Rev-erbβ Knock-out Affectsthe Proliferation, Migration and Invasion Ability of Hepatocellular Carcinoma HepG2 Cell Line in vitro
Abstract
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.
Keywords: CRISPR/Cas9, HepG2 cell lines, Rev-erbβ, Gene knockout, Proliferation, Migration
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