Role and mechanism of hepatic stellate cells in regulating the apoptosis of hepatocellular carcinoma cells through cystathionine γ-lyase/hydrogen sulfide
10.12449/JCH241117
- VernacularTitle: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
- Author:
Hongwei SHANG
1
;
Yanan MA
;
Xin LU
;
Lingna LYU
;
Huiguo DING
Author Information
1. 首都医科大学基础医学院,北京 100069
- Keywords:
Carcinoma,Hepatocellular;
Hepatic Stellate Cells;
Hydrogen Sulfide
- From:
Journal of Clinical Hepatology
2024;40(11):2238-2245
- CountryChina
- Language:Chinese
-
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.
