Studies Of The Mechanism Of EMT Induced By TGF-β1 In Neuroblastoma Cells

Neuroblastoma is the most common extracranial malignant solid tumor that occurs in childhood.Neuroblastoma is a high-grade malignancy.Many of the patients are diagnosed as middle an advanced stage due to appearring insidiously.The long-term outcome of children with neuroblastoma which are classified as high risk remains as poor as 30%,despite the administration of aggressive multimodal therapy.Metastasis is one of the major causes of death in neuroblastoma patients.Metastasis of tumor has been shown to be associated with the acquisition of epithelial-mesenchymal transition(EMT)which can promote the loss of tight junctions between cancer cells,increase the metastasis and invasion of cancer cells and inhibition of cells apoptosis.The regulatory mechanism of EMT by tumor cells is complex,and it is involved in cell microenvironment and various cytokines signal transduction.EMT induced by TGF-β(transforming growth factor-β)may be through Smad dependent signaling pathways and non Smad dependent signaling pathways.However,the regulatory mechanism of EMT induced by TGF-β is not yet clear,thus why we conducted the following research:Objective: To analyse the correlation of clinical features and prognosis inchildhood neuroblastoma.To investigate the molecular mechanism of transcription factor Gli in EMT induced by TGF-β1 in SK-N-SH cells.Study on the ability of subcutane transplantation in nude mice by SK-N-SH cells induced by TGF-β1.Methods: 59 patients with neuroblastoma that were diagnosed between July 1,2008 and June 30,2013 at Shanghai Children’s Hospital were enrolled in the present study.The follow up was performed until December 31,2013.The patients were divided into low risk,middle risk and high risk groups depended on age and tumor stage,treated with surgery,chemotherapy and 13-cis-retinoic acid.Analysing the clinical features and the expression of E-cadherin in the pathology with the prognosis in these patients.TGF-β1 was applied to SK-N-SH,and changes in cell morphology were observed.Cellular immunofluorescence was used to detect the expression of EMT-related markers.Real time PCR assays and Western Blots were used to detect the mRNA and protein expression levels of EMT-related genes and proteins.A scratch test and migration assay were performed to verify that the EMT could alter the SK-N-SH cell migration capacity.Elucidate the mechanism of Smad signaling pathway in EMT induced by TGF-β1 through over expression and interference of Smad2/3 in SK-N-SH cells.Detected the expression of transcription factor Gli in SK-N-SH cells and the expression changes of cell proliferation,cell apoptosis,EMT key molecules induced by TGF-β1 after SK-N-SH cells treated with GANT61 and siRNA.Human neuroblastoma SK-N-SH cells were used to establish transplanted subcutaneously tumor in nude mice as control group,while SK-N-SH cells induced by TGF-β1 as experimental group.The tumorigenicity rates and capability of tumors between two groups were compared.Results:(1)It was found that 44 patients(44/59;74.58%)achieved CR or PR,8 patients(8/59;13.56%)experienced relapse or metastasis and 7 patients(7/59;11.86%)discontinued treatment or follow up.The 3-year overall survival(OS)rate of patients with stage Ⅰ, Ⅱ, Ⅲ,Ⅳ and Ⅳs disease was 100,100,65.6,34.8 and 85.7%,respectively(P=0.0201).Univariate statistical analysis revealed that the factors significantly associated with prognosis were patient age,tumor stage and risk group(P=0.004,P=0.013,P=0.001).The expression of E-cadherin decreased in stage IV neuroblastoma patients.The expression of E-cadherin was significantly decreased in association with patients’ recurrence/ metastasis(P=0.047).(2)SK-N-SH cells exhibited irregular shapes,including spindle,circular,and polygonal shapes.After 14 days of treatment,most of the cells exhibited a pronounced spindle shape.Immunofluorescence detection revealed that SK-N-SH cells expressed the EMT markers E-cadherin and α-smooth muscle actin(α-SMA).After SK-N-SH cells were treated with different concentration of TGF-β1 for three days,Real-time PCR assays and Western Blots showed that the mRNA and protein expression levels of E-cadherin were significantly decreased(P=0.0353,P=0.0006),while the mRNA and protein expression levels of α-SMA were significantly increased(P<0.0001,P=0.0007).After SK-N-SH cells were treated with TGF-β1 for three days,a scratch test and transwell migration assay revealed that cell migration was increased significantly with increasingly higher concentrations of TGF-β1.(3)The expression of Smad2 and Smad3 were not increased in EMT induced by TGF-β1 in SK-N-SH cells.The EMT phenotype didn’t been promoted by transfected with Smad2 and Smad3.Interference the expression of Smad2 and Smad3 did not affect the EMT induced by TGF-β1 in SK-N-SH cells.SK-N-SH cells existed the positive expression of transcription factor Gli.GANT61 was a kind of targeted inhibitor which targetting transcription factor Gli1/2,reducing the cell proliferation and promoting the apoptosis of SK-N-SH cells.The expression of transcription factor Gli in EMT induced by TGF-β1 increased significantly,while over expression or interference of Smad2/3 did not affect the increasing expression.Interfering with the expression of Gli1/Gli2 can inhibit the EMT induced by TGF-β1 in SK-N-SH cells.(4)A total observation of two months after SK-N-SH cells were used to establish transplanted subcutaneously tumor in nude mice.The tumorigenicity rate of control group was 25%(2/8)and 85.71%(6/7)of experimental group respectively.There were significant differences in the tumorigenicity rate of two groups(P=0.019).The sizes of subcutaneous tumor were bigger in experimental group than control group.The tumor sizes between the two groups at seventh and eighth weeks were significant difference(P=0.02、P=0.019).The tumor tissue showed small blue round cell consistently by HE staining.The positive neuron specific enolase(NSE)staining showed brown yellow particles mainly in the cytoplasm.The diagnosis of neuroblastoma was confirmed by pathology.Conclusion: The present study identified that patient age,tumor stage and risk group are important prognostic factors for neuroblastoma patients.Relapse and metastasis are the main causes of death in neuroblastoma patients.The decrease expression of E-cadherin was significantly associated with patients’ recurrence/ metastasis.SK-N-SH cells express the EMT markers E-cadherin and α-SMA,and TGF-β1 can induce the EMT in SK-N-SH cells to increase SK-N-SH cell migration.The EMT induced by TGF-β1 in SK-N-SH cells does not depend on the Smad signaling pathway.Transcription factor Gli participates in the EMT induced by TGF-β1 in SK-N-SH cells through non Smad dependent signaling pathways.TGF-β1 significantly enhanced the ability of human neuroblastoma SK-N-SH cell tumorigenicity in nude mice.