摘要
目的探究N-乙酰转移酶10(NAT10)对Erastin诱导结直肠癌(CRC)细胞铁死亡敏感性的影响及其机制。方法使用公共数据库明确NAT10在CRC中的表达及其与Erastin利用度的关系;应用荧光实时定量聚合酶链反应(RT-qPCR)探索Erastin对NAT10 mRNA表达的影响;细胞计数试剂盒(CCK-8)检测NAT10抑制剂Remodelin联合Erastin对CRC细胞SW620活力的影响。构建NAT10稳转敲低细胞株,蛋白质印迹法(Western blot)验证敲减效率成功后,进行RNA-Seq和ac4C-RNA-Seq的测序,分析与对照组比较,NAT10敲低组出现ac4C修饰水平降低和mRNA表达水平下降的基因,再和铁死亡抑制因子集取交集,最后筛选出NAT10调控铁死亡的潜在下游靶基因。RT-qPCR或Western blot检测对照组和NAT10敲低组或Remodelin处理细胞中的转录调节因子核蛋白1(NUPR1)表达水平,验证NAT10对NUPR1的调控作用。两组间比较采取t检验。结果NAT10表达越高,Erastin的药物利用度越低(R=-0.16,P<0.05),SW620细胞Erastin作用组响应性升高(1.440±0.035比1.000±0.096,t=-7.423,P<0.05),Remodelin+Erastin组细胞活力明显低于Erastin组(0.293±0.017比0.906±0.048,t=60.680,P<0.05)。通过铁死亡抑制剂Fer-1干预实验,Fer-1+Erastin+Remodelin细胞组比Erastin+Remodelin组比较,细胞活力出现下降(0.489±0.011比0.412±0.036,t=3.599,P<0.05)。成功构建NAT10稳转敲减细胞系(1.105±0.065比0.309±0.104,t=11.220,P<0.05),测序结果联合铁死亡数据库抑制基因集筛选出NUPR1是NAT10调控铁死亡的下游潜在靶基因。公共数据库证实NAT10与NUPR1呈明显正相关(R=0.40,P<0.05)。RT-qPCR证实NUPR1的mRNA表达在NAT10敲低组中低于对照组(0.087±0.007比0.198±0.005,t=20.758,P<0.05)。Western blot结果表明用Remodelin抑制NAT10表达后,NUPR1在SW480和SW620实验组中蛋白表达水平低于对照组(SW480:0.611±0.175比1.245±0.350,t=6.267,P<0.05;SW620:0.745±0.230比1.201±0.343,t=6.826,P<0.05)。结论结肠癌SW620细胞系中NAT10在Erastin作用下响应性升高,NUPR1可能作为其潜在靶基因介导铁死亡抵抗。
Objective To investigate the effect and mechanism of N-acetyltransferase 10(NAT10)on Erastin induction of ferroptosis sensitivity in colorectal cancer(CRC)cells.Methods The public database Cancer Therapeutics Response Portal(CTRP)was utilized to analyze the correlation between NAT10 expression and Erastin sensitivity.The real-time quantitative polymerase chain reaction(RT-qPCR)was used to explore the expression of NAT10 mRNA upon Erastin application.The cell counting kit-8(CCK-8)was used to detect the effect of the NAT10 inhibitor Remodelin in combination with Erastin on cell viability of SW620 cells.The NAT10 stable knockdown cell line was constructed with the efficiency verification by Western blotting,followed by ac4C RIP-Seq and RNA-Seq sequencing analysis.Genes with reduced ac4C modification level and decreased mRNA expression in the NAT10 knockdown group were analyzed.Then,we took the intersection of these genes and the ferroptosis inhibitor gene set obtained from public database.The regulatory effect of NAT10 on this target gene was verified by public database,RT-qPCR and Western blotting.T-test was used for comparisons between groups.Results The higher the expression of NAT10,the lower the drug utilization of Erastin(R=-0.16,P<0.05).NAT10 increased upon the stimulation of Erastin in SW620 cells(1.440±0.035 vs.1.000±0.096,t=-7.423,P<0.05).The cell viability of the Remodelin+Erastin group was significantly lower than that in the Erastin group(0.293±0.017 vs.0.906±0.048,t=60.680,P<0.05).The addition of Fer-1 into Erastin+Remodelin could effectively inhibit the decrease in cell viability(0.489±0.011 vs.0.412±0.036,t=3.599,P<0.05)compared with the Erastin+Remodelin group.The NAT10 stable knockdown cell line was successfully constructed(1.105±0.065 vs.0.309±0.104,t=11.220,P<0.05),and finally gene transcription regulator nuclear protein 1(NUPR1)was selected as the potential downstream target by sequencing analysis.The public database confirmed a significantly positive correlation between NAT10 and NUPR1(R=0.40,P<0.05).RT-qPCR confirmed that the mRNA expression of NUPR1 was lower in the NAT10 knockdown group than in the control group(0.087±0.007 vs.0.198±0.005,t=20.758,P<0.05).Moreover,Western blotting showed that after inhibiting the NAT10 expression with Remodelin,the protein expression levels of NUPR1 in SW480 and SW620 experimental groups were lower than those in the control group(SW480:0.611±0.175 vs.1.245±0.350,t=6.267,P<0.05;SW620:0.745±0.230 vs.1.201±0.343,t=6.826,P<0.05).Conclusion NAT10 mediates resistance of the ferroptosis inducer Erastin,probably by regulating the expression of ferroptosis suppressor NUPR1.
作者
王晨红
宋丹
郑勇斌
Wang Chenhong;Song Dan;Zheng Yongbin(Department of Gastrointestinal Surgery,Renmin Hospital of Wuhan University,Wuhan 430060,China)
出处
《中华实验外科杂志》
CAS
北大核心
2023年第3期421-423,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金(82203713)
中央高校基本科研业务费专项资金(2042022kf1089)
湖北省陈孝平科技发展基金会(CXPJJH121003-2103)。
关键词
结直肠癌
铁死亡
乙酰化修饰
Colorectal cancer
Ferroptosis
Acetylation modification