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Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑β/Smad signaling pathway

  • Authors:
    • Xueji Wang
    • Jing Wang
    • Wei Cui
    • Jingxia Wang
    • Xueyan Zhang
    • Bingliang Zhang
  • View Affiliations / Copyright

    Affiliations: Department of Nuclear Medicine, People's Hospital of Ningxia Hui Autonomous Region, Yinchuan, Ningxia Hui Autonomous Region 750011, P.R. China, Department of Nuclear Medicine, People's Hospital of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750011, P.R. China, Department of Oncology, People's Hospital of Ningxia Hui Autonomous Region, Yinchuan, Ningxia Hui Autonomous Region 750011, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 322
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    Published online on: September 29, 2026
       https://doi.org/10.3892/etm.2026.13317
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Abstract

Papillary thyroid cancer (PTC) is the most common type of malignant tumor in the endocrine system, with an incidence rate that is increasing year by year. In addition, the mortality rate of advanced PTC is also on the rise, posing a threat to human health. Programmed cell death protein‑1 (PD‑1)/programmed death‑ligand 1 (PD‑L1) signaling has been reported to be abnormally expressed in PTC, while the possible effects and underlying mechanisms remain to be further explored. In the present study, the expression levels of PD‑1 and PD‑L1 were upregulated in clinical PTC tissues as well as TPC‑1 and BCPAP cells as detected by reverse transcription‑quantitative PCR and western blotting analysis. Notably, PD‑1 expression in tissue samples may originate from both tumor cells and tumor‑infiltrating immune cells, whereas PD‑1 detected in cell lines solely represents tumor‑intrinsic expression. Functionally, downregulation of PD‑1 or PD‑L1 notably inhibited the viability, proliferation and induced the apoptosis of TC cells, as measured by Cell Counting Kit‑8, EdU, colony formation, flow cytometry and western blotting analysis. In addition, downregulation of PD‑1 or PD‑L1 markedly suppressed the migration, invasion and epithelial‑mesenchymal transition progress of TC cells, as measured by wound healing and Transwell migration and invasion assays, as well as western blotting analysis. Furthermore, downregulation of PD‑1 or PD‑L1 inactivated the TGF‑β/Smad signaling pathway. Exogenous TGF-β partially restored the effects of short hairpin (sh)‑PD‑1 or sh‑PD‑L1 on the viability, proliferation, apoptosis, migration, invasion and EMT progress of PTC cells. To conclude, the present study suggests PD‑1/PD‑L1 may represent potential therapeutic targets for the treatment of patients with PTC, though further studies across additional thyroid cancer subtypes are warranted.
View Figures

Figure 1

PD-1 and PD-L1 expression levels were
upregulated in clinical PTC tissues and cell lines. (A) RT-qPCR and
(B) western blotting were adopted to evaluate the expression levels
of PD-1/PD-L1 in clinical PTC tissues. (C) RT-qPCR and (D) western
blotting was adopted to evaluate the expression levels of
PD-1/PD-L1 in thyroid cancer cells. (E) IHC analysis was adopted to
evaluate the expression levels of PD-1/PD-L1 in clinical PTC
tissues. **P<0.01 vs. Normal or Nthy-ori3-1 group.
RT-qPCR, reverse transcription-quantitative PCR; PD-1, programmed
death-1; PD-L1, programmed death-ligand 1; PTC, papillary thyroid
cancer.

Figure 2

Downregulation of PD-1/PD-L1
inhibited the proliferation of TC cells. (A) RT-qPCR and (B)
western blotting were used to assess the transfection efficiency.
(C) Western blotting was carried out to evaluate the effects of
PD-1/PD-L1 on the expression of NIS. (D) A Cell Counting Kit-8
assay was performed to evaluate the effects of PD-1/PD-L1 on the
viability of TC cells. (E) An EdU assay was performed to evaluate
the effects of PD-1/PD-L1 on the proliferation of TC cells (scale
bar, 100 µm). (F) Colony formation assay was performed to evaluate
the effects of PD-1/PD-L1 on the colony formation ability of TC
cells. **P<0.01 vs. Blank group. RT-qPCR, reverse
transcription-quantitative PCR; PD-1, programmed death-1; PD-L1,
programmed death-ligand 1; TC, thyroid cancer; sh, short hairpin;
NC, negative control; OD, optical density; NIS, sodium/iodine
symporter.

Figure 3

Downregulation of PD-1/PD-L1 induced
the apoptosis of TC cells. (A) Flow cytometry analysis was adopted
to evaluate the effects of PD-1/PD-L1 on the apoptosis of TC cells.
(B) Western blotting was performed to evaluate the effects of
PD-1/PD-L1 on the protein expression levels associated with the
apoptosis of TC cells. **P<0.01 vs. the Blank group.
RT-qPCR, reverse transcription-quantitative PCR; PD-1, programmed
death-1; PD-L1, programmed death-ligand 1; TC, thyroid cancer; sh,
short hairpin; NC, negative control.

Figure 4

Downregulation of PD-1/PD-L1
inhibited the migration and invasion of TC cells. (A) Wound healing
(scale bar, 100 µm), (B) Transwell migration (scale bar, 50 µm) and
(C) invasion assays (scale bar, 50 µm) were performed to analyze
the effects of PD-1/PD-L1 on the migration and invasion of TC
cells. (D) Western blotting assay was performed to evaluate the
effects of PD-1/PD-L1 on the protein expressions associated with
EMT progress of TC cells. **P<0.01 vs. the Blank
group. RT-qPCR, reverse transcription-quantitative PCR; PD-1,
programmed death-1; PD-L1, programmed death-ligand 1; TC, thyroid
cancer; EMT, epithelial-mesenchymal transition; sh, short hairpin;
NC, negative control.

Figure 5

Downregulation of PD-1/PD-L1
inhibited the expression of TGF-β/Smad signaling pathway of TC
cells. (A) Western blotting was performed to evaluate the effects
of PD-1/PD-L1 on the expression levels of TGF-β/Smad signaling
pathway of TC cells. (B) Western blotting was performed to evaluate
the expression levels of TGF-β/Smad signaling pathway of TC cells.
**P<0.01 vs. Blank or sh-NC group,
##P<0.01 vs. sh-PD-1 group and
&&P<0.01 vs. PD-L1 group. PD-1, programmed
death-1; PD-L1, programmed death-ligand 1; TC, thyroid cancer; sh,
short hairpin; NC, negative control; p-, phosphorylated.

Figure 6

Downregulation of PD-1/PD-L1
inhibited the proliferation and induced the apoptosis of TC cells
through regulating TGF-β/Smad signaling pathway. (A) A Cell
Counting Kit-8 assay was performed to evaluate the viability of TC
cells. (B) Western blotting was carried out to evaluate the
expression of NIS. (C) An EdU assay was performed to evaluate the
proliferation of TC cells (scale bar, 100 µm). (D) A colony
formation assay was performed to evaluate the colony formation
ability of TC cells. (E) Flow cytometry analysis was performed to
evaluate the apoptosis of TC cells. **P<0.01 vs.
Blank or sh-NC group, ##P<0.01 vs. sh-PD-1 group and
&&P<0.01 vs. PD-L1 group. PD-1, programmed
death-1; PD-L1, programmed death-ligand 1; TC, thyroid cancer; sh,
short hairpin; NC, negative control; NIS, sodium/iodine symporter;
OD, optical density.

Figure 7

Downregulation of PD-1/PD-L1
inhibited the migration and invasion of TC cells regulating
TGF-β/Smad signaling pathway. (A) Wound healing (scale bar, 100
µm), (B) Transwell migration (scale bar, 50 µm) and (C) invasion
assays (scale bar, 50 µm) were performed to analyze the migration
and invasion of TC cells. (D) Western blotting was performed to
evaluate the expression levels associated with EMT progress of TC
cells. **P<0.01 vs. Blank or sh-NC group,
##P<0.01 vs. sh-PD-1 group and
&&P<0.01 vs. PD-L1 group. PD-1, programmed
death-1; PD-L1, programmed death-ligand 1; TC, Thyroid cancer; sh,
short hairpin; NC, negative control; EMT, epithelial-mesenchymal
transition.
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Copy and paste a formatted citation
Spandidos Publications style
Wang X, Wang J, Cui W, Wang J, Zhang X and Zhang B: Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway. Exp Ther Med 32: 322, 2026.
APA
Wang, X., Wang, J., Cui, W., Wang, J., Zhang, X., & Zhang, B. (2026). Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway. Experimental and Therapeutic Medicine, 32, 322. https://doi.org/10.3892/etm.2026.13317
MLA
Wang, X., Wang, J., Cui, W., Wang, J., Zhang, X., Zhang, B."Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway". Experimental and Therapeutic Medicine 32.5 (2026): 322.
Chicago
Wang, X., Wang, J., Cui, W., Wang, J., Zhang, X., Zhang, B."Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway". Experimental and Therapeutic Medicine 32, no. 5 (2026): 322. https://doi.org/10.3892/etm.2026.13317
Copy and paste a formatted citation
x
Spandidos Publications style
Wang X, Wang J, Cui W, Wang J, Zhang X and Zhang B: Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway. Exp Ther Med 32: 322, 2026.
APA
Wang, X., Wang, J., Cui, W., Wang, J., Zhang, X., & Zhang, B. (2026). Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway. Experimental and Therapeutic Medicine, 32, 322. https://doi.org/10.3892/etm.2026.13317
MLA
Wang, X., Wang, J., Cui, W., Wang, J., Zhang, X., Zhang, B."Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway". Experimental and Therapeutic Medicine 32.5 (2026): 322.
Chicago
Wang, X., Wang, J., Cui, W., Wang, J., Zhang, X., Zhang, B."Effects of PD‑1/PD‑L1 on the malignant behavior of thyroid cancer cells through the TGF‑&beta;/Smad signaling pathway". Experimental and Therapeutic Medicine 32, no. 5 (2026): 322. https://doi.org/10.3892/etm.2026.13317
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