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. 2019 Apr 8;10:207. doi: 10.3389/fendo.2019.00207

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Copyright © 2019 Liu, Fu, Li, Liang, Li, Li, Sui, Dionigi and Sun.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Silencing FOXD2-AS1 inhibits cancer stem cell characteristics in thyroid cancer cells. (A) Real-time PCR analysis of FOXD2-AS1 expression in normal thyroid follicular epithelial cells PTFE and seven thyroid cancer cells, including four PTC cell lines, B-CPAP, BHT101, KTC-1 and K1, and two ATC cell lines, CAL-62, and 8305C, and one thyroid duct cell carcinoma cells, TT. GAPDH was used as endogenous controls. *P < 0.05. (B) FOXD2-AS1 expression in the scramble, FOXD2-AS1 shRNA#1 and FOXD2-AS1 shRNA#2 thyroid cancer cells using real-time PCR. Transcript levels were normalized by GAPDH expression. *P < 0.05. (C) Representative images of spheroids formed by the scramble, FOXD2-AS1 shRNA#1 and FOXD2-AS1 shRNA#2 thyroid cancer cells at 200-fold magnification were counted. Histograms showed the mean number of spheroids formed. *P < 0.05. (D) The effect of silencing FOXD2-AS1 on side population in the indicated thyroid cancer cells by Hoechst 33342 dye exclusion assay. Histograms showed the fraction of side population in thyroid cancer cells.*P < 0.05. (E) The effect of silencing FOXD2-AS1 on the CD133⁺ population in the indicated thyroid cancer cells by flow cytometric analysis. Histograms showed the CD133⁺ percentage of thyroid cancer cells. *P < 0.05. (F,G) The effect of silencing FOXD2-AS1 on multiple stemness markers, including ABCG2, SOX2, NANOG, BMI-1, ALHD1A1, POU5F1, and KLF4, by real-time PCR analysis in the indicated thyroid cancer cells. Transcript levels were normalized by GAPDH expression. *P < 0.05. (H) The effect of silencing FOXD2-AS1 on ABCG2, SOX2, NANOG, and BMI-1 by Western blot in the indicated thyroid cancer cells. α-Tubulin was detected as a loading control.