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Role and mechanism of miR‑222‑5p in endothelial cell apoptosis

  • Authors:
    • Shimeng Wang
    • Boxin Zhao
    • Ying Cui
    • Lin Gui
    • Jingyao Fan
    • Lijuan Huang
  • View Affiliations / Copyright

    Affiliations: Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China, Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 76
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    Published online on: December 31, 2025
       https://doi.org/10.3892/mmr.2025.13786
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Abstract

Atherosclerosis (AS) is a chronic, multifactorial condition strongly associated with the onset and progression of cardiovascular disease, and it remains one of the leading causes of mortality worldwide. Endothelial cell apoptosis is an important event in the initiation and development of AS. MicroRNAs (miRNAs/miRs) have been extensively studied and perform roles at various stages of AS. Among them, miR‑222‑5p has been implicated in the regulation of AS; however, its precise mechanistic involvement remains to be fully elucidated. Therefore, the present study aimed to determine the functional role and underlying mechanism of miR‑222‑5p in AS. To this end, human umbilical vein endothelial cells (HUVECs) were treated with oxidized low‑density lipoprotein (ox‑LDL) to establish an endothelial cell apoptosis model. Reverse transcription‑quantitative polymerase chain reaction was used to assess mRNA and miRNA levels, and transfection efficiency. Cell viability was measured using the Cell Counting Kit‑8 assay and apoptosis was determined by flow cytometry. The protein expression levels of Bax, Bcl‑2 and integrin subunit α5 (ITGA5) were determined by western blotting. The results revealed that ox‑LDL stimulation significantly increased miR‑222‑5p expression in HUVECs. Overexpression of miR‑222‑5p significantly promoted apoptosis, whereas its knockdown reduced apoptosis and improved cell viability. Further analysis identified ITGA5 as a potential downstream target of miR‑222‑5p. In ox‑LDL‑induced apoptosis models, ITGA5 expression was significantly downregulated, and transfection with small interfering RNA targeting ITGA5 (si‑ITGA5) enhanced apoptotic activity. Furthermore, an inverse relationship was observed between ITGA5 and miR‑222‑5p expression. Co‑transfection experiments revealed that si‑ITGA5 partially reversed the anti‑apoptotic effects of the miR‑222‑5p inhibitor. In summary, the present study demonstrated that miR‑222‑5p may regulate endothelial cell apoptosis by targeting ITGA5, potentially contributing to AS progression.
View Figures

Figure 1

Ox-LDL induces apoptosis in
endothelial cells. (A) Western blotting analyzed the expression of
(B) anti-apoptotic Bcl-2 (n=3) and (C) pro-apoptotic Bax in
ox-LDL-treated endothelial cells (n=3). (D) Apoptosis was measured
by flow cytometry and (E) quantified (n=3). (F) Cell viability was
determined using the Cell Counting Kit-8 assay (n=3). *P<0.05,
**P<0.01 and ***P<0.001 vs. 0 mg/l ox-LDL. ns, not
significant; ox-LDL, oxidized low-density lipoprotein.

Figure 2

Expression of miR-222-5p in
ox-LDL-stimulated endothelial cells was measured by reverse
transcription-quantitative PCR (n=3). ****P<0.0001 vs. control.
miR, microRNA; ox-LDL, oxidized low-density lipoprotein.

Figure 3

Effect of miR-222-5p overexpression
on endothelial cells. (A) Reverse transcription-quantitative PCR
was used to quantify the overexpression efficiency of transfection
with a miR-222-5p mimic (n=3). (B) Cell Counting Kit-8 assay was
used to assess the effects of miR-222-5p and NC mimics on cell
viability (n=3). (C) Quantitative analysis of apoptotic rates
determined by flow cytometry (n=3). (D) Representative flow
cytometry dot plots for evaluating endothelial cell apoptosis. (E)
Western blotting was used to determine (F) Bcl-2 (n=3) and (G) Bax
protein levels after mimic transfection (n=3). *P<0.05,
**P<0.01 and ***P<0.001. miR, microRNA; NC, negative control;
ns, not significant.

Figure 4

Effects of miR-222-5p inhibitor on
endothelial cells. (A) Reverse transcription-quantitative PCR was
used to measure knockdown efficiency (n=3). (B) Cell Counting Kit-8
assay was performed to determine cell viability (n=3). (C)
Quantitative analysis of apoptotic rates determined by flow
cytometry (n=3). (D) Representative flow cytometry dot plots for
evaluating endothelial cell apoptosis. (E) Western blotting was
conducted to evaluate (F) Bcl-2 and (G) Bax protein levels
post-inhibitor transfection (n=3). *P<0.05, **P<0.01,
***P<0.001 and ****P<0.0001. miR, microRNA; NC, negative
control; ns, not significant.

Figure 5

miR-222-5p directly binds to the
3′UTR region of ITGA5 and negatively regulates its expression. (A)
Predicted miR-222-5p binding site in the ITGA5 3′UTR. (B)
Bioinformatics databases (TargetScan, miRDB, miRWalk and
miRTarBase) identified ITGA5 as a candidate target. Reverse
transcription-quantitative PCR to quantify ITGA5 expression
following (C) mimic (n=3) or (D) inhibitor transfection (n=3).
Western blotting was used to detect the expression levels of ITGA5
protein after transfection with (E) a miR-222-5p mimic and NC
mimic, (F) which was semi-quantified (n=3). (G) Western blotting
was used to detect the expression levels of ITGA5 protein after
transfection with a miR-222-5p inhibitor and NC, (H) which was
semi-quantified (n=3). *P<0.05, **P<0.01, ***P<0.001 and
****P<0.0001. ITGA5, integrin subunit α5; miR, microRNA; NC,
negative control; ns, not significant; UTR, untranslated
region.

Figure 6

ITGA5 expression levels in
ox-LDL-stimulated endothelial cells. (A) ITGA5 expression in
ox-LDL-stimulated endothelial cells was quantified by reverse
transcription-quantitative PCR (n=3). (B) Western blotting and (C)
semi-quantification of results used to detect the protein
expression levels of ITGA5 in ox-LDL-stimulated endothelial cells
(n=3). **P<0.01 and ***P<0.001 vs. control. ITGA5, integrin
subunit α5; ox-LDL, oxidized low-density lipoprotein.

Figure 7

Effects of si-ITGA5 on endothelial
cells. (A) Reverse transcription-quantitative PCR was used to
measure knockdown efficiency (n=3). (B) Cell Counting Kit-8 assay
was performed to determine cell viability (n=3). (C) Quantitative
analysis of apoptotic rates determined by flow cytometry (n=3). (D)
Representative flow cytometry dot plots for evaluating endothelial
cell apoptosis. (E) Western blotting detection of (F) Bcl-2 and (G)
Bax protein expression after transfection with si-ITGA5 (n=3).
*P<0.05, **P<0.01 and ****P<0.0001. ITGA5, integrin
subunit α5; NC, negative control; ns, not significant; si, small
interfering RNA.

Figure 8

Si-ITGA5 partially reverses the
inhibition of apoptosis by a miR-222-5p inhibitor. (A) Western
blotting was used to determine (B) Bcl-2 and (C) Bax protein levels
after co-transfection (n=3). (D) Cell Counting Kit-8 assay was used
to assess cell viability after co-transfection with miR-222-5p
inhibitor and si-ITGA5 (n=3). (E) Flow cytometry was performed to
evaluate (F) apoptosis under co-transfection conditions (n=3). (G)
Schematic diagram of the molecular mechanism: miR-222-5p mediates
endothelial cell apoptosis and atherosclerotic progression by
targeting the ITGA5 pathway. *P<0.05, **P<0.01 and
***P<0.001. HUVECs, human umbilical vein endothelial cells;
ITGA5, integrin subunit α5; miR, microRNA; NC, negative control;
ns, not significant; si, small interfering; UTR, untranslated
region.
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Spandidos Publications style
Wang S, Zhao B, Cui Y, Gui L, Fan J and Huang L: Role and mechanism of miR‑222‑5p in endothelial cell apoptosis. Mol Med Rep 33: 76, 2026.
APA
Wang, S., Zhao, B., Cui, Y., Gui, L., Fan, J., & Huang, L. (2026). Role and mechanism of miR‑222‑5p in endothelial cell apoptosis. Molecular Medicine Reports, 33, 76. https://doi.org/10.3892/mmr.2025.13786
MLA
Wang, S., Zhao, B., Cui, Y., Gui, L., Fan, J., Huang, L."Role and mechanism of miR‑222‑5p in endothelial cell apoptosis". Molecular Medicine Reports 33.2 (2026): 76.
Chicago
Wang, S., Zhao, B., Cui, Y., Gui, L., Fan, J., Huang, L."Role and mechanism of miR‑222‑5p in endothelial cell apoptosis". Molecular Medicine Reports 33, no. 2 (2026): 76. https://doi.org/10.3892/mmr.2025.13786
Copy and paste a formatted citation
x
Spandidos Publications style
Wang S, Zhao B, Cui Y, Gui L, Fan J and Huang L: Role and mechanism of miR‑222‑5p in endothelial cell apoptosis. Mol Med Rep 33: 76, 2026.
APA
Wang, S., Zhao, B., Cui, Y., Gui, L., Fan, J., & Huang, L. (2026). Role and mechanism of miR‑222‑5p in endothelial cell apoptosis. Molecular Medicine Reports, 33, 76. https://doi.org/10.3892/mmr.2025.13786
MLA
Wang, S., Zhao, B., Cui, Y., Gui, L., Fan, J., Huang, L."Role and mechanism of miR‑222‑5p in endothelial cell apoptosis". Molecular Medicine Reports 33.2 (2026): 76.
Chicago
Wang, S., Zhao, B., Cui, Y., Gui, L., Fan, J., Huang, L."Role and mechanism of miR‑222‑5p in endothelial cell apoptosis". Molecular Medicine Reports 33, no. 2 (2026): 76. https://doi.org/10.3892/mmr.2025.13786
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