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. 2024 Apr;51(4):57.
doi: 10.3892/or.2024.8716. Epub 2024 Feb 23.

Effect of hypoxia‑HIF‑1��‑periostin axis in thyroid cancer

Ye Yang #  1 , Junyi Wu #  2 , Huiqin Zhu #  3 , Xiaoqin Shi  4 , Jun Liu  2 , Yang Li  2 , Min Wang  2
Affiliations

Effect of hypoxia‑HIF‑1α‑periostin axis in thyroid cancer

Ye Yang et al. Oncol Rep. 2024 Apr.

Abstract

The incidence of thyroid carcinoma (TC) has exhibited a rapid increase in recent years. A proportion of TCs exhibit aggressive behavior. The present study aimed to investigate the potential role of hypoxia‑hypoxia inducible factor 1 subunit α (HIF‑1α)‑periostin axis in the progression of TC. The upregulation of periostin and HIF‑1α expression levels was detected in 95 clinical TC tissues as compared with normal thyroid tissues. Hypoxia promoted the viability and invasion of TC cells and this effect was inhibited by the downregulation of periostin. Hypoxia also induced the Warburg effect in TC and this effect was inhibited by the silencing of periostin. Further investigations revealed that hypoxia activated HIF‑1α, which in turn regulated the expression of periostin. Immunoprecipitation and dual luciferase reporter assays demonstrated that HIF‑1α upregulated the expression of periostin by binding to the promoter of periostin. On the whole, these findings suggest the existence of a hypoxia‑HIF‑1α‑periostin axis in TC and indicate the role of this axis in the progression of TC.

Keywords: Warburg effect; hypoxia; hypoxia inducible factor 1 subunit alpha; periostin; thyroid cancer.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1.
Figure 1.
Periostin and HIF-1α expression in thyroid cancer. (A) Periostin protein was localized in the cytoplasm of the cancer cells and in some normal thyroid cells, but with strong staining in the epithelial cancer cells. Brown color indicates periostin positivity, Scale bars, 25 µm. (B) PTC tissues exhibited a significantly higher expression of periostin as compared to normal thyroid tissues (P<0.01). Brown color indicates periostin positivity. (C) HIF-1α was localized in the cytoplasm of cancer cells and some normal thyroid cells, but with strong staining in the cancer cells. Brown color indicates HIF-1α positivity. Scale bars, 20 µm. (D) PTC tissues present with significantly higher expression of HIF-1α, as compared to normal thyroid tissues (P<0.01). (E) Images depicting the staining for HIF-1α and periostin were obtained from adjacent sections of the same tissue at varying magnifications. Scale bars, 25 µm. HIF-1α, hypoxia inducible factor 1 subunit α; PTC, papillary thyroid cancer.
Figure 2.
Figure 2.
Hypoxia upregulates the expression of periostin in TC cells via HIF-1α. (A) Results of RT-qPCR analysis: the expression of HIF-1α mRNA in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Compared to transfection with periostin shRNA under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells significantly increased with periostin shRNA transfection under hypoxic conditions (#P<0.05). n=3 biological replicates. Error bars represent standard deviation. (B) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (**P<0.01). The mRNA expression of periostin significantly decreased following transfection with periostin shRNA under normoxic conditions (*P<0.05). As compared to transfection with periostin shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following periostin shRNA transfection under hypoxic conditions (#P<0.01). n=3 biological replicates. Error bars represent standard deviation. (C) Results of RT-qPCR analysis: The expression of HIF-1α mRNA in BCPAPs under hypoxic conditions for 24 h is significantly greater as compared normoxia (**P<0.01). The expression of HIF-1α mRNA significantly decreased following transfection with HIF-1α shRNA under normoxic conditions (*P<0.01). Compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of HIF-1α in BCPAP cells increased following transfection with HIF-1α shRNA under hypoxic conditions (#P<0.01). n=3 biological replicates. Error bars represent standard deviation. (D) Results of RT-qPCR analysis: The mRNA expression of periostin in BCPAP cells under hypoxic conditions for 24 h was significantly greater than under normoxic conditions (**P<0.01). The expression of periostin mRNA significantly decreased following the transfection of HIF-1α shRNA under normoxic conditions (*P<0.05). As compared to transfection with HIF-1α shRNA under normoxic conditions, the mRNA expression of periostin in the BCPAP cells significantly increased following transfection with HIF-1α shRNA under hypoxic conditions (#P<0.01). n=3 biological replicates. Error bars represent standard deviation. (E) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without periostin shRNA transfection. (F) Western blot analysis of HIF-1α, N-cadherin, E-cadherin, periostin and NIS under normoxic or hypoxic conditions, with or without HIF-1α shRNA transfection. TC, thyroid cancer; HIF-1α, hypoxia inducible factor 1 subunit α; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NIS, solute carrier family 5 member 5.
Figure 3.
Figure 3.
Effects of hypoxia and periostin on the proliferation of TC cells. (A and B) Effects of hypoxia and periostin shRNA on proliferation were evaluated using CCK-8 assays in BCPAP and TPC-1 cells at different time points (24, 48 and 72 h). Results of CCK-8 assay at 72 h show significantly an increased cell proliferation of TC cells (BCPAP and TPC-1) under hypoxic conditions vs. normoxic conditions (P<0.05); transfection with Periostin shRNA inhibited the proliferation of cells (P<0.05). Hypoxia combined with transfection with periostin shRNA inhibited the proliferation of TC cells compared to the cells subjected only to hypoxia (P<0.05). n=3 biological replicates; error bars represent standard deviation. (C-F) Results of MTT assay: Both the BCPAP and TPC-1 cells exhibited a significantly greater proliferation under hypoxic compared with normoxic conditions for 3 and 5 days (*P<0.05); transfection with periostin shRNA inhibited cell proliferation following culture for 3 and 5 days (**P<0.01). Hypoxia combined with periostin shRNA transfection inhibited the proliferation of TC cells compared with the cells subjected only to hypoxia (#P<0.01). n=3 biological replicates; error bars represent standard deviation. TC, thyroid cancer; CCK-8, Cell Counting Kit-8 assay.
Figure 4.
Figure 4.
Effects of hypoxia and periostin on the invasiveness and migratory ability of TC cells. (A and B) Transwell migration and invasion assays. BCPAP and TPC-1 cells under normoxic or hypoxic conditions plus transfection with or without periostin shRNA (c-shRNA). The number of cells that migrated or invaded through Matrigel-coated inserts was determined. Invasiveness capability of cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA inhibited the invasiveness capability of cells (**P<0.01). Hypoxia combined with periostin shRNA transfection restored the invasiveness capability of cells compared to only periostin shRNA transfection (##P<0.01). The migratory capability of cells under hypoxic conditions for 24 h was significantly greater than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA inhibited the migration capability of cells (**P<0.01). Hypoxia combined with periostin shRNA transfection restored the migration capability of cells compared to only periostin shRNA transfection (##P<0.01). Scale bars, 50 µm; n=3 biological replicates. (C and D) Wound healing assay. BCPAP and TPC-1 cells under normoxia or hypoxia plus transfection with or without periostin shRNA. Wound healing assays suggested BCPAP cells under hypoxic conditions achieved an increased healing rate as compared with that under normoxic conditions (*P<0.05); periostin knockdown significantly inhibited this effect (**P<0.05). Hypoxia combined with periostin shRNA remitted the mobility of cells (#P<0.05). Scale bars, 100 µm; n=3 biological replicates. TC, thyroid cancer.
Figure 5.
Figure 5.
Molecular mechanisms of the regulation of periostin expression by HIF-1α. (A) The human periostin promoter contains four HIF-1α consensus sequences for potential binding sites. (B) DNA agarose gel electrophoresis results of input, control IgG and amplified products by HIF-1α overexpression plasmid from primer 1 to primer 4. (C) The results of reverse transcription-quantitative PCR are presented as regions amplified with four different primer pairs (primer 1-primer 4). The results revealed that binding of HIF-1α in the region probed with primer 2 and primer 3 amplified significantly compared to the control group after HIF-1α overexpression (***P<0.001), and fold enrichment of primer 2 was higher. (D) Luciferase reporter assays results. Compared to the blank control group, periostin-promoter-WT co-transfection with HIF-1α overexpression plasmid significantly increased the luciferase activity (***P<0.001). Co-transfection of mutant periostin-promoter with primer 2 (Δ2 mutant-type) or primer 3 (Δ3 mutant-type) with a HIF-1α overexpression plasmid demonstrated a significantly decreased luciferase activity, as compared with periostin-promoter-WT (#P<0.05 and ###P<0.001). Co-mutant of primer 2 and primer 3 (Δ2+3 mutant-type) resulted the luciferase activity being most significantly decreased ###P<0.001). HIF-1α, hypoxia inducible factor 1 subunit α; WT, wild-type.
Figure 6.
Figure 6.
Hypoxia induced the Warburg effect by upregulating the expression of periostin. (A) Results of phosphomolybdic acid colorimetry. ATP expression in BCPAP cells under hypoxic conditions was significantly lower than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA under normoxic conditions increased the ATP expression in BCPAPs (**P<0.01). As compared to the hypoxic group, transfection using periostin shRNA under hypoxic conditions significantly increased the ATP expression in BCPAP cells (##P<0.01). (B) Results of the LDH test. The LDH expression in BCPAPs under hypoxic conditions was significantly higher than that under normoxic conditions (*P<0.05). Transfection with periostin shRNA under normoxic conditions decreased LDH expression in BCPAPs (**P<0.01). In comparison with the hypoxic group, transfection with periostin shRNA under hypoxic conditions significantly decreased LDH expression in BCPAP cells (##P<0.01). (C and D) The ECAR and OCR of BCPAP cells under different conditions were measured using the Seahorse analyzer. The ECAR of BCPAP cells under hypoxic conditions or with HIF-1α overexpression was significantly higher than that under nomoxia (***P<0.001). Transfection with periostin shRNA under normoxic conditions significantly decreased the ECAR of BCPAP cells (**P<0.01). Transfection using periostin shRNA under hypoxic conditions or co-transfection with HIF-1α overexpression plasmids increased the ECAR of BCPAP cells (###P<0.001). By contrast, the OCR of BCPAPs with HIF-1α overexpression was significantly decreased (**P<0.01). Transfection with periostin shRNA significantly elevated the OCR of BCPAP cells (*P<0.05). Transfection with periostin shRNA under hypoxic conditions or co-transfection with HIF-1α overexpression plasmids decreased the OCR of BCPAP cells (##P<0.01 and ###P<0.001, respectively). LDH, lactate dehydrogenase; ECAR, extracellular acidification rate; OCR, oxygen consumption rate; HIF-1α, hypoxia inducible factor 1 subunit α.
Figure 7.
Figure 7.
Schematic representation of the effect of hypoxia-HIF-1α-periostin axis in thyroid cancer. HIF-1α, hypoxia inducible factor 1 subunit α.
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Grants and funding

The present study was funded by the Shanghai General Hospital Characteristic Research Project (grant no. CTCCR-2021C19).