DNA去甲基化酶TET1促进银纳米颗粒诱导的LO2细胞中长链非编码RNA HULC的表达  

Upregulation of TET1 contributes to the activation of lncRNA HULC in LO2 cells treated with silver nanoparticles

作　　者：焦群芳 李惠芳 郑冬燕 黄豪 钟清华 蔡晓楠 凌晓璇 刘林华 JIAO Qunfang;LI Huifang;ZHENG Dongyan;HUANG Hao;ZHONG Qinghua;CAI Xiaonan;LING Xiaoxuan;LIU Linhua(Dongguan Key Laboratory of Environmental Medicine,School of Public Health,Guangdong Medical University,Dongguan 523808,Guangdong Province,China)

机构地区：[1]广东医科大学公共卫生学院,东莞市环境医学重点实验室,广东东莞523808

出　　处：《热带病与寄生虫学》2024年第5期294-300,共7页Journal of Tropical Diseases and Parasitology

基　　金：国家自然科学基金项目(82204134);广东省自然科学基金项目(2023A1515140163);广东省医学科研基金项目(A2018225)

摘　　要：目的 探讨银纳米颗粒(silver nanoparties, AgNPs)对DNA去甲基化酶10-11易位蛋白(ten-eleven translocation proteins, TETs)家族表达的影响及其对长链非编码RNA(long non-coding RNA, lncRNA)肝癌高表达转录本(high up-regulated in liver cancer, HULC)表达调控的分子机制。方法 选取浓度为0(空白对照组)、5、10和20μg/mL的AgNPs处理人正常肝细胞系(LO2细胞),同时选用10μg/mL的AgNPs分别与DNA甲基化酶抑制剂5-氮杂胞嘧啶核苷(5-azacitidine, 5-azaC)和组蛋白去乙酰化酶抑制剂曲古抑菌素A(trichostatin A, TSA)联合处理LO2细胞24 h。采用qRT-PCR检测lncRNA HULC、HOTAIRM1、H19、MALAT1以及DNA甲基转移酶(DNA methyltransferases, DNMTs)家族、TETs家族的m RNA表达情况,免疫印迹实验(western blot, WB)检测DNMTs家族、TETs家族的蛋白表达情况,并且利用siRNA干扰技术沉默TET1的表达,进一步探讨TET1与lncRNA HULC的调控关系。结果 qRT-PCR结果显示,相较于空白对照组,各浓度组中H19的mRNA表达均下调(t=7.250、6.876、5.077,P均<0.05),20μg/mL AgNPs组lncRNA HULC的mRNA表达上调,HOTAIRM1表达下调(t=12.250、12.850,P均<0.05)。TSA干预后lncRNA HULC的表达上调,H19表达下调(t=12.970、12.950,P均<0.05)。相较于对照组,20μg/mL AgNPs组中的TET1和TET3表达上调(t=6.909、15.551,P均<0.05)。TSA干预后TET1的表达上调,TET3表达下调(t=17.224、3.602,P<0.05)。WB结果显示,相较于空白对照组,各浓度组的DNMT1和DNMT3a蛋白表达均上调,而DNMT3b蛋白表达均下调(t=5.968、2.518、4.010,t=8.983、16.230、14.260,t=23.000、41.630、49.300;P均<0.05)。5-azaC和TSA组分别干预后DNMT1蛋白表达均下调,DNMT3a蛋白表达均上调(t=3.111、3.695,t=30.740、62.790;P均<0.05),而DNMT3b表达分别下调和上调(t=7.024、3.372,P均<0.05)。各浓度组TET1的蛋白表达均上调(t=5.869、7.519、10.470,P均<0.05)。成功构建沉默TET1的细胞模型,si-TET1-3组(TET1基因沉默组)TET1蛋白和lncRNA HULC的mRNA表达均较sObjective To explore the impact of silver nanoparticles(AgNPs)on the expression of DNA demethylation enzyme,specifically ten-eleven-translocation proteins(TETs)family and the molecular mechanism underlying the regulation of long non-coding RNA HULC.Methods Human normal liver cells(LO2)were treated with silver nanoparticles(AgNPs)at concentrations of 0(blank control group),5,10 and 20μg/mL.Additionally,LO2 cells were treated with 10μg/mL AgNPs in combination with the DNA methyltransferase inhibitor 5-azacitidine(5-azaC)and the histone deacetylase inhibitor trichostatin A(TSA)for 24 hours.qRT-PCR was employed to assess the mRNA expression levels of long non-coding RNAs(lncRNAs)HULC,HOTAIRM1,H19 and MALAT1,as well as the expression of the DNA methyltransferases(DNMTs)and TETs families.Western blot(WB)was utilized to evaluate the protein expression levels of the DNMTs and TETs families.Furthermore,RNA interference(siRNA)technology was employed to silence the expression of TET1 in order to further investigate the regulatory relationship between TET1 and lncRNA HULC.Results qRT-PCR results showed that compared to the blank control group,the mRNA expression of H19 was downregulated in all concentration groups(t=7.250,6.876,5.077,all P<0.05).In the 20μg/mL Ag-NPs group,the mRNA expression of lncRNA HULC was upregulated,while HOTAIRM1 expression was downregulated(t=12.250,12.850,both P<0.05).After TSA intervention,the expression of lncRNA HULC was upregulated,and H19 expression was downregulated(t=12.970,12.950,both P<0.05).Compared with the control group,the expression of TET1 and TET3 in the 20μg/mL AgNPs group was up-regulated(t=6.909,15.551,both P<0.05).After TSA intervention,TET1 expression was upregulated and TET3 was down-regulated(t=17.224,3.602,both P<0.05).WB analysis revealed that compared to the blank control group,the protein expression of DNMT1 and DNMT3a was upregulated in all concentration groups,while DNMT3b protein expression was downregulated(t=5.968,2.518,4.010;t=8.983,16.230,14.260;t=23.000,41.630

关 键 词：银纳米颗粒 长链非编码RNA 肝癌高表达转录本 DNA去甲基化酶 

分 类 号：R34[医药卫生—基础医学]