南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (8): 1125-1130.doi: 10.12122/j.issn.1673-4254.2021.08.01

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小核非编码RNA 7SK 128-179截短体通过下调CDC6抑制胚胎干细胞的体外增殖

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   

  1. 西安医学院第一附属医院，陕西 西安 710077；西安医学院基础医学部, 陕西 西安 710021；西安交通大学第二附属医院，陕西 西安 710004；西安交通大学基础医学部生理与病理生理学系，陕西 西安 710061；西安医学院临床医学院，陕西 西安 710021
  • 出版日期:2021-08-20 发布日期:2021-09-07

7SK truncation at 128-179 nt suppresses embryonic stem cell proliferation in vitro by downregulating CDC6

CHEN Rui, ZHANG Yurong, CHEN Peng, PANG Yixin, LI Hongbao, CHEN Ziwei, ZHANG Xiaoyong, ZHANG Hongyi, LI Wujun   

  1. 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
  • Online:2021-08-20 Published:2021-09-07

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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。

关键词: 7SK截短体；胚胎干细胞；增殖；周期蛋白依赖激酶9；细胞分裂周期蛋白6

Abstract: 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.

Key words: 7SK truncation; embryonic stem cell; proliferation; cyclin-dependent kinase 9; cell division cycle 6