Divergent chondro/osteogenic transduction laws of fibrocartilage stem cell drive temporomandibular joint osteoarthritis in growing mice / 国际口腔科学杂志·英文版

The anterior disc displacement (ADD) leads to temporomandibular joint osteoarthritis (TMJOA) and mandibular growth retardation in adolescents. To investigate the potential functional role of fibrocartilage stem cells (FCSCs) during the process, a surgical ADD-TMJOA mouse model was established. From 1 week after model generation, ADD mice exhibited aggravated mandibular growth retardation with osteoarthritis (OA)-like joint cartilage degeneration, manifesting with impaired chondrogenic differentiation and loss of subchondral bone homeostasis. Lineage tracing using Gli1-CreER+; Tmfl/-mice and Sox9-CreER+;Tmfl/-mice showed that ADD interfered with the chondrogenic capacity of Gli1+ FCSCs as well as osteogenic differentiation of Sox9+ lineage, mainly in the middle zone of TMJ cartilage. Then, a surgically induced disc reposition (DR) mouse model was generated. The inhibited FCSCs capacity was significantly alleviated by DR treatment in ADD mice. And both the ADD mice and adolescent ADD patients had significantly relieved OA phenotype and improved condylar growth after DR treatment. In conclusion, ADD-TMJOA leads to impaired chondrogenic progenitor capacity and osteogenesis differentiation of FCSCs lineage, resulting in cartilage degeneration and loss of subchondral bone homeostasis, finally causing TMJ growth retardation. DR at an early stage could significantly alleviate cartilage degeneration and restore TMJ cartilage growth potential.

NCTD Retards AML HL60 Cell Proliferation via Targeting Hedgehog/SOX2 axis / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of norcantharidin (NCTD) to proliferation of leukemia cells through disrupting key regulators of sonic Hedgehog (SHH) pathway and its downstream transcription factor SOX2.@*METHODS@#CCK8 was used to detected the HL60 and NB4 cells after inhibited by NCTD, SMO and GLI1 inhibitor for 24 hours. Expression level of SMO, GLI1 and SOX2 in HL60 cells with NCTD treatment was detected by immunoblot. HL60 cells were transfected with pcDNA3.1 plasmid expressing GLI1 or SOX2. Empty vector and pcDNA3. 1-EGFP were divided into negative and positive control group, respectively. The expression of exogenous GLI1 or SOX2 in HL60 cells was confirmed by immunoblot, and growth curve of HL60 cell was checked by CCK8. Proliferation of genetic modified HL60 cells treated by various dose of NCTD was detected.@*RESULTS@#NCTD, SMO/GLI1 inhibitors could inhibit the proliferation of NB4 and HL60 cells in a dose-dependent manner. Compared with solvent (DMSO)-treated control group, NCTD remarkably decreased protein level of SMO, GLI1 and SOX2. GLI1 and SOX2 were overexpressed in HL60 cells as compared with pcDNA3.1 empty vector-transfected group. Growth curve demonstrated significant proliferative advantage of GLI1/SOX2-transfected cells. CCK8 assay indicated that GLI1/SOX2-overexpressed HL60 cells were more resistant to NCTD treatment.@*CONCLUSION@#NCTD attenuates HL60 proliferation via targeting the Hedgehog/SOX2 axis.

Integrative molecular characterization of Chinese prostate cancer specimens / 亚洲男科学杂志(英文版)

Prostate cancer (PCa) exhibits epidemiological and molecular heterogeneity. Despite extensive studies of its phenotypic and genetic properties in Western populations, its molecular basis is not clear in Chinese patients. To determine critical molecular characteristics and explore correlations between genomic markers and clinical parameters in Chinese populations, we applied an integrative genetic/transcriptomic assay that combines targeted next-generation sequencing and quantitative real-time PCR (qRT-PCR) on samples from 46 Chinese patients with PCa. Lysine (K)-specific methyltransferase 2D (KMT2D), zinc finger homeobox 3 (ZFHX3), A-kinase anchoring protein 9 (AKAP9), and GLI family zinc finger 1 (GLI1) were frequently mutated in our cohort. Moreover, a clinicopathological analysis showed that RB transcriptional corepressor 1 (RB1) deletion was common in patients with a high risk of disease progression. Remarkably, four genomic events, MYC proto-oncogene (MYC) amplification, RB1 deletion, APC regulator of WNT signaling pathway (APC) mutation or deletion, and cyclin-dependent kinase 12 (CDK12) mutation, were correlated with poor disease-free survival. In addition, a close link between KMT2D expression and the androgen receptor (AR) signaling pathway was observed both in our cohort and in The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data. In summary, our results demonstrate the feasibility and benefits of integrative molecular characterization of PCa samples in disease pathology research and personalized medicine.

Expression Level and Clinical Significance of Gli1 in Patients with Myelodysplastic Syndrome / 中国实验血液学杂志

OBJECTIVE@#To study the expression level and clinical significance of Gli1 gene in patients with myelodysplastic syndrome(MDS).@*METHODS@#The positive rate of bone marrow CD34 cells was detected by flow cytometry in 53 patients with MDS.Magnetic beads were used to separate CD34 cells. The expression of Gli1 on CD34 cells was detected by RT-qPCR, 25 patients with iron deficiency anemia were selected as controls. The relationship of Gli1 expression with clinical characteristics were analyzed.@*RESULTS@#The expression of Gli1 in patients with MDS (0.73±1.26) was significantly higher than that in the control group (0.07±0.46) (P＜0.05). The expression of Gli1 significantly correlated with platelet count, chromosome grouping and IPSS risk stratification (P＜0.05). The median overall survival time of patients in high and low expression groups were 7 and 20 months respectively (P＜0.05). Multivariate analysis showed that Gli1 and chromosome grouping were 2 independent poor prognostic factors (P＜0.05).@*CONCLUSION@#The expression of Gli1 is high in MDS. Abnormal expression of Gli1 positively correlates with clinical characteristics and prognosis of patients.Gli1 may be involved in the occurrence and development of MDS.

Muscarinic acetylcholine receptor M1 mediates prostate cancer cell migration and invasion through hedgehog signaling / 亚洲男科学杂志(英文版)

The autonomic nervous system contributes to prostate cancer proliferation and metastasis. However, the exact molecular mechanism remains unclear. In this study, muscarinic acetylcholine receptor M1 (CHRM1) expression was measured via immunohistochemical analysis in human prostate cancer tissue array slides. PC-3, LNCaP, and A549 cells were treated with pirenzepine or carbachol, and the cell migration and invasion abilities were evaluated. Western blotting and quantitative real-time PCR were performed to measure GLI family zinc finger 1 (GLI1), patched 1 (PTCH1), and sonic hedgehog (SHH) expression levels. High expression of CHRM1 was found in early-stage human prostate cancer tissues. In addition, the selective CHRM1 antagonist pirenzepine inhibited PC-3, LNCaP, and A549 cell migration and invasion, but the agonist carbachol promoted the migration and invasion of these three cell lines. Muscarinic signaling can be relayed by hedgehog signaling. These data show that CHRM1 is involved in the regulation of prostate cancer migration and invasion through the hedgehog signaling pathway.

Pentoxifylline inhibits liver fibrosis via hedgehog signaling pathway / 华中科技大学学报（医学）（英德文版）

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.

Autophagy Accompanied with Bisdemethoxycurcumin-induced Apoptosis in Non-small Cell Lung Cancer Cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the effects of bisdemethoxycurcumin (BDMC) on non-small cell lung cancer (NSCLC) cell line, A549, and the highly metastatic lung cancer 95D cells.</p><p><b>METHODS</b>CCK-8 assay was used to assess the effect of BDMC on cytotoxicity. Flow cytometry was used to evaluate apoptosis. Western blot analysis, electron microscopy, and quantification of GFP-LC3 punctuates were used to test the effect of BDMC on autophagy and apoptosis of lung cancer cells.</p><p><b>RESULTS</b>BDMC inhibited the viability of NSCLC cells, but had no cytotoxic effects on lung small airway epithelial cells (SAECs). The apoptotic cell death induced by BDMC was accompanied with the induction of autophagy in NSCLC cells. Blockage of autophagy by the autophagy inhibitor 3-methyladenine (3-MA) repressed the growth inhibitory effects and induction of apoptosis by BDMC. In addition, BDMC treatment significantly decreased smoothened (SMO) and the transcription factor glioma-associated oncogene 1 (Gli1) expression. Furthermore, depletion of Gli1 by siRNA and cyclopamine (a specific SMO inhibitor) induced autophagy.</p><p><b>CONCLUSION</b>Aberrant activation of Hedgehog (Hh) signaling has been implicated in several human cancers, including lung cancers. The present findings provide direct evidence that BDMC-induced autophagy plays a pro-death role in NSCLC, in part, by inhibiting Hedgehog signaling.</p>

Expression of Gli1 in Adult Acute Lymphoblastic Leukemia and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the expression and clinical significance of Hedgehog signaling transcription factor Gli1 in acute lymphoblastic leukemia (ALL) patients.</p><p><b>METHODS</b>The clinical specimens were obtained from 32 newly diagnosed and 6 relapsed ALL patients. Normal bone marrow cells from 15 healthy donors were used as controls. Real-time qPCR and Western blot were applied to detect Gli1 mRNA and protein expression in bone marrow mononuclear cells (BMMNC) of these samples respectively. The relation of Gli1 mRNA levels with clinical parameter was also evaluated.</p><p><b>RESULTS</b>The expression level of Gli1 mRNA in de novo and relapsed ALL patients was significantly higher than that in the normal controls (P < 0.05). There was no stalistically significant difference of the Gli1 mRNA expression between de novo and relapsed ALL cases (P > 0.05). In 24 de novo ALL patients with complete remission (CR) after induction chemotherapy, the levels of Gli1 mRNA were significantly reduced as compared with levels before treatment (P < 0.05). However, in 4 ALL patients without remission, no obvious difference of Gli1 mRNA levels were observed as compared with levels of Gli1 before treatment (P > 0.05). A positive correlation between the Gli1 mRNA expression level and white blood cell count (WBC) was found in the BMMNC of ALL patients (R = 0.725, P < 0.05). Similarly, Gli1 protein expression was significantly higher in the de novo and relapsed ALL cases compared with normal controls. The Gli1 protein level was down-regulated when the ALL patients was in CR.</p><p><b>CONCLUSION</b>The expression of Gli1 mRNA and protein has been found to be high in de novo and relapsed ALL patients, and the change of Gli1 expression maybe relate to therapeutic efficacy and prognosis of ALL patients.</p>

The role of Gli1 in the invasion and migration of pancreatic cancer cells / 中华外科杂志

<p><b>OBJECTIVE</b>To study the role and possible mechanism of glioma-associated oncogene-1 (Gli1) in regulating the cell invasion and migration of pancreatic cancer cells.</p><p><b>METHODS</b>Quantitative real-time (qRT) -PCR was used to detect the effect of siRNA interference on Gli1, murine double minute 2 (MDM2) and p53 genes. Cell invasion and migration assays were used to observe the effect of Gli1, MDM2 and p53 silence on cell invasion and migration in p53 wild-type Capan-2 pancreatic cancer cells, respectively. Meanwhile, immunoblotting (IB) was used to detect the protein level of matrix metalloproteinase (MMP) -9, phospho-excelluar signal-regulated kinase (pERK) and phosphorylation protein kinase B (pAKT) in Gli1-silencing Capan-2 cells. The data were analyzed by paired t-test.</p><p><b>RESULTS</b>qRT-PCR showed that the expression of Gli1, MDM2 and p53 is down-regulated 70.5% and 74.5%, 61.8% and 65.3%, and 73.8% and 78.2% after siRNA interference, compared with the mock and siRNA control groups, respectively. Cell invasion (94 ± 8) and migration (143 ± 8) in p53 wild-type Capan-2 cells transfected with Gli1siRNA were significantly decreased, compared with the siRNA control group (150 ± 7, 190 ± 10) (t = 6.584, P = 0.022; t = 8.266, P = 0.014) , while MDM2 silence inhibited cell invasion (experiment group:85 ± 12, control group: 138 ± 6) and migration (experiment group: 127 ± 9, control group:180 ± 10) in the same cells, respectively (t = 5.097, P = 0.036;t = 4.860, P = 0.040). However, cell invasion (experiment group: 153 ± 11, control group: 106 ± 7) and migration (experiment group: 209 ± 13, control group: 164 ± 8) in p53-silencing Capan-2 cells were significantly enhanced (t = 4.669, P = 0.043; t = 4.990, P = 0.038). IB showed that Gli1 silence down-regulated MMP-9 but not pERK and pAKT protein expression.</p><p><b>CONCLUSION</b>Gli1 might contribute to the cell invasion and migration in pancreatic cancer via the regulation of MDM2, p53 and MMP-9 expression.</p>

Expression of sonic hedgehog signaling pathw ay and its inhibition by cyclopamine in rat liver with chronic fluorosis / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of sonic hedgehog (Shh) signaling pathway in liver fluorosis and to explore related mechanism.</p><p><b>METHODS</b>To establish animal model, 48 normal SD rats (aged 4-5 weeks) were randomly divided into 4 groups (12 each): control group, fluoriosis group, blocking group and blocking control group. After 6 months, the blocking group and blocking control group were injected intraperitoneally once every 2 days for 3 times with 10 mg/kg cyclopamine or dimethysulfoxide, respectively. Rats were sacrificed at the end of the experiment and the fluoride content in urine and liver function was determined. The expression of Shh and Gli1 protein and mRNA in hepatocytes was detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively.</p><p><b>RESULTS</b>The fluoride contents in the urine and the incidence of dental fluorosis increased in the fluoride and blocking control groups as compared with those in the control group, but decreased in the blocking group compared with those of the fluoride and blocking control group. Compared with the control group, the titers of aspartate transaminase (AST) and alanine transaminase (ALT) significantly increased, while the activity of total protein and albumin decreased in the fluoride and blocking control groups. Compared with the fluoride and blocking control groups, the activity of the ALT slightly declined and the AST, total protein and albumin slightly increased in the blocking group. Histologically, the cells were disorganized and swollen with cytoplasmic clearing (balloon cells), compared with the control group. The expression of Shh and Gli1 significantly increased in all but the control group. Compared with the fluoride and blocking control groups, the expression of Shh and Gli1 declined in the blocking group.</p><p><b>CONCLUSIONS</b>The overexpression and cyclopamine inhibition of the Shh signaling pathway are closely related to the content of fluoride in the liver. The Shh signaling pathway plays an important role in the pathogenesis of liver injury caused by fluorosis, suggesting a preventive and therapeutic target of the disease.</p>

Effect of Helicobacter pylori lipopolysaccharide on expression of Gli and Ptch-1 proteins in sonic hedgehog signaling pathway of gastric mucosa GES-1 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of Helicobacter Pylori lipopolysaccharide (Hp-LPS) on expression of Gli and Ptch-1 proteins in sonic hedgehog (Shh) signaling pathway of gastric mucosa GES-1 cells.</p><p><b>METHODS</b>The LPS was extracted from Hp by hot phenol water method, and then the concentration of LPS was detected by the kinetic turbidimetric assay. GES-1 cells were stimulated by different concentrations of Hp-LPS (0, 1, 10, 20, 30 and 40 μg/ml). The inhibition rates of cell growth were measured by MTT assay after treated with Hp-LPS for 24 h. The expression of Gli and Ptch-1 proteins were determined by Western Blot.</p><p><b>RESULTS</b>MTT assay showed that the inhibition rates of GES-1 cell growth after treatment by different concentrations of Hp-LPS (1, 10, 20, 30 and 40μg/ml) were 25.8% ± 2.7%, 34.2% ± 3.1 %, 46.3% 3.4%, 60.8% ± 2.1% and 82.9% ± 2.8% respectively (r=0.985, P<0.001). Western blot showed that the expressions of Gli and Ptch-1 proteins were decreased after Hp-LPS treatment (0, 1, 10, 20, 30 and 40 μg/ml): the relative expression values of Gli were 1.286 ± 0.180, 0.963 ± 0.067, 0.850 ± 0.085, 0.566 ± 0.058, 0.549 ± 0.056 and 0.377 ± 0.047, respectively (r=-0.945, P<0.001); those of Ptch-1 were 1.688 ± 0.088, 1.466 ± 0.061, 1.170 ± 0.065, 1.042 ± 0.064, 0.648 ± 0.057 and 0.482 ± 0.074, respectively (r=-0.985, P<0.001).</p><p><b>CONCLUSION</b>Hp-LPS can decrease the related protein expression of Shh signaling pathway, which indicates that Hp may interfere with the function of Shh signaling pathway in gastric mucosa via the effect of its LPS.</p>

Down-regulation of Smoothened gene expression inhibits proliferation of breast cancer stem cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.</p><p><b>METHODS</b>Human SMO shRNA was designed, synthesized chemically, and transfected into MCF-7 cells to down-regulate SMO gene. By using G418, stable cells with down-regulated SMO were selected. In vitro proliferation of these cells was measured by CCK8 assay. The proportion of CD44(+)/CD24(-) cells was detected by flow cytometry and the mammospheres formation was determined by suspension sphere culture. The expression of SMO, GLI1 and Oct4 was detected by Western blot. In vivo, the volume of tumor was measured every 3 days and the expression of SMO, GLI1 and Oct4 detected by Western blot.</p><p><b>RESULTS</b>In vitro, the cells were transfected with SMO-shRNA and selected by G418 after 21 days. SMO-shRNA effectively down-regulated the expression of SMO gene and protein, and inhibited the proliferation of MCF-7 and markedly reduced the proportion of CD44(+)/CD24(-) cells and mammospheres. In vivo, SMO-shRNA treatment of MCF-7 significantly inhibited the volume of tumor. The positive rate of SMO in negative control and SMO-shRNA group was 5/5 and 2/5, respectively. The expression of SMO, GLI1 and Oct4 in different groups were 0.72 ± 0.17 and 0.21 ± 0.09, 1.21 ± 0.21 and 0.47 ± 0.12, 0.83 ± 0.13 and 0.25 ± 0.07. SMO, GLI1 and Oct4 down-regulation significantly suppressed at protein levels (P < 0.05).</p><p><b>CONCLUSION</b>The shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.</p>

Correlation between Gli1 expression and clinicopathological significance in human pancreatic cancer / 中华外科杂志

<p><b>OBJECTIVE</b>To study the clinicopathological significance and relationship of Gli1, MDM2 and p53 expression in human pancreatic cancer.</p><p><b>METHODS</b>The expression of Gli1, MDM2 and p53 proteins in 57 paired paraffin embedded pancreatic ductal adenocarcinoma (PDAC) specimens and adjacent non-cancerous pancreatic tissues was detected by immunohistochemistry. The relationship between their expression and clinicopathological characters was analyzed. Quantitative real-time PCR (qRT-PCR) was used to examine the expression of Gli1 mRNA level in 14 paired fresh PDAC specimens and adjacent non-cancerous pancreatic tissues. siRNA interference were used to further detect the close relationship among them.</p><p><b>RESULTS</b>IHC showed the expression of Gli1 (50.9%), MDM2 (57.9%) and p53 (56.1%) was increased in 57 cases of pancreatic cancer compared to that in paired normal pancreatic tissues (33.3%, 26.3% and 17.5% respectively, t = 2.413, 2.848 and 2.960, all P < 0.05). Gli1 expression was positively associated with tumor TNM stage (χ(2) = 8.211, P = 0.004), invasion depth (χ(2) = 4.247, P = 0.039) and MDM2 expression (r = 0.299, χ(2) = 5.105, P = 0.024), while expression of MDM2 and p53 was associated with tumor invasion depth (χ(2) = 5.182, P = 0.023) and TNM stage (χ(2) = 5.696, P = 0.017), respectively. Univariate and multivariate analysis revealed that Gli1 was an independent adverse prognostic indicator for patients with PDAC (RR = 2.290, 95%CI: 1.051-4.992, P = 0.037), and patients with Gli1 and MDM2 co-expression had a significantly poorer overall survival than patients with their negative expression (P = 0.034). Gli1 mRNA expression was much higher in 14 cases of PDAC than that in adjacent normal pancreatic tissues (t = 2.926, P = 0.012). In p53 mutant AsPC-1 cells, Gli1 knockdown down regulated MDM2, but had no effect on p53 expression, whereas Gli1 knockdown down regulated MDM2 and up regulated p53 protein levels in p53 wild-type Capan-2 cells.</p><p><b>CONCLUSIONS</b>Gli1, MDM2 and p53 are overexpressed in PDAC, and are benefit for predicting patients' prognosis. Gli1can regulate MDM2 and wild-type p53 expression. Their co-expression might coordinately contribute to the development and progression of PDAC.</p>

Identification of Nedd4 as a novel regulator in Hedgehog signaling / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hedgehog (Hh) signaling plays an important role in both embryonic development and postnatal tissue homeostasis. Aberrant Hh activation results in a large variety of cancers. This study was designed to discover novel modulators in Hh signaling pathway.</p><p><b>METHODS</b>We performed yeast-two-hybrid screening and immunoprecipitation to identify the interaction of Nedd4 and Smo. To verify whether Nedd4 is involved in the regulation of Hh signaling, we monitored the activation of Gli-luciferase reporter by overexpressing Nedd4 together with Gli-luciferase reporter. In order to examine the role of endogenous Nedd4 in regulating Hh signaling, we used a short hairpin RNA (shRNA) interference strategy to silence the Nedd4 expression, and then perform dual-luciferase reporter assay. Statistical comparisons were performed by Student's t tests.</p><p><b>RESULTS</b>We showed that Nedd4 binds to Smo in the transfected HEK293 cells. Overexpression of Nedd4 alone did not significantly activate the Gli reporter compared to pcDNA3 control (Nedd4 group: dimethyl sulfoxide (DMSO), relative luciferase unit (RLU) 1.87 ± 0.41). However, Smo agonist (SAG)-stimulated activation of Gli-luciferase reporter was markedly potentiated in Nedd4 transfected cells (Nedd4 group: SAG, RLU 13.49 ± 1.04, P < 0.05), indicating that overexpression of Nedd4 increases Gli luciferase reporter activity and Nedd4-induced activation of Hh signaling is activity dependent. In Nedd4 knockdown NIH 3T3 cells, the luciferase reporter activity was measured basally and after SAG treatment. In scrambled cells, compared to DMSO, SAG could activate reporter activity by (4.16 ± 0.84)-fold. In Nedd4 knockdown cells, the luciferase reporter activation by SAG was significantly inhibited (SAG, RLU 1.72 ± 0.24, P < 0.05); knockdown of Nedd4 did not change the basal activity of luciferase activity (DMSO, RLU 0.86 ± 0.11), suggesting that the loss of Nedd4 expression diminishes Gli-dependent activity in the Hh pathway and the regulation of Nedd4 in the Hh signaling pathway is activity-dependent.</p><p><b>CONCLUSION</b>Nedd4 positively regulates the Hh pathway and provides a potential target for inhibiting Hh signaling in cancer therapy.</p>

Inhibitory effect of cyclopamine on metastatic ability of EC109 cells and its mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of cyclopamine on metastatic ability of human esophageal cancer EC109 cells and explore the possible mechanism.</p><p><b>METHODS</b>Transwell chamber assay and angiogenesis assay were used to examine the metastatic ability, invasiveness and angiogenesis of EC109 cells treated with cyclopamine for 48 h. The expression of Gli-1 mRNA was detected using RT-PCR, and Western blotting was used to examine the protein expressions of Gli-1, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF).</p><p><b>RESULTS</b>Inhibition of the hedgehog signaling pathway by cyclopamine suppressed the migration, invasion, and angiogenesis of EC109 cells. Cyclopamine treatment significantly lowered the expression of Gli-1 mRNA (P<0.05) and the protein expressions of Gli-1, MMP-9 and VEGF (P<0.05).</p><p><b>CONCLUSION</b>Cyclopamine can significantly inhibit the metastatic capacity of EC109 cells possibly by down-regulating MMP-9 and VEGF expression as a result of Gli-1 inhibition.</p>

Effect of Gli1 gene silencing on proliferation of K562 cells and its mechanisms / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Gli1 gene silencing by RNA interference (RNAi) on proliferation of K562 cells and its mechanisms.</p><p><b>METHODS</b>The small interference RNA (siRNA) was synthesized in vitro. K562 cells were transfected with Gli1 siRNA by the way of lipofection (lipofectamine 2000). Non-specific siRNA transfected cells were used as control. Transfection efficiencies of different siRNA concentrations were detected by flow cytometry and the best siRNA concentration was selected. The silencing effect of siRNA was demonstrated by real time PCR and Westem blot analysis. Cell proliferation was measured by MTT method, cell cycle by PI assay, c-myc and p21 mRNA level was detected by real time PCR analysis.</p><p><b>RESULTS</b>Transfection efficiency of siRNA was increased in a dose-dependent manner when siRNA concentration was below 200 pmol, and the highest transfection efficiency reached (80.11 ± 5.63)%. Both the mRNA and protein level of Gli1 was down-regulated in Gli1 specific siRNA group, the mRNA level was (52.60 ± 3.57)% of that of control group after 24 h (t = 20.33, P < 0.01) and the protein level was (79.31 ± 5.58)% of that of control group after 48 h (t = 6.54, P < 0.01). The cell proliferation rate in Gli1 siRNA group was (94.41 ± 3.58)% (t = 2.40, P = 0.05) and (90.22 ± 3.34)% (t = 4.37, P < 0.01) of that of control group after 24 h and 48 h, respectively. G(2)/M cell cycle arrest was observed, the mRNA level of c-myc was down-regulated while p21 was up-regulated in Gli1 siRNA group after 24 h and 48 h (P < 0.05).</p><p><b>CONCLUSIONS</b>Targeted silencing of Gli1 gene by RNAi inhibits the proliferation of K562 cells, which acts through the down-regulation of c-myc and up-regulation of p21 expression.</p>

GLI-1 is involved in EGF-regulated enhancement of the invasiveness of prostate cancer ARCaP(E) cells in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the role of the hedgehog (HH) signaling pathway transcription factor glioma-associated oncogene hoinolog 1 (GLI-1) in EGF-regulated enhancement of the invasiveness of the prostate cancer ARCaP(E) cell line in vitro.</p><p><b>METHODS</b>The expressions of EGFR and GLI-1 in prostate cancer ARCaP(E) cells were analyzed by immunofluorescence staining. ARCaP(E) cells were treated with EGF at 100 ng/ml, followed by detection of the changes in cell morphology and invasiveness, as well as in the expressions of p-ERK, ERK and GLI-1. Migration transwell assay was used to determine the effects of 100 ng/ml EGF and GLI-1 antagonist GANT61 on the invasiveness of the ARCaP(E) cells.</p><p><b>RESULTS</b>Both EGFR and GLI-1 were expressed in the ARCaP(E) cells. EGF induced morphological transition of epithelial-like ARCaP(E) cells to mesenchymal-like cells, increased their in vitro invasiveness, and significantly upregulated the expressions of p-ERK and GLI-1 in the ARCaP(E) cells (P<0.05). GANT61 significantly inhibited the in vitro invasiveness of the ARCaP(E) cells and reduced the enhancing effect of EGF on their invasiveness (P<0.05).</p><p><b>CONCLUSION</b>The results from ARCaP(E) cells shed light on the cross-talk of the HH pathway with the EGF/ERK signaling pathway. GLI-1 might be responsible for EGF-regulated enhancement of the invasiveness of ARCaP(E) cells in vitro.</p>

Expression and clinical significances of hedgehog signaling pathway in non-Hodgkin's lymphoma / 中国实验血液学杂志

This study was aimed to investigate the expression and clinicopathologic significance of Gli1 and Gli2, 2 factors of Hedgehog(Hh) signaling pathway, in non-Hodgkin's lymphoma (NHL). Gli1 and Gli2 mRNA and protein in 18 cases of NHL and 10 cases of reactive lymphadenitis were amplified and identified by real-time PCR, and were assayed by immunohistochemical staining respectively. The results showed that (1) Gli1 and Gli2 mRNA in NHL group (RQ 2.05, 2.31) were expressed higher than that in reactive lymphadenitis group (RQ 0.82, 0.89). Gli1 mRNA activated level was positively related with Gli2 (r = 0.63, p < 0.01). In addition, Gli2 also positively correlated to clinical stages of NHL (p = 0.03), but the expressions of Gli1 and Gli2 mRNA had no significant correlation to B symptoms, blood β(2)-microglobulin, age and sex. (2) The positive expression rate of Gli1 and Gli2 protein in NHL group were 80% and 68% respectively, which were extremely higher than that in reactive lymphadenitis group. Gli1 protein level was positively related with Gli2 (r = 0.62, p < 0.05). Both Gli1 and Gli2 protein expression positively correlated to clinical staging of NHL (p = 0.05, p = 0.01). It is concluded that the Gli1 and Gli2 of Hh signaling pathway have been found to higher express in patients with NHL, and have significance for clinical staging and predicting prognosis of NHL. To further investigate the role of Hh signaling pathway in NHL will contribute to elucidate the occurrence and development of NHL, and provide a favorable method for therapy of NHL.

Hedgehog signaling pathway activates in gastric carcinoma and promotes the proliferation through GLI1 in MKN28 cell / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of Hedgehog (HH) pathway on proliferation and in vitro tumorigenicity of gastric cancer cell lines.</p><p><b>METHODS</b>The expression of SHH, PTCH, SMO, SUFU and GLI1 in seven cell lines were tested by RT-PCR. siRNA targeting GLI1 mRNA was transfected into MKN28 cells. Cell proliferation and in vitro tumorigenicity were examined by CCK8 and soft agar colony formation test.</p><p><b>RESULTS</b>SHH in six gastric cancer cell lines was up-regulated. Expression of PTCH in KATOIII cell lines and expression of SUFU in MKN28 and KATOIII were reduced. GLI1 siRNA significantly inhibited the expression of GLI1 in MKN28 cell line. Growth rate and colony formation rate of MKN28 cells treated with GLI1 siRNA were significantly lower than those of control cells (all P <0.001).</p><p><b>CONCLUSIONS</b>HH signaling pathway is widely activated in gastric cancer cell lines. The activation of HH signaling pathway promotes the growth of MKN28 cells.</p>

The effects of Hedgehog-Gli 1 signaling pathway on proliferation and apoptosis of hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of Hedgehog-Gli1 signaling pathway on proliferation, apoptosis and activation of hepatic stellate cells (HSCs) in vitro.</p><p><b>METHODS</b>The expression of Shh, Smo, Ptc and Gli-1 in HSC-T6 cells was analyzed by RT-PCR. HSC-T6 cells were incubated with various concentration of cyclopamine (0, 50, 100, 150, 200, 250 mumol/L) for 24 hours, cell viability was checked by MTT colorimetric assay, cell cycle was analyzed by flow cytometry, apoptosis was assayed by agarose electrophoresis of DNA and PI-Annexin V fluorescent staining, and the mRNA levels of Gli-1, TGF beta 1, PDGF and Bcl-2 were quantified by real-time RT-PCR.</p><p><b>RESULTS</b>RT-PCR indicated that the components of the Hedgehog-Gli1 signaling pathway were expressed in HSC-T6 cells. MTT assay indicated that cyclopamine inhibited cell viability in a concentration dependant manner (F = 636.81, P less than 0.01). Flow cytometry indicated that cells were piled up at G0/G1 phase in cyclopamine treated cells (65.08%+/-1.50%) as compared to control cells (55.41%+/-2.54%, t = -8.05, P less than 0.01). Cyclopamine treatment resulted in apoptosis as indicated by DNA fragmentation and PI-Annexin V staining. The mRNA levels of Gli-1, TGF beta 1, PDGF and Bcl-2 in cyclopamine treated cells were significantly lower than that in control cells (P less than 0.01).</p><p><b>CONCLUSION</b>Cyclopamine may inhibit the Hedgehog-Gli1 signaling, and hence repress proliferation and promote apoptosis in hepatic stellate cells.</p>