Hypoxia Inhibitor Improves Iodine Uptake Disorder in Thyroid Cancer Through the hsa_circ_0023990/miR-448/DNMT1/NIS Axis

Abstract

This research seeks to investigate how hypoxia inhibitors enhance iodine uptake in thyroid cancer cells. Clinical samples were gathered and assessed for hsa_circ_0023990, DNMT1, NIS, and their promoter methylation levels using RT-PCR, western blot, and methylation-specific PCR (MSP) techniques. The study involved examining the impact and mechanism of hsa_circ_0023990 on iodine uptake in differentiated thyroid carcinoma (DTC) cells through genetic manipulation. Luciferase reporter gene experiments were conducted to validate the interaction between hsa_circ_0023990, DNMT1, and miR-448. Xenograft tumors were established in nude mice for in vivo validation. The results showed that hsa_circ_0023990 was notably elevated in DTC and RAIR-DTC tissues. It was found that hsa_circ_0023990 could modulate NIS promoter methylation via the miR-448/DNMT1 signaling pathway, thereby influencing NIS expression. Hypoxia inhibitors effectively suppressed hsa_circ_0023990 expression in DTC cells, leading to increased NIS expression and enhanced iodine uptake. Subcutaneous transplantation experiments in animals further confirmed that hypoxia inhibitors could boost iodine absorption in tumor tissue and inhibit tumor growth through the hsa_circ_0023990/miR-448/DNMT1/NIS signaling axis. In conclusion, hypoxia inhibitors ameliorate iodine uptake dysfunction in thyroid cancer by acting on the hsa_circ_0023990/miR-448/DNMT1/NIS signaling pathway.

Keywords: NIS; hsa_circ_0023990; hypoxia inhibitor; iodine uptake disorder; thyroid cancer.

FIGURE 1

Aberrant expression of hsa_circ_0023990 in radioiodine‐refractory thyroid cancer tissues affects cell activity. (A) The hsa_circ_0023990 expression in thyroid cancer tissues and paracancerous tissues was detected by RT‐PCR. (B) The hsa_circ_0023990 expression in thyroid cancer cells was detected by RT‐PCR. (C) The hsa_circ_0023990 overexpression efficiency in TPC1 cells was detected by RT‐PCR. (D) The hsa_circ_0023990 knockdown efficiency in KMH‐2 cells was detected by RT‐PCR. (E) The TPC1 cell viability after hsa_circ_0023990 overexpression was detected by CCK8 assay. (F) The KMH‐2 cell viability after hsa_circ_0023990 knockdown was detected by CCK8 assay. (G) The TPC1 cell radioactive iodine uptake after hsa_circ_0023990 overexpression was detected by Gamma counter. (H) The KMH‐2 cell radioactive iodine uptake after hsa_circ_0023990 knockdown was detected by Gamma counter. **p < 0.01, ***p < 0.001.

FIGURE 2

hsa_circ_0023990 regulates NIS expression through affecting its methylation level. (A, B) The DNMT1 and NIS protein expressions in thyroid cancer tissues and paracancerous tissues were detected by western blot. (C) The NIS promoter methylation in RAIR‐DTC/paracancerous tissues was detected by MSP assay. TPC1 cells and KMH‐2 cells were transfected with hsa_circ_0023990 overexpression plasmid and si‐hsa_circ_0023990, respectively. (D–G) The DNMT1 mRNA and protein expressions in cells were detected by RT‐PCR and western blot. (H–K) The NIS mRNA and protein expressions in cells were detected by RT‐PCR and western blot. (L) The NIS promoter methylation in cells was detected by MSP assay. **p < 0.01, ***p < 0.001.

FIGURE 3

hsa_circ_0023990 regulates NIS expression through the miR‐448/DNMT1 signaling axis. (A) The sequences of hsa_circ_0023990/miR‐448 indicating complementary regions. (B) Luciferase activity in 293 T cells co‐transfected with control luciferase reporter plasmid or the reporter plasmids containing hsa_circ_0023990 WT or MUT, and miR‐448 mimic or mimic NC after 48 h. (C) The sequences of DNMT1/miR‐448 indicating complementary regions. (D) Luciferase activity in 293 T cells co‐transfected with control luciferase reporter plasmid or the reporter plasmids containing DNMT1 WT 3′ UTR or MUT 3′ UTR, and miR‐448 mimic or mimic NC after 48 h. KMH‐2 cells were transfected with si‐hsa_circ_0023990 and miR‐448 inhibitor. (E–G) The DNMT1 mRNA and protein expressions in cells were detected by RT‐PCR and western blot. (H–J) The NIS mRNA and protein expressions in cells were detected by RT‐PCR and western blot. **p < 0.01, ***p < 0.001.

FIGURE 4

Hypoxia inhibitor reduces hsa_circ_0023990 to increase iodine uptake in thyroid cancer cells. KMH‐2 cells were treated with hypoxia inhibitor (BAY87‐2243), DMSO treatment as the control group, under normoxic conditions. (A) The hsa_circ_0023990 expression in cells treated with BAY87‐2243 was detected by RT‐PCR. (B) The cell viability after BAY87‐2243 treatment was detected by CCK8 assay. (C–E) The NIS mRNA and protein expressions in cells treated with BAY87‐2243 were detected by RT‐PCR and western blot. (F) The cell radioactive iodine uptake after BAY87‐2243 treatment was detected by Gamma counter. **p < 0.01, ***p < 0.001.

FIGURE 5

Hypoxia inhibitors increase iodine uptake and inhibit tumor growth. (A) PET/CT image showing ¹⁸F‐FMISO distribution in tumor‐bearing mice. The cross‐sectional, coronal, and sagittal planes sequentially from left to right. (B) Concentration of ¹⁸F‐FMISO in tumor tissues. “% ID/g” denotes the proportion of radioactive counts per gram of tissue relative to the total radioactive counts administered. “% ID/g‐mean” signifies the mean value across the entire tumor specimen. (C) Tumor images. (D) Tumor growth curve. (E) The cell radioactive iodine uptake in tumor tissue cell suspension was detected by Gamma counter. *p < 0.05, **p < 0.01.

FIGURE 6

Hypoxia inhibitors increase iodine uptake in tumor tissue through the has_circ_0023990/DNMT1/NIS signaling axis to inhibit tumor growth. (A) H&E staining. Scale bar = 50 μm. (B, C) The cell proliferation in tumor tissues was detected by Ki‐67 immunohistochemistry. “IOD/Area” indicating the mean optical density of Ki‐67 expression. Scale bar = 50 μm. (D, E) The cell apoptosis in tumor tissues was detected by TUNEL staining. Scale bar = 50 μm. (F) The hsa_circ_0023990 expression in tumor tissues was detected by RT‐PCR. (G–I) The DNMT1 and NIS protein expressions in tumor tissues were detected by western blot. *p < 0.05, **p < 0.01, ***p < 0.001.