大黄素调控组蛋白乙酰化促进HpG2肝癌细胞焦亡及凋亡的发生

doi:10.11958/20230775

天津医药 ›› 2024, Vol. 52 ›› Issue (1): 56-60.doi: 10.11958/20230775

大黄素调控组蛋白乙酰化促进HpG2肝癌细胞焦亡及凋亡的发生

刘国旗¹^,²(), 李程程¹, 刘声菊¹^,², 朱丽英¹^,^△()

1.贵州医科大学附属医院临床检验中心（邮编550004）
2.贵州医科大学附属医院临床检验中心输血科

收稿日期:2023-08-16 出版日期:2024-01-15 发布日期:2024-01-18
通讯作者: ^△E-mail：179128466@qq.com
作者简介:刘国旗（1989），男，初级检验技师，主要从事肿瘤及糖尿病并发症相关方面研究。E-mail：794104973@qq.com
基金资助:
贵州省科技厅项目(黔科合基础-ZK[2021]一般483);贵州省科技厅项目(黔科合基础-ZK[2021]一般366);贵州省卫生健康委科学技术基金(GZWKJ2021-360);贵州省卫生健康委科学技术基金(GZWJKJ2020-2-006)

Emodin regulates histone acetylation to promote pyroptosis and apoptosis of HpG2 hepatoma cells

LIU Guoqi¹^,²(), LI Chengcheng¹, LIU Shengju¹^,², ZHU Liying¹^,^△()

1. Center for Clinical Laboratories, the Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China
2. Department of Blood Transfusion, the Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China

Received:2023-08-16 Published:2024-01-15 Online:2024-01-18
Contact: ^△E-mail：179128466@qq.com

刘国旗, 李程程, 刘声菊, 朱丽英. 大黄素调控组蛋白乙酰化促进HpG2肝癌细胞焦亡及凋亡的发生[J]. 天津医药, 2024, 52(1): 56-60.

LIU Guoqi, LI Chengcheng, LIU Shengju, ZHU Liying. Emodin regulates histone acetylation to promote pyroptosis and apoptosis of HpG2 hepatoma cells[J]. Tianjin Medical Journal, 2024, 52(1): 56-60.

摘要/Abstract

摘要：

目的研究天然产物大黄素是否能够影响HpG2肝癌细胞中组蛋白乙酰化水平，进而加速肝癌细胞焦亡和凋亡，为肝癌的治疗提供新的靶点。方法 CCK-8法检测不同浓度大黄素对HpG2细胞活力的影响；生物信息学分析TCGA数据库中肝癌患者组蛋白乙酰化相关基因表达情况，验证候选基因赖氨酸乙酰基转移酶2A（KAT2A）与细胞凋亡通路的相关性；实时荧光定量PCR（qPCR）检测HepG2细胞与L02细胞KAT2A mRNA水平；酶联免疫吸附试验（ELISA）检测大黄素对HpG2细胞中组蛋白乙酰转移酶（HAT）、组蛋白去乙酰转移酶（HDAC）、白细胞介素（IL）-1β、IL-18的影响；流式细胞术检测大黄素对肝癌细胞凋亡的影响；Western blot检测细胞凋亡、细胞焦亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2-相关X蛋白质（Bax）、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、胱天蛋白酶1（Caspase-1）、Gasdermin家族成员D N端（GSDMD-N）及KAT2A的表达情况。结果大黄素能降低HpG2细胞活性，半抑制浓度（IC₅₀）95%置信区间为58.12~66.52 μmol/L。与正常肝组织相比，组蛋白乙酰化相关基因mRNA水平在肝癌组织中表达增高，且KAT2A变化倍数最大[log₂（Fold Change）=2.010，P<0.01]；在肝癌组织中，KAT2A mRNA的表达与细胞凋亡通路呈负相关（r_s=-0.230，P<0.01）。与L02细胞相比，KAT2A mRNA在HepG2中表达升高（P<0.05）；与对照组相比，大黄素干预组HAT和HDAC的表达水平下降，IL-18、IL-1β表达水平水平增高，细胞凋亡率升高，KAT2A、BAX的表达降低，Bcl-2、NLRP3、GSDMD-N及Caspase-1表达水平升高（P<0.05）。结论大黄素可抑制肝癌细胞活力，加速细胞凋亡和焦亡，其机制可能与调控KAT2A表达相关。

关键词: 大黄素, 癌, 肝细胞, 乙酰化作用, 乙酰基转移酶类, 组蛋白类, 细胞焦亡, 细胞凋亡, KAT2A

Abstract:

Objective To study whether emodin, a natural product, can affect the level of histone acetylation in HpG2 hepatocellular carcinoma cells (HCC), and then accelerate the occurrence of pyroptosis and apoptosis in hepatocellular carcinoma cells, so as to provide a new target for the treatment of liver cancer. Methods CCK-8 method was used to detect the effect of different concentrations of emodin on the viability of HpG2 cells. Bioinformatics was used to analyze histone acetylation-related genes in patients with liver cancer in TCGA database. The correlation between the candidate gene lysine acetyltransferase 2A (KAT2A) and the apoptosis pathway was verified. qPCR method was used to detect the mRNA level of KAT2A in HepG2 cells and L02 cells. The effects of emodin on histone acetyltransferase (HAT) and histone deacetyltransferase (HDAC), interleukin 1β (IL-1β) and interleukin 18 (IL-18) in HpG2 cells were detected by ELISA. The effect of emodin on the apoptosis of liver cancer cells was detected by flow cytometry. The expression level of cell apoptosis, pyroptosis-associated protein B lymphocytoma-2 (Bcl-2), Bcl-2-related X protein (Bax), NOD-like receptor thermal protein domain-related protein 3 (NLRP3), Caspase-1, Gasdermin family member DN terminal (GSDMD-N) and KAT2A were detected by Western blot assay. Results Emodin could reduce the activity of HpG2 cells, and the confidence interval of IC₅₀ 95% was 58.12-66.52 μmol/L. Compared with normal liver tissue, the expression of histone acetylation related gene mRNA was increased in HCC tissue, and the change of KAT2A was the highest [log₂(Fold Change)=2.010, P<0.01]. In HCC tissue, the expression of KAT2A mRNA was negatively correlated with apoptosis pathway (r_s=-0.230, P<0.01). Compared with L02 cells, the expression of KAT2A mRNA in HepG2 was higher (P<0.05). Compared with the control group, expression levels of HAT and HDAC decreased in the 60 μmol/L emodin intervention group, expression levels of IL-18 and IL-1β increased, the apoptosis rate increased, expression levels of KAT2A and BAX decreased, and expression levels of Bcl-2, NLRP3, GSDMD-N and Caspase-1 increased (P<0.05). Conclusion Emodin could inhibit the viability of hepatoma cells, accelerate apoptosis and pyroptosis, and its mechanism may be related to the regulation of KAT2A expression.

Key words: Emodin, carcinoma, hepatocellular, acetylation, acetyltransferases, histones, pyroptosis, apoptosis, KAT2A

中图分类号:

R285，R735.7

图/表 10

图1 大黄素作用肝癌细胞IC50值

Fig.1 Emodin affected IC50 of hepatocellular carcinoma cells

图2 肝癌组织与健康人肝组织组蛋白乙酰化相关差异基因火山图

Fig.2 Volcano map of histone acetylation-related differential genes between hepatocellular carcinoma tissue and healthy liver tissue

图3 KAT2A在肝癌组织及健康人肝组织mRNA表达量比较

Fig.3 Comparison of mRNA expression of KAT2A between hepatocellular carcinoma tissue and normal tissue

图4 Spearman分析KAT2A mRNA与细胞凋亡通路的相关性

Fig.4 Spearman analysis of the correlation between KAT2A mRNA and the apoptotic pathway

表1 对照组与大黄素干预组HAT、HDAC、IL-18、IL-1β水平的比较

Tab.1 Comparison of HAT, HDAC, IL-18 and IL-1β levels between the control group and the 60 μmol/L emodin intervention group （n=5，OD450，$\bar{x}±s$）

组别	HAT	HDAC	IL-18	IL-1β
对照组	1.37±0.20	1.29±0.12	0.14±0.01	0.18±0.04
大黄素干预组	0.69±0.13	0.89±0.07	0.31±0.05	0.40±0.05
t	6.043^＊＊	6.386^＊＊	7.077^＊＊	6.753^＊＊

图5 流式细胞术检测细胞凋亡

Fig.5 Flow cytometry detection of apoptosis

图6 Western blot检测KAT2A表达情况 A：对照组；B：60 μmol/L大黄素干预组；图7、8同。

Fig.6 Western blot detection of KAT2A expression

图7 Western blot检测细胞凋亡相关蛋白

Fig.7 Western blot detection of apoptosis-associated proteins

图8 Western blot检测细胞焦亡相关蛋白

Fig.8 Western blot detection of pyroptosis related proteins

表2 对照组和60 μmol/L大黄素干预组KAT2A等各种蛋白表达水平的比较

Tab.2 Comparison of KAT2A, Bcl-2, BAX, NLRP3, GSDMD-N and Caspase-1 protein expression levels between the control group and the 60 μmol/L emodin intervention group （n=3，$\bar{x}±s$）

组别	KAT2A	Bcl-2	BAX
对照组	0.91±0.26	0.26±0.06	1.04±0.04
大黄素干预组	0.35±0.12	1.08±0.17	0.65±0.12
t	3.426^＊	7.909^＊＊	5.211^＊＊
组别	NLRP3	GSDMD-N	Caspase-1
对照组	0.57±0.02	0.58±0.13	0.52±0.07
大黄素干预组	0.97±0.16	1.03±0.18	0.98±0.04
t	4.392＊	3.632＊	10.507＊＊

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大黄素调控组蛋白乙酰化促进HpG2肝癌细胞焦亡及凋亡的发生

Emodin regulates histone acetylation to promote pyroptosis and apoptosis of HpG2 hepatoma cells

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