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Article Open Access

MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells

  • Authors:
    • Chaiyaboot Ariyachet
    • Archittapon Nokkeaw
    • Pisit Tangkijvanich
  • View Affiliations / Copyright

    Affiliations: Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand
    Copyright: © Ariyachet et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 167
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    Published online on: August 12, 2025
       https://doi.org/10.3892/ijmm.2025.5608
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Abstract

MicroRNAs (miRNAs) are small non‑coding RNAs that regulate gene expression by targeting messenger RNAs for translational repression or degradation. Dysregulation of miRNAs has been implicated in liver cancer development, including hepatocellular carcinoma (HCC). The present study identified miR‑885‑5p as a novel tumor-suppressor miRNA in liver cancer. Analysis of miRNA expression profiles from The Cancer Genome Atlas Program and Gene Expression Omnibus databases demonstrated a consistent downregulation of miR‑885‑5p in HCC tissues. Overexpression of miR‑885‑5p via lentiviral transduction significantly suppressed liver cancer cell proliferation, supporting its tumor‑suppressive role. To investigate the underlying mechanism, transcriptomic profiling of miR‑885‑5p‑overexpressing liver cancer cells was performed. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses highlighted the cell cycle as the most significantly affected pathway. Specifically, miR‑885‑5p downregulated key G1/S transition‑promoting genes, including CDK6, E2F Transcription Factor 2 and Origin Recognition Complex Subunit 1 (ORC1), in liver cancer cells. To examine if miR‑885‑5p regulates the G1/S transition, a bromodeoxyuridine labeling assay and cell cycle analysis were performed. Corroborating the transcriptomic data, liver cancer cells with overexpressed miR‑885‑5p exhibited reduced bromodeoxyuridine incorporation and G1 phase arrest. To gain further mechanistic insights, bioinformatics tools were used to predict gene targets of miR‑885‑5p in the G1/S transition. Dual luciferase assays were conducted, which identified the direct interaction of miR‑885‑5p with the 3' untranslated regions of CDK6 and ORC1 messenger RNAs. Given its inhibitory effect on the G1/S transition, the therapeutic potential of miR‑885‑5p was assessed. miR‑885‑5p overexpression sensitized liver cancer cells to the CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib. The present findings collectively demonstrated that miR‑885‑5p induces cell cycle arrest and enhances CDK4/6 inhibitor sensitivity in liver cancer, suggesting its potential as a therapeutic target.
View Figures

Figure 1

miR-885-5p shows tumor-suppressing
effects in liver cancer cells. (A) Reanalysis of microRNA profiling
from three datasets curated by the Database of Differentially
Expressed MiRNAs in Human Cancers to identify downregulated miRNAs
in HCC tissues. (B) Expression of miR-885-5p in normal liver
tissues, low-grade and high-grade HCC tissues from Database of
Differentially Expressed MiRNAs in Human Cancers datasets.
Statistical significance was determined using the Mann-Whitney test
for clinical data. (C) Kaplan-Meier survival analysis of patients
with HCC with low and high expression of miR-885-5p from TCGA
database. (D) RT-qPCR analysis of miR-885-5p in normal hepatocyte
cell line (THLE-2) and liver cancer cell lines (HepG2, JHH-4 and
SNU-449). Statistical significance was determined using the one-way
ANOVA with Tukey's multiple comparison test. (E) RT-qPCR analysis
of miR-885-5p in liver cancer cell lines with 885-OE. (F) MTT-based
proliferation assays in control and 885-OE liver cancer cells. (G)
Colony formation assay of control and 885-OE liver cancer cells
with representative images and quantification of colony
numbers/well. Data are shown as the mean ± standard deviation from
n ≥3 replicates from at least two independent experiments.
Statistical significance was determined using Student's t-test for
in vitro assays. *P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001. miR,
microRNA; HCC, hepatocellular carcinoma; 885-OE, miR-885-5p
overexpressed; TCGA, The Cancer Genome Atlas; LIHC, Liver
Hepatocellular Carcinoma Collection; RT-qPCR, reverse
transcription-quantitative PCR.

Figure 2

RNA-sequencing analysis of JHH-4
cells with miR-885-5p overexpression. (A) The volcano plot
represents log2 fold change and statistical significance
(-log10 P-value) for gene expression of control and
JHH-4 cell lines with overexpression of miR-885-5p. DEGs were
identified based on an absolute fold change >1.5 and P-value
<0.05. Significantly upregulated and downregulated DEGs are
shown as red and blue dots, respectively. (B) Overall functional
enrichment analysis of downregulated DEGs using Kyoto Encyclopedia
of Genes and Genomes, Reactome, Gene Ontology, and WikiPathways
with an adjusted P<0.05. (C) Gene Set Enrichment Analysis
identified a downregulation of DEGs involved in the cell cycle and
G1/S transition. (D) The heatmap visualized gene
expression patterns of cell cycle-related genes where red and green
represent upregulated and downregulated levels, respectively. (E)
RT-qPCR validation of cell cycle-related genes identified in
RNA-seq analysis from three replicates. RT-qPCR experiments were
normalized to the control group and analyzed using Student's
t-test. Data are presented as mean ± standard deviation, with
significance levels. miR, microRNA; NES, Normalized enrichment
score; Differentially expressed genes, DEGs; 885-OE, miR-885-5p
overexpression; RT-qPCR, reverse transcription-quantitative
PCR.

Figure 3

Overexpression of miR-885-5p promotes
G1 arrest in the cell cycle. (A) BrdU incorporation
assay in control and liver cancer cell lines with overexpression of
miR-885-5p with representative images and quantification of
BrdU-positive cells from 10 randomly selected fields. (B) Cell
cycle analysis of control and liver cancer cell lines with
overexpression of miR-885-5p (n=3). All experiments were performed
in three independent liver cancer lines. Fluorescent images were
captured under identical exposure settings for comparison. Data are
presented as mean ± standard deviation and analyzed by Student's
t-test for statistical significance. *P<0.05,
**P<0.01 and ***P<0.001. Scale bar, 100
μm. miR, microRNA; BrdU, Bromodeoxyuridine; DAPI,
4′,6-diamidino-2-phenylindole; 885-OE, miR-885-5p
overexpression.

Figure 4

miR-885-5p directly regulates
CDK6 and ORC1. (A) Hybridization patterns predicted
by the RNAhybrid tool for binding sites of miR-885-5p with WT and
MT 3′ UTRs of CDK6, ORC1, E2F1 and
E2F2. (B) Dual luciferase reporter assay for miR-885-5p and
the 3′ UTR of genes involved in the G1/S transition.
Relative luciferase activity was normalized to the no-insert group
and analyzed using one-way ANOVA with Tukey's multiple comparison
test (n=3). Data are presented as mean ± standard deviation, with
significance levels indicated as ***P<0.001 and
****P<0.0001. (C) Spearman correlation (r) assessing
the relationship between miR-885-5p expression (x-axis) and genes
involved in the G1/S transition (y-axis) in
hepatocellular carcinoma tissue samples from The Cancer Genome
Atlas database. miR, microRNA; UTR, untranslated region; WT,
wild-type; MT, mutated.

Figure 5

Overexpression of miR-885-5p
sensitized liver cancer cells to CDK4/6 inhibitors. JHH-4 and
SNU-449 liver cancer cells were treated with (A) abemaciclib, (B)
palbociclib and (C) ribociclib with final drug concentrations
ranging from 0 to 50 μM. The half-maximal inhibitory
concentration of each drug was determined in both control and liver
cancer cell lines with overexpression of miR-885-5p using the MTT
assay. Dose-response curves were generated, and the half-maximal
inhibitory concentration values were calculated using non-linear
regression analysis. Each data point is presented as mean ±
standard deviation from at least eight technical replicates from
three independent experiments. miR, microRNA; 885-OE, miR-885-5p
overexpression.

Figure 6

Proposed mechanistic model of the
tumor-suppressive role of miR-885-5p in liver cancer. In normal
cells (left panel), miR-885-5p is expressed at normal levels,
leading to the translational repression of CDK6 and
ORC1 mRNA. This results in controlled levels of cell
cycle-related genes and normal cell proliferation. In liver cancer
cells (right panel), miR-885-5p expression is downregulated,
leading to the loss of translational repression of CDK6 and
ORC1 mRNAs. Consequently, there is an increase in the levels
of cell cycle-related genes, resulting in increased cell
proliferation. Red and green lines represent CDK6 and
ORC1 mRNAs, respectively. Blue lines represent miR-885-5p.
The 'X' in the liver cancer cell panel represents the loss of
translational repression. miR, microRNA; mRNA, messenger RNAs.
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Copy and paste a formatted citation
Spandidos Publications style
Ariyachet C, Nokkeaw A and Tangkijvanich P: MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells. Int J Mol Med 56: 167, 2025.
APA
Ariyachet, C., Nokkeaw, A., & Tangkijvanich, P. (2025). MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells. International Journal of Molecular Medicine, 56, 167. https://doi.org/10.3892/ijmm.2025.5608
MLA
Ariyachet, C., Nokkeaw, A., Tangkijvanich, P."MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells". International Journal of Molecular Medicine 56.5 (2025): 167.
Chicago
Ariyachet, C., Nokkeaw, A., Tangkijvanich, P."MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells". International Journal of Molecular Medicine 56, no. 5 (2025): 167. https://doi.org/10.3892/ijmm.2025.5608
Copy and paste a formatted citation
x
Spandidos Publications style
Ariyachet C, Nokkeaw A and Tangkijvanich P: MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells. Int J Mol Med 56: 167, 2025.
APA
Ariyachet, C., Nokkeaw, A., & Tangkijvanich, P. (2025). MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells. International Journal of Molecular Medicine, 56, 167. https://doi.org/10.3892/ijmm.2025.5608
MLA
Ariyachet, C., Nokkeaw, A., Tangkijvanich, P."MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells". International Journal of Molecular Medicine 56.5 (2025): 167.
Chicago
Ariyachet, C., Nokkeaw, A., Tangkijvanich, P."MicroRNA‑885‑5p regulates cell cycle progression in liver cancer cells". International Journal of Molecular Medicine 56, no. 5 (2025): 167. https://doi.org/10.3892/ijmm.2025.5608
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