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. 2018 Dec;28(12):1627-1641.
doi: 10.1089/thy.2017.0626. Epub 2018 Oct 27.

MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

Chunxiao Yu  1 , Li Zhang  1   2 , Dandan Luo  1   3 , Fang Yan  4 , Jia Liu  1 , Shanshan Shao  1 , Lifang Zhao  1 , Tong Jin  5 , Jiajun Zhao  1 , Ling Gao  1   6
Affiliations

MicroRNA-146b-3p Promotes Cell Metastasis by Directly Targeting NF2 in Human Papillary Thyroid Cancer

Chunxiao Yu et al. Thyroid. 2018 Dec.

Abstract

Background: MiR-146b has been reported to be overexpressed in papillary thyroid cancer (PTC) tissues and associated with aggressive PTC. MiR-146b is regarded as a relevant diagnostic marker for this type of cancer. MiR-146b-5p has been confirmed to increase cell proliferation by repressing SMAD4. However, detailed functional analysis of another mature form of miR-146b, miR-146b-3p, has not been carried out. This study aimed to identify the differential expression of miR-146b-5p and miR-146b-3p in more aggressive PTC associated with lymph node metastasis, and further elucidate the contribution and mechanism of miR-146b-3p in the process of PTC metastasis. Methods: Expression of miR-146b-5p and miR-146b-3p was assessed in formalin-fixed paraffin-embedded tissue samples from PTC patients, and the relationship with lymph node metastasis was analyzed. A variety of PTC cells, including BHP10-3, BHP10-3SCmice, and K1 cells, were cultured and treated with miR-146b-5p or miR-146b-3p mimics/inhibitors. The cell migration and invasion abilities were characterized by the real-time cell analyzer assay and Transwell™ assay. PTC xenograft models were used to examine the effect of miR-146b-3p on PTC metastatic ability in vivo. Direct downstream targets of miR-146b-3p were analyzed by luciferase reporter assay and Western blotting. The mechanism by which miR-146b-3p affects cell metastasis was further characterized by co-transfection with merlin, the protein product of the NF2 gene. Results: MiR-146b-5p and miR-146b-3p expression was significantly higher in thyroid cancer tissues and cell lines than in normal thyroid tissue and cells. Moreover, expression of miR-146b-5p and miR-146b-3p was further increased in thyroid metastatic nodes than in thyroid cancer. After overexpression of miR-146b-5p or miR-146b-3p in BHP10-3 or K1 cells, PTC migration and invasion were increased. Notably, miR-146b-3p increased cell migration and invasion more obviously than did miR-146b-5p. Overexpression of miR-146b-3p also significantly promoted PTC tumor metastasis in vivo. Luciferase reporter assay results revealed that NF2 is a downstream target of miR-146b-3p in PTC cells, as miR-146b-3p bound directly to the 3' untranslated region of NF2, thus reducing protein levels of NF2. Overexpression of merlin reversed the enhanced aggressive effects of miR-146b-3p. Conclusions: Overexpression of miR-146b-5p and miR-146b-3p is associated with PTC metastasis. MiR-146b-3p enhances cell invasion and metastasis more obviously than miR-146b-5p through the suppression of the NF2 gene. These findings suggest a potential diagnostic and therapeutic value of these miRNAs in PTC metastasis.

Keywords: NF2; PTC; metastasis; miR-146b-3p; miR-146b-5p.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

<b>FIG. 1.</b>
FIG. 1.
MiR-146b-5p and miR146b-3p expression is significantly increased in PTC tissues and further enhanced in MLN. (A) The histological changes of different thyroid tissues and LN tissues stained with H&E (magnification × 100). (B) Relative expression levels of mature miR-146b-5p (left panels) and miR-146b-3p (right panels) in 72 samples, including PTC, matched normal thyroid tissue, MLN, and LN, were determined by qRT-PCR and normalized against an endogenous control RUN6B. Data were analyzed using a ΔΔCt approach and expressed as miR-146b/RUN6B ratio (−2ΔCt[miR-146b-RUN6B]). (C) Fold change of expression of mature miR-146b-5p (left panels) and miR-146b-3p (right panels) in 14 pairs of MLN and their corresponding primary PTC. Data were analyzed using log2 fold change (ΔΔCt [MLN/each corresponding PTC]). Significant upregulation of miR-146b in paired samples was defined as log2 fold change >1, which means twofold higher. (D) Relative expression levels of miR-146b-5p and miR-146b-3p in PTC patients with different aggressive abilities (PTC with LNM [PTC-M] vs. PTC without LNM [PTC-NM]) by qRT-PCR and normalized against an endogenous control RUN6B. NT, normal thyroid tissues, PTC, papillary thyroid cancer; MLN, metastatic lymph nodes; LN, lymph node; H&E, hematoxylin and eosin; qRT-PCR, quantitative reverse transcription polymerase chain reaction. #p < 0.05; ##p < 0.005. Color images available online at www.liebertpub.com/thy
<b>FIG. 2.</b>
FIG. 2.
Expression of miRNA-146b is significantly increased in PTC cell lines and positively correlated with the ability of cell invasion. (A) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3, BHP10-3SC mouse and K1 cells. Representative images showed migrated or invasive cells in the lower chamber stained with hematoxylin. (B) Cell count of Transwell™ assay for above three cells was statistically analyzed. (C) Fold changes of miR-146b-5p and -3p expressions in PTC cells with different invasive abilities (BHP10-3 and BHP10-3SCmice) were calculated relative to miR-146b-5p expression in PHT by qRT-PCR and normalized against an endogenous control RUN6B. PHT, primary normal human thyrocytes. ##p < 0.005 vs. BHP10-3SCmice; **p < 0.005 vs. PHT. Color images available online at www.liebertpub.com/thy
<b>FIG. 3.</b>
FIG. 3.
MiR-146b-3p enhances the migratory and invasive abilities more obviously than miR-146b-5p in BHP10-3 cells. Inhibiting miR-146b-3p expression can reduce the ability of metastasis in BHP10-3SC mouse cells. (A) Expression of miR-146b-5p or -3p was confirmed by real-time PCR after BHP10-3 cells were transfected with miR146b-5p mimics or -3p mimics. (B and C) BHP10-3 cells were transiently transfected with miR-146b-3p mimics (50 nM), miR-146b-5p mimics (50 nM), or negative control (miR-NC; 50 nM). Real-time measurements were performed in 24 h for the migration assay or 96 h for the invasion assay using the real-time cell analyzer system. (D) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3 cells after being transfected with miR-146b-3p mimics, miR-146b-5p mimics, or miR-NC for 24 or 48 h. Representative images showed migrated or invasive BHP10-3 cells in the lower chamber stained with hematoxylin. (E) Cell count of Transwell™ assay for the above three treatments was statistically analyzed. (F) Expression of miR-146b-5p or -3p was confirmed by real-time PCR after BHP10-3SC mouse cells were transfected with miR146b-5p inhibitor or -3p inhibitor. (G) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3SC mouse cells after transfection with miR-146b-3p inhibitor, miR-146b-5p inhibitor, or anti-NC for 24 or 48 h. Representative images show migrated or invasive BHP10-3SC mouse cells in the lower chamber stained with hematoxylin. (H) Cell count of Transwell™ assay for the above two treatments was statistically analyzed. #p < 0.05, ##p < 0.005 vs. miR-NC or anti-NC. Color images available online at www.liebertpub.com/thy
<b>FIG. 4.</b>
FIG. 4.
NF2 is a direct downstream target for miR-146b-3p. (A) Diagram of miR-146b-3p putative seed sequences in the 3′ UTR of candidate mRNA. Based on bioinformation software, NF2 and MTSS1 were found to contain two possible candidate miR-146b-3p binding sites on 3′ UTR separately. (B and C) Relative luciferase activity analysis in BHP10-3 cells transiently co-transfected with 3′ UTR of wild-type or mutant recombinant NF2 or MTSS1 luciferase plasmids with miR-NC or miR-146b-3p mimics. The two wild-type recombinant plasmids of NF2 (NF2-P1 and NF2-P2) and two wild-type recombinant plasmids of MTSS1 (MTSS1-P1 and MTSS1-P2) were co-transfected into cells (B). The wild type of two NF2 3′ UTR and mutant of these NF2 3′ UTR were transfected into cells (C). (D) Merlin protein expression was detected by Western blot analysis after transfection of miR-146b-3p mimics in BHP10-3 cells. (E) Relative luciferase activity analysis in BHP10-3SC mouse cells transiently co-transfected with 3′ UTR of wild-type or mutant recombinant NF2 luciferase plasmids with anti-NC or miR-146b-3p inhibitor. (F) Merlin protein expression was detected by Western blot analysis after miR-146b-3p inhibition in BHP10-3SC mouse cells. Renilla luciferase vector was used as an internal control. The relative luciferase activities of group transfected with miR-NC or anti-NC were set as 1. UTR, untranslated region. #p < 0.05; ##p < 0.005 vs. miR-NC or anti-NC. Color images available online at www.liebertpub.com/thy
<b>FIG. 5.</b>
FIG. 5.
MiR-146b-3p promotes tumor metastasis in vivo. (A) Schematic of male BALB/c PTC intramuscular xenograft nude mice model. (B) Body weight of miR-146b-3p agomir or NC agomir (control) treated BHP10-3SCmluc inoculated nude mice (n = 5, 10). (C) For spontaneous tumor metastasis images were taken from three representative miR-146b-3p agomir or NC agomir (control) treated model separately. Bioluminescence images were obtained from the chest side of the mice in vivo (upper), and fluorescence images were obtained from the lungs and lymph nodes of sacrificed mice ex vivo (lower) at the end time point. (D) The size and morphology of the miR-146b-3p agomir or NC agomir treated BHP10-3SCmluc xenografts in nude mice. (E) Ratio of metastasis of miR-146b-3p agomir or NC agomir (control) treated BHP10-3SCmluc inoculated nude mice. (F) Histopathological characteristics of a representative intramuscular xenograft tumor, pulmonary and lymph node metastases of sacrificed mouse stained with H&E (magnification × 100). The expression of miR-146b-3p (G) and protein levels of merlin (H) were determined by qRT-PCR and Western blot assay separately. The tissues were derived from xenograft mouse tumors treated with miR-146b-3p agomir or NC agomir. **p < 0.001. Color images available online at www.liebertpub.com/thy
<b>FIG. 6.</b>
FIG. 6.
Reintroduction of NF2 abrogates miR-146b-3p induced metastasis enhancement of BHP10-3 cells. BHP10-3 cells were co-transfected with miR-146b-3p mimics and pcDNA3 merlin or pcDNA3 (empty vector control), miR-NC (mimics control), or pcDNA3 merlin was respectively transfected into cells as control. (A) The protein level of endogenous merlin was detected by Western blot analysis after transfection. (B) The densitometric histogram of protein bands and results were expressed as ratio of corresponding protein to GAPDH. (C) Transwell™ assay was performed to determine the migration or invasive ability of BHP10-3 cells after being transfected for 24 or 48 h. Representative images show migrated or invasive BHP10-3 cells in the lower chamber stained with hematoxylin. (D) Cell count of Transwell™ assay for different treatment was statistically analyzed. (E and F) The migratory and invasive capacities of BHP10-3 cells were performed by RTCA assay after being transfected. #p < 0.05; ##p < 0.005 vs. miR-NC. Color images available online at www.liebertpub.com/thy
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