利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

LMNA基因编码A型和C型核纤层蛋白,参与细胞核核膜的组织,影响基因组稳定性并对细胞分化产生影响。人类肿瘤中LMNA表达异常普遍存在,其突变造成多种核纤层蛋白病,如Emery-Dreifuss肌营养不良症(Emery-Dreifussmusculardystrophy,EDMD)、扩张型心肌病(dilatedcardiomyopathy,DCM)和儿童早老症(Hutchinson-Glifordprogeriasyndrome,HGPS)等。为进一步研究LMNA在细胞内的功能,本研究利用CRISPR/Cas9技术对体外培养的293T与HepG2细胞株的LMNA基因进行编辑,获得两株LMNA基因敲除(LMNA KO)的稳定细胞系。与野生型相比,LMNAKO细胞系增殖能力相对减弱,凋亡增加。同时,细胞形态上也发生显著改变,核膜凹凸不平。本研究首次报道了LMNA KO永生细胞系构建和形态研究结果,为后续LMNA基因功能研究和致病突变体研究奠定基础。

Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines

The LMNA gene encodes the nuclear Lamin A and Lamin C proteins,and is related to nuclear membrane organization,genome stability and cell differentiation.Abnormal expression of LMNA is ubiquitous in human tumors,and its mutation leads to various forms of laminopathies,including Emery-Dreifuss muscular dystrophy(EDMD),dilated cardiomyopathy(DCM),and Hutchinson-Gliford progeria syndrome(HGPS).To further determine the functions of the LMNA gene in cellular physiology,the present study used the CRISPR/Cas9 technique to edit the LMNA gene of 293T and HepG2 cells in vitro,which resulted in two stable LMNA gene knockout(LMNA KO)cell lines.Compared to the respective wild type cells,the LMNA KO cell lines showed decrease in proliferation ability,increase in apoptosis,alteration in cellular morphology and uneven structures in the nucleus membrane.In this study,we report for the first time the results on the construction of LMNA KO immortalized cell lines and characterization of their morphological changes,thereby laying the foundation for the further studies of the LMNA gene functions and pathogenic mutations.