Rev-erbβ基因敲除对肝癌HepG2细胞增殖和迁移侵袭的影响
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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.
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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[9] CONG L,RAN FA,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339(6121):819-823.
[10] YANG H,WANG HY,SHIVALILA CS,et al.One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.Cell,2013,154(6):1370-1379.
[11] WANG T,WEI JJ,SABATINI DM,et al.Genetic screens in human cells using the CRISPR-Cas9 system.Science,2014,343(6166):80-84.
[12] XIAO D,ZHANG WF,LI Y,et al.A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene.J Biotechnol,2016,219:110-116[2018-09-02].https://doi.org/10.1016/j.jbiotec.2015.12.026.
[13] 陈芳,张伟锋,赵俊丽,等.应用CRISPR/Cas9系统构建Rev-erbβ基因敲除的 HEK293细胞系.细胞与分子免疫学杂志,2016,32(11):1446-1452.
[14] WADE M.High-throughput silencing using the CRISPR-Cas9 system:a review of the benefits and challenges.J Biomol Screen,2015,20(8):1027-1039.
[15] WANG F,GUO T,JIANG HM,et al.A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.Mol Genet Genomics,2018,293(3):769-783.
[16] XU ZW,YAN SX,WU HX,et al.The influence of TNF-α and Ang II on the proliferation,migration and invasion of HepG2 cells by regulating the expression of GRK2.Cancer Chemother Pharmacol,2017,79(4):747-758.
[17] ROOMI MW,MONTERREY JC,KALINOVSKY T,et al.Patterns of MMP-2 and MMP-9 expression in human cancer cell lines.Oncol Rep,2009,21(5):1323-1333.
[18] INGRAHAM CA,PARK GC,MAKARENKOVA HP,et al.Matrix metalloproteinase (MMP)-9 indused by Wnt signal increases the proliferation and migration of embryonic neural stem cells at low O2 levels.J Biol Chem,2011,286(20):17649-17657.
[19] FUJIMOTO N,TERLIZZI J,AHO S,et al.Extracellular matrix protein1 inhibits the activity of matrix metalloproteinase9 through high-affinity protein/protein interactions.Exp Dermatol,2006,15(4):300-307.
[20] FARINA AR,MACKAY AR.Gelatinase B/MMP-9 in tumour pathogenesis and progression.Cancers,2014,6(1):240-296.
[21] POMO JM,TAYLOR RM,GULLAPALLI RR.Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture:a model system to understand cancer cell growth mechanics.Cancer Cell Int,2016,16(1):44-58.
[2] DUMAS B,HARDING HP,CHOI HS,et al.A new orphan member of the nuclear hormone receptor superfamily closely related to Rev-Erb .Mol Endocrinol,1994,8(8):996-1005.
[3] RETNAKARAN R,FLOCK G,GIGUèRE V.Identification of RVR,a novel orphan nuclear receptor that acts as a negative transcriptional regulator.Mol Endocrinol,1994,8(9):1234-1244.
[4] WANG JD,LIU NS,LIU ZL,et al.The orphan nuclear receptor Rev-erbβ recruits Tip60 and HDAC1 to regulate apolipoproteinCⅢ promoter Biochim Biophys Acta,2008,1783(2):224-236.
[5] BUGGE A,FENG D,EVERETT LJ,et al.Rev-erbα and Rev-erbβ coordinately protect the circadian clock and normal metabolic function.Genes Dev,2012,26(7):657-667.
[6] RAMAKRISHNAN SN,LAU P,BURKE LJ,et al.Rev-erbbeta regulates the expression of genes involved in lipid absorption in skeletal muscle cells:evidence for cross-talk between orphan nuclear receptors and myokines.J Biol Chem,2005,280(10):8651-8659.
[7] DE MEI C,ERCOLANI L,PARODI C,et al.Dual inhibition of REV-ERBβ and autophagy as a novel pharmacological approach to induce cytotoxicity in cancer cells.Oncogene,2015,34(20):2597-2608.
[8] WANG YJ,KOJETIN D,BURRIS TP.Anti-proliferative actions of a synthetic Rev-erbα/β agonist in breast cancer cells.Biochem Pharmacol,2015,96(4):315-322.
[9] CONG L,RAN FA,COX D,et al.Multiplex genome engineering using CRISPR/Cas systems.Science,2013,339(6121):819-823.
[10] YANG H,WANG HY,SHIVALILA CS,et al.One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.Cell,2013,154(6):1370-1379.
[11] WANG T,WEI JJ,SABATINI DM,et al.Genetic screens in human cells using the CRISPR-Cas9 system.Science,2014,343(6166):80-84.
[12] XIAO D,ZHANG WF,LI Y,et al.A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene.J Biotechnol,2016,219:110-116[2018-09-02].https://doi.org/10.1016/j.jbiotec.2015.12.026.
[13] 陈芳,张伟锋,赵俊丽,等.应用CRISPR/Cas9系统构建Rev-erbβ基因敲除的 HEK293细胞系.细胞与分子免疫学杂志,2016,32(11):1446-1452.
[14] WADE M.High-throughput silencing using the CRISPR-Cas9 system:a review of the benefits and challenges.J Biomol Screen,2015,20(8):1027-1039.
[15] WANG F,GUO T,JIANG HM,et al.A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines.Mol Genet Genomics,2018,293(3):769-783.
[16] XU ZW,YAN SX,WU HX,et al.The influence of TNF-α and Ang II on the proliferation,migration and invasion of HepG2 cells by regulating the expression of GRK2.Cancer Chemother Pharmacol,2017,79(4):747-758.
[17] ROOMI MW,MONTERREY JC,KALINOVSKY T,et al.Patterns of MMP-2 and MMP-9 expression in human cancer cell lines.Oncol Rep,2009,21(5):1323-1333.
[18] INGRAHAM CA,PARK GC,MAKARENKOVA HP,et al.Matrix metalloproteinase (MMP)-9 indused by Wnt signal increases the proliferation and migration of embryonic neural stem cells at low O2 levels.J Biol Chem,2011,286(20):17649-17657.
[19] FUJIMOTO N,TERLIZZI J,AHO S,et al.Extracellular matrix protein1 inhibits the activity of matrix metalloproteinase9 through high-affinity protein/protein interactions.Exp Dermatol,2006,15(4):300-307.
[20] FARINA AR,MACKAY AR.Gelatinase B/MMP-9 in tumour pathogenesis and progression.Cancers,2014,6(1):240-296.
[21] POMO JM,TAYLOR RM,GULLAPALLI RR.Influence of TP53 and CDH1 genes in hepatocellular cancer spheroid formation and culture:a model system to understand cancer cell growth mechanics.Cancer Cell Int,2016,16(1):44-58.