Dendritic cells(DCs)are the most important antigen-presenting cells due to their professional and extremely efficient antigen-presenting function.In view of their exceptional ability to present antigen and to interact...Dendritic cells(DCs)are the most important antigen-presenting cells due to their professional and extremely efficient antigen-presenting function.In view of their exceptional ability to present antigen and to interact with T cells,DC play distinct roles in linking innate and adaptive immune responses and thus become logical targets for cancer immunotherapy.Evidences show that tumor-derived cytokines could impair DCs’biomechanics properties,which lead to inefficacy of DCs-based immune therapies.Our previous studies found that IL-10,as one of the widespread suppressive cytokines from tumor microenvironment(TME),could deteriorate DCs’motility and biomechanics properties while the underlying mechanism is unknown.In this study,CD14+monocytes were induced to differentiate into mature dendritic cells after isolation in vitro using recombinant cytokines IL-4,GM-CSF,LPS,and IFN-γ.And we also have compared the proteomic changes of mDCs treated by IL-10 and control group via two-dimensional electrophoresis combine with MALDI-TOF/TOF MS.Then we analyzed the function of differentially expressed proteins through bioinformatics methods include GO analysis that clarified the biological functions of differential proteins and KEGG analysis which enriched signal pathways of differential proteins to explore the molecular mechanism of IL-10 which has inhibitory effect on mDCs.The results showed that IL-10 significantly affected the morphology of mDCs,especially reducing the number and length of filopodia.Different expressed proteins were analyzed by two-dimensional electrophoresis combined with bioinformatics analysis to enrich for glycolytic signaling pathway,HIF-1 signaling pathway and cytoskeletal binding protein expression changes.The results of two-dimensional electrophoresis were verified by Western blot,and the results showed that the data were reliable.In addition,the intracellular ROS levels were significantly higher in mDCs treated with IL-10,validating the previously enriched HIF-1 signaling pathway.In summary,it indicated that IL-10 may interfered with the oxidative metabolic process,glycolytic metabolism,and expression of cytoskeleton-related proteins in mDCs,and disturbances in these physiological processes resulted in reduced biomechanics properties and motility of mDCs and subsequently impaired their immune functions,making DC-based tumor vaccines less effective than which we desired.Our study reveals alterations in the physiological metabolism of mDCs under IL-10 treatment from the proteome,which lays the foundation for further exploration of the altered state of mDCs in the tumor microenvironment.展开更多
OBJECTIVE To investigate the effect of microR NA-10a on the development of granulosa cells tumor(GCT).METHODS FISH was used to detect the miR-10a expression in tissues from GCT patients.Several functional assays were ...OBJECTIVE To investigate the effect of microR NA-10a on the development of granulosa cells tumor(GCT).METHODS FISH was used to detect the miR-10a expression in tissues from GCT patients.Several functional assays were performed to investigate the effect of miR-10a on proliferation,migration,invasion,spheroid formation and repressed anticancer drug-induced apoptosis of GCT in vitro.CRISPR-Cas9 system mediated miR-10a knockout in cancer GC and two mice GCT models were constructed to show the knockdown effect of miR-10a on cancer GC both in vitro and in vivo.RNA-seq,Western blot,luciferase reporter assay and FISH were used to identify potential direct functional targets and related pathways of miR-10a in cancer GC.RESULTS Strong miR-10a signal was detected in tissues from malignant GCT patients.And amplification of miR-10a negatively correlated with overall survival rate of ovarian cancer patients.In addition,ectopic expression of miR-10a significantly promoted cell proliferation,migration,invasion,spheroid formation and repressed anticancer drug-induced apoptosis in vitro.CRISPR-Cas9 system mediated miR-10a knockout in cancer GC showed opposite phenotype compared to miR-10a overexpressed cancer GC.By using xenograft and orthotropic models,the oncogenic role of miR-10a was further confirmed in vivo.RNA-seq,Western blot,luciferase reporter assay and FISH were used to identified PTEN/TET2 as direct functional targets of miR-10a in cancer GC;Akt and Wnt were found as two associated signaling pathways of miR-10a in cancer GC.CONCLUSION Taken together,our results demonstrate that the miR-10a is positively involved indevelopment of GCT.展开更多
基金funded by the National Natural Science Foundation of China ( 31660258,31771014, 31860262,11762006)the Science and Technology Foundation of Guizhou Province ( 2019-2787,2018-1412,2016-5676, 2017-5718)+2 种基金the Science and Technology Innovative Talent Team of Guizhou Province ( 2015-4021)the 2011 Collaborative Innovation Program of Guizhou Province ( 2015-04)the Cell and Gene Engineering Innovative Research Groups of Guizhou Province ( KY-2016-031)
文摘Dendritic cells(DCs)are the most important antigen-presenting cells due to their professional and extremely efficient antigen-presenting function.In view of their exceptional ability to present antigen and to interact with T cells,DC play distinct roles in linking innate and adaptive immune responses and thus become logical targets for cancer immunotherapy.Evidences show that tumor-derived cytokines could impair DCs’biomechanics properties,which lead to inefficacy of DCs-based immune therapies.Our previous studies found that IL-10,as one of the widespread suppressive cytokines from tumor microenvironment(TME),could deteriorate DCs’motility and biomechanics properties while the underlying mechanism is unknown.In this study,CD14+monocytes were induced to differentiate into mature dendritic cells after isolation in vitro using recombinant cytokines IL-4,GM-CSF,LPS,and IFN-γ.And we also have compared the proteomic changes of mDCs treated by IL-10 and control group via two-dimensional electrophoresis combine with MALDI-TOF/TOF MS.Then we analyzed the function of differentially expressed proteins through bioinformatics methods include GO analysis that clarified the biological functions of differential proteins and KEGG analysis which enriched signal pathways of differential proteins to explore the molecular mechanism of IL-10 which has inhibitory effect on mDCs.The results showed that IL-10 significantly affected the morphology of mDCs,especially reducing the number and length of filopodia.Different expressed proteins were analyzed by two-dimensional electrophoresis combined with bioinformatics analysis to enrich for glycolytic signaling pathway,HIF-1 signaling pathway and cytoskeletal binding protein expression changes.The results of two-dimensional electrophoresis were verified by Western blot,and the results showed that the data were reliable.In addition,the intracellular ROS levels were significantly higher in mDCs treated with IL-10,validating the previously enriched HIF-1 signaling pathway.In summary,it indicated that IL-10 may interfered with the oxidative metabolic process,glycolytic metabolism,and expression of cytoskeleton-related proteins in mDCs,and disturbances in these physiological processes resulted in reduced biomechanics properties and motility of mDCs and subsequently impaired their immune functions,making DC-based tumor vaccines less effective than which we desired.Our study reveals alterations in the physiological metabolism of mDCs under IL-10 treatment from the proteome,which lays the foundation for further exploration of the altered state of mDCs in the tumor microenvironment.
基金supported by Natural Science Foundation of Anhui Province for Young Scholars(1708085QH200)
文摘OBJECTIVE To investigate the effect of microR NA-10a on the development of granulosa cells tumor(GCT).METHODS FISH was used to detect the miR-10a expression in tissues from GCT patients.Several functional assays were performed to investigate the effect of miR-10a on proliferation,migration,invasion,spheroid formation and repressed anticancer drug-induced apoptosis of GCT in vitro.CRISPR-Cas9 system mediated miR-10a knockout in cancer GC and two mice GCT models were constructed to show the knockdown effect of miR-10a on cancer GC both in vitro and in vivo.RNA-seq,Western blot,luciferase reporter assay and FISH were used to identify potential direct functional targets and related pathways of miR-10a in cancer GC.RESULTS Strong miR-10a signal was detected in tissues from malignant GCT patients.And amplification of miR-10a negatively correlated with overall survival rate of ovarian cancer patients.In addition,ectopic expression of miR-10a significantly promoted cell proliferation,migration,invasion,spheroid formation and repressed anticancer drug-induced apoptosis in vitro.CRISPR-Cas9 system mediated miR-10a knockout in cancer GC showed opposite phenotype compared to miR-10a overexpressed cancer GC.By using xenograft and orthotropic models,the oncogenic role of miR-10a was further confirmed in vivo.RNA-seq,Western blot,luciferase reporter assay and FISH were used to identified PTEN/TET2 as direct functional targets of miR-10a in cancer GC;Akt and Wnt were found as two associated signaling pathways of miR-10a in cancer GC.CONCLUSION Taken together,our results demonstrate that the miR-10a is positively involved indevelopment of GCT.
