CRISPR/Cas9介导的KIFC1基因敲除对宫颈癌细胞增殖及凋亡的影响  

CRISPR/Cas9-mediated Knockout of KIFC1 Inhibits Proliferation and Induces Apoptosis of Cervical Cancer Cells

作　　者：范晓静 魏雅岚 叶洲杰 朱丽萍 王心睿 FAN Xiaojing;WEI Yalan;YE Zhoujie;ZHU Liping;WANG Xinrui(Medical Research Center,Fujian Children’s Hospital(Fujian Branch of Shanghai Children’s Medical Center),Fuzhou 350011,China;NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-human Primate(Fujian Maternity and Child Health Hospital),Fuzhou 350013,China;Medical Research Center,Fujian Maternity and Child Health Hospital,Fuzhou 350001,China)

机构地区：[1]福建省儿童医院(上海儿童医学中心福建医院)医学研究中心,福州350011 [2]国家卫健委非人灵长类生育调节技术评价重点实验室(福建省妇幼保健院),福州350013 [3]福建省妇幼保健院医学研究中心,福州350001

出　　处：《肿瘤防治研究》2023年第11期1051-1058,共8页Cancer Research on Prevention and Treatment

基　　金：国家自然科学基金(82101678);福建省科技创新联合资金(2021Y9160);福建省卫生健康科技计划(2020QNA018);福建省自然科学基金(2020J05278)。

摘　　要：目的探讨KIFC1基因在宫颈癌细胞中的功能及对宫颈癌HeLa细胞增殖的影响。方法设计靶向KIFC1基因的sgRNAs并基于pSpCas9(BB)-2A-GFP载体构建重组质粒,共转染至He La细胞。通过流式细胞分选、有限稀释和测序验证筛选单克隆敲除细胞株。采用RT-qPCR、Western blot和免疫荧光法检测敲除细胞的KIFC1转录及蛋白表达水平,利用相差显微成像、荧光共聚焦显微成像观察细胞核、微管骨架等细胞表型。通过生长曲线绘制、EdU标记、吖啶橙染色分析细胞增殖、细胞周期及细胞凋亡情况。结果KIFC1基因缺失导致He La细胞形态结构发生显著变化,多核、微核及异常微管骨架的细胞明显增加,细胞周期紊乱,细胞晚期凋亡数量显著升高,细胞增殖能力下降(均P<0.05)。结论KIFC1基因缺失影响He La细胞微管骨架的组装并形成异常延伸和细胞分裂,进而导致细胞核形态异常、染色质丢失、细胞周期停滞、凋亡增加。Objective To investigate the functions of the KIFC1 gene in tumor cells and its effect on the proliferation of cervical cancer cells.Methods We designed sgRNAs targeting the KIFC1 gene and constructed a recombinant plasmid based on the pSpCas9(BB)-2A-GFP vector,which was co-transfected into HeLa cells.We screened monoclonal knockout cell lines through flow cytometry sorting,limited dilution inoculation of cells,and sequencing.RT-qPCR,Western blot,and immunofluorescence were used to detect the transcription and protein expression levels of KIFC1 in knockout cells.Cell phenotypes such as nucleus and microtubule cytoskeleton were observed using phase-contrast microscopy and fluorescence confocal microscopy.Cell proliferation,cell cycle,and apoptosis were analyzed by growth curve plotting,EdU labeling,and acridine orange staining.Results The deletion of the KIFC1 gene resulted in the abnormal phenotypes of HeLa cells,with increased numbers of multinuclei,micronucleus,and disordered microtubules.The cell cycle was disrupted,accompanied with a significant increase in the ratio of late apoptotic cells and a decrease in cell proliferation(all P<0.05).Conclusion KIFC1 gene deletion affects the assembly of microtubules and cell division in HeLa cells,leading to abnormal nuclear morphology,chromatin elimination,cell cycle arrest,and increased cell apoptosis.

关 键 词：CRISPR/Cas9 KIFC1 宫颈癌 He La细胞 细胞凋亡 细胞分裂 

分 类 号：R737.33[医药卫生—肿瘤]