目的:探讨碱性核蛋白1(BNC1)对食管鳞状细胞癌(ESCC)细胞增殖、迁移、侵袭、细胞周期和凋亡的影响及其作用机制。方法:通过q PCR法检测ESCC细胞和正常食管上皮细胞中BNC1 m RNA的表达水平,免疫组织化学染色法检测10例ESCC患者癌及癌旁...目的:探讨碱性核蛋白1(BNC1)对食管鳞状细胞癌(ESCC)细胞增殖、迁移、侵袭、细胞周期和凋亡的影响及其作用机制。方法:通过q PCR法检测ESCC细胞和正常食管上皮细胞中BNC1 m RNA的表达水平,免疫组织化学染色法检测10例ESCC患者癌及癌旁组织中BNC1的蛋白表达水平。利用si RNA敲低BNC1在KYSE-150和KYSE-30细胞中的表达,CCK-8法、划痕愈合实验、Transwell实验和流式细胞术检测BNC1对细胞增殖、迁移、侵袭、细胞周期和凋亡等的影响。通过CHIP-seq实验和GEPIA在线网站数据分析并结合敲低BNC1后的转录组测序数据分析筛选BNC1调控的下游靶基因,q PCR法验证BNC1敲低后靶基因的表达变化,并用双荧光素酶报告基因实验验证BNC1对靶基因的调控作用。结果:BNC1 m RNA和蛋白水平在ESCC组织中较癌旁组织高表达(均P<0.01)。敲低BNC1可明显抑制KYSE-150、KYSE-30细胞的增殖、迁移和侵袭能力(P<0.05或P<0.01),将细胞阻滞于G1期并促进细胞的凋亡(均P<0.01)。CHIP-seq实验结果和在线网站GEPIA数据分析结合敲低BNC1后的转录组测序数据显示,G蛋白通路抑制因子1(GPS1)可能为BNC1正向调控的致癌靶基因。q PCR法和双荧光素酶报告基因实验结果显示,BNC1对GPS1有调控作用(P<0.01)。结论:BNC1在ESCC组织和细胞中高表达,干扰BNC1可显著抑制ESCC细胞的增殖、迁移和侵袭能力,阻滞细胞于G1期并促进细胞凋亡,其机制可能是BNC1通过靶向GPS1调控ESCC细胞的恶性生物学行为。展开更多
Basonuclin(BNC1)is expressed primarily in proliferative keratinocytes and gametogenic cells.However,its roles in spermatogenesis and testicular aging were not dear.Previously we discovered a heterozygous BNC1 truncati...Basonuclin(BNC1)is expressed primarily in proliferative keratinocytes and gametogenic cells.However,its roles in spermatogenesis and testicular aging were not dear.Previously we discovered a heterozygous BNC1 truncation mutation in a premature ovarian insufficiency pedigree.In this study,we found that male mice carrying the truncation mutation exhibited progressively fertility loss and testicular premature aging.Genome-wide expression profiling and direct binding studies(by chromatin immunoprecipitation sequencing)with BNC1 in mouse testis identified several spermatogenesis-specific gene promoters targeted by BNC1 including kelch-like family member 10(Klhl1O),testis expressed 14(Tex14)9 and spermatogenesis and centriole associated 1(Spatcl).Moreover,biochemical analysis showed that BNC1 was associated with TATA-box binding protein-associated factor 7 like(TAF7L),a germ cell-specific paralogue of the transcription factor IID subunit TAF7,both in vitro and in testis,suggesting that BNC1 might directly cooperate with TAF7L to regulate spermatogenesis.The truncation mutation disabled nuclear translocation of the BNC1/TAF7L complex,thus,disturbing expression of related genes and leading to testicular premature aging.Similarly,expressions of Y-box-binding protein 2(YBX2),outer dense fiber of sperm tails 1(ODfl),and glyceraldehyde-3-phosphate dehydrogenase,spermatogenic(GAPDHS)were significantly decreased in the testis of men with non-obstructive azoospermia.The present study adds to the understanding of the physiology of male reproductive aging and the mechanism of spermatogenic failure in infertile men.展开更多
文摘目的:探讨碱性核蛋白1(BNC1)对食管鳞状细胞癌(ESCC)细胞增殖、迁移、侵袭、细胞周期和凋亡的影响及其作用机制。方法:通过q PCR法检测ESCC细胞和正常食管上皮细胞中BNC1 m RNA的表达水平,免疫组织化学染色法检测10例ESCC患者癌及癌旁组织中BNC1的蛋白表达水平。利用si RNA敲低BNC1在KYSE-150和KYSE-30细胞中的表达,CCK-8法、划痕愈合实验、Transwell实验和流式细胞术检测BNC1对细胞增殖、迁移、侵袭、细胞周期和凋亡等的影响。通过CHIP-seq实验和GEPIA在线网站数据分析并结合敲低BNC1后的转录组测序数据分析筛选BNC1调控的下游靶基因,q PCR法验证BNC1敲低后靶基因的表达变化,并用双荧光素酶报告基因实验验证BNC1对靶基因的调控作用。结果:BNC1 m RNA和蛋白水平在ESCC组织中较癌旁组织高表达(均P<0.01)。敲低BNC1可明显抑制KYSE-150、KYSE-30细胞的增殖、迁移和侵袭能力(P<0.05或P<0.01),将细胞阻滞于G1期并促进细胞的凋亡(均P<0.01)。CHIP-seq实验结果和在线网站GEPIA数据分析结合敲低BNC1后的转录组测序数据显示,G蛋白通路抑制因子1(GPS1)可能为BNC1正向调控的致癌靶基因。q PCR法和双荧光素酶报告基因实验结果显示,BNC1对GPS1有调控作用(P<0.01)。结论:BNC1在ESCC组织和细胞中高表达,干扰BNC1可显著抑制ESCC细胞的增殖、迁移和侵袭能力,阻滞细胞于G1期并促进细胞凋亡,其机制可能是BNC1通过靶向GPS1调控ESCC细胞的恶性生物学行为。
基金This work was supported by the National Key Research and Development Program of China(2018YFC1005003,2017YFC1001003,and 2017YFC1001303)the National Natural Science Foundation of China(81471421,81401219,and 81701461)+3 种基金The Fundamental Research Funds for the Central Universities,Natural Science Foundation of Zhejiang Province(Q19H040040)Key Research Program of Zhejiang Provincial Natural Science Foundation(LZ18H040001)Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai(2018YQ39)Zhejiang University Education Foundation Global Partnership Fund.
文摘Basonuclin(BNC1)is expressed primarily in proliferative keratinocytes and gametogenic cells.However,its roles in spermatogenesis and testicular aging were not dear.Previously we discovered a heterozygous BNC1 truncation mutation in a premature ovarian insufficiency pedigree.In this study,we found that male mice carrying the truncation mutation exhibited progressively fertility loss and testicular premature aging.Genome-wide expression profiling and direct binding studies(by chromatin immunoprecipitation sequencing)with BNC1 in mouse testis identified several spermatogenesis-specific gene promoters targeted by BNC1 including kelch-like family member 10(Klhl1O),testis expressed 14(Tex14)9 and spermatogenesis and centriole associated 1(Spatcl).Moreover,biochemical analysis showed that BNC1 was associated with TATA-box binding protein-associated factor 7 like(TAF7L),a germ cell-specific paralogue of the transcription factor IID subunit TAF7,both in vitro and in testis,suggesting that BNC1 might directly cooperate with TAF7L to regulate spermatogenesis.The truncation mutation disabled nuclear translocation of the BNC1/TAF7L complex,thus,disturbing expression of related genes and leading to testicular premature aging.Similarly,expressions of Y-box-binding protein 2(YBX2),outer dense fiber of sperm tails 1(ODfl),and glyceraldehyde-3-phosphate dehydrogenase,spermatogenic(GAPDHS)were significantly decreased in the testis of men with non-obstructive azoospermia.The present study adds to the understanding of the physiology of male reproductive aging and the mechanism of spermatogenic failure in infertile men.
