The Experimental Study On The Role And The Mechanism Of FOXO1 In Fusion Negtive Rhabdomyosarcoma(FNRMS)

ObjectiveRhabdomyosarcoma(RMS),a malignant cancer with a mesenchymal origin,is one of the most common soft-tissue sarcomas,accounting for 5–8% of malignant tumours in children and adolescents.According to gene mutation and expression,RMS can be broadly classified as PAX3/7-FOXO1 fusion-positive(FPRMS)or fusion-negative tumours(FNRMS).FPRMS is associated with poor prognosis.Despite better prognosis in FNRMS,traditional chemotherapy and radiotherapy for children and adolescents have a severe impact on their physical and mental health.In addition,patients with recurrence and metastasis have a poor prognosis.However,the underlying molecular mechanisms and driving factors are unclear.This research aims to study the biological role of FOXO1 in FNRMS and its related molecular mechanisms,so as to develop novel molecule-targeted therapies in FNRMS in the future.Methods1.To assess the expression of FOXO1 in FNRMS tissues and cells by immunohistochemistry,RT-qPCR and Western Blot immunoblotting.To determine localization of FOXO1 by immunofluorescence staining and Western Blot immunoblotting followed by subcellular fractionation;2.To investigate the effect of FOXO1 in FNRMS using sh RNA expressing lentiviral vectors,FOXO1ER-overexpressing lentiviral vectors and FOXO1 inhibitor AS1842856.To address cell-cycle progression and apoptosis by flow cytometry as well;3.To determine the effect of FOXO1 on tumor growth in vivo by mouse xenograft assay after genetic and pharmacological inactivation of FOXO1;4.To explore molecular mechanisms and the target of FOXO1,we conducted RNA sequence to analyze gene expression of RD and SMS-CTR cell lines exposed to AS1842856 for 72 hours and after FOXO1 knockdown to identify transcriptome changes;5.We choosed several genes significantly downregulated in RD and SMS-CTR by the FOXO1 inactivation;then,some of these genes were overexpressed to observe whether the biological effects after knockdown of FOXO1 or pharmacological inhibition of FOXO1 could be rescued.Results1.The expression of FOXO1 was relatively high in FNRMS tissues and cells compared with normal tissues and human skeletal muscle cells.Then,we confirmed presence of FOXO1 mainly in the nuclei of FNRMS cells;2.Genetic inactivation or overexpression of FOXO1 in FNRMS cell lines produced a strong antiproliferative effect associated with G0/G1 cell cycle arrest.In addition,pharmacological inactivation of FOXO1 in FNRMS cell lines produced a strong antiproliferative effect associated with G0/G1 cell cycle arrest and increased cell apoptosis;3.Genetic and pharmacological inactivation of FOXO1 inhibited FNRMS growth in vivo;4.VGLL2 was downregulated after genetic and pharmacological inactivation of FOXO1 in FNRMS;5.Overexpression of VGLL2 partially rescued the effects after FOXO1 inactivation in FNRMS cell lines,including proliferation,cell cycle arrest and cell apoptosis.ConclusionThe expression of FOXO1 was relatively high in FNRMS tissues and cells compared with normal tissues and human skeletal muscle cells.Genetic and pharmacological inactivation of FOXO1 in FNRMS produced a strong antiproliferative effect through VGLL2 downregulation.In addition,overexpression of FOXO1 also induces growth inhibition in FNRMS.These results suggested the significance of a tight regulation of FOXO1 activity in FNRMS,and FOXO1 may become the new molecularly thrapeutic target for patients with FNRMS.