Lysine-specific demethylase 4A(KDM4A)catalyzes demethylation of histone lysine residues,which regulates chromatin state and transcription.In drosophila and mice,KDM4A plays an important role in multiple biological pro...Lysine-specific demethylase 4A(KDM4A)catalyzes demethylation of histone lysine residues,which regulates chromatin state and transcription.In drosophila and mice,KDM4A plays an important role in multiple biological processes including development,aging,metabolism,and immunity,however the functions of KDM4A in fish are still unclear.There are two copies of the kdm4a gene in zebrafish,namely kdm4aa and kdm4ab,kdm4aa was edited using CRISPR/Cas9 technology in the present study,then homozygous kdm4aa mutants(kdm4aa^(-/-))were obtained,and loss of kdm4aa was confirmed by sequencing and increased H3K9me3.Whole-mount in situ hybridization showed that kdm4aa is widely expressed during the embryonic development of zebrafish.Compared with WT zebrafish,kdm4aa^(-/-)zebrafish showed no significant difference in gamete formation and fertilization,but the survival rate of kdm4aa^(-/-)embryos dramatically reduced to 21%at 26 hpf.Further observation showed that about 80%of survived kdm4aa^(-/-)zebrafish experienced disruption in stripe formation,and 10%of survived kdm4aa^(-/-)zebrafish underwent vertebral malformation.Alizarin red S staining demonstrated the abnormal spinal development in kdm4aa^(-/-)zebrafish.These results indicated that kdm4aa is required for normal embryonic development of zebrafish,loss of kdm4aa function leads to decreased survival during the early stages of zebrafish development and morphological variation in adult zebrafish.展开更多
Background:Cancer stem-like cells(CSCs)are a small subset of cells in tumors that exhibit self-renewal and differentiation properties.CSCs play a vital role in tumor formation,progression,relapse,and therapeutic resis...Background:Cancer stem-like cells(CSCs)are a small subset of cells in tumors that exhibit self-renewal and differentiation properties.CSCs play a vital role in tumor formation,progression,relapse,and therapeutic resistance.B7-H3,an immunoregulatory protein,has many protumor functions.However,little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer(GC)stemness.Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism.Methods:GC stemness influenced by B7-H3 was detected both in vitro and in vivo.The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction,Western blotting,and flow cytometry.Sphere formation assay was used to detect the sphere-forming ability.The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments.The signaling pathway(Protein kinase B[Akt]/Nuclear factor erythroid 2-related factor 2[Nrf2]pathway)of B7-H3 on the regulation of glutathione(GSH)metabolism was examined by Western blotting assay.Multi-color immunohistochemistry(mIHC)was used to detect the expression of B7-H3,cluster of differentiation 44(CD44),and Nrf2 on human GC tissues.Student’s t-test was used to compare the difference between two groups.Pearson correlation analysis was used to analyze the relationship between two molecules.The Kaplan-Meier method was used for survival analysis.Results:B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo.Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells,which was further confirmed by the experimental results.Meanwhile,stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism.Furthermore,Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway,and inhibition of AKT signaling pathway could suppress not o展开更多
基金from Shanghai Agriculture Applied Technology Development Program,China(Grant No.G20190102)National Natural Science Foundation of China(Grant No.81770165 to BH)Shanghai Municipal Education Commission(Project for Gaofeng Discipline of Fishery).
文摘Lysine-specific demethylase 4A(KDM4A)catalyzes demethylation of histone lysine residues,which regulates chromatin state and transcription.In drosophila and mice,KDM4A plays an important role in multiple biological processes including development,aging,metabolism,and immunity,however the functions of KDM4A in fish are still unclear.There are two copies of the kdm4a gene in zebrafish,namely kdm4aa and kdm4ab,kdm4aa was edited using CRISPR/Cas9 technology in the present study,then homozygous kdm4aa mutants(kdm4aa^(-/-))were obtained,and loss of kdm4aa was confirmed by sequencing and increased H3K9me3.Whole-mount in situ hybridization showed that kdm4aa is widely expressed during the embryonic development of zebrafish.Compared with WT zebrafish,kdm4aa^(-/-)zebrafish showed no significant difference in gamete formation and fertilization,but the survival rate of kdm4aa^(-/-)embryos dramatically reduced to 21%at 26 hpf.Further observation showed that about 80%of survived kdm4aa^(-/-)zebrafish experienced disruption in stripe formation,and 10%of survived kdm4aa^(-/-)zebrafish underwent vertebral malformation.Alizarin red S staining demonstrated the abnormal spinal development in kdm4aa^(-/-)zebrafish.These results indicated that kdm4aa is required for normal embryonic development of zebrafish,loss of kdm4aa function leads to decreased survival during the early stages of zebrafish development and morphological variation in adult zebrafish.
基金the National Natural Science Foundation of China(U20A2009,41991234,42077422 and 41725003)the National Key Research and Development Program of China(2022YFF1301801)the Major Science and Technology Projects in Tibet(XZ202101ZD0007G and XZ202101ZD0003N).
基金supported by Suzhou Special Project on Clinical Key Diseases Treatment Technology of Suzhou Commission of Health(No.LCZX201803)People’s Livelihood and Science and Technology project of Department of Science and Technology of Suzhou(No.SS2019059)+5 种基金Jiangsu Provincial Medical Key Discipline(No.ZDXK202246)Key Project of Jiangsu Provincial Health Commission(No.zd2021050)the Natural Science Foundation of the Jiangsu Higher Education Institutions of China(No.20KJA310005)Key Project of Medical Research of Jiangsu Commission of Health(No.ZDA2020008)National Natural Science Foundation of China(No.81802843)Social Development Project of Department of Science and Technology of Jiangsu Province(No.BE2019667)
文摘Background:Cancer stem-like cells(CSCs)are a small subset of cells in tumors that exhibit self-renewal and differentiation properties.CSCs play a vital role in tumor formation,progression,relapse,and therapeutic resistance.B7-H3,an immunoregulatory protein,has many protumor functions.However,little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer(GC)stemness.Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism.Methods:GC stemness influenced by B7-H3 was detected both in vitro and in vivo.The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction,Western blotting,and flow cytometry.Sphere formation assay was used to detect the sphere-forming ability.The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments.The signaling pathway(Protein kinase B[Akt]/Nuclear factor erythroid 2-related factor 2[Nrf2]pathway)of B7-H3 on the regulation of glutathione(GSH)metabolism was examined by Western blotting assay.Multi-color immunohistochemistry(mIHC)was used to detect the expression of B7-H3,cluster of differentiation 44(CD44),and Nrf2 on human GC tissues.Student’s t-test was used to compare the difference between two groups.Pearson correlation analysis was used to analyze the relationship between two molecules.The Kaplan-Meier method was used for survival analysis.Results:B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo.Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells,which was further confirmed by the experimental results.Meanwhile,stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism.Furthermore,Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway,and inhibition of AKT signaling pathway could suppress not o
