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Article Open Access

Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response

  • Authors:
    • Naoko Sekiguchi
    • Hidenori Takahashi
    • Shogo Kobayashi
    • Kazuki Sasaki
    • Shinichiro Hasegawa
    • Yoshifumi Iwagami
    • Daisaku Yamada
    • Yoshito Tomimaru
    • Hirofumi Akita
    • Tadafumi Asaoka
    • Takehiro Noda
    • Junzo Shimizu
    • Koji Tanaka
    • Ryota Chijimatsu
    • Yuichiro Doki
    • Hidetoshi Eguchi
  • View Affiliations / Copyright

    Affiliations: Department of Gastroenterological Surgery, Graduate School of Medicine, The University of Osaka, Suita, Osaka 565‑0871, Japan, Center for Comprehensive Genomic Medicine, Okayama University Hospital, Kita‑Ku, Okayama 700‑8558, Japan
    Copyright: © Sekiguchi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 36
    |
    Published online on: January 16, 2026
       https://doi.org/10.3892/ijo.2026.5849
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Abstract

Circular RNAs (circRNAs) are associated with various biological features of cancer, including chemosensitivity and the structural characteristics of circRNAs indicate their potential as liquid biomarkers. Gemcitabine is a cornerstone treatment for pancreatic cancer (PC). A deeper understanding of gemcitabine sensitivity and the exploration of clinically valuable liquid biomarkers that are predictive of gemcitabine sensitivity may contribute to the development of improved‑tailored treatment strategies for PC. The aim of the present study was to identify a candidate circRNA associated with gemcitabine sensitivity, investigate its biological functions and evaluate its potential as a liquid biomarker in predicting gemcitabine sensitivity. circRNA sequencing analysis was conducted to identify candidate circRNAs and the function of a candidate circRNA in modulating gemcitabine sensitivity was investigated in vitro. Further, the potential of this circRNA in predicting gemcitabine sensitivity in patients with PC who received gemcitabine‑based neoadjuvant chemotherapy was evaluated using pre‑treatment serum samples. circ72309 was identified as the candidate circRNA and its overexpression in gemcitabine‑resistant PC cell lines increased gemcitabine‑induced apoptosis and markedly increased gemcitabine sensitivity in vitro. Furthermore, circ72309 decreased cytidine deaminase by increasing reactive oxygen species activity and increasing human equilibrative nucleoside transporter 1 expression via regulation of target miRNAs. Patients with high serum circ72309 had markedly improved progression‑free survival (PFS) and high serum circ72309 was an independent prognostic predictor of a favorable PFS in patients with PC. circ72309 affected multiple steps in the gemcitabine metabolic pathway and its overexpression resulted in markedly increased gemcitabine sensitivity. Therefore, circ72309 expression in the pre‑treatment serum samples may serve as a predictor of gemcitabine sensitivity in patients with PC.

View Figures

Figure 1

Selection of candidate circRNAs
associated with gemcitabine resistance. (A) Heatmap of the
differentially expressed circRNAs for the gemcitabine-sensitive and
gemcitabine-resistant PC cells. (B) Schematic diagram showing the
circ72309 back-splicing junction and the primer-target overlap. (C)
Changes in the Cq values of GAPDH and circ72309 following
RNase R treatment based on RT-qPCR. (D) Differential expression of
circ72309 in gemcitabine-resistant cell lines (GR3/GR8/GR10)
compared to MIAPaCa-2 via RT-qPCR, (E) Differential expression of
circ72309 in the cell culture supernatant between MIAPaCa-2 and GR3
cells using RT-qPCR. *P<0.05. circRNA, circular RNA;
PC, pancreatic cancer; RT-qPCR, reverse transcription-quantitative
PCR.

Figure 2

Effects of circ72309 overexpression
on cellular function in gemcitabine-resistant cell lines. (A)
Vector schema for overexpression of circ72309. (B) Dose-response
curves of gemcitabine in GR_NC and GR_circ72309OE clones.
GR_circ72309OE clones showed a concentration-dependent decrease in
viability with a typical sigmoidal fitting (IC50 values:
GR3, 13.3 ng/ml, R2=0.974; GR8, 130 ng/ml,
R2=0.951; GR10, 16.9 ng/ml, R2=0.984),
whereas GR_NC cells retained high viability even at maximal
gemcitabine concentrations, consistent with the resistant
phenotype. Fitted curves are shown as solid lines, while resistant
clone values without a typical sigmoidal decline are represented as
dashed lines. (C) MTT assays were used to assess the effects of
si-circ72309 in PC cell lines (MIAPaCa-2, PSN-1 and Panc-1).
Dose-response curves were analyzed using non-linear regression with
a four-parameter logistic model; all fitted curves showed excellent
agreement with the experimental data (R2>0.95).
Fitted curves are shown as solid lines. (D) Apoptosis assays
following treatment with 20 ng/ml gemcitabine.
*P<0.05. circRNA, circular RNA; PC, pancreatic
cancer; GR, gemcitabine resistant; NC, negative control; siRNA,
small interfering RNA.

Figure 3

Mechanism by which circ72309
overexpression increases gemcitabine resistance. (A) Heatmap of the
differentially expressed mRNAs for the three pairs of GR3_NC and
GR3_circ72309OE cells. (B) Gene Set Enrichment Analysis of mRNA
sequencing. (C) ROS activity assay comparing GR_NC and
GR_circ72309OE. (D) Relative MFI of DCF normalized to the control
group. (E) Effect of N-acetylcysteine, an ROS scavenger, on
gemcitabine chemosensitivity in GR_circ72309OE cells. Fitted
dose-response curves are shown as solid lines, and non-sigmoidal
resistant responses are represented as dashed lines.
*P<0.05. circRNA, circular RNA; ROS, reactive oxygen
species; MFI, mean fluorescence intensity; DCF,
2′,7′-Dichlorofluorescein.

Figure 4

Changes in the expression of genes
involved in the gemcitabine metabolic pathway following circ72309
overexpression. (A) Difference in CDA expression between
GR_NC and GR_circ72309OE cells. (B) Differences in SLC29A1
expression between GR_NC and GR_circ72309OE cells. (C)
Representative western blot images of CDA and hENT1. (D and E)
Quantification of western blot band intensity; data are presented
as the mean ± SE from at least three independent experiments (n=3).
Protein expression levels of CDA and hENT1. *P<0.05.
circRNA, circular RNA; hENT1, human equilibrative nucleoside
transporter 1; CDA, cytidine deaminase.

Figure 5

Clinical results. (A) circ72309
levels in pre-treatment serum in patients with PC. Δ, outlier. (B)
Waterfall plot of the tumor change rate. Red, high circ72309 group
(n=15); blue, low circ72309 group (n=15). (C) Kaplan-Meier curve of
progression-free survival between circ72309-high (n=15) and -low
(n=15) groups. circRNA, circular RNA; PC, pancreatic cancer.
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Copy and paste a formatted citation
Spandidos Publications style
Sekiguchi N, Takahashi H, Kobayashi S, Sasaki K, Hasegawa S, Iwagami Y, Yamada D, Tomimaru Y, Akita H, Asaoka T, Asaoka T, et al: <p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>. Int J Oncol 68: 36, 2026.
APA
Sekiguchi, N., Takahashi, H., Kobayashi, S., Sasaki, K., Hasegawa, S., Iwagami, Y. ... Eguchi, H. (2026). <p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>. International Journal of Oncology, 68, 36. https://doi.org/10.3892/ijo.2026.5849
MLA
Sekiguchi, N., Takahashi, H., Kobayashi, S., Sasaki, K., Hasegawa, S., Iwagami, Y., Yamada, D., Tomimaru, Y., Akita, H., Asaoka, T., Noda, T., Shimizu, J., Tanaka, K., Chijimatsu, R., Doki, Y., Eguchi, H."<p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>". International Journal of Oncology 68.3 (2026): 36.
Chicago
Sekiguchi, N., Takahashi, H., Kobayashi, S., Sasaki, K., Hasegawa, S., Iwagami, Y., Yamada, D., Tomimaru, Y., Akita, H., Asaoka, T., Noda, T., Shimizu, J., Tanaka, K., Chijimatsu, R., Doki, Y., Eguchi, H."<p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>". International Journal of Oncology 68, no. 3 (2026): 36. https://doi.org/10.3892/ijo.2026.5849
Copy and paste a formatted citation
x
Spandidos Publications style
Sekiguchi N, Takahashi H, Kobayashi S, Sasaki K, Hasegawa S, Iwagami Y, Yamada D, Tomimaru Y, Akita H, Asaoka T, Asaoka T, et al: <p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>. Int J Oncol 68: 36, 2026.
APA
Sekiguchi, N., Takahashi, H., Kobayashi, S., Sasaki, K., Hasegawa, S., Iwagami, Y. ... Eguchi, H. (2026). <p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>. International Journal of Oncology, 68, 36. https://doi.org/10.3892/ijo.2026.5849
MLA
Sekiguchi, N., Takahashi, H., Kobayashi, S., Sasaki, K., Hasegawa, S., Iwagami, Y., Yamada, D., Tomimaru, Y., Akita, H., Asaoka, T., Noda, T., Shimizu, J., Tanaka, K., Chijimatsu, R., Doki, Y., Eguchi, H."<p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>". International Journal of Oncology 68.3 (2026): 36.
Chicago
Sekiguchi, N., Takahashi, H., Kobayashi, S., Sasaki, K., Hasegawa, S., Iwagami, Y., Yamada, D., Tomimaru, Y., Akita, H., Asaoka, T., Noda, T., Shimizu, J., Tanaka, K., Chijimatsu, R., Doki, Y., Eguchi, H."<p>Circ72309 modulates gemcitabine metabolism and gemcitabine sensitivity in pancreatic cancer: Serum Circ72309 levels as a potential predictor of treatment response</p>". International Journal of Oncology 68, no. 3 (2026): 36. https://doi.org/10.3892/ijo.2026.5849
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