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. 2024 May;28(10):e18317.
doi: 10.1111/jcmm.18317.

Integrating single-cell and multi-omic approaches reveals Euphorbiae Humifusae Herba-dependent mitochondrial dysfunction in non-small-cell lung cancer

Chengcheng Zhang  1 , Xiaoxue Zhao  1 , Feng Li  2 , Jingru Qin  1 , Lu Yang  1 , Qianqian Yin  1 , Yiyi Liu  1 , Zhiyao Zhu  1 , Fei Zhang  3 , Zhongqi Wang  1 , Haibin Liang  3
Affiliations

Integrating single-cell and multi-omic approaches reveals Euphorbiae Humifusae Herba-dependent mitochondrial dysfunction in non-small-cell lung cancer

Chengcheng Zhang et al. J Cell Mol Med. 2024 May.

Erratum in

Abstract

Euphorbiae Humifusae Herba (EHH) is a pivotal therapeutic agent with diverse pharmacological effects. However, a substantial gap exists in understanding its pharmacological properties and anti-tumour mechanisms. This study aimed to address this gap by exploring EHH's pharmacological properties, identifying NSCLC therapy-associated protein targets, and elucidating how EHH induces mitochondrial disruption in NSCLC cells, offering insights into novel NSCLC treatment strategies. String database was utilized to explore protein-protein interactions. Subsequently, single-cell analysis and multi-omics further unveiled the impact of EHH-targeted genes on the immune microenvironment of NSCLC, as well as their influence on immunotherapeutic responses. Finally, both in vivo and in vitro experiments elucidated the anti-tumour mechanisms of EHH, specifically through the assessment of mitochondrial ROS levels and alterations in mitochondrial membrane potential. EHH exerts its influence through engagement with a cluster of 10 genes, including the apoptotic gene CASP3. This regulatory impact on the immune milieu within NSCLC holds promise as an indicator for predicting responses to immunotherapy. Besides, EHH demonstrated the capability to induce mitochondrial ROS generation and perturbations in mitochondrial membrane potential in NSCLC cells, ultimately leading to mitochondrial dysfunction and consequent apoptosis of tumour cells. EHH induces mitochondrial disruption in NSCLC cells, leading to cell apoptosis to inhibit the progress of NSCLC.

Keywords: EHH; NSCLC; immune; mitochondrial dysfunction; multi‐omics; single‐cell.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic diagram. (**p < 0.01, ***p < 0.001).
FIGURE 2
FIGURE 2
Screening for common genes. (A) The common 133 target genes for EHH and NSCLC. (B) Number of genes, regarding EHH target genes, and differential genes in NSCLC.
FIGURE 3
FIGURE 3
Interaction networks of EHH‐targeted genes and core genes (A) Protein–protein interactions exhibits the networks of EHH‐targeted genes. (B) 5 Core networks of PPI.
FIGURE 4
FIGURE 4
KEGG and signalling pathways. (A) Regulatory networks of EHH‐targeted genes. (B) Seven active components and 10 core target proteins of EHH. (C) Core signalling pathway induced by EHH.
FIGURE 5
FIGURE 5
Single‐cell sequencing analysis in NSCLC. (A) UMAP diagram showing cell subpopulations. (B) Up‐regulated KEGG genesets. (C) 10‐core genes expression levels in the cell subtypes of NSCLC. (D) Up‐regulated immunologic genesets in NSCLC. (E) Down‐regulated immunologic genesets in NSCLC.
FIGURE 6
FIGURE 6
Single‐cell sequencing demonstrates expression patterns of 10 key genes in metastatic NSCLC.
FIGURE 7
FIGURE 7
Gene Ontology (GO) and KEGG Pathway Analysis. (A) Top 20 of pathway enrichment involved by EHH‐targeted genes. (B) The association between pathways. (**p < 0.01, ***p < 0.001).
FIGURE 8
FIGURE 8
Patterns of immune infiltration. (A) Relationship between AKT1 and immune cell infiltration. (B) Relationship between AKT1 and immune checkpoint expression. (C, D) Expression levels of 10 key genes and the prediction ability of immune response in NSCLC. (E, F) GSEA enrichment shows the key immune pathways. (***p < 0.001).
FIGURE 9
FIGURE 9
Molecular docking between seven bioactive compounds of EHH and the receptor proteins.
FIGURE 10
FIGURE 10
In vitro anti‐NSCLC effects of EHH. (A, B) CCK‐8 assay. (C, D) Western blot detected protein expression after treating with EHH in NCI‐H1299 cell line. (E) ROS level after treating with EHH in NCI‐H1299 cell line. (F) Mitochondrial membrane potential in NCI‐H1299 cells after treating with EHH. (*p < 0.05, **p < 0.01, ***p < 0.001).
FIGURE 11
FIGURE 11
The relationship between CASP 3 and survival in patients with NSCLC. (A) Survival analysis indicates that CASP 3 is indicative of improved survival outcomes in patients with NSCLC. (B) Among the 10 central genes analysed, CASP3 stands out as the sole gene correlated with prognosis.
FIGURE 12
FIGURE 12
Association between CASP3 and immune cell infiltration levels. (A, B) Immune infiltration landscapes in NSCLC patients exhibiting high versus low CASP3 expression. (C) Correlation of CASP3 expression levels with immune checkpoint expression (*p < 0.05, **p < 0.01, ***p < 0.001).
FIGURE 13
FIGURE 13
In vivo anti‐NSCLC effects of EHH. (A, B) Subcutaneous tumour formation test. (C) HE staining of heart, kidney and liver. (EHH‐H: high‐dose EHH; EHH‐L: low‐dose EHH; N.C: control group). (D) Immunohistochemistry confirms the anti‐tumour effect of EHH (*p < 0.05, **p < 0.01, ***p < 0.001).
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