The flavonoid glycoside vaccarin inhibits adipogenesis and stimulates lipolysis via Hedgehog signaling in 3T3-L1 adipocytes

Vaccarin,a flavonoid glycoside isolated from Vaccaria segetalis,is non-toxic to 3T3-L1 cells up to concentrations of 200μM.Accordingly,we investigated the effects of this natural product on adipogenesis and lipolysis in 3T3-L1 adipocytes.Our results revealed that vaccarin significantly inhibited lipid accumulation by suppressing the adipogenesis-related transcription factors peroxisome proliferator-activated receptorγ(PPARγ)and the CCAAT/enhancer-binding proteinα(C/EBPα).Specifically,lipid accumulation decreased by up to 27.7±2.7%when 3T3-L1 adipocytes were treated with a 10μM concentration of vaccarin.Mechanistic studies showed that the compound inhibited adipogenesis through activation of the Hedgehog(Hh)signaling pathway and so restoring Smo and Gli1 expression at an early stage of differentiation.In mature 3T3-L1 cells,vaccarin significantly increased the secretion of glycerol into the surrounding medium and thus indicating that it accelerated the degradation of triglycerides.In addition,vaccarin,was shown to enhance lipolysis through stimulation of the transcription levels of lipoprotein lipase,monoglycerides lipase,adipose triacylglyceride lipase,hormone-sensitive lipase and adipose differentiated-related protein.All told,vaccarin suppressed lipid accumulation and enhanced lipolysis during adipocyte differentiation by restoring Hh signaling.As such,it is a phytochemical capable of halting adipocyte hyperplasia and,thereby,ameliorating the effects of obesity.

Anethole improves the developmental competence of porcine embryos by reducing oxidative stress via the sonic hedgehog signaling pathway

Background Anethole(AN)is an organic antioxidant compound with a benzene ring and is expected to have a positive impact on early embryogenesis in mammals.However,no study has examined the effect of AN on porcine embryonic development.Therefore,we investigated the effect of AN on the development of porcine embryos and the underlying mechanism.Results We cultured porcine in vitro-fertilized embryos in medium with AN(0,0.3,0.5,and 1 mg/mL)for 6 d.AN at 0.5 mg/mL significantly increased the blastocyst formation rate,trophectoderm cell number,and cellular survival rate compared to the control.AN-supplemented embryos exhibited significantly lower reactive oxygen species levels and higher glutathione levels than the control.Moreover,AN significantly improved the quantity of mitochondria and mitochondrial membrane potential,and increased the lipid droplet,fatty acid,and ATP levels.Interestingly,the levels of proteins and genes related to the sonic hedgehog(SHH)signaling pathway were significantly increased by AN.Conclusions These results revealed that AN improved the developmental competence of porcine preimplantation embryos by activating SHH signaling against oxidative stress and could be used for large-scale production of high-quality porcine embryos.

CK2αcauses stemness and chemotherapy resistance in liver cancer through the Hedgehog signaling pathway

Background:Liver cancer is one of the major causes of cancer-related deaths globally.Cancer cell stem-ness and chemotherapy resistance contribute to the high mortality.Although evidence indicates that the alpha subunit of protein kinase 2(CK2α)is involved in several human cancers,its function in liver cancer remains unknown.In the present study,we aimed to elucidate the role of CK2αin liver cancer.Methods:We examined the role of CK2αregulation in stemness and chemotherapy resistance capacity of liver cancer cells.MTT assays,tumor sphere formation assays,RT-PCR,flow cytometry,Western blotting assay,clonogenicity assay,matrigel invasion assay and bioinformatics were conducted in this study.Results:CK2αexpression in the liver cancer tissues was notably upregulated compared with that in the corresponding non-tumorous tissues.The overexpression of CK2αpromoted tumor sphere formation,increased the percentage of CD133(+)and side population cells,caused the resistance of liver cancer cells to 5-FU treatment,increased the expression levels of NANOG,OCT4,SOX2,Gli1 and Ptch1,and enhanced the ability of CD133(+)cell clone formation and invasion.Consistently,the downregulation of CK2αhad the opposite effects.CK2αsilencing inhibited the Hedgehog pathway by reducing the expression of Gli1 and Ptch1.Mechanistically,CK2αregulation on liver cancer cell stemness and chemotherapy resistance was found to be involved in the Hedgehog signaling pathway.Conclusions:Our study may bring some new insights into the occurrence of liver cancer.Furthermore,these findings suggest that targeting CK2αmay be a novel therapeutic strategy for patients with liver cancer.

Effect of Sonic hedgehog gene-modified bone marrow mesenchymal stem cells on graft-induced retinal gliosis and retinal ganglion cells survival in diabetic mice

AIM:To investigate the effects of Sonic hedgehog(Shh)gene-modified bone marrow mesenchymal stem cells(MSCs)on graft-induced retinal gliosis and retinal ganglion cells(RGCs)survival in diabetic mice.METHODS:Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs(MSC-Shh).Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs(MSC-Gfp;control)were administered in diabetic mice.After 4wk,the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography,and markers of gliosis were examined by immunofluorescence and Western blotting.The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence.The mechanisms underlying the effects of MSC-Shh was investigated.RESULTS:A significant reduction of proliferative vitreoretinopathy(PVR)was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp.Significant downregulation of glial fibrillary acidic protein(GFAP)was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp.The extracellular signal-regulated kinase 1/2(ERK1/2),protein kinase B(AKT)and phosphatidylin-ositol-3-kinase(PI3K)pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp.Brain-derived neurotrophic factor(BDNF)and ciliary neurotrophic factor(CNTF)levels were significantly increased in the host retina,and RGCs loss was significantly prevented after MSC-Shh administration.CONCLUSION:MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice.The ERK1/2,AKT and PI3K pathways are involved in this process.MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.

Hedgehog signaling pathway and ovarian cancer

Epithelial ovarian carcinoma （EOC） is the most common form of ovarian malignancies and the most lethal gynecologic malignancy in the United States. To date, in spite of treatment to it with the extensive surgical debulking and chemotherapy, the prognosis of EOC remains dismal. Recently, it has become increasingly clear that in many instances, the signaling and molecular players that control development are the same, and when inappropriately regulated, drive tumorigenesis and cancer development. Here, we discuss the possible involvement of Hedgehog （Hh） pathway in the cellular regulation and development of cancer in the ovaries. Using the in vitro and in vivo assays developed has facilitated the dissection of the mechanisms behind Hh-driven ovarian cancers formation and growth. Based on recent studies, we propose that the inhibition of Hh signaling may interfere with spheroid-like structures in ovarian cancers. The components of the Hh signaling may provide novel drug targets, which could be explored as crucial combinatorial strategies for the treatment of ovarian cancers.

Expression of Gli1 in the hedgehog signaling pathway and breast cancer recurrence

The hedgehog （Hh） signaling pathway plays an essential role in the embryonic development and homeostasis of diverse adult tissues, and its deregulation has been implicated in the tumorigenesis and metastasis of various malignancies including breast cancer. Aberrant activation of the Hh pathway includes the following mechanisms： （I） Hh ligand-independent mechanism - Loss of function mutations in the Hh receptor Patched 1 （PTCH1） or gain of function mutations in the Smoothened （SMO） lead to constitutive activation of this pathway; （II） Autocrine signaling- Ith ligand produced by tumor cells stimulates the Hh signaling in tumor cells; （III） Paracrine signaling - tumor cell produced-Hh ligand activates stromal and endothelial cells that produce growth factors in microenvironment for supporting tumor growth and survival; and （IV） Reverse paracrine signaling - Hh ligand produced by stromal cells support tumor growth and survival. Upon the pathway activation, the Gli transcription factors, effectors of the Hh signaling, activate or inhibit transcription by binding to their responsive genes and interacting with the transcriptional complex. The Gli transcription factor family includes Glil, Gli2, and Gli3 （1）. Glil is a transcriptional activator whose expression has been recognized as an activation state of the Hh signaling pathway, Gli2 is either an activator or repressor, and Gli3 is a strong repressor of transcriptional activities. To date, a ligand-dependent autocrine model of activating the Hh signaling has been described in breast cancer, and both an autocrine and paracrine mechanisms in colorectal cancer, pancreatic cancer and prostate cancer （2,3）. Notably, a ligand-independent mechanism （mutationsin PTCHI and SMO） of the signaling has been well demonstrated in basal cell carcinoma and medulloblastoma （4,5）.

Role of aberrant Sonic hedgehog signaling pathway in cancers and developmental anomalies

Development is a sophisticated process maintained by various signal transduction pathways,including the Hedgehog(Hh)pathway.Several important functions are executed by the Hh signaling cascade such as organogenesis,tissue regeneration,and tissue homeostasis,among various others.Considering the multiple functions carried out by this pathway,any mutation causing aberrant Hh signaling may lead to myriad developmental abnormalities besides cancers.In the present review article,we explored a wide range of diseases caused by aberrant Hh signaling,including developmental defects and cancers.Finally,we concluded this mini-review with various treatment strategies for Hh-induced diseases.

Inhibition of colorectal cancer by ursolic acid via noncanonical Hedgehog signaling pathway

OBJECTIVE To identify the inhibitory effect of ursolic acid on the colorectal cancer HCT116 cells in vitro and in vivo,and to explore the underlying mechanism.METHODS The smoothened(SMO)gene-silenced human colorectal cancer HCT116^(hSMO)cell line was established by transfection with the lentivirus carrying SMO shRNA.The cytotoxic effect of ursolic acid on HCT116^(hSMO)cells was determined by MTT assay.The effect of ursolic acid on the migration of HCT116^(hSMO)cells was studied by wound healing assay.The effect of ursolic acid on apoptosis of HCT116^(hSMO)cells was explored by Hoechst33342/PI double staining and flow cytometry.The effects of ursolic acid on the expressions of apoptotic marker gene Bcl-2,Bax,caspase-3 and caspase-9 were measured by real-time quantitative RT-PCR(RT-qPCR)and Western blotting(WB)analysis.RT-qPCR and WB were used to examine the relationship between GLI1,c-Myc expression and PI3K/Akt pathway to further investigate the mechanism of GLI1 activation in HCT116^(hSMO)cells.The effects of ursolic acid on the expressions of GLI1,p-Akt,Akt,c-Myc,SHH and SUFU of noncanonical Hedgehog pathway were evaluated by RT-qPCR and WB assays.Xenograft nude mouse model bearing HCT116^(hSMO)cells was established and intraperitoneally treated with ursolic acid to investigate the effect on tumor growth in vivo.The body weight and tumor size of mice were assessed regularly every 2 d.The effect of ursolic acid on the apoptosis of tumor tissue was determined by TUNEL assay.The expressions of Bcl-2,Bax,GLI1,p-Akt,Akt,c-Myc,SHH,SUFU mRNA and proteins were measured by RT-qPCR and WB.The levels of Bcl-2,Bax,GLI1,p-Akt,c-Myc and SHH proteins in tumor tissues were also evaluated by immunohistochemistry.RESULTS Ursolic acid significantly inhibited the growth and migration of HCT116^(hSMO)cells in vitro,compared with the control(P<0.05).Meanwhile,ursolic acid also induced apoptosis of HCT116^(hSMO)cells in vitro(P<0.05).Furthermore,SC79(Akt activator)enhanced the expressions of p-Akt,GLI1 and c-Myc,which could be abolished by ursolic acid,and the effect was equal to Akt inhibitor LY294002.The expressions of Bcl-2,GLI1,p-Akt,c-Myc,SHH mRNA and proteins were reduced by ursolic acid,while the levels of Bax and SUFU were increased.Ursolic acid could inhibit the growth and induce the apoptosis of colorectal cancer xenograft in vivo.Similarly,lower levels of Bcl-2,GLI1,p-Akt,c-Myc and SHH,and higher expression of Bax and SUFU were noted in ursolic acid-treated mice.CONCLUSION Ursolic acid can inhibit the growth and induce apoptosis of HCT116^(hSMO)cells both in vitro and in vivo.And the mechanism is related to the suppression of PI3K/Akt-mediated noncanonical Hedgehog signaling pathway.

Genetic variants in the Hedgehog signaling pathway genes are associated with gastric cancer risk in a Chinese Han population

The Hedgehog signaling pathway participates in the occurrence and progression of cancers including gastric cancer.We conducted this study to evaluate whether genetic variants in the Hedgehog signaling pathway genes would affect gastric cancer risk.Multi-marker Analysis of GenoMic Annotation(MAGMA)was used to investigate the aggregated genetic effects of single nucleotide polymorphisms(SNPs)assigned to candidate genes.The relationship between SNPs and gastric cancer risk was estimated by multivariate logistic regression analyses.Gene expression was calculated using databases obtained from The Cancer Genome Atlas(TCGA)and The Gene Expression Omnibus(GEO).Kaplan‐Meier plotter was used to evaluate the association between gene expression with gastric cancer survival.Tumor Immune Estimation Resource 2.0(TIMER 2.0)was applied to determine the correlation between selected gene expression and the immune cell infiltration degree.We identified that the G allele of rs2990912 in KIF27 was associated with higher gastric cancer risk,especially in the young and male subgroups.The expression of KIF27 in gastric cancer tissues was higher than that in normal tissues,leading to poor survival in gastric cancer patients.Besides,KIF27 expression was related to immune cell infiltration and positively correlated with PD-L1 expression.Our findings highlight the key role of genetic variation in the Hedgehog signaling pathway genes in gastric cancer susceptibility,which may provide important insights into the diagnosis,prognosis,and treatment of gastric cancer.

Inhibitory action of berberine on colorectal cancer HCT116 cells by regulation of Hedgehog signaling pathway

OBJECTIVE To investigate the inhibition and mechanism of berberine on human colorectal cancer HCT116 cells through canonical Hedgehog signaling pathway.METHODS The effect of berberine on cell morphology was observed by microscopy.MTT colorimetric assay,cell scratch experiment,colony formation assay and Hoechest/PI staining were utilized to detect the activities of berberine on cell viability,cell migration and cell apoptosis.Flow cytometry was applied to examine the cell apoptosis.The effects of berberine on caspase-3 and caspase-9 were detected by caspase activity detection kit.The expressions of Hedgehog signaling pathway-related proteins SHH,GLI1,PTCH1,SMO,SUFU,apoptosis-related proteins Bax and Bcl-2 as well as cell cycle-related proteins cyclin D1 were detected by Western blotting.Additionally,quantitative real time RT-PCR was employed to assess the mRNA expression levels of Hedgehog signaling pathway-related genes SHH,GLI1,PTCH1,SMO,SUFU,apoptosis-related genes Bax and Bcl-2 as well as cell cycle-related genes cyclin D1.RESULTS Berberine sharply altered the morphology of human colorectal cancer HCT116 cells,demonstrated by that migration ability of HCT116 cells was reduced significantly and the nuclei were densely stained.Berberine could induce apoptosis in a dose-dependent manner.The activities of caspase-3 and caspase-9 were increased prominently.The expression levels of Hedgehog signaling pathway-related protein SUFU and apoptosis-related protein Bax were augmented substantially.The expression levels of Hedgehog signaling pathway-related proteins SHH,GLI1,PTCH1,SMO,apoptosis-related protein Bcl-2 as well as cell cycle-related genes cyclin D1 were markedly lessened.Besides,the mRNA expression levels of Hedgehog signaling pathway-related gene SUFU and apoptosis-related gene Bax were augmented substantially.The mRNA expression levels of Hedgehog signaling pathway-related genes SHH,GLI1,PTCH1,SMO,apoptosis-related gene Bcl-2 as well as cell cycle-related gene cyclin D1 were markedly lessened.CONCLUSION Berberine,which is the main component of coptidis rhizoma,can remarkably restrain the growth and proliferation,promote apoptosis of human colorectal cancer cells HCT116,and the underlying mechanism may be involved in suppressing the activity of the Hedgehog signaling pathway.

Inhibition of self-renewal and differentiation of HT-29 cells-derived cancer stem-like cells by scutellarin via Hedgehog signaling pathway

OBJECTIVE To investigate the inhibitory effect of scutellarin on the self-renewal and differentiation of HT-29 cells-derived cancer stem-like cells(HT-29CSC)in vitro and in vivo,and to explore its mechanism.METHODS The effect of scutellarin on the growth of HT-29CSC was determined by 3D Culture assay.The effect of scutellarin on growth and transformation of HT-29CSC was probed by soft agar colony formation assay.The effect of scutellarin on the differentiation of HT-29CSC was determined by serum induction differentiation assay in vitro.The effects of scutellarin on the expressions of marker gene Lgr5,target gene c-Myc,proliferation gene CK20 and Nanog gene were measured by quantitative real-time RT-PCR.Investigate the effect of scutellarin on the expression of c-Myc,Gli1,and Lgr5 protein by Western blotting.A subcutaneous xenograft model of colon cancer in nude mice was established and administered by intraperitoneal injection.The change of body weight and tumor size of nude mice were observed every two days.Investi⁃gate the effects of scutellarin on the growth of xenograft tumors in nude mice.The expression of CD133,Lgr5,Gli1,Ptch1,c-Myc,Ki67,CK20,Nanog gene in tumors were measured by quantitative real-time RT-PCR.The expression of c-Myc,Gli1,Lgr5,CD133,Ki67 protein were measured by Western blotting.RESULTS Scutellarin can inhibit the growth of HT-29CSC in 3D culture.Compared with the solvent control group,scutellarin can significantly inhibit the growth and transformation and differentiation of HT-29CSC in vitro(P<0.01).The expression levels of marker genes Lgr5,target gene c-Myc,proliferation gene CK20 and Nanog in HT-29CSC were down-regulated by scutellarin.Scutellarin can reduce the expression of c-Myc,Gli1,and Lgr5 protein in HT-29CSC.Scutellarin can inhibit the growth of colon cancer xenografts,lower CD133,Lgr5,Gli1,Ptch1,c-Myc,Ki67,CK20,and Nanog mRNA level of xenograft tumors,reduce the expression of c-Myc,Gli1,Lgr5,CD133,and Ki67 protein of xenograft tumors in nude mice.CONCLUSION Scutellarin,which is the main component of scutellaria barbata,can inhibit the differentiation of HT-29CSC and the mechanism is to inhibit the activity of Hedgehog signaling pathway.

Activation of Sonic Hedgehog Signaling Pathway in S-type Neuroblastoma Cell Lines

The effects of Sonic hedgehog(Shh) signaling pathway activation on S-type neuroblastoma(NB) cell lines and its role in NB tumorigenesis were investigated.Immunohistochemistry was used to detect the expression of Shh pathway components— Patched1(PTCH1) and Gli1 in 40 human primary NB samples.Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Gli1 in three kinds of S-type NB cell lines(SK-N-AS,SK-N-SH and SHEP1),respectively.Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway.S-type NB cell lines were treated with different concentrations of Shh or/and cyclopamine for different durations.Cell viability was measured by using MTT method.Apoptosis rate and cell cycle were assayed by flow cytometry.The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice.High-level expression of PTCH1 and Gli1 was detected in both NB samples and S-type NB cell lines.Cyclopamine decreased the survival rate of the three cell lines while Shh increased it,and the inhibition effects of cyclopamine could be partially reversed by shh pre-treatment.Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase,while Shh induced the reverse effects and could partially prevent effects of cyclopamine.Cyclopamine could also inhibit the growth of NB in vivo.Our studies revealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle,suggesting a new therapeutic approach to NB.

Pentoxifylline Inhibits Liver Fibrosis via Hedgehog Signaling Pathway

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 m RNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The m RNA 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.

Research Progress of PTCH1 Gene in Lung Cancer

Background: The PTCH1 gene, also known as Patched 1, is located on the long arm of human chromosome 9 (9q22.3). It encodes the PTCH1 protein, which is a critical transmembrane receptor within the Hedgehog signaling pathway (Hh), playing a pivotal role in cellular communication and developmental processes. Recent studies have highlighted the significance of mutations in PTCH1 in the pathogenesis of lung cancer, positioning it as a crucial molecule for investigation in oncology. Purpose: This review aims to elucidate the role of the PTCH1 and the Hedgehog pathway in the initiation, progression, and potential treatment of lung cancer, thereby providing a theoretical foundation for personalized and precise therapeutic strategies. Method: To ensure a comprehensive review, this study systematically searched for literature related to the PTCH1, lung cancer, and the Hedgehog pathway across multiple databases including PubMed, Web of Science, and CNKI (China National Knowledge Infrastructure). The search strategy involved using specific keywords and advanced filtering options to include the most relevant and recent studies. Initial screening excluded irrelevant articles, followed by a detailed evaluation of the selected studies based on their scientific quality and relevance. Results: This review indicated that specific mutations in the PTCH1 gene are closely associated with the onset and progression of lung cancer. These mutations impede normal Hedgehog signaling, leading to unregulated cell proliferation and tumor growth. Targeting PTCH1, including vismodegib, have shown efficacy in clinical cases, particularly in SCCL with specific PTCH1 mutations, leading to complete remissions. Furthermore, the interaction between PTCH1 and microRNA-212 suggests potential therapeutic approaches by targeting miRNA to regulate PTCH1 expression. In addition, the investigation of traditional Chinese medicines such as Ginsenosides and Cordyceps sinensis extracts has shown their potential to modulate the Hedgehog pathway and reverse drug resistance. Conclusions: An in-depth understanding of the precise mechanisms by which PTCH1 mutations promote lung cancer could facilitate the development of targeted therapies. This study highlights the potential of PTCH1 as a biomarker for diagnosis and a target for precision medicine in lung cancer treatment, advocating for further research into its molecular pathways and therapeutic applications.

Blocking Ihh Signaling Pathway Inhibits the Proliferation and Promotes the Apoptosis of PSCs

Summary： The roles of Indian hedgehog （Ihh） signaling pathway in the proliferation and apoptosis of precartilaginous stem cells （PSCs） were investigated. PSCs, labeled with fibroblast growth factor receptor 3 （FGFR-3）, were isolated from neonatal rats by immunomagnetic separation. After identification with FGFR-3 and Col II, the cells were incubated with different concentrations of cyclopamine （cyclo）, the specific inhibitor of Ihh signaling pathway. The morphologic changes of the cells were observed under the inverted phase contrast microscope. The mRNA expression levels of Ihh, parathyroid hormonerelated peptide （PTHrP）, protein Patched （Ptch）, Bcl-2 and p21 were detected by RT-PCR. The protein expression levels of Ihh and Ptch were measured by Western blot. MTT assay was used to examine the effects of cyclo on proliferation of PSCs. Apoptosis rate of PSCs was examined by AnnexinV/PI assay of flow cytometric analyses. After PSCs were incubated with cyclo, obvious morphologic changes were observed as compared with the control group. The mRNA expression levels of PTHrP, Ptch and Bcl-2 were decreased to varying degrees in a cyclo dose-dependent manner. However, the expression levels of Ihh and p21 mRNA were increased. The protein expression of Ptch and Ihh had the same change as the mRNA expression. Meanwhile, cyclo could obvi- ously inhibit the proliferation and promote the apoptosis of PSCs. The results indicated that Ihh signaling pathway plays an important role in regulating the proliferation and apoptosis of PSCs, which is probably mediated by Bcl-2 and p21.

Sonic hedgehog elevates N-myc gene expression in neural stem cells

Proliferation of neural stem cells is regulated by the secreted signaling molecule sonic hedgehog. In this study, neural stem cells were infected with recombinant adeno-associated virus expressing sonic hedgehog-N-enhanced green fluorescent protein. The results showed that overexpression of sonic hedgehog in neural stem cells induced the increased expression of Gill and N-myc, a target gene of sonic hedgehog. These findings suggest that N-myc is a direct downstream target of the sonic hedgehog signal pathway in neural stem cells. Sonic hedgehog and N-myc are important mediators of sonic hedgehog-induced proliferation of neural stem cells.

The role of microRNAs during the genesis of medulloblastomas induced by the hedgehog pathway

Constitutive hedgehog （Hh） signaling is associated with the genesis of medulloblastomas （MB）. The objective of this study is to identify special microRNAs （miRNAs） regulated by the Hh pathway, and to clarify the role of miRNAs during the genesis of MB induced by sustained Hh activation. In the primary screening, we used stemloop RT-PCR to test the expression of 90 different miRNAs in the wildtype （WT） and Ptc-/- MEF cell lines. In the secondary screening, the miRNAs screened from the first screening were validated in the Sufu-/- MEF cell lines. We then verified the expression of miRNAs both in the normal cerebellar tissues and the MB induced by activated Hh pathway, and examined the expression of the other 21 miRNA members of the miR-154 cluster in the MB and normal cerebellum. In the first screening, 13 miRNAs showed significant differential expression in WT and Ptc-/- MEF cell lines, while 10 of them had significant difference in the Sufu-/- MEF cell line. Compared to the normal mouse cerebellum, only 2 miRNAs in 15 miRNAs were differentially expressed between the MB and normal cerebellar tissues. Among 21 members of the miR-154 cluster, 6 miRNAs were downregulated in the MB. Our study demonstrated that miR-154 may be regulated by the Hh pathway, and the activation of the Hh pathway led to the downregulation of the miR-154 cluster, resulting in the genesis of MB.

Research Progress of PTCH1 Gene in Lung cancer

Background:The PTCH1 gene,also known as Patched 1,is located on the long arm of human chromosome 9(9q22.3).It encodes the PTCH1 protein,which is a critical transmembrane receptor within the Hedgehog signaling pathway(Hh),playing a pivotal role in cellular communication and developmental processes.Recent studies have highlighted the significance of mutations in PTCH1 in the pathogenesis of lung cancer,positioning it as a crucial molecule for investigation in oncology.Purpose:This review aims to elucidate the role of the PTCH1 and the Hedgehog pathway in the initiation,progression,and potential treatment of lung cancer,thereby providing a theoretical foundation for personalized and precise therapeutic strategies.Method:To ensure a comprehensive review,this study systematically searched for literature related to the PTCH1,lung cancer,and the Hedgehog pathway across multiple databases including PubMed,Web of Science,and CNKI(China National Knowledge Infrastructure).The search strategy involved using specific keywords and advanced filtering options to include the most relevant and recent studies.Initial screening excluded irrelevant articles,followed by a detailed evaluation of the selected studies based on their scientific quality and relevance.Results:This review indicated that specific mutations in the PTCH1 gene are closely associated with the onset and progression of lung cancer.These mutations impede normal Hedgehog signaling,leading to unregulated cell proliferation and tumor growth.Targeting PTCH1,including vismodegib,have shown efficacy in clinical cases,particularly in SCCL with specific PTCH1 mutations,leading to complete remissions.Furthermore,the interaction between PTCH1 and microRNA-212 suggests potential therapeutic approaches by targeting miRNA to regulate PTCH1 expression.In addition,the investigation of traditional Chinese medicines such as Ginsenosides and Cordyceps sinensis extracts has shown their potential to modulate the Hedgehog pathway and reverse drug resistance.Conclusions:An in-depth understanding of the precise mechanisms by which PTCH1 mutations promote lung cancer could facilitate the development of targeted therapies.This study highlights the potential of PTCH1 as a biomarker for diagnosis and a target for precision medicine in lung cancer treatment,advocating for further research into its molecular pathways and therapeutic applications.

Sonic hedgehog signaling:Alternative splicing and pathogenic role in medulloblastoma

Alternative splicing(AS)produces the different mRNA splicing bodies,which are then translated into multiple protein isoforms and participate in various biological functions.With a deeper understanding of alternative splicing through the study of transcriptomes using high-throughput sequencing-based methods,the correlation between aberrant AS and diseases triggered a great concern,especially abnormal AS and cancer.Medulloblastoma(MB)is an intracranial tumor in children.Sonic hedgehog MB(SHH-MB)accounted for approximately 30%of MB,which is associated with the activation of SHH signaling.Growing evidence shows that aberrant AS is closely related to the tumorigenesis of MB.Here,we briefly introduced the AS and its mechanism.Next,we described canonical/noncanonical hedgehog signaling and its correlation with MB.The main description focused on AS of various regulators in canonical hedgehog signaling in MB.In addition,we also described AS of various regulators in noncanonical hedgehog signaling.Meanwhile,activated hedgehog signaling also induces AS in MB.Then,we pointed out that aberrant AS of hedgehog signaling is associated with different MB subgroups.Finally,we summarized the therapeutic applications of targeted AS in cancer treatment.In summary,further understanding of AS in SHH-MB could develop therapeutic targets for splicing factors which may be a novel therapeutic strategy.

Inhibition of the Hedgehog Signaling Pathway Depresses the Cigarette Smoke-Induced Malignant Transformation of 16HBE Cells on a Microfluidic Chip

Background：The hedgehog signaling system （HHS） plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases.However,little is known about the involvement of HHS in the malignant transformation of cells.This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial （16HBE） cells.Methods：In this study,two microfluidic chips were designed to investigate cigarette smoke extract （CSE）-induced malignant transformation of cells.Chip A contained a concentration gradient generator,while chip B had four cell chambers with a central channel.The 16HBE cells cultured in chip A were used to determine the optimal concentration of CSE for inducing malignant transformation.The 16HBE cells in chip B were cultured with 12.25% CSE （Group A）,12.25% CSE ± 5 μmol/L cyclopamine （Group B）,or normal complete medium as control for 8 months （Group C）,to establish the in vitro lung inflammatory-cancer transformation model.The transformed cells were inoculated into 20 nude mice as cells alone （Group 1） or cells with cyclopamine （Group 2） for tumorigenesis testing.Expression of HHS proteins was detected by Western blot.Data were expressed as mean ± standard deviation.The t-test was used for paired samples,and the difference among groups was analyzed using a one-way analysis of variance.Results：The optimal concentration of CSE was 12.25%.Expression of HHS proteins increased during the process of malignant transformation （Group B vs.Group A,F =7.65,P 〈 0.05）.After CSE exposure for 8 months,there were significant changes in cellular morphology,which allowed the transformed cells to grow into tumors in 40 days after being inoculated into nude mice.Cyclopamine could effectively depress the expression of HHS proteins （Group C vs.Group B,F =6.47,P 〈 0.05） and prevent tumor growth in nude mice （Group 2 vs.Group 1,t=31.59,P〈 0.01）.Conclusions：The activity of HHS is upregulated during the CSE-induced malignant transformation of 16HBE cells.Cyclopamine can effectively depress expression of HHS proteins in vitro and prevent tumor growth of the transformed cells in vivo.