Detection of SMO gene mutations in odontogenic keratocyst / 中华口腔医学杂志

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.

Exploring parent-of-origin effects for non-syndromic cleft lip with or without cleft palate on PTCH1, PTCH2, SHH, SMO genes in Chinese case-parent trios / 北京大学学报(医学版)

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) (<i>P</i> < 0.000 1). The process was done using Plink (version 1.07, <a href="http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml" target="_blank">http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml</a>). 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.

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>

Sonic hedgehog signaling enhanced the expression of histone demethylase, lysine-specific demethylase 8 in the head and neck squamous cell carcinoma cell line SCC-6 / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To determine whether the sonic hedgehog (Shh) signaling could regulate the expression of histone demethylases in the head and neck squamous cell carcinoma(SCC).</p><p><b>METHODS</b>Human recombinant SHH-N protein or over-expression of the mutant 2 smoothened (M2-SMO) was applied to activate the Shh signaling in tongue squamous cell carcinoma cell line-SCC-6 in this study. Cyclopamine was used to block the Shh signaling in SCC-6. The real-time reverse transcription (RT)-PCR was used to detect the expression of histone demethylases at the mRNA level.</p><p><b>RESULTS</b>The data showed that activation of the Shh signaling up-regulated the expression of histone demethylase, lysine-specific demethylase 8 (KDM-8) at the mRNA level by human recombinant SHH-N protein (1.841 ∼ 3.591 fold compare with untreated group; P < 0.01), over-expression of the M2-SMO also increased the expression of KDM-8 (1.358 ∼ 3.013 fold compared with empty vector group; P < 0.05), and after the Shh signaling was blocked by Cyclopamine, the expression of KDM-8 was down regulated (decreased 25.6% ∼ 66.6% compared with control cells, P < 0.05).</p><p><b>CONCLUSIONS</b>Histone demethylase KDM-8 was downstream target gene of Shh signaling in head and neck squamous cell carcinoma cell line SCC-6, and its expression was positively regulated by the Shh signaling.</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>

Mouse models of medulloblastoma / 癌症

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.

Tumor shrinkage by cyclopamine tartrate through inhibiting hedgehog signaling / 癌症

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.

Analysis on the expression of the stem cells related genes in hepatocellular carcinoma cell lines / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>RT-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.</p><p><b>RESULTS</b>There 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).</p><p><b>CONCLUSIONS</b>The stem cell-related genes are differentially expressed in different hepatocellular carcinoma cell lines.</p>

Relationship between hedgehog signaling pathway molecules and HPV16 infection in uterine cervical cancers / 中华病理学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Totally 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.</p><p><b>RESULTS</b>Immunohistochemical 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).</p><p><b>CONCLUSIONS</b>Shh, 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.</p>

Effects of specific small interfering RNA on Smoothened expression and LoVo cell proliferation and apoptosis / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effects of specific small interfering RNA (siRNA) on Smoothened (Smo) gene expression and the proliferation and apoptosis of colorectal cancer LoVo cells.</p><p><b>METHODS</b>Three 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.</p><p><b>RESULTS</b>Forty-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.</p><p><b>CONCLUSION</b>Specific siRNA can significantly decrease Smo mRNA expression and inhibit the proliferation while inducing apoptosis of LoVo cells.</p>

Changes of Smoothened expression during retinofugal pathway development in mouse embryos / 南方医科大学学报

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Smo expression in the chiasm and growth cones was observed by fluorescent immunostaining and retinal explant culture.</p><p><b>RESULTS</b>On 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.</p><p><b>CONCLUSION</b>Smo 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.</p>

Expression patterns of sonic hedgehog signaling molecules in human fetal prostate development / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate whether the sonic hedgehog signaling pathway is involved in the development of human fetal prostate, and to evaluate the changing staining patterns of its molecules, sonic hedgehog (SHH), patchedl (PTC1), smoothened (SMO), and GLI1, in the human fetal prostate at various gestation stages.</p><p><b>METHODS</b>Fifteen human fetal prostate specimens at various developmental stages (10 - 39 weeks) were included in this study. SHH, PTC1, SMO and GLI1 were detected in all the specimens by immunohistochemical technique. All the slides were observed and assessed under the light microscope.</p><p><b>RESULTS</b>SHH, PTC1, SMO and GLI1 could be detected in human fetal prostate tissues, and their expression formed two surges, the former at week 16, and the latter at week 28. The staining of SHH and SMO was distributed only in the ductal epithelium but not in the stroma. The expression of PTC1 and GLI1 could be found mainly in the epithelium, with minimal staining in the stroma.</p><p><b>CONCLUSION</b>The sonic hedgehog signaling pathway is involved in the development of the human fetal prostate. The high expression of its molecules at early gestation stages might be associated with the induction of prostatic buds, while their abundant expression at later gestation stages might be related to the prostate ductal branching, growth, differentiation and morphogenesis.</p>

Expression of Smo protein and the downstream transcription factor Gli1 protein in Sonic hedgehog signal transduction pathway in gastric carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the expression of Smo protein and the downstream transcription factor Gli1 protein in Sonic hedgehog signal transduction pathway in gastric carcinoma.</p><p><b>METHODS</b>A tissue microarray was constructed using 85 gastric carcinoma and 25 normal gastric mucosa specimens. The expression of Smo and Gli1 proteins were detected immunohistochemically and the correlation between their expression in gastric carcinoma was analyzed.</p><p><b>RESULTS</b>Only weak expression, if any, of Smo and Gli1 proteins was detected in normal gastric mucosa, but in papillary adenocarcinoma, tubular adenocarcinoma and poorly differentiated adenocarcinoma, their expressions were significant increased as the differentiation degree was lowered. Smo protein expression in gastric carcinoma was significantly correlated with that of Gli1 protein with correlation coefficient of 0.989 (P<0.001).</p><p><b>CONCLUSION</b>The abnormal activity of Sonic hedgehog signal transduction pathway may play an important role in the occurrence of papillary adenocarcinoma, tubular adenocarcinoma and poorly differentiated adenocarcinoma, and this abnormality is associated with Smo protein overexpression, which upregulates the expression of the downstream transcription factor Gli1 protein.</p>