摘要
目的探讨小核非编码RNA 7SK在胚胎干细胞(ESCs)增殖中的作用,为原始侏儒症疾病的早期诊治提供靶点。方法利用CRISPR/Cas9系统转染ESCs,通过PCR产物测序和甘油梯度分析鉴定每个单克隆细胞系获得7SK 128-179突变体。使用慢病毒系统敲除CDK9,然后通过Western blotting分析敲除效率和相关通路蛋白的变化。结果CRISPR/Cas9系统转染后,ESCs中出现128-179 nt缺失突变的7SK,该突变导致ESCs增殖缺陷。7SK 128-179 nt突变的ESCs中LARP7和CDC6的蛋白水平显著下调,通过干预CDK9的活性发现7SK对ESCs增殖的调控作用依赖于CDK9的活性。结论7SK 128-179 nt截短体可通过下调CDC6严重影响ESCs的增殖,且这一过程依赖于CDK9活性,提示7SK截短突变可作为侏儒症的早期筛查和治疗靶点。
Objective To explore the role of small nuclear noncoding RNA 7SK in embryonic stem cell(ESCs)proliferation and the value of 7SK as a target for early diagnosis and treatment for primordial dwarfism(PD).Methods ESC line R1 was transfected with the CRISPR/Cas9 system,and sequencing of the PCR product and glycerol gradient analysis were performed to identify novel 7SK deletion mutations.A lentivirus system was used to knock down cyclin-dependent kinase 9(CDK9)in clones with 7SK deletion mutations,and the effect of CDK9 knockdown on the protein level of cell division cycle 6(CDC6)was analyzed with Western blotting.Results We identified a novel deletion mutation of 7SK at 128-179 nt in the ESCs,which resulted in deficiency of cell proliferation.7SK truncation at 128-179 nt significantly reduced the protein expressions of La-related protein 7(LARP7)and CDC6.Conclusions 7SK truncation at 128-179 nt can significantly impair proliferation of ESCs by downregulating CDC6.7SK is a key regulator of proliferation and mediates the growth of ESCs through a mechanism dependent on CDK9 activity,suggesting the value of 7SK truncation at 128-179 nt as a potential target for early diagnosis and treatment of PD.
作者
陈蕊
张玉荣
陈鹏
庞依心
李宏宝
陈子溦
张小用
张鸿毅
李武军
CHEN Rui;ZHANG Yurong;CHEN Peng;PANG Yixin;LI Hongbao;CHEN Ziwei;ZHANG Xiaoyong;ZHANG Hongyi;LIWujun(First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China;Institute of Basic Medical Science,Xi'an Medical University,Xi'an 710021,China;Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710004,China;Department of Physiology and Pathophysiology,Xi'an Jiaotong University School of Basic Medical Sciences,Xi'an 710061,China;School of Clinical Medicine,Xi'an Medical University,Xi'an 710021,China)
出处
《南方医科大学学报》
CSCD
北大核心
2021年第8期1125-1130,共6页
Journal of Southern Medical University
基金
陕西省西安市科技计划项目(2019115213YX007SF040[7])
陕西省卫生健康科研项目基金(2018D075)。
