increased and prolonged (P<0.01), and the mRNA and protein expression levels of Shh and Gli1 and the number of myelinated axons were obviously decreased in the model group (P<0.01). In comparison with the model group, the escape latency was apparently shortened (P<0.05), while the mRNA and protein expression levels of Shh and Gli1 as well as the number of myelinated axons were strikingly increased in both moxibustion and medication groups (P<0.01). Results of TCM showed that in the model group, the arrangement of myelin coil structures was sparse and fuzzy, and some structures were bulged and disbanded. The oligodendrocytes were irregular, and the number of myelin sheath was rare. These situations were relatively milder in both moxibustion and medication groups. CONCLUSION: "Huayu Tongluo" moxibustion can promote the differentiation and maturation of oligodendrocyte precursor cells after cerebral ischemia by regulating the expressions of Shh and Gli1 in Shh signaling pathway, thus promoting the regeneration of cerebral white matter myelin sheaths in VD rats, which may contribute to improving learning-memory ability.

SAG-dihydrochloride enhanced the expression of germ cell markers in the human bone marrow- mesenchymal stem cells (BM-MSCs) through the activation of GLI-independent hedgehog signaling pathway.

Different studies indicated that the enhancing the expression of germ cell markers improved the efficiency of stem cells in the generation of germ line cells. The aim of the present study was to investigate the effect of SAG-dihydrochloride on the expression of germ cell markers in the human bone marrow-mesenchymal stem cells (BM-MSCs). For this purpose, the human BM-MSCs were cultured in the medium containing different concentrations of SAG-dihydrochloride (10, 20 and 30 µM). After RNA extraction and cDNA synthesis, the expression level of PTCH1, GLI1, PLZF, DDX4 and STRA8 genes were determined by using SYBR Green Real time PCR. The analysis of the results obtained from PTCH1 and GLI1 expression indicated that SAG-dihydrochloride had the ability to enhance the expression of germ cell markers in a Gli-independent manner. Furthermore, the significant increased expression of STRA8 was observed in the BM-MSCs treated by 10 µM SAG-dihydrochloride for 4 and 6 days (p < 0.05). There was also the up-regulation of DDX4 in the BM-MSCs following treatment with 20 µM SAG-dihydrochloride for 4 and 6 days. The obtained results suggested that treatment with SAG-dihydrochloride increased the expression of germ cell markers in the human BM-MSCs through the activation of non-canonical sonic hedgehog signaling pathway.

Ursolic acid inhibits the activation of smoothened-independent non-canonical hedgehog pathway in colorectal cancer by suppressing AKT signaling cascade.

It is being brought to light that smoothened (SMO)-independent non-canonical Hedgehog signaling is associated with the pathogenesis of various cancers. Ursolic acid (UA), a pentacyclic triterpenoid present in many medicinal herbs, manifests potent effectiveness against multiple malignancies including colorectal cancer (CRC). In our previous study, UA was found to protect against CRC in vitro by suppression of canonical Hedgehog signaling cascade. Here, the influence of UA on SMO-independent non-canonical Hedgehog signaling in CRC was investigated in the present study, which demonstrated that UA hampered the proliferation and migration, induced the apoptosis of HCT-116hSMO- cells with SMO gene knockdown, accompanied by the augmented expression of the suppressor of fused (SUFU), and lessened levels of MYC (c-Myc), glioma-associated oncogene (GLI1) and Sonic Hedgehog (SHH), and lowered phosphorylation of protein kinase B (PKB, AKT), suggesting that UA diminished non-canonical Hedgehog signal transduction in CRC. In HCT-116hSMO- xenograft tumor, UA ameliorated the symptoms, impeded the growth and caused the apoptosis of CRC, with heightened SUFU expression, and abated levels of MYC, GLI1, and SHH, and mitigated phosphorylation of AKT, indicating that UA down-regulated non-canonical Hedgehog signaling cascade in CRC. Taken together, UA may alleviate CRC by suppressing AKT signaling-dependent activation of SMO-independent non-canonical Hedgehog pathway.

ISL1 Promotes Human Glioblastoma-Derived Stem Cells' Self-Renewal by Activation of Sonic Hedgehog/GLI1 Function.

Glioblastoma (GBM), the most aggressive primary heterogeneous primary brain tumor, is a glioma subtype that originates from the glial cells of the central nervous system. Glioblastoma stem cells (GSCs), situated at the top of the hierarchy, initiate and maintain the tumor and are largely accountable for GBM resistance to the mainstay treatment and recurrence. The LIM homeobox transcription factor islet 1 (ISL1) induces tumorigenicity in various tumors; however, its function in GSCs has been less reported. We aimed to generate GSCs from surgical specimens of human GBM and investigate the effect of ISL1 knockdown on GSCs. We established patient-derived GSCs, determined cancer stem cell marker expression, and immunostained GSCs to assess cell viability and apoptosis. We demonstrated that ISL1 deletion decreased the GSC viability and proliferation, and upregulated apoptosis. Moreover, we performed enzyme-linked immunosorbent assay and western blotting and found that ISL1 knockdown affected the expression of sonic hedgehog (SHH) and its downstream regulator GLI1, and further validated these results by supplementing the cells with recombinant SHH. Our results suggested that ISL1 played a critical role in regulating GBM growth and that an ISL1/SHH/GLI1 pathway was required for the maintenance of GBM progression and malignancy. The regulation of GSC growth through ISL1 might be a mechanism of interest for future therapeutic studies.

Saikosaponin B1 and Saikosaponin D inhibit tumor growth in medulloblastoma allograft mice via inhibiting the Hedgehog signaling pathway.

Medulloblastoma (MB), accounting for nearly 10% of all childhood brain tumors, are implicated with aberrant activation of the Hedgehog (Hh) signaling pathway. Saikosaponin B1 (SSB1) and Saikosaponin D (SSD), two bioactive constituents of Radix Bupleuri, are reported to have many biological activities including anticancer activities. In our work, we evaluated the inhibition of SSB1 and SSD on MB tumor growth in allograft mice and explored the underlying mechanisms. The associated biological activity was investigated in Shh Light II cells, an Hh-responsive fibroblast cell line, using the Dual-Glo® Luciferase Assay System. First, SSB1 (IC50, 241.8 nM) and SSD (IC50, 168.7 nM) inhibited GLI-luciferase activity in Shh Light II cells stimulated with ShhN CM, as well as Gli1 and Ptch1 mRNA expression. In addition, both compounds suppressed the Hh signaling activity provoked by smoothened agonist (SAG) or excessive Smoothened (SMO) expression. Meanwhile, SSB1 and SSD did not inhibit glioma-associated oncogene homolog (GLI) luciferase activity activated by abnormal expression of downstream molecules, suppressor of fuse (SUFU) knockdown or GLI2 overexpression. Consequently, SSB1 (30 mg/kg, ip) and SSD (10 mg/kg, ip) displayed excellent in vivo inhibitory activity in MB allografts, and the tumor growth inhibition ratios were approximately 50% and 70%, respectively. Our findings, thus, identify SSB1 and SSD significantly inhibit tumor growth in MB models by inhibiting the Hedgehog pathway through targeting SMO.

Curcumin inhibits the invasion and metastasis of triple negative breast cancer via Hedgehog/Gli1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, there is a long history that curcuma longa L is used to treat distending pain of chest and belly, arthralgia of shoulder and arm aggravated by cold. Traditional Chinese medicine holds that breast cancer is caused by cold congelation, stagnation of qi and blood stasis. It is usually treated with some pungent and warm Chinese herbs, such as Curcuma longaL and Curcuma zedoaria (Christm.) Rosc, which are effective in promoting blood circulation for removing blood stasis, activating qi-flowing and relieving pain. Curcumin, a polyphenolic compound, is the main pharmacological component extracted from the rhizome of Curcuma longa L. Modern pharmacological studies have found that curcumin has many kinds of pharmacological activities of anti-inflammatory, anti-tumor, anti-angiogenesis, anti-metastasis and anti-multidrug resistance. AIM OF THE STUDY: To explore the mechanism of curcumin and Glioma-associated oncogene homolod-1 (Gli1) on invasion and metastasis of triple negative breast cancer (TNBC) cells through the Hedgehog (Hh)/Gli signaling pathway. MATERIAL AND METHODS: The effect of curcumin on TNBC cells was detected by colony formation, wound healing and transwell assay. Breast cancer stem cells (BCSCs) were cultured in serum-free medium and its stemness was detected by flow cytometry and subcutaneous xenografted tumor assay. The formation of mammospheres was used to detect the effect of curcumin and GANT61 (Gli inhibitor)on the formation ability of BCSCs. Gli1 overexpressed was conducted in MDA-MB-231 cells by lentivirus vector HBLV-h-Gli1-3xflag-ZsGreen-PURO. RT-qPCR and Western blot were detected the mRNA and protein level of genes of Hh pathway, Epithelial-mesenchymal transition (EMT) and stemness. The nuclear localization and expression of Gli1 was observed by laser confocal microscope scanning. Co-IP was investigated the key genes interacted with Gli1. RESULTS: The abilities of proliferation, invasion, migration and the formation of mammospheres in TNBC cells were inhibited by curcumin. Furthermore, curcumin reduced the invasion and migration abilities in stable Gli1-overexpressing MDA-MB-231 cell. Moreover, curcumin down-regulated the expression of genes related Hh pathway, EMT and stemness in MDA-MB-231 mammospheres. Observation of laser confocal microscope showed that Gli1 were expressed mainly in nucleus in MDA-MB-231 adherent cells and completely in nucleus in BCSCs, which was significantly reduced in the nucleus and cytoplasm after curcumin treatment. Besides, our results suggested that vimentin was interacted with Gli1. CONCLUSIONS: Curcumin can inhibit the proliferation and metastasis of TNBC cells, EMT and characteristics of BCSC by Hedgehog/Gli1 pathway.

Berberine inhibits chemotherapy-exacerbated ovarian cancer stem cell-like characteristics and metastasis through GLI1.

Despite the remarkable clinical response in ovarian cancer therapy, the distinctively high metastasis rate is still a barrier to achieve satisfying prognosis. Our study aimed to decipher the role of berberine in inhibiting chemotherapy-exacerbated ovarian cancer metastasis. We found that chemotherapy exacerbated the migration and cancer stem cell (CSC)-like characteristics through transcriptional factor GLI1, which regulated the pluripotency-associated gene BMI1 and the epithelial-mesenchymal transition (EMT) markers Vimentin and Snail. Berberine could not only down-regulate CSC-like characteristics but also reverse EMT and migration through inhibiting chemotherapy-activated GLI1/BMI1 signaling pathway. Together, our study revealed the pivotal role of berberine in overcoming chemotherapy-exacerbated ovarian cancer metastasis, thereby provided a potential adjuvant therapeutic agent in combination with chemotherapeutics to prevent metastasis during ovarian cancer chemotherapy.

Clinical Significance of Glioma-associated Oncogene 1 Expression in Patients With Locally Advanced Gastric Cancer Administered Adjuvant Chemotherapy With S-1 After Curative Surgery.

BACKGROUND/AIM: Glioma-associated oncogene 1 (GLI1) is an important transcription factor in the hedgehog signalling pathway and tumour formation. We evaluated the clinical significance of GLI1 expression as a prognostic factor in patients with locally advanced gastric cancer (GC). PATIENTS AND METHODS: GLI1 expression levels were measured by quantitative real-time polymerase chain reaction analysis of cancerous and adjacent normal mucosa specimens obtained from 142 patients with Stage II/III GC administered adjuvant chemotherapy with S-1 after curative resection. The associations of GLI1 expression with clinicopathological features and survival were evaluated. RESULTS: Clinicopathological features and GLI1 expression showed no association. Overall survival was significantly poorer in the high compared to the low GLI1 expression group (p=0.04). Multivariate analysis revealed that GLI1 expression was a significant independent prognostic factor [p=0.019, hazard ratio (HR)=1.94, 95% confidence interval (CI)=1.70-3.38]. CONCLUSION: GLI1 expression may be a useful prognostic marker in patients with locally advanced GC.

Garcinone C suppresses colon tumorigenesis through the Gli1-dependent hedgehog signaling pathway.

BACKGROUND: Although garcinone C, a natural xanthone derivative identified in the pericarp of Garcinia mangostana, has been demonstrated to exert different health beneficial activities in oxidative stress and ß-amyloid aggregation, the role of garcinone C in colon tumorigenesis has not been investigated. In addition, aberrant Hedgehog (Hh) signaling activation is associated with tumorigenesis including colon cancer. Here, we hypothesized that garcinone C can prevent colon tumorigenesis through regulating the Hh signaling pathway. METHOD: Colony formation assay and flow cytometry were used to evaluate the effect of garcinone C on the proliferation and cell cycle progression of colon cancer cells. Protein expression of cell cycle related markers and Hh/Gli1 signaling mediators were determined. The regulatory effect of orally administered garcinone C on the Hh/Gli1 signaling pathway and colon tumorigenesis was evaluated in an azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colon cancer animal model. RESULTS: Garcinone C suppressed the proliferation of colon cancer cells, induced G0/G1 cell cycle arrest, as well as regulated the expression of cell cycle-related markers such as cyclin D1, cyclin E, CDK6, and p21. Garcinone C inhibited the expression of Gli1, a key mediator of Hedgehog signaling, and protein kinase B (AKT) phosphorylation in Smo-independent colon cancer cells. In the AOM/DSS-induced colon tumorigenesis model, garcinone C significantly inhibited tumor development, regulated the expression of cell cycle markers and Gli1, and reduced AKT phosphorylation in colon tumor tissues, which is consistent with our in vitro results. CONCLUSION: Garcinone C can suppress colon tumorigenesis in vitro and in vivo through Gli1-dependent non-canonical Hedgehog signaling, suggesting that it may serve as a potent chemopreventive agent against colon tumorigenesis.

Prognostic and predictive value of a five-molecule panel in resected pancreatic ductal adenocarcinoma: A multicentre study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a devastating prognosis. The performance of clinicopathologic parameters and molecules as prognostic factors remains limited and inconsistent. The present study aimed to construct a multi-molecule biomarker panel to more accurately predict post-resectional prognosis of PDAC patients. METHODS: Firstly, a novel computational strategy integrating prognostic evidence from omics and literature on the basis of bioinformatics prediction (CIPHER) to generate the network, was designed to systematically identify potential high-confidence PDAC-related prognostic candidates. After specimens from 605 resected PDAC patients were retrospectively collected, 23 candidates were detected immunohistochemically in tissue-microarrays for the development cohort to construct a multi-molecule panel. Lastly, the panel was validated in two independent cohorts. FINDINGS: According to the constructed five-molecule panel, disease-specific survival (DSS) was significantly poorer in high-risk patients than in low-risk ones in development cohort (HR 2.15, 95%CI 1.51-3.05, P<0.0001; AUC 0.67). In two validation cohorts, similar significant differences between the two groups were also observed (HR 3.18 and 3.31, 95%CI 1.89-5.37 and 1.78-6.16, All P<0.0001; AUC 0.72 and 0.73). In multivariate analyses, this panel was the sole prognosticator that was significant in each cohort. Furthermore, its predictive power for long-term survival, higher than its individual constituents, could be largely enhanced by combination with traditional clinicopathological variables. Finally, adjuvant chemotherapy (ACT) correlated with better DSS only in high-risk patients, uni- and multi-variately, in all the cohorts. INTERPRETATION: The novel prognostic panel developed by a systematically network-based strategy presents strong ability in prediction of post-resectional survival of PDAC patients. Furthermore, panel-defined high-risk patients might benefit more from ACT.

Heparanase promotes myeloma stemness and in vivo tumorigenesis.

Heparanase is known to enhance the progression of many cancer types and is associated with poor patient prognosis. We recently reported that after patients with multiple myeloma were treated with high dose chemotherapy, the tumor cells that emerged upon relapse expressed a much higher level of heparanase than was present prior to therapy. Because tumor cells having stemness properties are thought to seed tumor relapse, we investigated whether heparanase had a role in promoting myeloma stemness. When plated at low density and grown in serum-free conditions that support survival and expansion of stem-like cells, myeloma cells expressing a low level of heparanase formed tumor spheroids poorly. In contrast, cells expressing a high level of heparanase formed significantly more and larger spheroids than did the heparanase low cells. Importantly, heparanase-low expressing cells exhibited plasticity and were induced to exhibit stemness properties when exposed to recombinant heparanase or to exosomes that contained a high level of heparanase cargo. The spheroid-forming heparanase-high cells had elevated expression of GLI1, SOX2 and ALDH1A1, three genes known to be associated with myeloma stemness. Inhibitors that block the heparan sulfate degrading activity of heparanase significantly diminished spheroid formation and expression of stemness genes implying a direct role of the enzyme in regulating stemness. Blocking the NF-κB pathway inhibited spheroid formation and expression of stemness genes demonstrating a role for NF-κB in heparanase-mediated stemness. Myeloma cells made deficient in heparanase exhibited decreased stemness properties in vitro and when injected into mice they formed tumors poorly compared to the robust tumorigenic capacity of cells expressing higher levels of heparanase. These studies reveal for the first time a role for heparanase in promoting cancer stemness and provide new insight into its function in driving tumor progression and its association with poor prognosis in cancer patients.

TKP, a serine protease extracted from Trichosanthes kirilowii, inhibits the migration and invasion of colorectal adenocarcinoma cells by targeting Wnt/ß-catenin and Hedgehog/Gli1 signalings.

Trichosanthes kirilowii, which is a type of Liana from cucurbitaceous family, possesses many bioactive constituents and therefore has multifarious pharmacological functions. TKP, which is a serine protease extracted from the fruit of Trichosanthes kirilowii, has been reported to possess potential anticancer activity. However, the effects of TKP on cancer cell migration and invasion are still unknown. Here, we reported that TKP could inhibit the migration and invasion abilities of colorectal cancer cells. In addition, the mRNA, protein expression levels, and activities of migration and invasion-related proteins MMP2 and MMP9 were decreased in TKP-treated cells. Mechanistically, TKP treatment repressed Wnt/ß-catenin and Hedgehog/Gli1 signaling cascades. However, the addition of lithium chloride or the transfection of plasmid pcDNA3.1-V5-HisA-Gli1 reversed the impacts of TKP on MMP2, MMP9, cell migration, and invasion. These results indicated that TKP suppressed the migration and invasion of colorectal cancer cells through blocking Wnt/ß-catenin and Hedgehog/Gli1 pathways-mediated MMP2 and MMP9.

Icariin-induced miR-875-5p attenuates epithelial-mesenchymal transition by targeting hedgehog signaling in liver fibrosis.

BACKGROUND: Hepatic fibrosis is the final endpoint for most chronic liver diseases and remains a significant public health problem worldwide. Icariin, a naturally occurring flavonol glucoside, has been reported to exhibit protective effects on liver injury and alleviate liver fibrosis. However, the underlying detail molecular mechanism is not fully revealed. METHODS: Mouse primary hepatic stellate cells (HSCs) and carbon tetrachloride (CCL4 )-induced liver fibrosis model in mice were used as in vitro and in vivo models in this study. The expression levels of miR-875-5p were detected by quantitative reverse transcription-PCR. The validation of the direct target of miR-875-5p was through dual-luciferase reporter assay and western blotting assay. The cell proliferation and cell mobility were determined using MTT assay and Transwell migration assay, respectively. RESULTS: We found that icariin inhibited epithelial-mesenchymal transition and collagen protein section of HSCs. Icariin exerted hepatoprotective effects on mice model of CCL4 -induced liver fibrosis. Our further results revealed that miR-875-5p was downregulated in human cirrhosis tissues and activated murine HSCs. Icariin induced miR-875-5p upregulation and subsequently decreased glioma-associated oncogene homolog 1 (GLI1) expression through direct binding to the three prime untranslated region of GLI1 mRNA. CONCLUSION: Our study highlighted the potential therapeutic application of icariin for liver fibrosis management.

Hesperetin derivative attenuates CCl4-induced hepatic fibrosis and inflammation by Gli-1-dependent mechanisms.

Hepatic fibrosis, a common pathological feature and leading cause of various chronic liver diseases, still lacks effective therapy. Hesperetin derivative (HD) is a derivative of Traditional Chinese Medicine monomer isolated from the fruit peel of Citrusaurantium L. (Rutaceae). In the present study, we revealed the anti-fibrotic effects of HD in CCl4-induced mouse hepatic fibrosis model and in TGF-ß1-activated LX-2 cells, in vivo and in vitro. Results showed that HD prevented CCl4-induced liver injury and histological damage. Consistently, HD inhibited the up-regulation of liver fibrogenesis markers α-SMA, Col1α1, Col3α1 and TIMP-1 in primary hepatic stellate cells (HSCs) and suppressed inflammatory responses in primary liver macrophages from hepatic fibrosis mice. Furthermore, HD promoted the apoptosis of activated HSCs, a key step in the onset of fibrosis regression. Mechanistically, the Hedgehog pathway was involved in HD-treated hepatic fibrosis, and HD specifically contributed to attenuate the aberrant expression of Glioma associated oncogene-1 (Gli-1). Interestingly, blockade of Gli-1 removed the inhibitory effect of HD on activated HSCs, indicating that Gli-1 may play a pivotal role in mediating the anti-fibrotic effect of HD in hepatic fibrosis. Collectively, our results suggest that HD may be a potential anti-fibrotic Traditional Chinese Medicine monomer for the treatment of hepatic fibrosis.

In vivo clonal analysis reveals spatiotemporal regulation of thalamic nucleogenesis.

The thalamus, a crucial regulator of cortical functions, is composed of many nuclei arranged in a spatially complex pattern. Thalamic neurogenesis occurs over a short period during mammalian embryonic development. These features have hampered the effort to understand how regionalization, cell divisions, and fate specification are coordinated and produce a wide array of nuclei that exhibit distinct patterns of gene expression and functions. Here, we performed in vivo clonal analysis to track the divisions of individual progenitor cells and spatial allocation of their progeny in the developing mouse thalamus. Quantitative analysis of clone compositions revealed evidence for sequential generation of distinct sets of thalamic nuclei based on the location of the founder progenitor cells. Furthermore, we identified intermediate progenitor cells that produced neurons populating more than one thalamic nuclei, indicating a prolonged specification of nuclear fate. Our study reveals an organizational principle that governs the spatial and temporal progression of cell divisions and fate specification and provides a framework for studying cellular heterogeneity and connectivity in the mammalian thalamus.

miR-132 weakens proliferation and invasion of glioma cells via the inhibition of Gli1.

OBJECTIVE: Transcriptional factor Gli1 in Hedgehog signal pathway facilitates epithelial mesenchymal transition (EMT) and is associated with invasion or proliferation of multiple tumor cells. The previous study showed the correlation between miR-132 down-regulation and glioma pathogenesis. We investigated the role of miR-132 in mediating Gli1 expression and in affecting proliferation or invasion of glioma cells. PATIENTS AND METHODS: Dual luciferase reporter gene assay was used to confirm the targeted regulation between miR-132 and Gli1. Tumor tissues at different pathological grades (grade II, III and IV) were collected from glioma patients, in parallel with brain tissues from contusion surgery. The expression of miR-132 and Gli1 was measured by RT-PCR. Glioma cell line U251 was treated with miR-132 or si-Gli1 followed by measuring the expression of Gli1, E-cadherin, Vimentin and Cyclin D1. In addition, flow cytometry and transwell assay were performed to evaluate cell invasion potency. RESULTS: Bioinformatics analysis showed the complementary binding sites between miR-132 and 3'-UTR of Gli1 mRNA. Transfection of miR-132 mimic significantly reduced luciferase activity, indicating the targeted regulatory relationship between miR-132 and Gli1 mRNA. Compared with control group, miR-132 expression was decreased and Gli1 level was elevated in glioma tissues, both of which were correlated with the pathological grade. Transfection of miR-132 mimic or si-Gli1 remarkably suppressed the expression of Gli1, Vimentin or Cyclin D1 in U251 cells, up-regulated E-cadherin expression, suppressed cell proliferation and invasion. CONCLUSIONS: Our data indicated that over-expression of miR-132 could inhibit proliferation or invasion of glioma cells via targeted inhibition of Gli1 expression.

Combined inhibition of atypical PKC and histone deacetylase 1 is cooperative in basal cell carcinoma treatment.

Advanced basal cell carcinomas (BCCs) circumvent Smoothened (SMO) inhibition by activating GLI transcription factors to sustain the high levels of Hedgehog (HH) signaling required for their survival. Unfortunately, there is a lack of efficacious therapies. We performed a gene expression-based drug repositioning screen in silico and identified the FDA-approved histone deacetylase (HDAC) inhibitor, vorinostat, as a top therapeutic candidate. We show that vorinostat only inhibits proliferation of BCC cells in vitro and BCC allografts in vivo at high dose, limiting its usefulness as a monotherapy. We leveraged this in silico approach to identify drug combinations that increase the therapeutic window of vorinostat and identified atypical PKC Ɩ/ʎ (aPKC) as a HDAC costimulator of HH signaling. We found that aPKC promotes GLI1-HDAC1 association in vitro, linking two positive feedback loops. Combination targeting of HDAC1 and aPKC robustly inhibited GLI1, lowering drug doses needed in vitro, in vivo, and ex vivo in patient-derived BCC explants. We identified a bioavailable and selective small-molecule aPKC inhibitor, bringing the pharmacological blockade of aPKC and HDAC1 into the realm of clinical possibility. Our findings provide a compelling rationale and candidate drugs for combined targeting of HDAC1 and aPKC in HH-dependent cancers.

MiR-150 alleviates EMT and cell invasion of colorectal cancer through targeting Gli1.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) is related to colorectal cancer invasion and metastasis. Glioma-associated oncogene homolog 1 (Gli1) abnormal expression is associated with EMT, invasion, and metastasis in various cancers. MiR-150 is found downregulated in colorectal cancer pathogenesis. Bioinformatics analysis shows the complementary targeted relationship between miR-150 and the 3'-UTR of Gli1 mRNA. This study explores the role of miR-150 in regulating Gli1 expression, colorectal cancer cell EMT, and invasion. MATERIALS AND METHODS: Dual luciferase assay confirmed the targeted relationship between miR-150 and Gli1 predicted by bioinformatics analysis. MiR-150 and Gli1 expressions were compared in NCM460, SW480, and SW620 cells. Cell colony formation and invasion were tested in SW480 and SW620 cells. Anip973 and AGYZ83-a cells were treated by 10 ng/mL TGF-ß1 to detect miR-150 and Gli1 expressions. SW620 cells were cultured in vitro and divided into five groups, including miR-NC, miR-150 mimic, si-NC, si-Gli1, and miR-150 mimic + si-Gli1 groups. RESULTS: MiR-150 specifically inhibited Gli1 expression. The level of miR-150 was significantly downregulated, while Gli1 was elevated in SW480 and SW620 cells compared with that in NCM460 cells. SW620 exhibited markedly stronger invasive and colony formation abilities than SW480. The level of miR-150 was apparently reduced, whereas Gli1 was increased in SW620 than that in SW480 cells after the treatment of TGFß1. MiR-150 mimic and/or si-Gli1 transfection markedly reduced Gli1 and Snail levels, upregulated E-cadherin expression, and attenuated cell colony formation and invasion. CONCLUSIONS: Downregulation of miR-150 and elevation of Gli1 promote the development and invasion of colorectal cancer cell EMT. MiR-150 attenuated the progression of colorectal cancer cell EMT via inhibiting Gli1.

Targeting of miR-150 on Gli1 gene to inhibit proliferation and cell cycle of esophageal carcinoma EC9706.

OBJECTIVE: Glioma-associated oncogene homolog 1 (Gli1) in Hedgehog signal pathway regulates Cyclin D1 expression, cell cycle or proliferation modulation. Esophageal cancer patients had significantly elevated Gli1 expression, which is related with survival and prognosis. It has been demonstrated that the level of miR-150 was decreased in esophageal cancer patients compared to normal control. As a complementary relationship exists between miR-150 and 3'-UTR of Gli1, this study investigated if miR-150 played a role in regulating Gli1 expression, and proliferation or cell cycle of esophageal cancer cells. PATIENTS AND METHODS: Esophageal squamous cell carcinoma (ESCC) patients from our hospital were recruited to collect tumor and adjacent tissues for miR-150 and Gli1 expression. Esophageal carcinoma cell line EC9706 and normal esophageal epithelial cell line HEEC were compared for expression of miR-150, Gli1 and Cyclin D1. Dual luciferase reporter gene assay examined the targeted relationship between miR-150 and 3'-UTR of Gli1. In vitro cultured EC9706 cells were treated with miR-150 mimic, si-Gli1 or the combination of miR-150 mimic and si-Gli1, respectively, to check their gene expression, cell cycle and proliferation. RESULTS: ESCC tissues had significantly higher Gli1 expression and lower miR-150 expression. EC9706 cell also had higher Gli1 expression than that in HEEC, whilst miR-150 was down-regulated. Via targeting 3'-UTR of Gli1 gene, miR-150 inhibited its expression. Transfection of miR-150 mimic, si-Gli1 or the combination of miR-150 mimic and si-Gli1, respectively, remarkably decreased expression of Gli1 and Cyclin D1 expression in EC9706 cells, whose cell cycle arresting at G0/G1 phase was enhanced with weakened proliferation. CONCLUSIONS: MiR-150 can induce G0/G1 cell cycle arresting and weaken proliferation of esophageal carcinoma cells via targeted inhibition on Gli1 and downstream expression of Cyclin D1.