Objective: To detect the SMO mutations in odontogenic keratocyst (OKC) and to explore the mechanism behind. Methods: Patients with OKC who received treatment in the Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology,Peking University, from September 2012 to June 2017 were enrolled. OKC samples from 10 patients diagnosed as naevoid basal cell carcinoma syndrome (NBCCS)-related OKC (4 females and 6 males) and 20 patients diagnosed as sporadic OKC (7 females and 13 males) were collected. Genomic DNAs were extracted from fibrous capsules and epithelial lining respectively. SMO mutations were detected and analyzed by Sanger sequencing. Results: Three SMO mutations were found in one NBCCS-associated OKC who carrying c.2081C>G (p.P694R) mutation) and two sporadic OKC who carrying c.907C>T (p.L303F) mutation and c.1247_1248delinsAA (p.G416E), respectively), among which the first two mutations were novel mutations that had not been reported before. Besides, two mutations in sporadic OKC were not paired with PTCH1 mutations. Conclusions: In addition to PTCH1 gene mutations, SMO gene mutations also exist in OKC which might be related to the development of OKC.
Basal Cell Nevus Syndrome/genetics* ; Female ; Humans ; Male ; Mutation ; Odontogenic Cysts/genetics* ; Odontogenic Tumors/genetics* ; Smoothened Receptor/genetics*

OBJECTIVE: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect, affecting 1.4 per 1 000 live births, and multiple genetic and environmental risk factors influencing its risk. All the known genetic risk factors accounted for a small proportion of the heritability. Several authors have suggested parent-of-origin effects (PoO) may play an important role in the etiology of this complex and heterogeneous malformation. To clarify the genetic association between PTCH1, PTCH2, SHH and SMO in hedgehog (HH) pathway and NSCL/P, as well as testing for potential PoO effects in Chinese case-parent trios. METHODS: We tested for transmission disequilibrium tests (TDT) and PoO effects using 83 common single nucleotide polymorphic (SNP) markers of HH pathway genes from 806 NSCL/P case-parent trios. These trios were drawn from an international consortium established for a genome-wide association studies (GWAS) of non-syndromic oral clefts of multiple ethnicities. DNA samples were collected from each trio. Single marker and haplotype based analysis were performed both in TDT tests and PoO effects. SNPs were excluded if they (ⅰ) had a call rate of < 95%, (ⅱ) had a minor allele frequency (MAF) of < 0.05, (ⅲ) had Mendelian errors over all trios of >5%, (ⅳ) had a genotype distribution in the parents that deviated from the Hardy-Weinberg equilibrium (HWE) (P < 0.000 1). The process was done using Plink (version 1.07, http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml). TDT test was performed in Plink v1.07. A log-linear model was used to explore PoO effects using Haplin v6.2.1 as implemented in R package v3.4.2. Significance level was assessed using the Bonferroni correction. RESULTS: A total of 18 SNPs were dropped due to low MAF, thus leaving 65 SNPs available for the analysis. Thus the Bonferroni threshold was 7.7×10^-4 (0.05/65). Nominal significant association with NSCL/P was found at a SNP (rs4448343 in PTCH1, P=0.023) and six haplotypes (rs10512249-rs4448343, rs1461208-rs7786445, rs10512249-rs4448343, rs16909865-rs10512249-rs4448343, rs1461208-rs7786445-rs12698335, and rs288756-rs288758-rs1151790, P < 0.05). A total of six haplotypes (rs288765-rs1233563, rs12537550-rs11765352, rs872723-rs288765-rs1233563, rs288765-rs1233563-rs288756, rs6459952-rs12537550-rs11765352, and rs12537550-rs11765352-rs6971211) showed PoO effect (P < 0.05). None of the results remained significant after the Bonferroni correction (P>7.7×10^-4). CONCLUSION Neither significant association between SNPs within HH pathway and the risk of NSCL/P nor PoO effects was seen in this study.
Asians ; Cleft Lip/genetics* ; Cleft Palate/genetics* ; Genome-Wide Association Study ; Hedgehog Proteins/genetics* ; Humans ; Patched-2 Receptor ; Smoothened Receptor

OBJECTIVETo investigate the influence of down-regulating Smoothened (SMO) gene expression through short hairpin RNA (shRNA) on the proliferation of breast cancer stem cells.

METHODSHuman 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.

RESULTSIn 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).

CONCLUSIONThe shRNA by chemical synthesis can effectively down-regulate SMO gene expression and inhibit the proliferation of breast cancer stem cells.

Animals ; Cell Proliferation ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hyaluronan Receptors ; metabolism ; MCF-7 Cells ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Neoplastic Stem Cells ; pathology ; Octamer Transcription Factor-3 ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Smoothened Receptor ; Transcription Factors ; metabolism ; Transfection ; Tumor Burden ; Zinc Finger Protein GLI1

BACKGROUNDHedgehog (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.

METHODSWe 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.

RESULTSWe 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.

CONCLUSIONNedd4 positively regulates the Hh pathway and provides a potential target for inhibiting Hh signaling in cancer therapy.

Animals ; Endosomal Sorting Complexes Required for Transport ; physiology ; HEK293 Cells ; Hedgehog Proteins ; physiology ; Humans ; Mice ; NIH 3T3 Cells ; Nedd4 Ubiquitin Protein Ligases ; Receptors, G-Protein-Coupled ; physiology ; Signal Transduction ; physiology ; Smoothened Receptor ; Transcription Factors ; physiology ; Ubiquitin-Protein Ligases ; physiology ; Zinc Finger Protein GLI1

The link of hedgehog (Hh) signaling activation to human cancer and synthesis of a variety of Hh signaling inhibitors raise great expectation that inhibiting Hh signaling may be effective in human cancer treatment. Cyclopamine (Cyc), an alkaloid from the Veratrum plant, is a specific natural product inhibitor of the Hh pathway that acts by targeting smoothened (SMO) protein. However, its poor solubility, acid sensitivity, and weak potency relative to other Hh antagonists prevent the clinical development of Cyc as a therapeutic agent. Here, we report properties of cyclopamine tartrate salt (CycT) and its activities in Hh signaling-mediated cancer in vitro and in vivo. Unlike Cyc, CycT is water soluble (5-10 mg/mL). The median lethal dose (LD50) of CycT was 62.5 mg/kg body weight compared to 43.5 mg/kg for Cyc, and the plasma half-life (T1/2) of CycT was not significantly different from that of Cyc. We showed that CycT had a higher inhibitory activity for Hh signaling-dependent motor neuron differentiation than did Cyc (IC50 = 50 nmol/L for CycT vs. 300 nmol/L for Cyc). We also tested the antitumor effectiveness of these Hh inhibitors using two mouse models of basal cell carcinomas (K14cre:Ptch1(neo/neo) and K14cre:SmoM2(YFP)). After topical application of CycT or Cyc daily for 21 days, we found that all CycT-treated mice had tumor shrinkage and decreased expression of Hh target genes. Taken together, we found that CycT is an effective inhibitor of Hh signaling-mediated carcinogenesis.
Animals ; Carcinoma, Basal Cell ; pathology ; Cell Differentiation ; drug effects ; Embryonic Stem Cells ; cytology ; Hedgehog Proteins ; antagonists & inhibitors ; metabolism ; Mice ; Motor Neurons ; cytology ; Plants, Medicinal ; chemistry ; Receptors, G-Protein-Coupled ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects ; Skin Neoplasms ; pathology ; Smoothened Receptor ; Solubility ; Tartrates ; blood ; pharmacology ; Tumor Burden ; drug effects ; Veratrum ; chemistry ; Veratrum Alkaloids ; blood ; isolation & purification ; pharmacology

Medulloblastoma is the most common malignant pediatric brain tumor. Despite its prevalence and importance in pediatric neuro-oncology, the genes and pathways responsible for its initiation, maintenance, and progression remain poorly understood. Genetically engineered mouse models are an essential tool for uncovering the molecular and cellular basis of human diseases, including cancer, and serve a valuable role as preclinical models for testing targeted therapies. In this review, we summarize how such models have been successfully applied to the study of medulloblastoma over the past decade and what we might expect in the coming years.
Animals ; Cerebellar Neoplasms ; genetics ; metabolism ; pathology ; Disease Models, Animal ; Genetic Engineering ; Hedgehog Proteins ; metabolism ; Humans ; Medulloblastoma ; genetics ; metabolism ; pathology ; Mice ; Mice, Transgenic ; Mutation ; Patched Receptors ; RNA Interference ; RNA, Small Interfering ; genetics ; Receptors, Cell Surface ; genetics ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Signal Transduction ; Smoothened Receptor ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo investigate the expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines of SMMC-7721, Bel-7402, HepG2, MHCC-97 and normal hepatocellular cell line of L02, and to compare the response of these cell lines to all-trans retinoic acid.

METHODSRT-PCR was used to detect expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines and normal hepatocellular cell line. Real time-PCR was used to quantify the expression of the genes.

RESULTSThere are different levels of expression of the stem cell-related gene in hepatocellular carcinoma cell lines and control cell line (P less than 0.05). There are significant differences in HepG2 and L-02 for the response to all-trans retinoic acid (P less than 0.05).

CONCLUSIONSThe stem cell-related genes are differentially expressed in different hepatocellular carcinoma cell lines.

Carcinoma, Hepatocellular ; metabolism ; pathology ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Nanog Homeobox Protein ; Octamer Transcription Factor-3 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptors, G-Protein-Coupled ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; SOXB1 Transcription Factors ; genetics ; metabolism ; Signal Transduction ; Smoothened Receptor ; Stem Cells ; drug effects ; metabolism ; Tretinoin ; pharmacology ; Wnt Proteins ; genetics ; metabolism ; beta Catenin ; genetics ; metabolism

OBJECTIVETo investigate the significance of sonic hedgehog (Shh), indian hedgehog (Ihh), smoothened (Smo) and patched (Ptch) expressions in uterine cervical lesions and their relationships with HPV type 16 infection.

METHODSTotally 183 cases of cervical lesions, including 32 non-neoplastic cervix, 71 cervical intraepithelial neoplasia (28 CINI, 18 CINII, and 25 CINIII) and 80 squamous cell carcinomas (SCC) were selected from the Department of Pathology, Yanbian University Hospital, Yanbian Women Hospital, and Yanbian Tumor Hospital. Shh, Ihh, Ptch and Smo proteins expression were investigated by immunohistochemistry using tissue microarry platform, and the presence of HPV type 16 was detected by PCR method.

RESULTSImmunohistochemical staining showed that the frequencies of Shh, Ihh, Ptch and Smo expression were rare in normal cervical epithelium, but were strongly expressed in cervical cancer and its precursor lesions (CINII/III) (P < 0.01, P < 0.01, P < 0.05, P < 0.05, respectively). In cervical cancer, the expression rate of Shh (95%) was higher than that of CIN (CINI to CINIII) (46.4%, 61.1%, 80.0%, respectively, P < 0.05). HPV16 was positive in 77.5% of SCC. In cervical cancer, the expression of Shh was related with HPV16 infection (P < 0.05), and the expression of Smo was correlated with lymph node metastasis (P < 0.05).

CONCLUSIONSShh, Ihh, Ptch, and Smo genes may play important roles in the development of cervical cancer. Detection of Hedgehog signaling pathway molecules seems helpful for the early diagnosis of cervical cancer and its precursor lesions, and are potentially therapeutic targets as well.

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; virology ; Cervical Intraepithelial Neoplasia ; metabolism ; pathology ; virology ; Female ; Hedgehog Proteins ; metabolism ; Human papillomavirus 16 ; Humans ; Lymphatic Metastasis ; Middle Aged ; Neoplasm Staging ; Papillomavirus Infections ; Patched Receptors ; Patched-1 Receptor ; Receptors, Cell Surface ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Signal Transduction ; Smoothened Receptor ; Uterine Cervical Neoplasms ; metabolism ; pathology ; virology ; Young Adult

OBJECTIVETo study the effects of specific small interfering RNA (siRNA) on Smoothened (Smo) gene expression and the proliferation and apoptosis of colorectal cancer LoVo cells.

METHODSThree different siRNAs (siRNA-1, siRNA-2, and siRNA-3, respectively) were transfected into LoVo cells via cationic liposome, and the changes of Smo mRNA level were determined using semi-quantitative RT-PCR 48 h after transfection. Flow cytometry and MTT assay were performed to assess the effect of the siRNAs on the proliferation and apoptosis of LoVo cells.

RESULTSForty-eight hours after Smo siRNA-1 transfection, Smo mRNA expression in LoVo cells decreased by about 63.56%, a reduction significantly greater than that in cells transfected with the other two siRNAs. The cell proliferation decreased significantly after Smo siRNA-1 transfection in comparison with the control cells, and 48 h after transfection, significantly higher apoptosis rate was observed in Smo siRNA-1-transfected cells than in the control cells.

CONCLUSIONSpecific siRNA can significantly decrease Smo mRNA expression and inhibit the proliferation while inducing apoptosis of LoVo cells.

Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; RNA, Small Interfering ; genetics ; Receptors, G-Protein-Coupled ; deficiency ; genetics ; Smoothened Receptor ; Time Factors ; Transfection

OBJECTIVETo examine the change of Smoothened (Smo) expression in the retinofugal pathway and in the growth cones during the period of embryonic day 13 (E13) to E15.

METHODSSmo expression in the chiasm and growth cones was observed by fluorescent immunostaining and retinal explant culture.

RESULTSOn E13 and E14, Smo was expressed moderately in the retina and optic disc, and in the corner of the retina, Smo expression was especially dense. On E13, Smo expression was detected in the optic nerves and ventral diencephalon, but only in the superficial region of the optic tract on E14. Smo was also detected in the stem and filopodia of the growth cones in the retinal explant culture during this period.

CONCLUSIONSmo expression changes in different developmental phases, suggesting that Smo might play a role in signal optic axon growth during the development of the retinofugal pathway.

Animals ; Fluorescent Antibody Technique ; Mice ; Mice, Inbred C57BL ; Optic Chiasm ; cytology ; embryology ; metabolism ; Optic Nerve ; cytology ; embryology ; metabolism ; Receptors, G-Protein-Coupled ; biosynthesis ; Retina ; cytology ; embryology ; metabolism ; Smoothened Receptor ; Visual Pathways ; cytology ; embryology ; metabolism