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ER‑α36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells

  • Authors:
    • Huanhuan He
    • Xuan Wang
    • Zhixuan Wei
    • An Wang
    • Xiangyue Fang
    • Hanbo He
    • Zhuorui Wu
    • Xiji Shu
    • Binlian Sun
    • Qiongxia Chen
    • Xuan Huang
    • Hongyan Zhou
    • Yuchen Liu
    • Zhengqi Fu
  • View Affiliations / Copyright

    Affiliations: Department of Pathology and Pathophysiology, School of Medicine, Jianghan University, Wuhan, Hubei 430056, P.R. China, Cancer Institute, School of Medicine, Jianghan University, Wuhan, Hubei 430056, P.R. China
    Copyright: © He et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 284
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    Published online on: August 11, 2025
       https://doi.org/10.3892/mmr.2025.13649
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Abstract

Estrogen receptor (ER)‑α36 and autophagy have each independently been reported to promote the proliferation of liver cancer cells; however, the association between them has not been explored. Therefore, the present study aimed to investigate the role and the underlying mechanism of ER‑α36 in the regulation of autophagy in liver cancer cells. The proliferation of liver cancer cell variants was examined by colony formation assay. A xenograft tumor model in nude mice was used to examine the role of ER‑α36 in malignant proliferation of liver cancer cells in vivo. Autophagic flux and lysosomal localization were assessed with immunofluorescence and confocal microscopy. The levels of ER‑α36, LAMP1, AKT, p62 and LC3‑Ⅱ/Ⅰ in liver cancer cell variants, and tumors formed by HepG2 cell variants in the nude mice were examined using Western blot and immunohistochemistry. The results revealed that ER‑α36 knockdown impaired autophagic flux by increasing lysosomal membrane permeabilization (LMP) and blocking lysosomal degradation. ER‑α36 knockdown also significantly inhibited the proliferation of liver cancer cells and orthotopic liver xenograft tumors. In addition, decreased AKT phosphorylation and the juxtanuclear clustering of lysosomes were observed in the liver cancer cells with ER‑α36 knockdown. In vitro experiments using the AKT inhibitor MK‑2206 indicated that AKT is involved in the ER‑α36 knockdown‑induced changes in LMP and lysosomal localization in liver cancer cells. In summary, the present study revealed that ER‑α36 plays a role in regulating the autophagy and proliferation of liver cancer cells, which is associated with the modulation of AKT signaling, LMP and lysosome localization. These findings highlight an important role of ER‑α36 in liver tumorigenesis.
View Figures

Figure 1

ER-α36 knockdown attenuates the
colony formation of liver cancer cells and promotes the
accumulation of autophagosomes. Expression of ER-α36 in stably
transfected HepG2 and Huh7 cells: (A) Representative western blots
and (B) quantitative analysis. (C) Representative images of colony
formation by the liver cancer cell variants and (D) quantitative
analysis. (E) Levels of LC3-II and LC3-I in the liver cancer cell
variants measured by western blotting and (F) quantitative analysis
of the LC3-II/LC3-I ratio. Data are presented as the mean ± SEM
(n=3). **P<0.01. (G) Analysis of the liver cancer cells by
immunofluorescence assay using an LC3 antibody and nuclear staining
with Hoechst 33342. Scale bar, 20 µm. ER, estrogen receptor; LC3,
microtubule-associated protein 1 light chain 3; Sh36, transfected
with ER-α36 specific short hairpin RNA expression vector; Vector,
transfected with empty vector.

Figure 2

ER-α36 knockdown blocks autophagic
flux and degradation. Expression of p62 in stably transfected liver
cancer cells with different levels of ER-α36 expression: (A)
Representative western blots and (B) quantitative analysis. (C)
Confocal microscopy images of liver cancer cells with and without
ER-α36 knockdown infected with pmCherry-enhanced GFP-LC3b
adenovirus (scale bar, 10 µm) and (D) quantification of yellow and
red puncta. (E) Levels of LC3-II and LC3-I assessed by western
blotting in transfected liver cancer cells treated with or without
CQ (20 µM) for 24 h, and (F) quantitative analysis of the
LC3-II/LC3-I ratio. (G) Western blots of ubiquitinated proteins in
the transfected liver cancer cells and (H) quantitative analysis of
Ub levels. **P<0.01. ER, estrogen receptor; GFP, green
fluorescence protein; LC3, microtubule-associated protein 1 light
chain 3; CQ, chloroquine; Ub, ubiquitin; Sh36, transfected with
ER-α36 specific short hairpin RNA expression vector; Vector,
transfected with empty vector.

Figure 3

ER-α36 knockdown induces lysosomal
damage and membrane permeabilization. (A) Co-localization of LC3
(green) and LAMP1 (red) in stably transfected liver cancer cells
with different levels of ER-α36 expression. Scale bar, 10 µm. (B)
Fluorescence images of the transfected liver cancer cells following
staining with Lyso-Tracker Red. Scale bar, 20 µm. (C)
Co-localization of Gal-3 and LAMP1 in the transfected liver cancer
cells. Scale bar, 10 µm. (D) Expression levels of LAMP1 in the
liver cancer cells evaluated by western blotting and (E)
quantitative analysis. Data are presented as the mean ± SEM.
**P<0.01. ER, estrogen receptor; LC3, microtubule-associated
protein 1 light chain 3; LAMP1, lysosome-associated membrane
protein 1; Gal-3, galectin-3; Sh36, transfected with ER-α36
specific short hairpin RNA expression vector; Vector, transfected
with empty vector.

Figure 4

ER-α36 knockdown suppresses the
malignant proliferation of liver cancer cells and induces lysosomal
membrane permeabilization. (A) Representative images of liver
tumors from nude mice intrahepatically injected with HepG2 cells
expressing different ER-α36 levels, harvested at 28 days
post-injection. (B) Quantitative analysis of liver tumor volume,
liver weight, body weight and the ratio of liver weight to body
weight. (C) Representative H&E staining images showing
pathological karyomitosis changes. Scale bar, 50 µm. (D)
Immunohistochemical staining of LC3, p62, LAMP1 and Ki67 with
corresponding IOD values. Scale bar, 50 µm. (E) Immunofluorescence
staining of Gal-3 in the tumors formed from the transfected HepG2
cells. Scale bar, 10 µm. (F) Western blotting results showing the
levels of LC3-II and LC3-II, p62, LAMP1, p-AKT and t-AKT in the
tumors, and (G) quantitative analyses of the LC3-II/LC3-I and
p-AKT/t-AKT ratios, and p62 and LAMP1 expression levels. β-actin
was used as the internal loading control. Data are presented as the
mean ± SEM. **P<0.01. ER, estrogen receptor; H&E,
hematoxylin and eosin; LC3, microtubule-associated protein 1 light
chain 3; LAMP1, lysosome-associated membrane protein 1; Gal-3,
galectin-3; IOD, integrated optical density; p-, phosphorylated;
t-, total; Sh36, transfected with ER-α36 specific short hairpin RNA
expression vector; Vector, transfected with empty vector.

Figure 5

ER-α36 knockdown decreases AKT
phosphorylation and influences lysosomal localization in liver
cancer cells. Stably transfected liver cancer cells with different
levels of ER-α36 expression were treated with or without MK-2206
(100 nM) for 6 h. (A) Representative western blots of p-AKT and
t-AKT, and (B) quantitative analysis. (C) Immunofluorescence
analysis of LAMP1. Cell peripheries and the juxtanuclear region are
indicated with dashed lines and solid lines, respectively. Scale
bar, 10 µm. (D) Quantification of the juxtanuclear percentage of
LAMP1. **P<0.01. ER, estrogen receptor; p-, phosphorylated; t-,
total; LAMP1, lysosome-associated membrane protein 1; Sh36,
transfected with ER-α36 specific short hairpin RNA expression
vector; Vector, transfected with empty vector.

Figure 6

AKT is involved in the lysosomal
localization and LMP induced by ER-α36 knockdown. Transfected liver
cancer cells with different levels of ER-α36 expression were
treated with MK-2206 (100 nM) for 6 h, followed by E2 (1 nM) for 30
min, and the same volume of alcohol was used as a vehicle control.
(A) Representative western blots of ER-α36, p-AKT and AKT, and (B)
quantitative analysis of ER-α36 expression and the p-AKT/t-AKT
ratio. In subsequent assays, the transfected liver cancer cells
were treated with MK-2206 (100 nM) for 6 h, followed by E2 (1 nM)
for 24 h. (C) Representative western blots of LAMP1 and (D)
quantitative analysis of LAMP1 expression. (E) Representative
images of colony formation by the liver cancer cells and (F)
quantitative analysis of colony forming ability. Data are presented
as the mean ± SEM. **P<0.01. (G) Fluorescence microscopy images
of the cells following staining with Lyso-Tracker Red. Scale bar,
20 µm. (H) Co-localization of Gal-3 (green) and LAMP1 (red) in the
cells as revealed by immunofluorescence staining. Scale bar, 10 µm.
ER, estrogen receptor; E2, 17β-estradiol; Gal-3, galectin-3; p-,
phosphorylated; t-, total; LAMP1, lysosome-associated membrane
protein 1; Sh36, transfected with ER-α36 specific short hairpin RNA
expression vector; Vector, transfected with empty vector.
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Copy and paste a formatted citation
Spandidos Publications style
He H, Wang X, Wei Z, Wang A, Fang X, He H, Wu Z, Shu X, Sun B, Chen Q, Chen Q, et al: ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells. Mol Med Rep 32: 284, 2025.
APA
He, H., Wang, X., Wei, Z., Wang, A., Fang, X., He, H. ... Fu, Z. (2025). ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells. Molecular Medicine Reports, 32, 284. https://doi.org/10.3892/mmr.2025.13649
MLA
He, H., Wang, X., Wei, Z., Wang, A., Fang, X., He, H., Wu, Z., Shu, X., Sun, B., Chen, Q., Huang, X., Zhou, H., Liu, Y., Fu, Z."ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells". Molecular Medicine Reports 32.4 (2025): 284.
Chicago
He, H., Wang, X., Wei, Z., Wang, A., Fang, X., He, H., Wu, Z., Shu, X., Sun, B., Chen, Q., Huang, X., Zhou, H., Liu, Y., Fu, Z."ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells". Molecular Medicine Reports 32, no. 4 (2025): 284. https://doi.org/10.3892/mmr.2025.13649
Copy and paste a formatted citation
x
Spandidos Publications style
He H, Wang X, Wei Z, Wang A, Fang X, He H, Wu Z, Shu X, Sun B, Chen Q, Chen Q, et al: ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells. Mol Med Rep 32: 284, 2025.
APA
He, H., Wang, X., Wei, Z., Wang, A., Fang, X., He, H. ... Fu, Z. (2025). ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells. Molecular Medicine Reports, 32, 284. https://doi.org/10.3892/mmr.2025.13649
MLA
He, H., Wang, X., Wei, Z., Wang, A., Fang, X., He, H., Wu, Z., Shu, X., Sun, B., Chen, Q., Huang, X., Zhou, H., Liu, Y., Fu, Z."ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells". Molecular Medicine Reports 32.4 (2025): 284.
Chicago
He, H., Wang, X., Wei, Z., Wang, A., Fang, X., He, H., Wu, Z., Shu, X., Sun, B., Chen, Q., Huang, X., Zhou, H., Liu, Y., Fu, Z."ER‑&alpha;36 knockdown is associated with lysosomal dysfunction and proliferation inhibition in liver cancer cells". Molecular Medicine Reports 32, no. 4 (2025): 284. https://doi.org/10.3892/mmr.2025.13649
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