CRISPR/Cas9敲低环氧合酶2表达对黄曲霉毒素B1诱导肝细胞核DNA损伤与脂质蓄积的影响

Effects of Cyclooxygenase 2 Knockdown Using CRISPR/Cas9 on Nuclear DNA Damage and Lipid Accumulation in Aflatoxin B1-induced Hepatocytes

为探讨黄曲霉毒素B1(AFB1)诱导的肝细胞核DNA损伤与脂质蓄积的关系,以慢病毒质粒为载体,采用CRISPR/Cas9技术构建含靶向环氧合酶2(COX-2)编码基因(PTGS2)小向导RNA(sgRNA)的重组质粒,经测序验证成功后,制备假病毒感染HepG2细胞,构建稳定敲低COX-2表达的细胞.通过实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(WB)检测mRNA和蛋白水平,结果显示与HepG2-Cas9-NC对照细胞相比,HepG2-Cas9-PTGS2敲低细胞中PTGS2 mRNA与COX-2蛋白表达水平分别减少至(49.1±1.8)%和(48.1±0.7)%.给予细胞AFB1处理,通过WB和免疫荧光(IF)法检测DNA损伤标志物γH2AX的表达水平,结果显示处理组敲低细胞中,γH2AX蛋白水平和荧光焦点形成数目显著低于对照细胞(p<0.05);进一步检测脂质合成相关指标,发现敲低细胞中PPARγ蛋白、总胆固醇(TC)、总甘油三脂(TG)水平以及油红O染色阳性脂滴的分布密度,均显著低于对照细胞中(p<0.05).在敲低细胞中回补COX-2后给予AFB1处理,γH2AX蛋白水平和脂质分布密度均显著升高(p<0.05).综上,成功构建了HepG2-Cas9-PTGS2敲低细胞,并发现敲低COX-2表达对AFB1诱导的肝细胞核DNA损伤和脂质蓄积有显著抑制作用,为进一步研究靶向干预COX-2在外源化学物诱导肝细胞毒性中的作用机制提供了细胞模型和依据.

To investigate the relationship between aflatoxin B1(AFB1)-induced nuclear DNA damage and lipid accumulation in he-patocytes,the lentiviral plasmids were used as the vector to construct recombinant plasmids containing the targeting cyclooxygenase 2(COX-2)gene(PTGS 2)small guide RNA(sgRNA)using CRISPR/Cas9 technique.The recombinant plasmids were packaged with pseudovirus to infect HepG2 cells for a stable COX-2 knockdown cell line.Quantitative real-time PCR(qRT-PCR)and Wes-tern blot(WB)detection showed that PTGS 2 mRNA and COX-2 protein levels in HepG2-Cas9-PTGS 2 cells reduced to(49.1±1.8)%and(48.1±0.7)%respectively,compared with HepG2-Cas9-NC control cells.After these cells were treated with AFB1,the expression level ofγH2AX,a DNA damage marker,was detected using WB and immunofluorescence(IF).The results showed that the protein level ofγH2AX and the fluorescence foci numbers were significantly lower in HepG2-Cas9-PTGS 2 cells than in HepG2-Cas9-NC cells(p<0.05).Lipid synthesis related experiments revealed that PPARγprotein expression,total cholesterol(TC),total triglyceride(TG)levels,and Oil Red O staining positive cell density were significantly lower in HepG2-Cas9-PTGS 2 cells than in HepG2-Cas9-NC cells(p<0.05).Furthermore,the expression levels of COX-2,γH2AX,and lipid density in AFB1-treated HepG2-Cas9-PTGS 2 cells increased after being rescued by wide type COX-2 expression(p<0.05).In summary,COX-2 knockdown HepG2-Cas9-PTGS 2 cells were successfully constructed using CRISPR/Cas9 technique,and COX-2 knockdown significantly inhibited AFB1-induced nuclear DNA damage and lipid accumulation in hepatocytes,which provides cell model and basis for studying the mechanism of COX-2 in hepatotoxicity induced by xenobiotics.