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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

尚宏伟 马亚楠 路欣 吕翎娜 丁惠国

引用本文:
Citation:

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
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)

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

Research funding: 

Sino-German Cooperation Group (GZ1517)

More Information
  • 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。

     

  • 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

    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

    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

    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

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