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Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis

  • Authors:
    • Yuankun Chen
    • Tiantian Zhu
    • Fang Chen
    • Mingyue Niu
    • Haifeng Wu
    • Qiuping Wu
    • Zheng Wang
    • Wenting Li
  • View Affiliations / Copyright

    Affiliations: Department of Tropical and Liver Diseases, The Second Affiliated Hospital of Hainan Medical University, Haikou, Hainan 570100, P.R. China, Department of Emergency Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P.R. China, Department of Respiratory and Critical Medicine, People's Hospital of Zhengzhou University, Zhengzhou, Henan 450003, P.R. China, Department of Infectious Diseases, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P.R. China
    Copyright: © Chen et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 97
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    Published online on: January 7, 2026
       https://doi.org/10.3892/ol.2026.15450
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Abstract

Liver cancer is a highly aggressive cancer and the regulatory roles of microRNAs (miRs) in its progression are still being explored. miR‑448, which is implicated in several types of cancer, remains to be fully characterized in liver cancer, particularly regarding its presence in exosomes. The aim of the present study was to examine the effects of exosomal miR‑448 (EXO‑miR‑448) on liver cancer cell behavior. The expression levels of miR‑448 in human liver cancer cell lines and its localization in exosomes were analyzed using reverse transcription‑quantitative PCR, transmission electron microscopy and nanoparticle tracking analysis, with western blotting performed to detect exosomal markers. Functional assays were conducted to assess the effects of EXO‑miR‑448 on cell proliferation, migration and invasion. The results demonstrated that miR‑448 expression was significantly downregulated in human liver cancer cell lines (HepG2, Hep3B and SK‑HEP‑1) compared with that in normal liver cells. Furthermore, exosomal analysis confirmed that miR‑448 was enriched within exosomes rather than being secreted into the supernatant. EXO‑miR‑448 also inhibited liver cancer cell proliferation, migration and invasion, as demonstrated using Cell Counting Kit‑8 and Transwell assays. Bioinformatics and functional assays further identified Ras‑related protein Rab‑7a (RAB7A) as a direct downstream target of miR‑448, with its overexpression rescuing the inhibitory effects of EXO‑miR‑448 on cell behavior. Furthermore, EXO‑miR‑448 suppressed glycolysis in liver cancer cells by targeting RAB7A, as indicated by reduced lactate production, glucose uptake, ATP levels and extracellular acidification rate. In conclusion, EXO‑miR‑448 inhibits liver cancer cell proliferation, migration, invasion and glycolysis by targeting RAB7A. These findings underscore the importance of miR‑448 in liver cancer biology and support its further evaluation in future translational studies.
View Figures

Figure 1

miR-448 expression in liver cancer
cell lines and exosome characterization. (A) Relative expression
levels of miR-448 in liver cancer cell lines (HepG2, Hep3B and
SK-HEP-1) and the normal liver cell line THLE-2, assessed using
reverse transcription-quantitative PCR. ***P<0.001 vs. THLE-2.
(B) Relative expression levels of miR-448 in exosomes treated with
RNase A or RNase A + Triton X-100. ***P<0.001 vs. control. (C)
Transmission electron microscopy image of exosomes isolated from
HepG2 cells (scale bar, 200 nm). (D) Size distribution of HepG2
derived exosomes determined by nanoparticle tracking analysis, with
a peak diameter of ~104 nm. (E) Western blotting of exosome markers
(CD9 and CD63) in HepG2 cell lysates and exosomes, with calnexin as
a negative control. miR, microRNA.

Figure 2

EXO-miR-448 inhibits liver cancer
cell proliferation, migration and invasion. (A) Relative expression
levels of miR-448 in exosomes derived from HepG2 cells transfected
with miR-448 mimics or mimics NC, assessed using reverse
transcription-quantitative PCR. **P<0.01 vs. EXO-NC. (B) Cell
viability assessed using the Cell Counting Kit-8 assay. **P<0.01
and ***P<0.001 vs. EXO-NC. (C) Migration evaluated using the
Transwell migration assays (scale bar, 100 µm). ***P<0.001 vs.
EXO-NC. (D) Invasion assessed using the Transwell invasion assays
(scale bar, 100 µm). ***P<0.001 vs. EXO-NC. miR, microRNA; NC,
negative control; EXO, exosomal.

Figure 3

RAB7A as a direct downstream target
of miR-448 in liver cancer. (A) Bioinformatics analysis predicted
potential downstream targets of miR-448, including RAB7A, TMSB4X,
VASH2, SGPP1 and MPC1. (B) Relative mRNA expression levels of
candidate targets in HepG2 cells treated with EXO-miR-448 or
EXO-NC, assessed using reverse transcription-quantitative PCR.
*P<0.05, **P<0.01 and ***P<0.001 vs. EXO-NC. (C) Predicted
binding sites of miR-448 in the 3′-UTR of RAB7A (WT and MUT). (D)
Dual-luciferase reporter assay validating the interaction between
miR-448 and RAB7A. **P<0.01 vs. mimic NC. (E) Western blotting
showing markedly increased RAB7A protein levels in HepG2 cells
transfected with the RAB7A OE construct alone. ***P<0.001 vs.
vector. (F) Western blotting showing reduced RAB7A protein levels
in HepG2 cells after EXO-miR-448 treatment. **P<0.01 vs. EXO-NC
+ vector. EXO, exosomal; miR, microRNA; MPC1, mitochondrial
pyruvate carrier 1; MUT, mutant; NC, negative control; OE,
overexpression; RAB7A, Ras-related protein Rab-7a; SGPP1,
sphingosine-1-phosphate phosphatase 1; TMSB4X, thymosin β 4
X-linked; VASH2, vasohibin 2; WT, wild-types.

Figure 4

EXO-miR-448 inhibits liver cancer
cell proliferation, migration and invasion by targeting RAB7A. (A)
Paired comparison of RAB7A expression between HCC tumor tissues and
their matched adjacent normal tissues (n=50 pairs). ***P<0.001.
(B) Unpaired comparison of all HCC tumor samples (n=424) vs. normal
liver tissues (n=50). ***P<0.001. (C) RAB7A mRNA expression in
normal liver cells (THLE-2) and liver cancer cell lines (HepG2,
Hep3B and SK-HEP-1). *P<0.05 and ***P<0.001 vs. THLE-2. (D)
RAB7A protein expression in normal liver cells (THLE-2) and liver
cancer cell lines (HepG2, Hep3B and SK-HEP-1). ***P<0.001 vs.
THLE-2. (E) Validation of RAB7A knockdown efficiency in HepG2 cells
using reverse transcription-quantitative PCR. ***P<0.001 vs. NC.
(F) Validation of RAB7A knockdown efficiency in HepG2 cells using
western blotting. ***P<0.001 vs. NC. (G) Cell Counting Kit-8
assay showing reduced proliferation of HepG2 cells after RAB7A
knockdown. *P<0.05 and **P<0.01 vs. NC. (H) EdU assay showing
reduced proliferation of HepG2 cells after RAB7A knockdown (scale
bar, 50 µm). ***P<0.001 vs. NC. (I) Transwell migration assay
showing suppressed migratory ability of HepG2 cells following RAB7A
knockdown (scale bar, 100 µm). ***P<0.001 vs. NC. (J) Transwell
invasion assay showing suppressed invasive ability of HepG2 cells
following RAB7A knockdown (scale bar, 100 µm). ***P<0.001 vs.
NC. (K) Rescue experiment showing that RAB7A OE reverses the
inhibitory effect of EXO-miR-448 on HepG2 cell proliferation. scale
bar, 50 µm. **P<0.01 vs. EXO-NC + vector; #P<0.05
vs. EXO-miR-448 + vector. (L) Rescue experiment showing that RAB7A
OE reverses the inhibitory effect of EXO-miR-448 on HepG2 cell
migration. Scale bar, 100 µm. ***P<0.001 vs. EXO-NC + vector;
##P<0.01 vs. EXO-miR-448 + vector. (M) Rescue
experiment showing that RAB7A OE reverses the inhibitory effect of
EXO-miR-448 on HepG2 cell invasion. Scale bar, 100 µm.
***P<0.001 vs. EXO-NC + vector; ###P<0.001 vs.
EXO-miR-448 + vector. EXO, exosomal; HCC, hepatocellular carcinoma;
miR, microRNA; NC, negative control; OE, overexpression; RAB7A,
Ras-related protein Rab-7a; siRNA, small interfering RNA; TPM,
transcripts per million.

Figure 5

EXO-miR-448 suppresses liver cancer
progression by targeting RAB7A to inhibit glycolysis. (A) Lactate
production in HepG2 cells treated with EXO-miR-448 or EXO-NC,
determined by a colorimetric assay. ***P<0.001 vs. EXO-NC. (B)
Glucose uptake in HepG2 cells treated with EXO-miR-448 or EXO-NC,
assessed using the 2-NBDG assay. ***P<0.001 vs. EXO-NC. (C)
Intracellular ATP levels in HepG2 cells treated with EXO-miR-448 or
EXO-NC, determined using an ATP assay kit. ***P<0.001 vs.
EXO-NC. (D) ECAR in HepG2 cells treated with EXO-miR-448 or EXO-NC,
measured using a Seahorse XF Analyzer. ***P<0.001 vs. EXO-NC.
(E) Lactate production in HepG2 cells transfected with si-RAB7A or
control siRNA. ***P<0.001 vs. NC. (F) Glucose uptake in HepG2
cells transfected with si-RAB7A or control siRNA. ***P<0.001 vs.
NC. (G) Intracellular ATP levels in HepG2 cells transfected with
si-RAB7A or control siRNA. ***P<0.001 vs. NC. (H) ECAR in HepG2
cells transfected with si-RAB7A or control siRNA. ***P<0.001 vs.
NC. (I) Lactate production in HepG2 cells following EXO-miR-448
treatment with or without RAB7A OE. ***P<0.001 vs. EXO-NC +
vector; ###P<0.001 vs. EXO-miR-448 + vector. (J)
Glucose uptake in HepG2 cells following EXO-miR-448 treatment with
or without RAB7A OE. ***P<0.001 vs. EXO-NC + vector;
###P<0.001 vs. EXO-miR-448 + vector. (K) ATP levels
in HepG2 cells following EXO-miR-448 treatment with or without
RAB7A OE. ***P<0.001 vs. EXO-NC + vector;
###P<0.001 vs. EXO-miR-448 + vector. (L) ECAR
analysis showing glycolytic activity in HepG2 cells following
EXO-miR-448 treatment with or without RAB7A OE. ***P<0.001 vs.
EXO-NC + vector; ###P<0.001 vs. EXO-miR-448 + vector.
miR, microRNA; NC, negative control; ECAR, extracellular
acidification rate; si, small interfering RNA; RAB7A, Ras-related
protein Rab-7a; NBDG,
N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino-2-deoxy-D-glucose; EXO,
exosomal; OE, overexpression; 2-DG, 2-deoxy-D-glucose.
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Copy and paste a formatted citation
Spandidos Publications style
Chen Y, Zhu T, Chen F, Niu M, Wu H, Wu Q, Wang Z and Li W: Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis. Oncol Lett 31: 97, 2026.
APA
Chen, Y., Zhu, T., Chen, F., Niu, M., Wu, H., Wu, Q. ... Li, W. (2026). Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis. Oncology Letters, 31, 97. https://doi.org/10.3892/ol.2026.15450
MLA
Chen, Y., Zhu, T., Chen, F., Niu, M., Wu, H., Wu, Q., Wang, Z., Li, W."Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis". Oncology Letters 31.3 (2026): 97.
Chicago
Chen, Y., Zhu, T., Chen, F., Niu, M., Wu, H., Wu, Q., Wang, Z., Li, W."Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis". Oncology Letters 31, no. 3 (2026): 97. https://doi.org/10.3892/ol.2026.15450
Copy and paste a formatted citation
x
Spandidos Publications style
Chen Y, Zhu T, Chen F, Niu M, Wu H, Wu Q, Wang Z and Li W: Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis. Oncol Lett 31: 97, 2026.
APA
Chen, Y., Zhu, T., Chen, F., Niu, M., Wu, H., Wu, Q. ... Li, W. (2026). Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis. Oncology Letters, 31, 97. https://doi.org/10.3892/ol.2026.15450
MLA
Chen, Y., Zhu, T., Chen, F., Niu, M., Wu, H., Wu, Q., Wang, Z., Li, W."Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis". Oncology Letters 31.3 (2026): 97.
Chicago
Chen, Y., Zhu, T., Chen, F., Niu, M., Wu, H., Wu, Q., Wang, Z., Li, W."Exosomal microRNA‑448 suppresses the malignant behaviors of liver cancer cells by targeting RAB7A and inhibiting glycolysis". Oncology Letters 31, no. 3 (2026): 97. https://doi.org/10.3892/ol.2026.15450
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