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Article Open Access

KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances

  • Authors:
    • Ming Liu
    • Xintong Zhang
    • Yanfen Hu
    • Li Niu
    • Linzhi Guo
    • Guoqing Zhao
    • Fan Lu
    • Fengfeng Jia
    • Li Li
    • Hao Wu
    • Jianjun Zhu
  • View Affiliations / Copyright

    Affiliations: Department of Medical Cell Biology and Genetics, School of Basic Medical Science, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China, Department of Pathophysiology, School of Basic Medical Science, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China, Laboratory of Morphology, School of Basic Medical Science, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China, Department of Basic Medicine, School of Basic Medical Science, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China, Department of Neurology, Shanxi Coal Central Hospital, Taiyuan 030001, P.R. China, Taiyuan Technology Transfer Promotion Center, Taiyuan, Shanxi 030006, P.R. China
    Copyright: © Liu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 33
    |
    Published online on: January 13, 2026
       https://doi.org/10.3892/ijo.2026.5846
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Abstract

Kynurenine (Kyn) 3‑monooxygenase (KMO) is a key enzyme of the tryptophan (Try)‑Kyn pathway and is located on the outer membrane of mitochondria. Notably, it has not yet been elucidated as to whether KMO is involved in hepatocellular carcinoma (HCC) progression by affecting mitochondria. In the present study, KMO was revealed to be downregulated in HCC patients and this downregulation was associated with a poor prognosis. Notably, the downregulation of KMO promoted the proliferation and migration of HCC cells and increased mitochondrial mass. The levels of the Try metabolite 3‑hydroxyanthranilic acid (3‑HAA) were elevated in HCC cells overexpressing KMO. The results indicated that 3‑HAA may inhibit HCC cell growth promoted by KMO downregulation and reverse the KMO downregulation‑induced increase in mitochondrial mass. Furthermore, KMO and 3‑HAA were shown to regulate the expression of the transcription factor nuclear receptor subfamily 4 group A member 1 (NR4A1) and reduce NR4A1 mitochondrial translocation, thus inhibiting the growth of HCC cells. In summary, the current study elucidated that low KMO expression in HCC affects mitochondrial mass and function by reducing the level of the Try metabolite 3‑HAA, downregulating the expression of NR4A1 and promoting its mitochondrial translocation, which in turn may promote the progression of HCC. These findings provide new insights into the treatment of HCC, potentially targeting the mitochondria and the Try‑Kyn pathway.

View Figures

Figure 1

Downregulation of KMO expression is
related to the poor prognosis in HCC. (A) mRNA expression of KMO in
HCC tissue in TCGA database. (B) mRNA expression of KMO in cancer
tissues and adjacent tissues of 31 HCC patients by using reverse
transcription-quantitative PCR. (C) Immunohistochemistry showed the
protein expression of KMO in HCC tissue and para-cancerous tissue.
Scale bar, 50 µm. Immunohistochemistry score was compared
between two groups. (D) Survival analysis of KMO in HCC patients in
TCGA database. (E) tSNE plot with annotations for cell types of HCC
single-cell sequencing data analysis and the proportion of
different cell types. (F) tSNE plot with KMO expression in tumor
and normal samples. Bubble chart of KMO expression in two groups.
(G) KMO expression in different cell types in HCC tumor and normal
samples. *P<0.05, ***P<0.001. KMO,
kynurenine 3-monooxygenase; HCC, hepatocellular carcinoma; TCGA,
the Cancer Genome Atlas.

Figure 2

Downregulation of KMO promotes HCC
cell growth. (A) Effect of KMO on proliferation activity of HCC
cells by using EdU detection. EdU labeled newly proliferated cells
(red) and Hoechst33342 labeled cell nucleus (blue). Positive cells
rate (red/blue) were counted and compared between control and
KMO-knockdown or overexpression groups. Scale bar, 100 µm.
(B) Effect of KMO on colony-forming ability of HCC cells. Colony
number was counted and compared between control and KMO-knockdown
or overexpression groups. **P<0.01. KMO, kynurenine
3-monooxygenase; HCC, hepatocellular carcinoma.

Figure 3

Downregulation of KMO promotes HCC
cell migration. (A) Effect of KMO on migration of HCC cells by
using wound healing assay. Scale bar, 200 µm. Healing
percentage was calculated and compared between control and
KMO-knockdown or overexpression groups. (B) Effect of KMO on HCC
cell migration by using Transwell chamber assay. Scale bar, 200
µm. Migrated cell number was counted and compared between
control and KMO-knockdown or overexpression groups.
**P<0.01. KMO, kynurenine 3-monooxygenase; HCC,
hepatocellular carcinoma; si, short interfering; EV, control group;
OE, overexpressing.

Figure 4

KMO locates in mitochondria and is
related to Try-Kyn metabolism. (A) Weighted gene co-expression
network analysis of KMO-related genes. Dendrogram shows the size of
different color modules. The numerical value represents the
correlation coefficient and the values in parentheses represent the
significance P-value. Genes in MEturquoise module have the
strongest correlation with KMO expression (correlation
coefficient=0.45). (B) GO enrichment analysis of KMO-related
co-expressed genes. The GO cellular component analysis showed that
these genes were enriched in different mitochondrial components
(Red box). (C) Subcellular distribution of KMO in HCC cells
observed by using confocal microscopy after immunofluorescence
staining. KMO is shown in green and mitochondria are stained red.
The yellow color indicates the co-localization of KMO with
mitochondria. Scale bar, 20 µm. (D) KEGG enrichment analysis
of KMO-related co-expressed genes. These genes can be enriched in
Try metabolism signaling pathway (Red box). (E) The protein-protein
interaction network and correlation between KMO and key genes
involved in Try metabolism pathway. KMO, kynurenine
3-monooxygenase; Try, tryptophan; Kyn, kynurenine; GO, Gene
Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5

Downregulation of KMO causes disorder
of mitochondrial mass and function. (A) Effect of KMO on
mitochondrial mass of HCC cells was observed by using confocal
microscopy after MitoTracker staining. 2D and corresponding 3D
images were collected and the parts in white box in the 2D image
were magnified. Scale bar, 20 µm. (B) Mitochondrial mass was
analyzed by using the value of average fluorescence intensity per
cell. (C) Effect of KMO on mtDNA copy number in HCC cells was
assayed by using reverse transcription-quantitative PCR for MT-ND1.
(D) Heatmap showed the expression of 13 genes encoded by the
mitochondrial genome in the high and low KMO expression groups.
Lollipop chart showed the correlation between KMO and these 13
genes. (E) Reverse transcription-quantitative PCR verification of
mRNA expression levels of MT-ATP6 and MT-CO1 genes in HCC cells
with KMO knockdown and overexpression. *P<0.05,
**P<0.01, ***P<0.001. KMO, kynurenine
3-monooxygenase; si, short interfering; EV, control group; OE,
overexpressing.

Figure 6

Downregulation of KMO induced the
decrease of 3-HAA level promotes HCC cells proliferation. (A)
Heatmap showed tryptophan metabolites levels in KMO overexpression
and control HCC cell groups. (B) Comparison of 3-HAA levels between
KMO overexpression group and control group. (C) mRNA expression of
KYNU in KMO knockdown and overexpression HCC cells by using reverse
transcription-quantitative PCR. (D) Effect of 3-HAA on cell
proliferation in KMO knockdown HCC cells by using EdU detection.
EdU labeled newly proliferated cells (red) and Hoechst33342 labeled
cell nucleus (blue). Positive cells rate (red/blue) were counted
and compared between KMO-knockdown cells treated with DMSO
(control) and KMO-knockdown cells treated with 3-HAA groups. Scale
bar, 100 µm. (E) Effect of 3-HAA on cell viability in KMO
knockdown HCC cells by using CCK8 test on 0, 24, 48, 72 and 96 h.
The cell proliferation rates of KMO knockdown cells treated with
3-HAA or DMSO at the same time were compared. Final working
concentration of 3-HAA: 100 µM. *P<0.05,
**P<0.01, ***P<0.001, ns, P>0.05.
KMO, kynurenine 3-monooxygenase; 3-HAA, 3-hydroxyanthranilic acid;
KYNU, kynureninase.

Figure 7

Downregulation of KMO promotes
disorder of mitochondrial mass and function via 3-HAA in HCC cells.
(A) Effect of 3-HAA on mitochondrial mass in KMO knockdown HCC
cells was observed by using confocal microscopy after MitoTracker
staining. The parts in white box were magnified. Scale bar, 20
µm. (B) Mitochondrial mass was analyzed by using the value
of average fluorescence intensity per cell. (C) Effect of 3-HAA on
mtDNA copy number in KMO knockdown HCC cells. (D) Effect of 3-HAA
on ATP production in KMO knockdown HCC cells. (E) Effect of 3-HAA
on ROS production in KMO knockdown HCC cells. (Final working
concentration of 3-HAA: 100 µM. *P<0.05,
**P<0.01, ***P<0.001. KMO, kynurenine
3-monooxygenase; 3-HAA, 3-hydroxyanthranilic acid; HCC,
hepatocellular carcinoma.

Figure 8

Downregulation of KMO promotes the
decrease of NR4A1 in HCC cells. (A) Venn diagram among KMO related
DEGs, transcription factors and mitochondrial related genes. The
overlapped four genes are the screened mitochondrial related
transcription factors from KMO related DEGs. (B) Correlation
analysis between KMO and four mitochondrial related transcription
factors screened using TCGA database. (C) mRNA expression of four
mitochondrial related transcription factors in KMO-knockdown,
overexpression and 3-HAA treated KMO-knockdown HCC cells and the
corresponding controls. (D) Survival analysis of NR4A1 in HCC
patients in TCGA database. (E) The correlation analysis between
NR4A1 and mitochondrial DNA-encoded genes in HCC. Final working
concentration of 3-HAA: 100 µM. *P<0.05,
**P<0.01, ns: P>0.05. KMO, kynurenine
3-monooxygenase; NR4A1, nuclear receptor subfamily 4 group A member
1; HCC, hepatocellular carcinoma; DEGs, differentially expressed
genes; TCGA, the Cancer Genome Atlas; 3-HAA, 3-hydroxyanthranilic
acid.

Figure 9

Downregulation of KMO promotes
translocation of NR4A1 to mitochondria by 3-HAA. Effect of KMO and
3-HAA on subcellular distribution of NR4A1 in (A) MHCC-97H and (B)
LM3 cells. NR4A1 is shown in green and mitochondria are stained
red. The yellow color indicates the co-localization of NR4A1 with
mitochondria. Scale bar, 20 µm. (C) The overlap rate of
NR4A1 and mitochondria (orange color rate) was quantified and
compared between the indicated HCC cells. Final working
concentration of 3-HAA: 100 µM. Confocal microscope
magnification, ×100. *P<0.05,
***P<0.001. KMO, kynurenine 3-monooxygenase; NR4A1,
nuclear receptor subfamily 4 group A member 1; 3-HAA,
3-hydroxyanthranilic acid; HCC, hepatocellular carcinoma.

Figure 10

KMO affects cell growth,
mitochondrial quality and ATP production through NR4A1. (A) Effect
of NR4A1 downregulation on cell viability in KMO overexpression HCC
cells was observed by using CCK8 test on 0, 24, 48, 72 and 96 h.
The cell viability rates between KMO overexpression cells
transfected with siKMO and transfected with siControl were
compared. (B) Effect of NR4A1 downregulation on cell proliferation
in KMO overexpression HCC cells was observed by using EdU test. EdU
labeled newly proliferated cells (red) and Hoechst33342 labeled
cell nucleus (blue). Scale bar, 100 µm. Positive cells rate
(red/blue) were counted and compared between KMO overexpression
cells and KMO overexpression cells with NR4A1 knockdown. (C) Effect
of NR4A1 downregulation on mitochondrial mass in KMO overexpression
HCC cells was observed by using confocal microscopy after
MitoTracker staining. The parts in white box were magnified. Scale
bar, 20 µm. Mitochondrial mass was analyzed by using the
value of average fluorescence intensity per cell. (D) Effect of
NR4A1 downregulation on ATP production in KMO overexpression HCC
cells. (E) The mechanism of KMO downregulation promoting tumor
growth in HCC: KMO downregulation reduced 3-HAA levels, which
suppressed expression of the transcription factor NR4A1 and drove
its mitochondrial translocation, thereby disrupting mitochondrial
homeostasis and promoting the proliferation of HCC cells.
*P<0.05, **P<0.01. KMO, kynurenine
3-monooxygenase; NR4A1, nuclear receptor subfamily 4 group A member
1; HCC, hepatocellular carcinoma; 3-HAA, 3-hydroxyanthranilic acid;
ROS, reactive oxygen species; 3-HK, 3-hydroxykynurenine QUIN,
quinolinic acid; Try, tryptophan; Kyn, kynurenine.
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Copy and paste a formatted citation
Spandidos Publications style
Liu M, Zhang X, Hu Y, Niu L, Guo L, Zhao G, Lu F, Jia F, Li L, Wu H, Wu H, et al: <p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>. Int J Oncol 68: 33, 2026.
APA
Liu, M., Zhang, X., Hu, Y., Niu, L., Guo, L., Zhao, G. ... Zhu, J. (2026). <p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>. International Journal of Oncology, 68, 33. https://doi.org/10.3892/ijo.2026.5846
MLA
Liu, M., Zhang, X., Hu, Y., Niu, L., Guo, L., Zhao, G., Lu, F., Jia, F., Li, L., Wu, H., Zhu, J."<p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>". International Journal of Oncology 68.3 (2026): 33.
Chicago
Liu, M., Zhang, X., Hu, Y., Niu, L., Guo, L., Zhao, G., Lu, F., Jia, F., Li, L., Wu, H., Zhu, J."<p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>". International Journal of Oncology 68, no. 3 (2026): 33. https://doi.org/10.3892/ijo.2026.5846
Copy and paste a formatted citation
x
Spandidos Publications style
Liu M, Zhang X, Hu Y, Niu L, Guo L, Zhao G, Lu F, Jia F, Li L, Wu H, Wu H, et al: <p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>. Int J Oncol 68: 33, 2026.
APA
Liu, M., Zhang, X., Hu, Y., Niu, L., Guo, L., Zhao, G. ... Zhu, J. (2026). <p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>. International Journal of Oncology, 68, 33. https://doi.org/10.3892/ijo.2026.5846
MLA
Liu, M., Zhang, X., Hu, Y., Niu, L., Guo, L., Zhao, G., Lu, F., Jia, F., Li, L., Wu, H., Zhu, J."<p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>". International Journal of Oncology 68.3 (2026): 33.
Chicago
Liu, M., Zhang, X., Hu, Y., Niu, L., Guo, L., Zhao, G., Lu, F., Jia, F., Li, L., Wu, H., Zhu, J."<p>KMO downregulation promotes hepatocellular carcinoma growth via 3‑HAA‑mediated mitochondrial mass and function imbalances</p>". International Journal of Oncology 68, no. 3 (2026): 33. https://doi.org/10.3892/ijo.2026.5846
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