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

doi:10.20199/j.issn.1672-2302.2024.05.008

摘要/Abstract

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20 μg/mL的AgNPs处理人正常肝细胞系（LO2细胞），同时选用10 μg/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mRNA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），20 μg/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，20 μg/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si-NC组（沉默对照组）降低（t=3.297、4.708，P均<0.05）。结论随着AgNPs浓度的增加，细胞中lncRNA HULC和DNA去甲基化酶TET1的表达均上调，添加5-azaC和TSA干预后可有效改变其表达水平，表明lncRNA HULC极大可能受到DNA甲基化的影响。

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

Abstract:

Objective 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 20 μg/mL. Additionally, LO2 cells were treated with 10 μg/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 20 μg/mL AgNPs 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 20 μg/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, 49.300, all P<0.05). After 5-azaC and TSA intervention, DNMT1 protein expression was downregulated, and DNMT3a protein expression was upregulated (t=3.111, 3.695; t=30.740, 62.790, all P<0.05), while DNMT3b expression showed a downregulation and upregulation trend respectively (t=7.024, 3.372, both P<0.05). The protein expression of TET1 was upregulated in all concentration groups (t=5.869, 7.519, 10.470, all P<0.05). Successful construction of TET1-silenced cell model, the expression of TET1 protein and lncRNA HULC mRNA in si-TET1-3 group (TET1 gene silenced group) was lower than that in si-NC group (silencing control group) (t=3.297, 4.708, both P<0.05). Conclusion With the increase in AgNPs concentration, the expression of lncRNA HULC and DNA demethylase TET1 in cells is significantly upregulated. The addition of 5-azaC and TSA can effectively alter their expression levels, indicating that lncRNA HULC is likely influenced by DNA methylation.

Key words: Silver nanoparticles (AgNPs), LncRNA, HULC, DNA demethylase

中图分类号:

焦群芳, 李惠芳, 郑冬燕, 黄豪, 钟清华, 蔡晓楠, 凌晓璇, 刘林华. 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[J]. 热带病与寄生虫学, 2024, 22(5): 294-300.

JIAO Qunfang, LI Huifang, ZHENG Dongyan, HUANG Hao, ZHONG Qinghua, CAI Xiaonan, LING Xiaoxuan, LIU Linhua. Upregulation of TET1 contributes to the activation of lncRNA HULC in LO2 cells treated with silver nanoparticles[J]. Journal of Tropical Diseases and Parasitology, 2024, 22(5): 294-300.

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基因	上游引物（5′-3′）	下游引物（5′-3′）
lncRNA HULC	AACAGACCAAAGCATCAAGCAA	CAAATTTGCCACAGGTTGAACAC
HOTAIRM1	GAACTGGCGAGAGGACGAAT	GTGGGGACTATGGCTGGTTT
H19	GAACTGGCGAGAGGACGAAT	GTGGGGACTATGGCTGGTTT
MALAT1	TTTGTTCATTTCTGGTGGTGGG	TAAGACCAAGGGAGGGGAGAG
TET1	GCCTCCATCAGACGAACCCCTAT	ACTCAATCAAAACCGAGCCGTG
TET2	GGAAGCCAGAATAGTCGTGTGAGTC	TCTGAAGGAGCCCAGAGAGAGAAG
TET3	AGCGCTAAAGCAAGGAAAGA	AAGCAAGTCTGGCTGGGTTT
GAPDH	GGAGTCAACGGATTTGGTCGTATTG	TCTCGCTCCTGGAAGATGGTGAT

细胞名称	siRNA序列（5′-3′）
si-NC	UUCUCCGAACGUGUCACGUdTdT
si-TET1-1	GGGCACAAUACAACAGAAAdTdT
si-TET1-2	GCAAAUCAACAGGAAGUUUCUdTdT
si-TET1-3	CAGUGUAACCAGCACAGUUdTdT

AgNPs浓度（μg/mL）	HULC	HOTAIRM1	H19	MALAT1
0	1.00±0.06	1.00±0.13	1.00±0.06	1.00±0.04
5	0.80±0.07	0.92±0.06	0.58±0.08^*	1.06±0.09
10	0.99±0.05	1.06±0.04	0.60±0.07^*	0.99±0.07
20	2.68±0.30^*	0.20±0.03^*	0.70±0.04^*	1.10±0.04

组别	HULC	HOTAIRM1	H19
AgNPs	1.00±0.09	1.00±0.09	1.00±0.03
AgNPs+5-azaC	2.05±0.24	1.16±0.12	0.86±0.05
AgNPs+TSA	7.25±0.80^*	1.19±0.18	0.46±0.03^*

AgNPs浓度（μg/mL）	TET1	TET2	TET3
0	1.00±0.28	1.00±0.07	1.00±0.01
5	0.82±0.04	0.86±0.06	0.73±0.12
10	1.33±0.31	1.20±0.14	2.01±0.02
20	4.86±1.29^*	1.15±0.13	25.87±3.92^*