Rui Chen1, Yurong Zhang1, Peng Chen2, Yixin Pang3, Hongbao Li4, Ziwei Chen5, Xiaoyong Zhang1, Hongyi Zhang1, Wujun Li1. 1. First Affiliated Hospital of Xi'an Medical University, Xi'an 710077, China. 2. Institute of Basic Medical Science, Xi'an Medical University, Xi'an 710021, China. 3. Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710004, China. 4. Department of Physiology and Pathophysiology, Xi'an Jiaotong University School of Basic Medical Sciences, Xi'an 710061, China. 5. School of Clinical Medicine, Xi'an MedicalUniversity, Xi'an710021, China.
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.
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.
Authors: F Ann Ran; Patrick D Hsu; Jason Wright; Vineeta Agarwala; David A Scott; Feng Zhang Journal: Nat Protoc Date: 2013-10-24 Impact factor: 13.491
Authors: Vincent V Pham; Carolina Salguero; Shamsun Nahar Khan; Jennifer L Meagher; W Clay Brown; Nicolas Humbert; Hugues de Rocquigny; Janet L Smith; Victoria M D'Souza Journal: Nat Commun Date: 2018-10-15 Impact factor: 14.919