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. 2020 May 3;21(5):412-423.
doi: 10.1080/15384047.2020.1721250. Epub 2020 Feb 9.

The stress-activated protein kinase pathway and the expression of stanniocalcin-1 are regulated by miR-146b-5p in papillary thyroid carcinogenesis

Abeer Al-Abdallah  1 , Iman Jahanbani  1 , Heba Mehdawi  1 , Rola H Ali  1 , Nabeel Al-Brahim  2 , Olusegun Mojiminiyi  1
Affiliations

The stress-activated protein kinase pathway and the expression of stanniocalcin-1 are regulated by miR-146b-5p in papillary thyroid carcinogenesis

Abeer Al-Abdallah et al. Cancer Biol Ther. .

Abstract

Papillary thyroid cancer (PTC) is the most common type of thyroid cancer. Deciphering the pathophysiological mechanisms that contribute to PTC development is essential to the discovery of optimal diagnostic and therapeutic approaches. MiR-146b-5p has been identified as a cancer-associated microRNA highly up-regulated in PTC. This study explores the hypothesis that miR-146b-5p contributes to papillary thyroid carcinogenesis through regulation of cell signaling pathways in a manner that overcomes the cellular growth suppressive events and provides survival advantage. The effect of miR-146b-5p inhibition on major cancer related signaling pathways and expression of Stanniocalcin-1 (STC1), an emerging molecule associated with stress response and carcinogenesis, was tested in cultured primary thyroid cells using luciferase reporter assays, quantitative real-time PCR, immunofluorescence staining, and flow cytometry. Our results demonstrated that miR-146b-5p inhibits the JNK/AP1 pathway activity and down-regulates the expression of STC-1 in thyroid-cultured cells and in thyroid tissue samples. In the presence of miR-146b-5p, PTC cells were resistant to cell death in response to oxidative stress. This is a novel report that miR-146b-5p directly targets STC1 and regulates the activity of JNK/AP1 pathway. Considering the importance of the JNK/AP1 pathway and STC1 in mediating many physiological and pathological processes like apoptosis, stress response and cellular metabolism, a biological regulator of these pathways would have a great scientific and clinical significance.

Keywords: activator protein-1; miRNA, papillary thyroid carcinoma; stanniocalcin-1; stress-activated protein kinase.

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Figures

Figure 1.
Figure 1.
Primary thyroid cancer cells culture and characterization. (a) Thyroid cells forming follicles in the first week of culture. (b)- Thyroid cells confluent growth. (c) Immunofluorescence staining with the epithelial marker, cytokeratin (green) and the mesenchymal marker, smooth muscle actin (red). (d) Immunofluorescence staining with p-ERK (green) and thyroglobulin (red) confirming that the cultured cells are thyroid follicular cells. Blue stain is the DAPI nuclear stain.
Figure 2.
Figure 2.
Cell signaling pathways endogenous activity in PTCs in relation to miR-146b-5p expression level. The activity of each signaling pathway is calculated by comparing the normalized luciferase activity in transfected experimental samples (PTC, n = 13) versus control (MNG, n = 3) all without miR-146b-5p inhibitor. Fold Change = (firefly/renilla ratio of PTC)/(firefly/renilla ratio of MNG). Samples in the luciferase assays were run in quadruplicates. JNK/AP1 is the only pathway that showed a significant inverse correlation with the level of miR-146b-5p expression (R = −0.91).
Figure 3.
Figure 3.
Immunofluorescence staining showing qualitatively the effect of miR-146b-5p inhibition on AP1 expression in a representative transfected PTC sample. (a) Representative image showing that the presence of miR-146b-5p inhibitor (green fluorescence) is accompanied by nuclear expression of AP1 (red arrow); in the same field the absence of the inhibitor (cell indicated by white arrow) has no nuclear AP1. (b) Representative image showing nuclear expression of AP1 (red fluorescence) in the thyroid epithelial cells (cytokeratin positive – green fluorescence) treated with miR-146b inhibitor, while no nuclear AP1 is detected in the cells transfected with negative control. Blue stain is the DAPI nuclear stain.
Figure 4.
Figure 4.
Effect of miR-146b-5p inhibition on the expression of phospho-JNK. (a) Nuclear/cytoplasmic p-JNK (Green) is detected in thyroid cells transfected with miR-146b-5p inhibitor while control cells are negative. (b) Immunoblot showing protein expression of p- JNK (three isoforms of 46, 54 and 67 kDa) and AP1 (39 kDa) in thyroid cells transfected with miR-146b-5p inhibitor with less or no expression in cells transfected with negative control.
Figure 5.
Figure 5.
Expression and localization of p-JNK in different thyroid lesions. (a) Immunofluorescence staining of p-JNK (green) is detected in the nuclei of thyroid follicular cells in NIFTP and in MNG. PTC show positive nuclear p-JNK stain in some cases (picture at the lower left) and negative staining in most of the cases (picture at lower right). Nuclei are stained blue with DAPI. (b) Bar graph showing that the PTC group with negative p-JNK express a significantly higher level of miR-146b-5p when compared to the group with positive p-JNK (P = .0001). (c) Bar graph showing that STC1 expression is significantly more downregulated in the PTC group with negative p-JNK compared to the group with positive p-JNK (P = .01).
Figure 6.
Figure 6.
miR-146b-5p protects PTC cells from cell death in response to oxidative stress (Representative of three independent experiments). (a) Primary thyroid cancer cells transfected with miR-146b inhibitor showed minimal increased number of dying cells (quadrants B1, B2 and B4; 18%) compared to cells transfected with negative control (6%). (b) Under oxidative stress conditions, cells transfected with miR-146b inhibitor showed increased number of dying cells (quadrants B1, B2 and B4; 46%) compared to cells transfected with negative control (9%). Comparing A to B, these results show that in the presence of high endogenous level of miR-146b-5p, PTC cells are resistant to cell death when exposed to oxidative stress (no significant difference between stress (9%) and no stress (6%) conditions).
Figure 7.
Figure 7.
Immunohistochemistry stain of STC1 in different thyroid lesions. (a) Kidney tissue used as a positive control. (b) MNG, (c) NIFTP, (d, e) PTC, these cases express low level of miR-146b-5p and show positive STC1 staining in the cytoplasm/nucleus of thyroid follicular cells. (F) PTC sample with high miR-146b-5p expression showing negative staining of STC1. Scale bar is 20 μM.
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This work is funded by Kuwait University, Research Sector, research grants # MG 02/13 and # MY 08/15; And by Kuwait Foundation for the Advancement of Sciences [P11563MM02]