Background:Neuroblastoma(NB)is a malignant pediatric tumor requiring new therapies.Accumulating evidence has confirmed that microRNAs play critical roles in NBmetastasis.Dihydroartemisinin(DHA)is capable of inhibiting...Background:Neuroblastoma(NB)is a malignant pediatric tumor requiring new therapies.Accumulating evidence has confirmed that microRNAs play critical roles in NBmetastasis.Dihydroartemisinin(DHA)is capable of inhibiting the growth of NB cells.The primary objective of the current investigation was to characterize a newly discovered microRNA,miR-32-5p,in terms of the functional role,underlying mechanism of action,and potential synergistic therapeutic impact in the context of NB metastasis.Materials and methods:Real-time quantitative polymerase chain reaction and Western blotting were employed to assess the expression levels of miR-32-5p and its target,vacuolar protein sorting 4B(VPS4B).Furthermore,Transwell assay was utilized to evaluate in vitro cell migration and invasion,whereas a metastasis xenograft model was established in nude mice via caudal vein injections.Results:Gene Expression Omnibus database and real-time quantitative polymerase chain reaction analysis showed that miR-32-5p was downregulated in human NB samples and NB cell lines,in comparison with the normal tissue and cell lines.Inhibiting miR-32-5p induced the migration and invasion of NB cells,whereas overexpression of miR-32-5p prevented the migration and invasion in NB cell lines.Furthermore,VPS4B was identified as the direct target ofmiR-32-5p and themiR-32-5p reduction associated with NB metastasis upregulated the expression of VPS4B.Conversely,overexpression of VPS4B reversed the suppressive effects ofmiR-32-5p onNB cells.Moreover,miR-32-5p increased the sensitivity to DHA both in NB cells and in the metastasis xenograft model of nude mice.Conclusions:The downregulation of miR-32-5p in NB regulates NB metastasis by targeting VPS4B.Moreover,miR-32-5b can improve the sensitivity of DHA in the xenograft mouse model.Our findings have important implications for the combined application of miR-32-5p and DHA in the treatment of NB.展开更多
Background:Nuclear Yes1-associated transcriptional regulator(YAP1)promotes tumor progression.However,the function of cytoplasmic YAP1 in breast cancer cells and its impact on the survival of breast cancer patients rem...Background:Nuclear Yes1-associated transcriptional regulator(YAP1)promotes tumor progression.However,the function of cytoplasmic YAP1 in breast cancer cells and its impact on the survival of breast cancer patients remain unclear.Our research aimed to explore the biological function of cytoplasmic YAP1 in breast cancer cells and the possibility of cytoplasmic YAP1 as a predictive marker of breast cancer survival.Methods:We constructed cell mutant models,including NLS-YAP15SA(nuclear localized),YAP1S94A(incapable of binding to the TEA domain transcription factor family)and YAP1S127D(cytoplasmic localized),and used Cell Counting Kit-8(CCK-8)assays,5-ethynyl-2’-deoxyuridine(EdU)incorporation assays,and Western blotting(WB)analysis to detect cell proliferation and apoptosis.The specific mechanism of cytoplasmic YAP1-mediated endosomal sorting complexes required for transport III(ESCRT-III)assembly was studied by co-immunoprecipitation,immunofluorescence staining,and WB analysis.Epigallocatechin gallate(EGCG)was used to simulate YAP1 retention in the cytoplasm in in vitro and in vivo experiments to study the function of cytoplasmic YAP1.YAP1 binding to NEDD4-like E3 ubiquitin protein ligase(NEDD4L)was identified using mass spectrometry and was verified in vitro.Breast tissue microarrays were used to analyze the relationship between cytoplasmic YAP1 expression and the survival of breast cancer patients.Results:YAP1 was mainly expressed in the cytoplasm in breast cancer cells.Cytoplasmic YAP1 promoted autophagic death of breast cancer cells.Cytoplasmic YAP1 bound to the ESCRT-III complex subunits charged multivesicular body protein 2B(CHMP2B)and vacuolar protein sorting 4 homolog B(VPS4B),promoting assembly of CHMP2B-VPS4B and activating autophagosome formation.EGCG retained YAP1 in the cytoplasm,promoting the assembly of CHMP2B-VPS4B to promote autophagic death of breast cancer cells.YAP1 bound to NEDD4L,and NEDD4L mediated ubiquitination and degradation of YAP1.Breast tissue microarrays revealed that high levels of cytoplasmic YAP1 were beneficial to the survival of breast cancer patients.Conclusions:Cytoplasmic YAP1 mediated autophagic death of breast cancer cells by promoting assembly of the ESCRT-III complex;furthermore,we established a new breast cancer survival prediction model based on cytoplasmic YAP1 expression.展开更多
基金supported by the Fundamental Research Funds for the Central PublicWelfare Research Institutes(grant ZZ13-YQ-100)the NationalNatural Science Foundation of China(grants 82274181 and 82141001)+1 种基金Innovation Fund of China Academy of Chinese Medical Sciences(grantsCI2021A05106,CI2021A04611,andZZ16-ND-10-01)Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences(CI2021B015,CI2023E001TS12).
文摘Background:Neuroblastoma(NB)is a malignant pediatric tumor requiring new therapies.Accumulating evidence has confirmed that microRNAs play critical roles in NBmetastasis.Dihydroartemisinin(DHA)is capable of inhibiting the growth of NB cells.The primary objective of the current investigation was to characterize a newly discovered microRNA,miR-32-5p,in terms of the functional role,underlying mechanism of action,and potential synergistic therapeutic impact in the context of NB metastasis.Materials and methods:Real-time quantitative polymerase chain reaction and Western blotting were employed to assess the expression levels of miR-32-5p and its target,vacuolar protein sorting 4B(VPS4B).Furthermore,Transwell assay was utilized to evaluate in vitro cell migration and invasion,whereas a metastasis xenograft model was established in nude mice via caudal vein injections.Results:Gene Expression Omnibus database and real-time quantitative polymerase chain reaction analysis showed that miR-32-5p was downregulated in human NB samples and NB cell lines,in comparison with the normal tissue and cell lines.Inhibiting miR-32-5p induced the migration and invasion of NB cells,whereas overexpression of miR-32-5p prevented the migration and invasion in NB cell lines.Furthermore,VPS4B was identified as the direct target ofmiR-32-5p and themiR-32-5p reduction associated with NB metastasis upregulated the expression of VPS4B.Conversely,overexpression of VPS4B reversed the suppressive effects ofmiR-32-5p onNB cells.Moreover,miR-32-5p increased the sensitivity to DHA both in NB cells and in the metastasis xenograft model of nude mice.Conclusions:The downregulation of miR-32-5p in NB regulates NB metastasis by targeting VPS4B.Moreover,miR-32-5b can improve the sensitivity of DHA in the xenograft mouse model.Our findings have important implications for the combined application of miR-32-5p and DHA in the treatment of NB.
基金National Natural Science Foundation of China,Grant/Award Numbers:81573001,81773295Haiyan Research Fund Project of Harbin Medical University Cancer Hospital,Grant/Award Number:JJZD2023-04Beijing Kechuang Medical Development Foundation,Grant/Award Number:KC2021-JF-0055-06。
文摘Background:Nuclear Yes1-associated transcriptional regulator(YAP1)promotes tumor progression.However,the function of cytoplasmic YAP1 in breast cancer cells and its impact on the survival of breast cancer patients remain unclear.Our research aimed to explore the biological function of cytoplasmic YAP1 in breast cancer cells and the possibility of cytoplasmic YAP1 as a predictive marker of breast cancer survival.Methods:We constructed cell mutant models,including NLS-YAP15SA(nuclear localized),YAP1S94A(incapable of binding to the TEA domain transcription factor family)and YAP1S127D(cytoplasmic localized),and used Cell Counting Kit-8(CCK-8)assays,5-ethynyl-2’-deoxyuridine(EdU)incorporation assays,and Western blotting(WB)analysis to detect cell proliferation and apoptosis.The specific mechanism of cytoplasmic YAP1-mediated endosomal sorting complexes required for transport III(ESCRT-III)assembly was studied by co-immunoprecipitation,immunofluorescence staining,and WB analysis.Epigallocatechin gallate(EGCG)was used to simulate YAP1 retention in the cytoplasm in in vitro and in vivo experiments to study the function of cytoplasmic YAP1.YAP1 binding to NEDD4-like E3 ubiquitin protein ligase(NEDD4L)was identified using mass spectrometry and was verified in vitro.Breast tissue microarrays were used to analyze the relationship between cytoplasmic YAP1 expression and the survival of breast cancer patients.Results:YAP1 was mainly expressed in the cytoplasm in breast cancer cells.Cytoplasmic YAP1 promoted autophagic death of breast cancer cells.Cytoplasmic YAP1 bound to the ESCRT-III complex subunits charged multivesicular body protein 2B(CHMP2B)and vacuolar protein sorting 4 homolog B(VPS4B),promoting assembly of CHMP2B-VPS4B and activating autophagosome formation.EGCG retained YAP1 in the cytoplasm,promoting the assembly of CHMP2B-VPS4B to promote autophagic death of breast cancer cells.YAP1 bound to NEDD4L,and NEDD4L mediated ubiquitination and degradation of YAP1.Breast tissue microarrays revealed that high levels of cytoplasmic YAP1 were beneficial to the survival of breast cancer patients.Conclusions:Cytoplasmic YAP1 mediated autophagic death of breast cancer cells by promoting assembly of the ESCRT-III complex;furthermore,we established a new breast cancer survival prediction model based on cytoplasmic YAP1 expression.
