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LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation

  • Authors:
    • Junjun Guo
    • Shenbo Fu
    • Jia Liu
    • Lina Li
    • Jin Zhao
    • Fenggang Wang
    • Wenan Wu
    • Xu Chen
    • Enxiao Li
  • View Affiliations / Copyright

    Affiliations: Department of Oncology, Shaanxi Provincial Tumor Hospital, Xi'an, Shaanxi 710061, P.R. China, Department of Radiation Oncology, Shaanxi Provincial Tumor Hospital, Xi'an, Shaanxi 710061, P.R. China, Department of Thoracic Surgery, Shaanxi Provincial Tumor Hospital, Xi'an, Shaanxi 710061, P.R. China, Department of Internal Medicine, Shaanxi Provincial Tumor Hospital, Xi'an, Shaanxi 710061, P.R. China, Department of Oncology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
    Copyright: © Guo et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 272
    |
    Published online on: April 28, 2026
       https://doi.org/10.3892/ol.2026.15628
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Abstract

Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer with poor clinical outcomes, highlighting the need for enhanced understanding of its molecular drivers. The present study investigated the functional role of long non‑coding RNA LINC00184 in ESCC progression. Using overexpression experiments in KYSE‑150 and TE‑1 cell lines, the present study demonstrated that LINC00184 significantly enhances ESCC cell proliferation and migration while inhibiting apoptosis. Mechanistic studies revealed that LINC00184 recruits DNA methyltransferase 1 (DNMT1) to the promoter of the tumor suppressor gene N‑Myc downstream regulated gene 2 (NDRG2), thereby catalyzing CpG island hypermethylation and transcriptional silencing of NDRG2. The subsequent downregulation of NDRG2 results in activation of the oncogenic PI3K/AKT signaling pathway. Notably, treatment with the DNMT1 inhibitor 5‑azacytidine reverses LINC00184‑induced NDRG2 promoter methylation, restores NDRG2 expression and attenuates PI3K/AKT pathway activation. The present study findings identified a novel LINC00184/DNMT1/NDRG2/PI3K‑AKT regulatory axis in ESCC and suggested that targeting this epigenetic pathway may represent a promising therapeutic strategy in the future.
View Figures

Figure 1

Overexpression of LINC00184 promotes
proliferation and migration while inhibiting apoptosis in
esophageal squamous cell carcinoma cells. (A) Expression level of
LINC00184 in KYSE-150 cells following transfection with OE-NC and
OE-LINC00184 plasmids. (B) Expression level of LINC00184 in TE-1
cells following transfection with OE-NC and OE-LINC00184 plasmids.
(C) Relative cell viability in KYSE-150 cells following
transfection with OE-NC and OE-LINC00184 plasmids. (D) Relative
cell viability in TE-1 cells following transfection with OE-NC and
OE-LINC00184 plasmids. (E) Representative images of migrated
KYSE-150 cells after transfection with OE-NC and OE-LINC00184
(magnification, ×200). (F) Quantitative analysis of migrated cell
numbers corresponding to (E). (G) Representative images of migrated
TE-1 cells after transfection with OE-NC and OE-LINC00184
(magnification, ×200). (H) Quantitative analysis of migrated cell
numbers corresponding to (G). (I) Flow cytometry dot plots showing
the apoptosis rate of KYSE-150 cells in the OE-NC and OE-LINC00184
groups. (J) Quantitative analysis of apoptosis rates corresponding
to (I). (K) Flow cytometry dot plots showing the apoptosis rate of
TE-1 cells in the OE-NC and OE-LINC00184 groups. (L) Quantitative
analysis of apoptosis rates corresponding to (K). Data are
presented as mean ± SEM (n=3). Comparisons between two groups were
performed using the unpaired Student's t-test. Statistical
significance is indicated as **P<0.01 and ***P<0.001. OE,
overexpression; NC, negative control.

Figure 2

LINC00184 negatively regulates NDRG2
and activate the PI3K/AKT pathway in esophageal squamous cell
carcinoma cells. (A) Western blotting analysis reveals the protein
expression levels of NDRG2, p-AKT (Ser473), AKT, p-PI3K (Tyr458)
and PI3K in KYSE-150 transfected with OE-NC or OE-LINC00184
plasmid. (B) Quantitative analysis of immunoblots of NDRG2
presented in (A). (C) Quantitative analysis of immunoblots of
pAKT/AKT presented in (A). (D) Quantitative analysis of immunoblots
of pPI3K/PI3K presented in (A). (E) RT-qPCR analysis of NDRG2 mRNA
levels in KYSE-150 cells under control conditions and following
overexpression of LINC00184. (F) Western blotting analysis
demonstrating the protein expression levels of NDRG2, p-AKT
(Ser473), AKT, p-PI3K (Tyr458) and PI3K in KYSE-150 and TE-1 cells
transfected with OE-NC or OE-LINC00184 plasmid. (G) Quantitative
analysis of immunoblots of NDRG2 presented in (F). (H) Quantitative
analysis of immunoblots of pAKT/AKT presented in (F). (I)
Quantitative analysis of immunoblots of pPI3K/PI3K presented in
(F). (J) RT-qPCR analysis of NDRG2 mRNA levels in TE-1 cells under
control conditions and following overexpression of LINC00184. Data
are presented as mean ± SEM (n=3). Comparisons between two groups
were performed using the Student's t-test. Statistical significance
is indicated as *P<0.05, **P<0.01 and ***P<0.001. OE,
overexpression; NC, negative control; RT-qPCR, reverse
transcription-quantitative PCR; NDRG2, N-Myc downstream regulated
gene.

Figure 3

Restoration of NDRG2 rescues
LINC00184-driven PI3K/AKT activation and proliferation in
esophageal squamous cell carcinoma cells. (A) Western blotting
analysis of NDRG2 and phosphorylated AKT (p-AKT) protein levels in
TE-1 cells under the following conditions: Control, overexpression
of LINC00184 alone (OE-LINC00184), and co-overexpression of
LINC00184 and NDRG2. GAPDH serves as the loading control. (B) Cell
viability measured by Cell Counting Kit-8 assay in TE-1 cells under
the same treatment conditions as in (A). Data in (B) are presented
as mean ± SEM (n=6). Comparisons among multiple groups were
analyzed by one-way analysis of variance. ***P<0.001. OE,
overexpression; NC, negative control; NDRG2, N-Myc downstream
regulated gene.

Figure 4

LINC00184 regulates NDRG2 expression
through DNMT1-mediated methylation of the NDRG2 promoter. (A)
Expression level of LINC00184 in KYSE-150 cells following treatment
with siRNA targeting LINC00184 (n=3). (B) Expression level of
LINC00184 in TE-1 cells following treatment with siRNA targeting
LINC00184 (n=3). (C) Methylation level of the NDRG2 promoter in
KYSE-150 cells detected via MSP assay after overexpression or
silencing of LINC00184. (D) Methylation level of the NDRG2 promoter
in TE-1 cells detected via MSP assay after overexpression or
silencing of LINC00184. (E) Enrichment of DNMT1 at the NDRG2
promoter region detected by chromatin immunoprecipitation assay and
quantified using RT-qPCR in KYSE-150 cells with overexpression or
silencing of LINC00184 (n=3). (F) Enrichment of LINC00184 bound to
DNMT1 detected by RNA immunoprecipitation assay and quantified
using RT-qPCR in KYSE-150 cells after overexpression or silencing
of LINC00184 (n=3). (G) Enrichment of LINC00184 bound to DNMT1
detected by RNA immunoprecipitation assay and quantified using
RT-qPCR in TE-1 cells after overexpression or silencing of
LINC00184 (n=3). (H) Methylation level of the NDRG2 promoter in
KYSE-150 cells measured by MSP assay following LINC00184
overexpression combined with 5-AZA treatment. (I) Methylation level
of the NDRG2 promoter in TE-1 cells measured by MSP assay following
LINC00184 overexpression combined with 5-AZA treatment. (J) Western
blotting analysis of NDRG2 protein expression in KYSE-150 cells
after LINC00184 overexpression and 5-AZA intervention. (K)
Quantitative analysis of NDRG2 protein grayscale values obtained
from the western blotting results in (J) (n=3). (L) Western
blotting analysis of NDRG2 protein expression in TE-150 cells after
LINC00184 overexpression and 5-AZA intervention. (M) Quantitative
analysis of NDRG2 protein grayscale values obtained from the
western blotting results in (L) (n=3). (N) Western blotting
detection of NDRG2 protein levels under control conditions, single
overexpression of LINC00184, and combined treatment with
OE-LINC00184 + si-DNMT1. (O) RT-qPCR detection of relative DNMT1
mRNA levels in control cells and LINC00184-overexpressing cells
(n=6). (P) Western blotting analysis of total DNMT1 protein levels
in control cells and LINC00184-overexpressing cells; GAPDH was used
as the loading control (n=3). Data are presented as mean ± SEM
(n=3). Comparisons between two groups were performed using the
unpaired Student's t-test. Comparisons among multiple groups were
analyzed by one-way analysis of variance. Statistical significance
is indicated as *P<0.05 and ***P<0.001. OE, overexpression;
NC, negative control; NDRG2, N-Myc downstream regulated gene;
RT-qPCR, reverse transcription-quantitative PCR; DNMT1, DNA
methyltransferase 1; si/siRNA, small interfering RNA; lnc/lncRNA,
long non-coding RNA; 5-AZA, 5-azacytidine; MSP,
methylation-specific PCR. M, methylation; U, unmethylation.

Figure 5

Inhibition of DNMT1 abrogates
LINC00184-induced PI3K/AKT pathway activation and functional
phenotypes. (A) Western blotting analysis showing the protein
expression level of pAKT, AKT, pPI3K and PI3K in KYSE-150 cells
following overexpression of LINC00184 and treatment with 5-AZA. (B)
Quantitative analysis of the pAKT/AKT protein expression level
derived from the immunoblots in (A). (C) Quantitative analysis of
the pPI3K/PI3K protein expression level derived from the
immunoblots in (A). (D) Western blotting analysis showing the
protein expression level of pAKT, AKT, pPI3K and PI3K in TE-1 cells
following overexpression of LINC00184 and treatment with 5-AZA. (E)
Quantitative analysis of the pAKT/AKT protein expression level
derived from the immunoblots in (D). (F) Quantitative analysis of
the pPI3K/PI3K protein expression level derived from the
immunoblots in (D). (G) Western blotting analysis of p-AKT and
total AKT levels in esophageal squamous cell carcinoma cells under
the following conditions: Control, OE-LINC00184 and OE-LINC00184 +
si-DNMT1. (H) Quantitative analysis of the pAKT/AKT protein
expression level corresponding to the immunoblots presented in (G).
Data are presented as mean ± SEM (n=3). Comparisons among multiple
groups were analyzed by one-way analysis of variance. Statistical
significance is indicated as *P<0.05, **P<0.01 and
***P<0.001. OE, overexpression; NC, negative control; NDRG2,
N-Myc downstream regulated gene; RT-qPCR, reverse
transcription-quantitative PCR; DNMT1, DNA methyltransferase 1;
si/siRNA, small interfering RNA; 5-AZA, 5-azacytidine.

Figure 6

DNMT1 inhibition reverses
LINC00184-induced malignant phenotypes in ESCC cells. (A) Cell
viability of KYSE-150 cells following LINC00184 overexpression and
treatment with 5-AZA (n=3). (B) Representative images of migrated
KYSE-150 cells following LINC00184 overexpression and treatment
with 5-AZA. (C) Representative flow cytometry dot plots showing the
apoptosis rate of KYSE-150 cells following LINC00184 overexpression
and treatment with 5-AZA. (D) Statistical counting of migrated
KYSE-150 cells corresponding to (B) (n=3). (E) Quantitative
analysis of the apoptosis rate of KYSE-150 cells corresponding to
(C) (n=3). (F) Cell viability of TE-1 cells following LINC00184
overexpression and treatment with 5-AZA (n=3). (G) Representative
images of migrated TE-1 cells following LINC00184 overexpression
and treatment with 5-AZA. (H) Representative flow cytometry dot
plots showing the apoptosis rate of TE-1 cells following LINC00184
overexpression and treatment with 5-AZA. (I) Statistical counting
of migrated TE-1 cells corresponding to (G) (n=3). (J) Quantitative
analysis of the apoptosis rate of TE-1 cells corresponding to (H)
(n=3). (K) Cell viability detected via Cell Counting Kit-8 assay in
ESCC cells under the conditions of control, OE-LINC00184 and
OE-LINC00184 + si-DNMT1. Data are presented as mean ± SEM (n=3),
Comparisons among multiple groups were analyzed by one-way analysis
of variance. Statistical significance is indicated as *P<0.05,
**P<0.01 and ***P<0.001. OE, overexpression; NC, negative
control; DNMT1, DNA methyltransferase 1; si/siRNA, small
interfering RNA; 5-AZA, 5-azacytidine; ESCC, esophageal squamous
cell carcinoma.
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Copy and paste a formatted citation
Spandidos Publications style
Guo J, Fu S, Liu J, Li L, Zhao J, Wang F, Wu W, Chen X and Li E: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation. Oncol Lett 32: 272, 2026.
APA
Guo, J., Fu, S., Liu, J., Li, L., Zhao, J., Wang, F. ... Li, E. (2026). LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation. Oncology Letters, 32, 272. https://doi.org/10.3892/ol.2026.15628
MLA
Guo, J., Fu, S., Liu, J., Li, L., Zhao, J., Wang, F., Wu, W., Chen, X., Li, E."LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation". Oncology Letters 32.1 (2026): 272.
Chicago
Guo, J., Fu, S., Liu, J., Li, L., Zhao, J., Wang, F., Wu, W., Chen, X., Li, E."LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation". Oncology Letters 32, no. 1 (2026): 272. https://doi.org/10.3892/ol.2026.15628
Copy and paste a formatted citation
x
Spandidos Publications style
Guo J, Fu S, Liu J, Li L, Zhao J, Wang F, Wu W, Chen X and Li E: LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation. Oncol Lett 32: 272, 2026.
APA
Guo, J., Fu, S., Liu, J., Li, L., Zhao, J., Wang, F. ... Li, E. (2026). LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation. Oncology Letters, 32, 272. https://doi.org/10.3892/ol.2026.15628
MLA
Guo, J., Fu, S., Liu, J., Li, L., Zhao, J., Wang, F., Wu, W., Chen, X., Li, E."LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation". Oncology Letters 32.1 (2026): 272.
Chicago
Guo, J., Fu, S., Liu, J., Li, L., Zhao, J., Wang, F., Wu, W., Chen, X., Li, E."LINC00184 promotes esophageal squamous cell carcinoma progression via DNMT1‑mediated methylation of the NDRG2 promoter and PI3K/AKT pathway activation". Oncology Letters 32, no. 1 (2026): 272. https://doi.org/10.3892/ol.2026.15628
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