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Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve

  • Authors:
    • Pengyu Huang
    • Suzhu Chen
    • Jianshu Cai
    • Caihong Jiang
    • Bichen Liu
    • Yun Fu
    • Dianliang Lin
  • View Affiliations / Copyright

    Affiliations: Fujian Provincial Human Sperm Bank, Fujian Maternity and Child Health Hospital, College of Clinical Medicine for Obstetrics and Gynecology and Pediatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China, Center of Reproductive Medicine, Fujian Maternity and Child Health Hospital, College of Clinical Medicine for Obstetrics and Gynecology and Pediatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China, Nursing Department, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310000, P.R. China, Department of Medical Ultrasonics, Fujian Maternity and Child Health Hospital, College of Clinical Medicine for Obstetrics and Gynecology and Pediatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China, Department of Laboratory Medicine, Fujian Medical University Union Hospital, Fuzhou, Fujian 350001, P.R. China
    Copyright: © Huang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 224
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    Published online on: September 18, 2025
       https://doi.org/10.3892/etm.2025.12974
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Abstract

Decreased ovarian reserve (DOR) refers to the decreased ability of the ovaries to produce eggs and the low quality of the follicles, which may lead to abnormal menstruation and infertility. However, the role of circular RNAs (circRNAs) in DOR remains poorly understood. Through circRNA sequencing, hsa_circ_0005379 was identified as the most significantly upregulated circRNA in the follicular fluid exosomes of patients with DOR. The present study aimed to characterize the circRNA expression profile and investigate the function of hsa_circ_0005379 in DOR. Exosomes were isolated from the follicular fluid of individuals with DOR and healthy controls, followed by sequencing using the Illumina HiSeq platform. Functional enrichment analysis, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis, was performed on the differentially expressed circRNAs. To further validate the results, the expression of five circRNAs was assessed in follicular fluid via reverse transcription‑quantitative PCR (RT‑qPCR). A DOR cell model was established by treating KGN granulosa cells with cyclophosphamide (CTX). Cell viability was evaluated using a Cell Counting Kit‑8 assay following CTX exposure. hsa_circ_0005379 was either overexpressed or knocked down in the CTX‑treated cells, and the levels of cell apoptosis, reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were analyzed by flow cytometry and biochemical assays. Eight differentially expressed circRNAs were identified in individuals with DOR compared with controls, including seven upregulated and one downregulated. RT‑qPCR confirmed that the expression trends of hsa_circ_0000344, hsa_circ_0001126, hsa‑circ_0005379, hsa_circ‑0005777 and hsa_circ_0007509 aligned with those from the sequencing results. Furthermore, overexpression and knockdown of hsa_circ_0005379 in KGN cells was verified by RT‑qPCR. Silencing hsa_circ_0005379 in CTX‑treated KGN cells promoted cell viability and reduced apoptosis, accompanied by reduced ROS and MDA levels and enhanced SOD activity. Overexpression of hsa_circ_0005379 yielded the opposite results. Collectively, the results suggested that hsa_circ_0005379 played a key role in regulating cell viability and oxidative stress in CTX‑treated granulosa cells.
View Figures

Figure 1

Characterization of the follicular
fluid exosomes of patients with decreased ovarian reserve. (A)
Representative transmission electron microscopy images of
follicular fluid exosomes (magnification, x60,000). (B) The main
range of size distribution of follicular fluid exosomes. (C)
Western blot analysis of the exosome positive markers CD9 and CD63.
The exosomes were from follicular fluid of DOR, and the negative
control was the follicular fluid of DOR.

Figure 2

Differentially expressed circRNAs
between patients with DOR and normal controls. (A) Volcano plots of
all circRNAs. The red dots indicate upregulated circRNAs compared
with normal controls, and the green dots indicate downregulated
circRNAs compared with normal controls. (B) Heat map of
differentially expressed circRNAs between patients with DOR and
normal controls. The darker the color, the higher the relative
expression level of circRNAs. NC, normal control; DOR, decreased
ovarian reserve; circRNA, circular RNA.

Figure 3

Functional analysis of differentially
expressed circular RNA host genes. (A) Top 10 ranked biological
process terms. (B) Top 10 ranked cellular component terms. (C) Top
10 ranked molecular function terms. (D) Enriched Kyoto Encyclopedia
of Genes and Genomes pathways. NegLog10, negative logarithm 10.

Figure 4

Reverse transcription-quantitative
PCR analysis of the selected circular RNAs. Validation of
hsa_circ_0000344, hsa_circ_0001126, hsa_circ_0005379,
hsa_circ_0005777 and hsa_circ_0007509. n=6, *P<0.05,
**P<0.01 and ***P<0.001. NC, normal
control; DOR, decreased ovarian reserve.

Figure 5

Function of hsa_circ_0005379 in
CTX-induced KGN ovarian granulosa cells. (A) Different gradient
concentrations (0, 10, 15, 20, 25 and 30 µM) of CTX were used to
induce ovarian granulosa cell damage, and Cell Counting Kit-8 assay
was used to detect KGN cell viability. (B) RT-qPCR analysis of
hsa_circ_0005379 expression after transfection with three shRNAs
and negative control sh-NC. (C) RT-qPCR analysis of
hsa_circ_0005379 expression after transfection with OE vector and
an empty OE-NC vector. (D) The apoptosis of sh-hsa_circ_0005379 or
hsa_circ_0005379-OE transfected KGN cells was detected using
annexin V/PI staining. (E) Flow cytometry was used to detect the
ROS level of cells after different treatments. The concentration of
(F) MDA and (G) SOD in KGN cells with different treatments was
determined using corresponding biochemistry detection kits. n=3,
*P<0.05, **P<0.01 and
***P<0.001 vs. 0 µΜ or as indicated. CTX,
cyclophosphamide; RT-qPCR, reverse transcription-quantitative PCR;
OE, overexpression; NC, negative control; sh, short hairpin; SOD,
superoxide dismutase; MDA, malondialdehyde; ROS, reactive oxygen
species; MFI, mean fluorescence intensity; ns, not significant.
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Copy and paste a formatted citation
Spandidos Publications style
Huang P, Chen S, Cai J, Jiang C, Liu B, Fu Y and Lin D: Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve. Exp Ther Med 30: 224, 2025.
APA
Huang, P., Chen, S., Cai, J., Jiang, C., Liu, B., Fu, Y., & Lin, D. (2025). Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve. Experimental and Therapeutic Medicine, 30, 224. https://doi.org/10.3892/etm.2025.12974
MLA
Huang, P., Chen, S., Cai, J., Jiang, C., Liu, B., Fu, Y., Lin, D."Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve". Experimental and Therapeutic Medicine 30.6 (2025): 224.
Chicago
Huang, P., Chen, S., Cai, J., Jiang, C., Liu, B., Fu, Y., Lin, D."Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve". Experimental and Therapeutic Medicine 30, no. 6 (2025): 224. https://doi.org/10.3892/etm.2025.12974
Copy and paste a formatted citation
x
Spandidos Publications style
Huang P, Chen S, Cai J, Jiang C, Liu B, Fu Y and Lin D: Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve. Exp Ther Med 30: 224, 2025.
APA
Huang, P., Chen, S., Cai, J., Jiang, C., Liu, B., Fu, Y., & Lin, D. (2025). Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve. Experimental and Therapeutic Medicine, 30, 224. https://doi.org/10.3892/etm.2025.12974
MLA
Huang, P., Chen, S., Cai, J., Jiang, C., Liu, B., Fu, Y., Lin, D."Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve". Experimental and Therapeutic Medicine 30.6 (2025): 224.
Chicago
Huang, P., Chen, S., Cai, J., Jiang, C., Liu, B., Fu, Y., Lin, D."Circular RNA expression profiling and the potential role of hsa_circ_0005379 in decreased ovarian reserve". Experimental and Therapeutic Medicine 30, no. 6 (2025): 224. https://doi.org/10.3892/etm.2025.12974
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