RNA测序联合CRISPR/Cas9基因编辑技术鉴定调控维甲酸诱导分化功能的miRNA  

Identify Myeloid Differentiation-Related MiRNAs Response to ATRA Induction by RNA Sequencing and CRISPR/Cas9 Gene Editing

作　　者：王凌燕[1] 林仁章 江佩芳 张雲 李佳蒸 陈钰雯 胡建达[1] WANG Ling-Yan;LIN Ren-Zhang;JIANG Pei-Fang;ZHANG Yun;LI Jia-Zheng;CHEN Yu-Wen;HU Jian-Da(Department of Hematology,Fujian Medical University Union Hospital,Fujian Institute of Hematology,Fujian Provincial Key Laboratory of Hematologic Disease,Fuzhou 350001,Fujian Province,China;Department of Molecular and Cellular Biology,Beckman Research Institute of City of Hope,Duarte 91010,CA,USA)

机构地区：[1]福建医科大学附属协和医院血液科,福建省血液病研究所,福建省血液病重点实验室,福建福州350001 [2]美国City of Hope贝克曼研究所分子与细胞生物学系,加州Duarte91010

出　　处：《中国实验血液学杂志》2021年第2期339-347,共9页Journal of Experimental Hematology

基　　金：国家自然科学基金(81870135);血液科国家及省临床重点专科建设项目(201130301);福建省自然科学基金项目(2020J05050);福建省卫健委青年基金项目(2015-1-39);福建医科大学启航基金项目(2018QH1036)。

摘　　要：目的:研究miRNA的敲除对全反式维甲酸(ATRA)介导的急性早幼粒细胞白血病髓系分化的影响,并鉴定下调的miRNA若干,设计合成针对miRNA前体基因序列的sgRNA,通过CRISPR/Cas9技术实现miRNA在编码基因水平上的敲除。实时荧光定量PCR验证miRNA水平的变化和流式细胞术检测miRNA敲除后对细胞分化水平的影55种成熟miRNA下调。应用CRISPR/Cas9进行基因敲除后,PCR及Surveyor assay检测可见明显的DNA剪切条带,后,细胞的髓系分化受到明显抑制,而敲除miR-155后髓系分化水平则有所上调。结论:应用CRISPR/Cas9可进行针对平的影响,其具体机制是通过对分化相关靶基因的综合调控。Objective:To identify differentiation related miRNA and evaluate roles of miRNA during ATRA induced myeloid differentiation.Methods:The small RNA sequencing was used to analyze differential expressed miRNAs in ATRA induced NB4 cells.Then the several up or down-regulated miRNA were selected as the research candidates.SgRNAs targeting the genome of each miRNA were designed and NB4 cells with inducible expression of Cas9 protein were generated.After transduced sgRNA into NB4/Cas9 cells,the mutation level by PCR and surveyor assay were evaluated.The cell differentiation level was investigated by surface CDllb expression via flow cytometry.Results:A total of 410 mature miRNAs which expressed in NB4 cells were detected out after treated by ATRA,74 miRNAs were up-regulated and 55 were down-regulated miRNAs with DNA cleavage generated by CRISPR/Cas9 was assayed directly by PCR or surveyor assay,quantitative PCR showed that the expression of miRNA was downregulated,which evaluated that gene edition successfully inhibitied the expression of mature miRNA.MiR-223 knockout showed the myeloid differentation of NB4 significantly inhibitied,while miRNA-155 knockout showed the myeloid differentation of NB4 cells significantly increased.Conclusion:CRISPR/Cas9 is a powerful tool for gene editing and can lead to miRNA knockout.Knockouts of miR-223 and miR-155 have shown a differentiation-related phenotype,and the potential mechanism is the integrative regulation of target genes.

关 键 词：CRISPR/Cas9 MICRORNA 急性早幼粒细胞白血病 全反式维甲酸 髓系分化 

分 类 号：R733[医药卫生—肿瘤]