Inhibition of Sonic Hedgehog signaling inhibits fibrous scar formation and adversely affects functional outcome after ischemic brain injury in rats / 南方医科大学学报

OBJECTIVE@#To investigate the effects of inhibiting Sonic Hedgehog (Shh) signaling on fibrous scar formation and functional outcome after ischemic brain injury.@*METHODS@#Adult SD rats were randomized into sham-operated group, middle cerebral artery occlusion (MCAO) and reperfusion (I/R) group, I/R with intraventricular empty adenoviral vector (rAd-NC) injection group, and I/R with adenovirus-mediated Shh knockdown (rAd-ShShh) group. After the treatments, the neurological deficits of the rats were assessed, and the protein and mRNA expressions of fibronectin (Fn), α-SMA, and Shh in the ischemic hemisphere were detected with immunofluorescence assay and qPCR; TUNEL staining was used for detecting neural cell apoptosis. In the cell experiment, primary meningeal fibroblasts isolated from neonatal SD rats were pretreated for 24 h with TGF-β1 or TGF-β1 plus cyclopamine (CYC) before oxygen-glucose deprivation for 150 min followed by reoxygenation for 72 h (OGD/R). CCK-8 assay and scratch test were performed to examine the changes in cell proliferation and migration, and immunofluorescence assay, qPCR and Western blotting were used for detecting cell transformation and the expressions of Shh, α-SMA, and Fn.@*RESULTS@#Cerebral I/R injury significantly increased the protein and mRNA expressions of Shh, α-SMA, and Fn in the ischemic hemisphere of the rats, but their expression levels were significantly lowered by intraventricular injection of rAd-Shshh (P < 0.05), which obviously increased cell apoptosis in the ischemic hemisphere (P < 0.05) and improved modified mNSS and modified Bederson scores of the rats (P < 0.05). In the cell experiment, pretreatment with TGF-β1 and TGF-β1+CYC both increased the viability of the primary meningeal fibroblasts after OGD/R. TGF-β1 significantly enhanced the migration ability and induced obvious transformation of the exposed cells (P < 0.05), but these effects were significantly attenuated by co-treatment with CYC (P < 0.05). The expressions of Shh, α-SMA and Fn in the TGF-β1 group were all significantly higher in TGF-β1-treated cells (P < 0.05) and were obviously lowered by co-treatment with CYC (P < 0.05).@*CONCLUSION@#Inhibition of Shh signaling may inhibit fibrous scar formation and functional recovery in rats after ischemic brain injury.

A hierarchical vascularized engineered bone inspired by intramembranous ossification for mandibular regeneration / 国际口腔科学杂志·英文版

Mandibular defects caused by injuries, tumors, and infections are common and can severely affect mandibular function and the patient's appearance. However, mandible reconstruction with a mandibular bionic structure remains challenging. Inspired by the process of intramembranous ossification in mandibular development, a hierarchical vascularized engineered bone consisting of angiogenesis and osteogenesis modules has been produced. Moreover, the hierarchical vascular network and bone structure generated by these hierarchical vascularized engineered bone modules match the particular anatomical structure of the mandible. The ultra-tough polyion complex has been used as the basic scaffold for hierarchical vascularized engineered bone for ensuring better reconstruction of mandible function. According to the results of in vivo experiments, the bone regenerated using hierarchical vascularized engineered bone is similar to the natural mandibular bone in terms of morphology and genomics. The sonic hedgehog signaling pathway is specifically activated in hierarchical vascularized engineered bone, indicating that the new bone in hierarchical vascularized engineered bone underwent a process of intramembranous ossification identical to that of mandible development. Thus, hierarchical vascularized engineered bone has a high potential for clinical application in mandibular defect reconstruction. Moreover, the concept based on developmental processes and bionic structures provides an effective strategy for tissue regeneration.

Advances in research of Musashi2 in solid tumors / 南方医科大学学报

RNA binding protein (RBP) plays a key role in gene regulation and participate in RNA translation, modification, splicing, transport and other important biological processes. Studies have shown that abnormal expression of RBP is associated with a variety of diseases. The Musashi (Msi) family of mammals is an evolutionarily conserved and powerful RBP, whose members Msi1 and Msi2 play important roles in the regulation of stem cell activity and tumor development. The Msi family members regulate a variety of biological processes by binding and regulating mRNA translation, stability and downstream cell signaling pathways, and among them, Msi2 is closely related to embryonic growth and development, maintenance of tumor stem cells and development of hematological tumors. Accumulating evidence has shown that Msi2 also plays a crucial role in the development of solid tumors, mainly by affecting the proliferation, invasion, metastasis and drug resistance of tumors, involving Wnt/β-catenin, TGF-β/SMAD3, Akt/mTOR, JAK/STAT, Numb and their related signaling pathways (Notch, p53, and Hedgehog pathway). Preclinical studies of Msi2 gene as a therapeutic target for tumor have achieved preliminary results. This review summarizes the molecular structure, physiological function, role of Msi2 in the development and progression of various solid tumors and the signaling pathways involved.

Transforming growth factor-β1 induces transformation of rat meningeal fibroblasts into myofibroblasts by upregulating Shh signaling / 南方医科大学学报

OBJECTIVE@#To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts.@*METHODS@#Primary meningeal fibroblasts were isolated from neonatal (24 h) SD rats and purified using type Ⅳ collagenase. The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20 μmol/L SB-431542 (a TGF-β1 receptor inhibitor) for 72 h, and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test. The expression of fibronectin (Fn) was detected with immunofluorescence assay, and Western blotting was performed to examine the expressions of Fn, α-SMA and Shh protein in the cells; the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR.@*RESULTS@#TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts (P < 0.05), and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn (P < 0.05). TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels (P < 0.05). Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts (P < 0.05).@*CONCLUSION@#TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.

Research advances on signaling pathways affecting sweat gland development and their involvement in the reconstitution of sweat adenoid cells in vitro / 中华烧伤杂志

The damage of sweat glands in patients with extensive deep burns results in the loss of thermoregulation, which seriously affects the quality of life of patients. At present, there are many researches on the repair of sweat gland function, but the mechanism of human sweat gland development has not been fully clarified. More and more studies have shown that the cascaded pathways of Wnt/β-catenin, ecto- dysplasin A/ectodysplasin A receptor/nuclear factor-κB, sonic hedgehog, and forkhead box transcription factor jointly affect the development of sweat glands, and it has been reported that the cascaded signaling pathways can be used to achieve the reconstruction of sweat adenoid cells in vitro. This article reviews the signaling pathways that affect the development of sweat glands and their involvement in the reconstruction of sweat adenoid cells in vitro.

Research progress on role of traditional Chinese medicine in hepatic fibrosis based on miRNA-mediated activation of NLRP3 inflammasome / 中国中药杂志

In recent years, liver fibrosis has become a hotspot in the field of liver diseases. MicroRNA(miRNA)-mediated Nod-like receptor pyrin domain containing 3(NLRP3) inflammasome activation is pivotal in the pathogenesis of liver fibrosis. The present study mainly discussed the role of miRNA-mediated NLRP3 inflammasome activation in the pathogenesis of liver fibrosis. Different miRNA molecules regulated liver fibrosis by mediating NLRP3 inflammasome activation, including miRNA-350-3 p(miR-350-3 p)/interleukin-6(IL-6)-mediated signal transducer and activator of transcription 3(STAT3)/c-myc signaling pathway, miR-148 a-induced autophagy and apoptosis of hepatic stellate cells via hedgehog signaling pathway, miR-155-mediated NLRP3 inflammasome by the negative feedback of the suppressor of cytokine signaling-1(SOCS-1), miR-181 a-mediated downstream NLRP3 inflammatory pathway activation through mitogen-activated protein kinase kinase(MEK)/extracellular signal-regulated kinase(ERK)/nuclear transcription factor κB(NF-κB) inflammatory pathway, miR-21-promoted expression of NF-κB and NLRP3 of RAW264.7 cells in mice by inhibiting tumor necrosis factor-α inducible protein 3(A20), and miR-20 b-promoted expression of IL-1β and IL-18 by activating NLRP3 signaling pathway. Additionally, the anti-liver fibrosis mechanism of different active components in Chinese medicines(such as Curcumae Rhizoma, Glycyrrhizae Radix et Rhizoma, Aurantii Fructus, Polygoni Cuspidati Rhizoma et Radix, Moutan Cortex, Paeoniae Radix Alba, Epimedii Folium, and Cinnamomi Cortex) was also explored based on the anti-liver fibrosis effect of miRNA-mediated NLRP3 inflammasome activation.

Research progress in mechanism of Chinese herbal compounds and monomers in delaying lumbar intervertebral disc degeneration / 中国中药杂志

Traditional Chinese medicine has unique advantages in the treatment of degenerative bone and joint diseases, and its widely used in clinical practice. In recent years, many scholars have conducted a large number of basic studies on the delay of intervertebral disc degeneration by herbal compound and monomeric components from different perspectives. In order to further elucidate its mechanism of action, this paper summarizes the in vivo and in vitro experimental studies conducted at the level of both herbal compound and single components, respectively, in order to provide references for the basic research on the treatment of lumbar intervertebral disc degeneration by Chinese medicine. A summary shows that commonly used herbal compound prescriptions include both classical prescriptions such as Duhuo Jisheng Decoction, as well as clinical experience prescriptions such as Yiqi Huoxue Recipe. Angelicae Sinensis Radix, Chuanxiong Rhizoma, Rehmanniae Radix Praeparata, Achyranthis Bidentatae Radix, and Eucommiae Cortex were used most frequently. Tonic for deficiency and blood stasis activators were used most frequently. The most utilized monomeric components include icariin, ginsenoside Re, salvianolic acid B and aucubin. The main molecular mechanisms by which herbal compound and monomeric components delay of lumbar intervertebral disc degeneration include improving the intervertebral disc microenvironment, promoting the synthesis of aggregated proteoglycans and type Ⅱ collagen in the intervertebral disc, reducing the degradation of the extracellular matrix, and inhibiting apoptosis in the nucleus pulposus cells, etc. The main signaling pathways involved include Wnt/β-catenin signaling pathway, MAPK-related signaling pathway, mTOR signaling pathway, Fas/FasL signaling pathway, PI3 K/Akt signaling pathway, NF-κB signaling pathway, JAK/STAT signaling pathway, and hedgehog signaling pathway, etc.

Vismodegib for treatment of periocular basal cell carcinoma - 6-year experience from a tertiary cancer center

Abstract Background: The treatment of advanced periocular basal cell carcinomas becomes a challenge as surgery may involve highly mutilating procedures. Vismodegib is the first selective hedgehog inhibitor approved for the treatment of locally advanced tumors or metastatic disease. Objective: Analyze the results of treatment with vismodegib for advanced periocular basal cell carcinomas in a real-life setting of a reference center between 2014 and 2020. Methods: Retrospective longitudinal study. The patient's demographic profile, comorbidities, tumor characteristics, and treatment outcomes were analyzed. Results: A total of 13 patients were included. Median follow-up and treatment duration were 15.9 and 10.5 months, respectively. Objective clinical response rate was 76.9%: 30.8% had a complete response and 46.2% a partial response. The median duration of response was 13 months. Progressive disease was observed in 38.5% of cases, with a median of 19 months after the beginning of treatment. Eighty-four percent of the patients had at least one adverse event, and 61.54% needed to interrupt treatment temporarily or permanently to increase tolerability. Study limitations: Being a retrospective study in a real-life setting, the evaluation of objective clinical response was subjective to physician appreciation. Conclusion: Vismodegib is a safe and effective treatment for locally advanced basal cell carcinoma. To prevent recurrences, the drug should be used continually when tolerated. The role of neoadjuvant vismodegib before surgery is being investigated and might add an important step in searching for a definitive treatment for these cases.

Effects of SET7 on angiotensin II-mediated proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms / 中南大学学报(医学版)

OBJECTIVES@#Silence of SET domain containing lysine methyltransferase 7 (SET7) alleviates myocardial tissue injury caused by ischemia-reperfusion. But the effects of SET7 on angiotensin II (Ang II)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear. The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.@*METHODS@#Myocardial fibroblasts were isolated and identified by immunofluorescence. Myocardial fibroblasts were randomly divided into 4 groups: a control group (cells were normally cultured), an Ang II group (cells were treated with 100 nmol/L Ang II for 24 h), a siCtrl group (cells were transfected with siRNA control and were then treated with 100 nmol/L Ang II for 24 h), and a siSET7 group (cells were transfected with siRNA SET7 and were then treated with 100 nmol/L Ang II for 24 h). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation. Real-time PCR was used to detect the mRNA levels of SET7, collagen I, collagen III, and α-smooth muscle actin (α-SMA). Western blotting was used to detect the protein expression of SET7, collagen I, collagen III, α-SMA, sonic hedgehog (Shh), ptched1 (Ptch1), and glioma-associated oncogene homolog 1 (Gli1).@*RESULTS@#Fluorescence microscopy showed positive vimentin staining, and myocardial fibroblasts were in good condition. As compared to the control group, the mRNA and protein levels of SET7 in the Ang II group were significantly upregulated; cell proliferation rate and EdU fluorescence intensity in the Ang II group were significantly increased; the mRNA and protein levels of collagen I, collagen III, and α-SMA were significantly upregulated (all @*CONCLUSIONS@#Silence of SET7 gene inhibits Ang II-induced proliferation and collagen synthesis of myocardial fibroblasts. Shh signaling pathway may be involved in this process.

Effects of Arsenic Disulfide Combined with Itraconazole on Proli- feration and Apoptosis and Hedgehog Pathway of Diffuse Large B-Cell Lymphoma Cells / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of arsenic disulfide (AS@*METHODS@#The human DLBCL cell OCI-LY3 was treated with different concentrations of AS@*RESULTS@#The DLBCL cell viability was decreased significantly at 24, 48 or 72 h as cultured with itraconazole. Along with the increasing of itraconazole concentration, the DLBCL cell viability was significantly reduced as compared with that in control group, and the results showed statistically significant(r=-0.690，r=-0.639, r=-0.833, r=-0.808, r=-0.578). The inhibitory and apoptosis rates of the cells were significantly increased as compared with those of the single drug-treated group after treated by the combination of itraconazole and AS@*CONCLUSION@#Itraconazole can inhibit proliferation of DLBCL cells in a concentration-and time-dependent manner. In addition, the combination of AS

Mechanism of ursolic acid in regulating colorectal cancer cell HCT116 autophagy through hedgehog signaling pathway / 中国中药杂志

To prove that ursolic acid(UA)could activate the autophagy of colorectal cancer HCT116 cells by inhibiting hedgehog signaling pathway. The effect of UA on the viability of HCT116 cells was determined by MTT assay. The effect of UA on the proliferation and migration of HCT116 cells was detected by crystal violet staining and scratch test. In the study on autophagy, the time points were screened out first: the autophagy fluorescence intensity of UA acting on HCT116 at different time points were detected by Cell Meter~(TM) Autophagy Assay Kit; Western blot was used to detect the expression of autophagy protein P62 at different time points. Then, Cell Meter~(TM) Autophagy Assay Kit was used to detect the effect of UA on autophagy fluorescence intensity of HCT116 cells. The effect of different doses of UA on the expressions of LC3Ⅱ and P62 proteins in HCT116 cells were detected by Western blot. Further, AdPlus-mCherry-GFP-LC3 B adenovirus transfection was used to detect the effects of UA on autophagy flux of HCT116 cells; UA combined with autophagy inhibitor chloroquine(CQ) was used to detect the expression of LC3Ⅱ by Western blot. In terms of mechanism, the effect of UA on hedgehog signaling pathway-related proteins in HCT116 cells was detected by Western blot. The results showed that UA inhibited the activity, proliferation and migration of HCT116 cells. UA enhanced the fluorescence intensity of autophagy in HCT116 cells, while promoting the expression of LC3Ⅱ and inhibiting the expression of P62, in a time and dose dependent manner. UA activated the autophagy in HCT116 cells, which manifested that UA resulted in the accumulation of fluorescence spots and strengthened the fluorescence intensity of autophagosomes; compared with UA alone, UA combined with autophagy inhibitor CQ promoted the expression of LC3Ⅱ. UA reduced the expressions of PTCH1, GLI1, SMO, SHH and c-Myc in hedgehog signaling pathway, while increased the expression of Sufu. In conclusion, our study showed that UA activated autophagy in colorectal cancer HCT116 cells, which was related to the mechanism in inhibiting hedgehog signaling pathway activity.

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.

Effect of polysaccharides from seeds of Vaccaria segetalis in alleviating urinary tract infection induced bladder injury by inhibiting NLRP3 inflammasome / 中国中药杂志

To study the mechanism of polysaccharides from seeds of Vaccaria segetalis( PSV) in the treatment of bacterial cystitis through the NLRP3 inflammasome pathway. The rat model of urinary tract infection was used and treated with PSV,and the urine and bladders were collected. The level of interleukin-10( IL-10) in rat urine was detected by enzyme linked immunosorbent assay( ELISA). Western blot and immunofluorescence staining were used to detect the expressions of sonic hedgehog( SHH) and NLRP3 inflammasome [NOD-like receptor thermoprotein domain 3( NLRP3),apoptosis associated speck like protein( ASC) and pro-caspase-1]. The expression of Toll-like receptor pathway was detected by RT-PCR. The death of 5637 cells induced by uropathogenic Escherichia coli( UPEC) and lactate dehydrogenase( LDH) release were evaluated using live/dead staining. The results showed that in the rat bladder,the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors were significantly up-regulated,and NLRP3 inflammasomes were significantly activated by UPEC infection. The administration with PSV could significantly increase the concentration of IL-10 in urine,inhibit the expressions of SHH,NLRP3 inflammasomes and Toll-like receptors in bladder,and inhibit the activation of NLRP3 inflammasomes. A large number of 5637 cells were dead after UPEC infection and caused LDH production. PSV could significantly inhibit the death of 5637 cells and the release of LDH. In conclusion,PSV could inhibit the expression and activation of NLRP3 inflammasomes by inhibiting the Toll-like receptor pathway,thereby mitigating the bladder injury.

Effect of dietary nutrition on tail fat deposition and evaluation of tail-related genes in fat-tailed sheep

BACKGROUND: The effects of dietary nutrition on tail fat deposition and the correlation between production performance and the Hh signaling pathway and OXCT1 were investigated in fat-tailed sheep. Tan sheep were fed different nutritional diets and the variances in tail length, width, thickness and tail weight as well as the mRNA expression of fat-related genes (C/EBPα, FAS, LPL, and HSL) were determined in the tail fat of sheep at three different growth stages based on their body weight. Furthermore, the correlations between tail phenotypes and the Hedgehog (Hh) signaling pathway components (IHH, PTCH1, SMO, and GLI1) and OXCT1 were investigated. RESULTS: C/EBPα, FAS, LPL, and HSL were expressed with differences in tail fat of sheep fed different nutritional diets at three different growth stages. The results of the two-way ANOVA showed the significant effect of nutrition, stage, and interaction on gene expression, except the between C/EBPα and growth stage. C/EBPα, FAS, and LPL were considerably correlated with the tail phenotypes. Furthermore, the results of the correlation analysis demonstrated a close relationship between the tail phenotypes and Hh signaling pathway and OXCT1. CONCLUSIONS: The present study demonstrated the gene-level role of dietary nutrition in promoting tail fat deposition and related tail fat-related genes. It provides a molecular basis by which nutritional balance and tail fat formation can be investigated and additional genes can be identified. The findings of the present study may help improve the production efficiency of fat-tailed sheep and identify crucial genes associated with tail fat deposition.

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.

Effect of Sonic Hedgehog Signal Pathway Inhibitor Jervine on Myelodysplastic Syndromes MUTZ-1 Cells / 中国实验血液学杂志

OBJECTIVE@#To study the effect of SMO inhibitor (Jervine) on proliferation, apoptosis and cell cycle of MDS cell line MUTZ-1, and its mechanism.@*METHODS@#The effect of different concentrations Jervine on proliferation of MUTZ-1 cells was detected by CCK-8 method. Apoptosis and cell cycle of MUTZ-1 cells were detected by flow cytometry. Western blot was used to detect the changes of Shh signaling pathway effecting proteins BCL2 and CyclinD1. The expression levels of Smo and Gli1 gene were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR).@*RESULTS@#Jervine inhibited MUTZ-1 cell proliferation in a concentration dependent manner (24 h, r=-0.977), the apoptosis rate of MUTZ-1 cells increased with the enhancement of concentration of Jervine in MUTZ-1 cells (P＜0.001), the cell proportion of G phase increased and the cell number of S phase decreased with enhancement of concentration (P＜0.001). The result of RT-qPCR and Western blot showed that the expression of Smo, Gli1 mRNA and BCL2, CyclinD1 proteins decreased (P＜0.05).@*CONCLUSION@#SMO inhibitor can effectively inhibit the growth of MDS cell line MUTZ-1 improve the cell apoptosis and induce cell cycle arrest. Its action mechanism may be related with dowm-regulating the expression of BCL2 and CyclinD1.

Analysis of single-nucleotide polymorphism of Sonic hedgehog signaling pathway in non-syndromic cleft lip and/or palate in the Chinese population / 北京大学学报(医学版)

OBJECTIVE@#To study the relationship between Sonic hedgehog (Shh) associated single-nucleotide polymorphism (SNP) and non-syndromic cleft lip and/or palate (NSCL/P), and to explore the risk factors of cleft lip and/or palate. Many studies suggest that the pathogenesis of NSCL/P could be related to genes that control early development, in which the Shh signaling pathway plays an important role.@*METHODS@#Peripheral blood was collected from 197 individuals (100 patients with NSCL/P and 97 healthy controls). Haploview software was used for haplotype analysis and Tag SNP were selected, based on the population data of Han Chinese in Beijing of the international human genome haplotype mapping project. A total of 27 SNP were selected for the 4 candidate genes of SHH, PTCH1, SMO and GLI2 in the Shh signaling pathway. The genotypes of 27 SNP were detected and analyzed by Sequenom mass spectrometry. The data were analyzed by chi-squared test and an unconditional Logistic regression model.@*RESULTS@#The selected SNP basically covered the potential functional SNP of the target genes, and its minimum allele frequency (MAF) was >0.05: GLI2 73.5%, PTCH1 91.0%, SMO 100.0%, and SHH 75.0%. It was found that the genotype frequency of SNP (rs12674259) located in SMO gene and SNP (rs2066836) located in PTCH1 gene were significantly different between the NSCL/P group and the control group. Linkage disequilibrium was also found on 3 chromosomes (chromosomes 2, 7 and 9) where the 4 candidate genes were located. However, in the analysis of linkage imbalance haplotype, there was no significant difference between the disease group and the control group.@*CONCLUSION@#In China, NSCL/P is the most common congenital disease in orofacial region. However, as it is a multigenic disease and could be affected by multiple factors, such as the external environment, the etiology of NSCL/P has not been clearly defined. This study indicates that Shh signaling pathway is involved in the occurrence of NSCL/P, and some special SNP of key genes in this pathway are related to cleft lip and/or palate, which provides a new direction for the etiology research of NSCL/P and may provide help for the early screening and risk prediction of NSCL/P.

Rapamycin Induces Apoptosis of K562 Cells through EZH2/Hedgehog Signaling Pathway / 中国实验血液学杂志

OBJECTIVE@#To investigate the mechanism of rapamycin-induced apoptosis of chronic myelogenous leukemia cells.@*METHODS@#The chronic granulocytic leukemia K562 cells were divided into 3 groups: A, B and C group were treated with rapamycin of 10, 15 and 20 nmol/L, repectively for 24 h, while the K562 cells in control group were not treated with rapamycin. The effect of rapamycin on the proliferation of K562 cells was detected by MTT, and the effect of rapamycin on the apoptosis of K562 cells was detected by AnnexinV-FITC/PI double staining. The expression level of EZH2/Hedgehog signaling pathway genes in K562 cells was detected by RT-PCR, and Western blot was used to detect the levels of apoptotic protein and the related signaling pathway proteins in K562 cells.@*RESULTS@#The MTT assay showed that the different concentration of rapamycin had obvious inhibitory effects on the cells, and the survival rate of cells in group C was 37.6%±3.4%, which was significantly lower than that of the other groups (P＜0.05). The apoptosis rate of cells in group C was 93.1%±8.1%, which was significantly higher than that of the other groups (P＜0.05). By Western blot, it was found that the relative expression levels of Caspase-3 and BAX protein in group C were 0.36 ± 0.04 and 0.39±0.06, respectively, which were significantly higher than those in other groups (P＜0.05), and the level of BCL-2 protein was 0.17±0.03, which was significantly lower than that of other groups (P＜0.05). By RT-PCR, it was found that the mRNA levels of EZH2 and Hedgehog genes in A, B and C groups were significantly lower than those in the control group (P＜0.05), but mRNA level of Ptch1 gene was significantly higher than that of the control (P＜0.05). By Western blot, it was found that the expression levels of EZH2 and Hedgehog protein in A, B and C groups were significantly lower than that in the control group (P＜0.05), but the level of Ptch1 protein was higher than that of the control (P＜0.05). The relative levels of EZH2 and Hedgehog protein in group C were 0.21 ±0.03 and 0.16±0.05 respectively, which were significantly lower than those in other groups (P＜0.05), and Ptch1 protein level were 0.46 ±0.06, significantly higher than that of other groups (P＜0.05).@*CONCLUSION@#Rapamycin can inhibit the protein expression of EZH2 in leukemic cells, thus interfere with the activation of Hedgehog signaling pathway, promote the expression of apoptotic protein, reduce the level of anti apoptotic protein, and eventually induce apoptosis of leukemia cells.

Expression and significance of Shh and Wnt5a genes in Cornelia de Lange syndrome / 中国当代儿科杂志

OBJECTIVE@#To study the expression of Shh and Wnt5a genes in the limb buds of NIPBL fetal rats and the association of these two genes with Cornelia de Lange syndrome (CdLS).@*METHODS@#A total of 72 NIPBL fetal rats were divided into an experimental group and a control group, with 36 rats in each group. The limb buds were collected from 12 fetal rats each on embryonic days 10, 11 and 12 (E10, E11 and E12) respectively. Real-time PCR and Western blot were used to measure the mRNA and protein expression of Shh and Wnt5a.@*RESULTS@#The mRNA and protein expression of Shh and Wnt5a was detected in the limb buds on E10, E11 and E12, and the experimental group had significantly lower expression than the control group (P<0.01). The mRNA and protein expression of Shh and Wnt5a in limb buds was at a low level on E10, followed by an increase on E11 and a reduction on E12, and the expression on E12 was still lower than that on E10 (P<0.01).@*CONCLUSIONS@#The mRNA and protein expression of Shh and Wnt5a are consistent. The pathogenesis of CdLS may be associated with the low mRNA and protein expression of Shh and Wnt5a inhibited by the low expression of NIPBL gene.