摘要
目的探讨核受体Rev-erbβ基因敲除对肝癌HepG2细胞增殖和迁移的影响。方法首先采用对靶向基因进行特定DNA修饰的CRISPR/Cas9基因组编辑技术构建Rev-erbβ基因敲除的HepG2细胞系。用Rev-erbβ打靶载体共转染到HepG2细胞,通过筛选、克隆化构建Rev-erbβ基因敲除HepG2细胞系,并采用PCR、测序和Western blot鉴定。以正常HepG2细胞为对照,采用实时荧光定量PCR(qRT-PCR)检测Rev-erbβ基因敲除对肿瘤迁移、侵袭相关基因表达的影响,采用MTT、细胞划痕、Transwell等实验检测Rev-erbβ基因敲除对HepG2细胞增殖、迁移及侵袭的影响。结果成功构建一株Rev-erbβ基因完全敲除单克隆细胞系(经PCR、测序和Western blot鉴定),命名为HepG2 C5(Rev-erbβ~(-/-))。qRT-PCR结果显示,Rev-erbβ基因敲除可以导致基质金属蛋白酶-2,-9基因(MMP2,MMP9)、细胞外基质蛋白1基因(ECM1)表达上调(P均<0.05),细胞黏附相关基因E-钙黏蛋白基因(CDH1)下降(P=0.05);MTT、细胞划痕、Transwell实验显示,并且HepG2 C5比对照细胞有更强的增殖、迁移与侵袭能力(P<0.05)。结论 Rev-erbβ基因敲除可以改变HepG2细胞与迁移、黏附相关基因的表达,进而影响HepG2细胞增殖、迁移与侵袭能力。
Objective To investigate the effect of nuclear receptor Rev-erbβ knockout on proliferation and migration ability of human hepatocellular carcinoma cell line HepG2. Methods The Rev-erbβ gene knockout HepG2 cell line was abtained by CRISPR/Cas9 genome editing technique with specific DNA modification of the target gene. The Rev-erbβ gene targeting vectors were co-transfected into HepG2 cells. Through cloning and screening, the Rev-erbβ gene knockout HepG2 cell line was constructed,PCR, sequencing and Western blot methods were carried out for the identification of the Rev-erbβ gene knockout HepG2 cell line. The expression level of tumor migration and invasion-associated gene in Rev-erbβ gene knockout cell was determined by real-time quantitative PCR(qRT-PCR) and was compared with normal cell as control.MTT, cell scratch and Transwell experiments were conducted in order to explore the effect of Rev-erbβ gene on HepG2 cell's ability of proliferation,migration and invasion. Results A Rev-erbβ gene knockout monoclonal cell line, which was identified by PCR, sequencing and Western blot, was successfully constructed and named HepG2 C5(Rev-erbβ~(-/-)). qRT-PCR results showed that Rev-erbβ knockout resulted in up-regulation of matrix metalloproteinase-2(MMP2), matrix metalloproteinase-9(MMP9) and extracellular matrix protein-1(ECM1) gene expression(P<0.05) and down-regulation of E-cadherin(CDH1) gene expression(P=0.05). Results of MTT, cell scratch and transwell experiments showed that HepG2 C5 had stronger proliferation, migration and invasion ability than control cells(P<0.05).Conclusion Rev-erbβ gene knockout could change the expression of migration and adhesion-associated genes in HepG2 cell, and then affect the proliferation, migration and invasion ability of HepG2 cells.
引文
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