HDAC6 基因敲除的人肝癌细胞系的构建及其生物学功能鉴定

Construction of HDAC6 knockout human liver cancer cell line and detection of its biological functions

背景肝癌的发病率和死亡率逐年攀升,对肝癌发生发展机制的研究尤为重要。目的应用CRISPR/Cas9技术在人肝癌HepG2细胞中敲除组蛋白去乙酰酶6(histone deacetylase 6,HDAC6)基因,构建HDAC6基因敲除的HepG2稳定表达细胞系,并检测其对人肝癌细胞生长的影响。方法根据CRISPR/Cas9技术靶点设计原则设计特异性识别HDAC6基因的单向导RNA(single guide RNA,sgRNA)序列,构建LentiCRISPR-HDAC6-sgRNA真核重组表达质粒,测序鉴定后包装慢病毒感染HepG2细胞,用嘌呤霉素(puromycin)抗性筛选得到敲除HDAC6基因稳定表达的细胞系,Western blot鉴定HDAC6基因的敲除效果,CCK-8生长曲线实验检测HDAC6基因敲除对细胞增殖能力的影响,划痕实验和Transwell迁移实验检测HDAC6基因敲除对细胞迁移能力的影响。Western blot鉴定HDAC6基因敲除对调节α-tubulin乙酰化的影响,细胞存活曲线实验检测HDAC6基因敲除后HepG2对HDAC6抑制剂ACY-1215的敏感度,采用流式细胞术检测HDAC6基因敲除对HepG2细胞周期分布和凋亡的影响。结果成功构建HDAC6基因敲除的HepG2稳定表达细胞系后,经实验证明HDAC6基因敲除能够显著抑制HepG2细胞的增殖和迁移,使α-tubulin的乙酰化表达增强且能降低HepG2对ACY-1215的敏感度,还能使HepG2细胞发生G1期阻滞,促进HepG2细胞的凋亡。结论敲除HDAC6基因可以抑制HepG2细胞增殖和迁移,使α-tubulin乙酰化程度增加,调节HepG2细胞对ACY-1215的敏感度,且影响HepG2细胞的周期和凋亡,为进一步研究HDAC6在肝癌中的功能机制奠定了基础。

Background The incidence and mortality rate of liver cancer are increasing year by year.It is particularly important to study the mechanism of hepatocarcinogenesis and its development.Objective To construct the histone deacetylase 6(HDAC6)gene knockout HepG2 stable expression cell line by using CRISPR/Cas9 genome engineering technology,and detect its effect on the growth of human hepatoma cells.Methods According to the CRISPR/Cas9 target design principles,a single guide RNA(sgRNA)sequence that specifically recognizing the HDAC6 was designed for construction of the eukaryotic recombinant expressional plasmids LentiCRISPR-HDAC6-sgRNA.After sequencing,lentivirus was produced with the recombinant plasmid and infected HepG2 cells.The stable HDAC6 knockout cells were screened through the stress of puromycin,and the knockout effect was detected by sequencing and Western blotting.CCK8 growth curve test was used to detect the effect of HDAC6 gene knockout on cell proliferation,scratch test and Transwell migration test for detecting the effect of HDAC6 gene knockout on cell migration.Western blot was used to identify the effect of HDAC6 gene knockout on the regulation ofα-tubulin acetylation.The sensitivity of HepG2 to HDAC6 inhibitor ACY-1215 after HDAC6 gene knockout was detected by cell survival curve experiment,and the effect of HDAC6 gene knockout on HepG2 cell cycle distribution and apoptosis was detected by flow cytometry.Results The stable HDAC6 knockout HepG2 cell line was constructed successfully,HDAC6 knockout could significantly inhibit cell proliferation and migration,enhance the expression ofα-tubulin acetylation and reduce the sensitivity of HepG2 cells to ACY-1215.And it could also cause the G1 phase block of HepG2 cells and promote the apoptosis.Conclusion Our experiment demonstrates that knockout of HDAC6 can inhibit HepG2 cell proliferation,migration,increase the acetylation ofα-tubulin,adjust the sensitivity of HepG2 cells to ACY-1215,and affect the cell cycle and apoptosis,which provides references on the biological function of HDAC6 in hepatocarcinogenesis.