基于CRISPR/Cas9技术的miR-374a低表达细胞系的构建及鉴定
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摘要
目的:运用CRISPR/Cas9技术构建miR-374a低表达细胞系。方法:设计针对miR-374a成熟体gRNA,合成DNA,插入pSpCas9(BB)-2A-Puro,将鉴定正确的载体转染入肺癌细胞系A549细胞,使用嘌呤霉素筛选转染的细胞;PCR方法扩增,序列测定分析细胞基因组中靶位点区域序列;定量PCR方法检测转染细胞中miR-374a及其靶基因TGFA的表达情况;细胞增殖实验检测miR-374a基因编辑对细胞生长情况的影响。结果:构建了靶向miR-374a成熟体基因序列的基因编辑载体,转染细胞经过加压筛选,序列分析结果提示靶向位点的基因发生了缺失或插入突变;定量PCR结果显示,转染基因编辑载体的细胞miR-374a表达水平较对照细胞明显下降(P<0.01),而靶基因TGFA表达则明显上升(P<0.05);细胞增殖实验显示miR-374a低表达的细胞增殖能力发生了明显改变(P<0.05～P<0.01)。结论:成功构建了miR-374a低表达细胞系,为后续研究该分子的功能提供了基础。
Objective:To establish the cell line with low-expression miR-374 a using CRISPR/CAS9 gene targeting technology.Methods:The mature gRNA targeting miR-374 a was designed to synthesize the DNA,which was inserted into the CRISPR/CAS9 vector to construct the pSpCas9(BB)-2 A-Puro.A549 cells were transfected with the constructed vector,and screened using puromycin.The gene sequences of target site were analyzed using PCR and sequencing method.The qPCR was used to detect the relative expression levels of miR-374 a and its target gene TGFA in transfected cells.The CCK8 was used to detect the proliferation activity of cells.Results:The gene targeting miR-374 a vector was successfully constructed.The transfected cells were screened under pressure,and the sequencing results showed that the inserting/deletion mutation was found.The results qPCR showed that the expression level of miR-374 a significantly decreased,and the expression level of TGFA significantly increased in transfecting gene editing vector compared with control cells(P<0.01 and P<0.05).The cell proliferation assay showed that the proliferation activity significantly changed in low-expressed miR-374 a cells(P<0.05 to P<0.01).Conclusions:The low-expressed miR-374 a cell line was successfully established,which provides the basis in studying the molecular function.
Objective:To establish the cell line with low-expression miR-374 a using CRISPR/CAS9 gene targeting technology.Methods:The mature gRNA targeting miR-374 a was designed to synthesize the DNA,which was inserted into the CRISPR/CAS9 vector to construct the pSpCas9(BB)-2 A-Puro.A549 cells were transfected with the constructed vector,and screened using puromycin.The gene sequences of target site were analyzed using PCR and sequencing method.The qPCR was used to detect the relative expression levels of miR-374 a and its target gene TGFA in transfected cells.The CCK8 was used to detect the proliferation activity of cells.Results:The gene targeting miR-374 a vector was successfully constructed.The transfected cells were screened under pressure,and the sequencing results showed that the inserting/deletion mutation was found.The results qPCR showed that the expression level of miR-374 a significantly decreased,and the expression level of TGFA significantly increased in transfecting gene editing vector compared with control cells(P<0.01 and P<0.05).The cell proliferation assay showed that the proliferation activity significantly changed in low-expressed miR-374 a cells(P<0.05 to P<0.01).Conclusions:The low-expressed miR-374 a cell line was successfully established,which provides the basis in studying the molecular function.
引文
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[2] XU Z,XU Z,XU J,et al.LARP1 is regulated by the XIST/miR-374a axis and functions as an oncogene in non-small cell lung carcinoma[J].Oncol Rep,2017,38(6):3659.
[3] LU T,ZHANG C,CHAI MX,et al.MiR-374a promotes the proliferation of osteosarcoma cell proliferation by targeting Axin2[J].Int J Clin Exp Pathol,2015,8(9):10776.
[4] WANG Y,XIN H,HAN Z,et al.MicroRNA-374a promotes esophageal cancer cell proliferation via Axin2 suppression[J].Oncol Rep,2015,34(4):1988.
[5] XIONG Y,QIU J,LI C,et al.Fortunelin-induced modulation of phosphatase and tensin homolog by microRNA-374a decreases inflammation and maintains intestinal barrier function in colitis[J].Front Immunol,2018,9(83):1.
[6] GONG W,QIE S,HUANG P,et al.Protective effect of mir-374a on chemical hypoxia-induced damage of PC12 cells in vitro via the GADD45α/JNK signaling pathway[J].Neurochem Res,2017,43(3):581.
[7] RAN FA,HSE PD,WRIGHT J,et al.Genome engineering using the CRISPR-Cas9 system[J].Nat Protoc,2013,8(11):2281.
[8] GAJ T,GERSBACH CA,BARBAS CF 3rd.ZFN,TALEN and CRISPR/Cas-based methods for genome engineering[J].Trends Biotechnol,2013,31(7):397.
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