肝星状细胞通过胱硫醚γ-裂解酶/硫化氢（CSE/H<sub>2</sub>S）调控肝细胞癌细胞凋亡的作用及其机制

尚宏伟; 马亚楠; 路欣; 吕翎娜; 丁惠国

doi:10.12449/JCH241117

肝星状细胞通过胱硫醚γ-裂解酶/硫化氢（CSE/H₂S）调控肝细胞癌细胞凋亡的作用及其机制

DOI: 10.12449/JCH241117

尚宏伟¹,
马亚楠^{2, 3},
路欣¹,
吕翎娜²,
丁惠国^2, , ,

1.
首都医科大学基础医学院，北京 100069
2.
首都医科大学附属北京佑安医院肝病消化中心，北京 100069
3.
山西白求恩医院肿瘤中心，太原 030000

基金项目:

中德研究小组项目 (GZ1517)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：尚宏伟负责实验指导，收集分析数据，撰写及修改论文；马亚楠负责实验设计，收集分析数据；路欣、吕翎娜负责收集分析数据；丁惠国负责课题设计，审核文章并最后定稿。尚宏伟和马亚楠对本文贡献等同，同为第一作者。

详细信息

通信作者:
丁惠国， dinghuiugo@ccmu.edu.cn （ORCID： 0000-0002-8716-4926）

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出版历程
- 收稿日期: 2024-08-07
- 录用日期: 2024-08-26
- 出版日期: 2024-11-25

Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionine γ-lyase/hydrogen sulfide

Hongwei SHANG¹,
Yanan MA^{2, 3},
Xin LU¹,
Lingna LYU²,
Huiguo DING^{2
, ,
,}

1.
School of Basic Medical Sciences，Capital Medical University，Beijing 100069，China
2.
Department of Hepatology and Gastroenterology，Beijing YouAn Hospital，Capital Medical University，Beijing 100069，China
3.
Oncology Center，Shanxi Bethune Hospital，Taiyuan 030000，China

Research funding:

Sino-German Cooperation Group (GZ1517)

More Information

Corresponding author: DING Huiguo， dinghuiugo@ccmu.edu.cn （ORCID： 0000-0002-8716-4926）

摘要

摘要: 目的肝星状细胞（HSC）和硫化氢（H₂S）信号分子作为肝细胞癌（HCC）微环境中重要的组分，参与调控HCC的发生发展等多种生物学进程。本研究通过HSC与肝癌细胞系共培养，探讨HSC通过分泌H₂S参与调控肝癌细胞凋亡的作用及其机制。方法以HSC细胞系（LX-2）及肝癌细胞系（HepG2、PLC/PRF/5）为研究对象。RT-qPCR和Western Blot（WB）法检测LX-2中H₂S关键合成酶——胱硫醚γ-裂解酶（CSE）mRNA及表达水平；ELISA测定上清液LX-2产生的H₂S浓度；二代测序、细胞免疫荧光、染色质免疫沉淀（ChIP）及WB检测H₂S（内源性和外源性）作用HepG2和PLC/PRF/5细胞后，JNK/JunB-TNFSF14信号通路基因、结合位点及相关蛋白。Transwell小室将LX-2分别与HepG2和PLC/PRF/5共培养，CCK-8和流式细胞术检测肝癌细胞的细胞活力、凋亡，WB测定H₂S-TNFSF14信号通路相关蛋白。所有细胞实验均重复3次。计量资料两组间比较采用成组t检验；多组间比较采用单因素方差分析或重复测量方差分析，进一步两两比较采用Dunnett-t检验。结果 LX-2主要通过CSE合成H₂S，LX-2培养上清液中H₂S浓度随着时间延长逐渐增加［（22.89±0.08）pg/mL vs （28.29±0.15）pg/mL vs （36.19±1.90）pg/mL，F=79.63，P<0.05］。LX-2中CSE mRNA水平显著高于CBS mRNA和MPST mRNA（1.008±0.13 vs 0.320±0.014 vs 0.05±0.02， F=80.84，P<0.05）。当CSE被炔丙基甘氨酸（PPG）抑制之后，随着PPG浓度增加，H₂S浓度下降（P<0.05）。LX-2分别与HepG2、PLC/PRF/5共培养，随着培养时间延长，HepG2（87.48%±0.82% vs 70.48%±0.641% vs 52.89%±0.57% vs 45.20%±0.69%， F=1 517.13，P<0.001）和PLC/PRF/5（92.41%±0.48% vs 74.10%± 0.73% vs 53.70%±0.60% vs 44.00%± 0.27%，F=2 626.21，P<0.001）细胞活力降低；凋亡增加（HepG2：12.88%±0.64% vs 15.5%±0.16% vs 18.43%±0.37% vs 13.01%±0.58%，F=142.15，P<0.001；PLC/PRF/5：8.51±0.05 vs 12.80±0.33 vs 15.59±0.21 vs 10.72±0.30，F=676.40，P<0.001），第3天最显著。二代测序显示，内源性H₂S（LX-2产生）和NaHS（外源性H₂S供体）参与调控HepG2中多种基因表达。NaHS和LX-2通过释放H₂S，激活肝癌细胞中JNK/JunB信号通路、上调凋亡基因TNFSF14表达，且p-JunB与TNFSF14基因转录调控区域结合增加。结论在肝癌微环境中，HSC通过信号分子CSE/H₂S，激活了肝癌细胞中JNK/JunB信号通路，TNFSF14表达增加，从而促进了肝癌细胞凋亡。
- 癌，肝细胞 /
- 肝星状细胞 /
- 硫化氢
Abstract: Objective As important components in the microenvironment of hepatocellular carcinoma （HCC）， hepatic stellate cells （HSCs） and hydrogen sulfide （H₂S） participate in various biological processes that regulate the development and progression of HCC. Through the co-culture of HSCs and HCC cells， this article aims to investigate the role and mechanism of HSCs in regulating the apoptosis of HCC cells by secreting H₂S. Methods The HSC cell line （LX-2） and HCC cell lines （HepG2 and PLC/PRF/5） were used for experiment. RT-qPCR and Western Blot （WB） were used to measure the mRNA and protein expression levels of cystathionine γ-lyase （CSE）， a key synthase for H₂S； ELISA was used to measure the concentration of H₂S in supernatant； next-generation sequencing， cell immunofluorescence assay， chromatin immunoprecipitation （ChIP）， and WB were used to measure the JNK/JunB-TNFSF14 signaling pathway genes， binding sites， and related proteins after HepG2 cells were treated by H₂S. LX-2 cells were co-cultured with HepG2 or PLC/PRF/5 cells in a Transwell chamber； CCK-8 assay and flow cytometry were used to measure the viability and apoptosis of HCC cells， and WB was used to measure the H₂S-TNFSF14 signaling pathway-related proteins. All cell experiments were repeated three times. The independent-samples t test was used for comparison of continuous data between two groups； a one-way analysis of variance or the analysis of variance with repeated measures was used for comparison between multiple groups， and the Dunnett-t test was used for further comparison between two groups. Results LX-2 cells synthesized H₂S mainly through CSE， and the concentration of H₂S in supernatant of LX-2 cells gradually increased over time （22.89±0.08 pg/mL vs 28.29±0.15 pg/mL vs 36.19±1.90 pg/mL， F=79.63， P<0.05）. In LX-2 cells， the mRNA expression level of CSE was significantly higher than that of CBS and MPST （1.008±0.13 vs 0.320±0.014 vs 0.05±0.02， F=80.84， P<0.05）. When CSE was inhibited by PPG， the concentration of H₂S decreased with the increase in the concentration of PPG （P<0.05）. LX-2 cells were co-cultured with HepG2 or PLC/PRF/5 cells， and over the time of culture， there were significant reductions in the viability of HepG2 cells （87.48%±0.82% vs 70.48%±0.641% vs 52.89%±0.57% vs 45.20%±0.69%， F=1 517.13， P<0.001） and PLC/PRF/5 cells （92.41%±0.48% vs 74.10%±0.73% vs 53.70%±0.60% vs 44.00%±0.27%， F=2626.21， P<0.001） and significant increases in the apoptosis of HepG2 cells （12.88%±0.64% vs 15.5%±0.16% vs 18.43%±0.37% vs 13.01%±0.58%， F=142.15， P<0.001） and PLC/PRF/5 cells （8.51±0.05 vs 12.80±0.33 vs 15.59±0.21 vs 10.72±0.30， F=676.40， P<0.001）， with the most significant changes on day 3. Next-generation sequencing showed that endogenous H₂S and NaHS （endogenous H₂S donor） were involved in regulating the expression of various genes in HepG2 cells. By releasing H₂S， NaHS and LX2 activated the JNK/JunB signaling pathway and upregulated the expression of the apoptosis gene TNFSF14 in HCC cells， with increased binding between p-JunB and the transcriptional regulatory regions of the TNFSF14 gene. Conclusion In the microenvironment of HCC， HSCs activate the JNK/JunB signaling pathway in HCC cells through the signal molecules CSE/H₂S， and there is an increase in the expression of TNFSF14， thereby promoting the apoptosis of HCC cells.
- Carcinoma， Hepatocellular /
- Hepatic Stellate Cells /
- Hydrogen Sulfide

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图 1 LX-2通过CSE途径产生H₂S

注： a，LX-2细胞产生H₂S的浓度；b，CSE、CBS和MPST的mRNA在LX-2细胞中的表达倍数；c，PPG处理后LX-2产生H₂S的浓度；d，CSE的mRNA表达；e，CSE蛋白的表达水平。

Figure 1. LX-2 produce H₂S mainly via the CSE

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图 2 LX-2与肝癌细胞系共培养对肝癌细胞活力及凋亡的影响

注： a，HepG2细胞活力；b，PLC/PRF/5细胞活力；c，HepG2细胞凋亡；d，PLC/PRF/5细胞凋亡。a、c，对照组为单独HepG2培养；b、d，对照组为单独PLC/PRF/5培养。与第1天比较，*P<0.05。

Figure 2. Effect of LX-2 co-culture with hepatoma cell on viability and apoptosis

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图 3 RNA-seq分析内源性和外源性H₂S对HepG2细胞转录基因的影响

注： a，聚类分析；b，差异倍数值变化最大的30个差异基因；c，TNFSF14基因表达变化。对照组，单独HepG2培养。

Figure 3. RNA-seq to analyse the transcriptome of endogenous and exogenous of H₂S on HepG2

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图 4 内源性H₂S和NaHS通过激活JNK/JunB信号通路调控TNFSF14转录

注： a，ChIP显示，在HepG2和PLC/PRF/5细胞中，p-JunB与TNFSF14启动子结合力增加；b，NaHS分别处理HepG2和PLC/PRF/5 0、1、2、8、24 h后，HepG2和PLC/PRF/5细胞核中p-JunB表达及核转位，DAPI（蓝色）和FITC（绿色）分别为对细胞核DNA及p-JunB进行染色；c，NaHS不同处理时间后，HepG2和PLC/PRF/5细胞核与细胞质中p-JunB的表达；d，NaHS处理组和LX-2共培养组HepG2及PLC/PRF/5细胞的p-JunB蛋白变化；e，PPG和SP600125抑制了H₂S及NaHS对HepG2与PLC/PRF/5中JNK/p-JNK、JunB/p-JunB、TNFSF14蛋白水平。

Figure 4. Endogenous H₂S and NaHS upregulated TNFSF14 via JNK/JunB signaling pathway

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表 1 RT-qPCR和ChIP-PCR所需要的引物序列

Table 1. Primers for Real-Time PCR and ChIP-PCR

项目	寡核苷酸序列
RT-qPCR引物
TNFSF14	F：5'-CGTGAGACCTTCGCTCTTGTAT-3'
	R：5'-CCCTCAGTGTTTGTGGTGGAT-3'
CSE	F：5'-AAGACGCCTCCTCACAAGGT-3'
	R：5'-ATATTCAAAACCCGAGTGCTGG-3'
GAPDH	F：5'-TGAAGGTCGGAGTCAACGGA-3'
	R：5'-CCTGGAAGATGGTGATGGGAT-3'
CBS	F：5'-AATGGTGACGCTTGGGAA-3' R：5'-TGAGGCGGATCTGTTTGA-3'
MPST	F：5'-GACCCCGCCTTCATCAAG-3' R：5'-CATGTACCACTCCACCCA-3'
ChIP-qPCR引物
TNFSF14	F：5'-TTGTTCATTGCTGCATCCCC-3'
	R：5'-CTCCTCTTCTTCCGGTACCC-3'

下载: 导出CSV

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