NCOA3 i s overexpressed and associated with poor prognosis in thyroid cancer
We first tested mRNA levels of NCOA3 in 21 pairs of primary thyroid cancer tissues and paired normal thyroid tissues by quantitative RT-PCR (qRT-PCR) assay. As shown in Fig. 1a, compared with matched normal thyroid tissues, NCOA3 was up-regulated in 11 of 21 (52.3%) thyroid cancer tissues. Then we randomly selected 4 thyroid cancer tissues to determine protein levels of NCOA3, as shown in Fig. 1b, compared with matched normal thyroid tissues, NCOA3 was up-regulated in PTCs. Immunohistochemistry experiment was also performed in 5 randomly selected thyroid cancer tissues, and confirmed this conclusion as shown in Fig. 1c. The protein levels of NCOA3 are up-regulated in PTCs, while it is not the same for mRNA levels, we hypothesized that post-transcriptional mechanisms may be involved in regulating NCOA3 expression. The data were presented as mean ± SEM. Statistically significant differences were indicated: *, P < 0.05; ***, P < 0.001.
NCOA3 down-regulation inhibits thyroid cancer cell growth and xenograft tumor growth
To determine the role of NCOA3 in thyroid tumorigenesis, we used two siRNAs (si-NCOA3-709 and si-NCOA3-2252) to knockdown NCOA3 in K1 and FTC133 cells, which is confirmed by Fig. 2a, and then MTT and colony formation assay were performed. As shown in the left panel of Fig. 2b, NCOA3 knockdown inhibited cell proliferation and colony formation compared to the control, in si-NCOA3-transfected cells were fewer than those formed in si-NC-transfected cells (Fig. 2c).
NCOA3 knockdown on thyroid cancer cell cycle contributions and apoptosis were also tested. As shown in Fig. 2d, compared to si-NC-transfected cells, cell cycle was arrested at the G0/G1 phase in si-NCOA3-transfected cells (61.2 ± 1.79 to 51.7 ± 0.62 in K1 cells, and 57.7 ± 1.46 to 51.0 ± 0.86 in FTC133 cells). In addition, as shown in Fig. 2e, we found that si-NCOA3-transfection showed an increase in both early and late apoptosis in comparison with si-NC transfection (13.4% ± 0.29–7.53% ± 0.33% in K1 cells, and 12.8% ± 1.09–7.01% ± 0.53% in FTC133 cells).
NCOA3 re-expression in 8505C cells was confirmed by western blot analysis (Fig. 2f), and ectopic expression of NCOA3 significantly promoted cell proliferation and colony formation compared to the control (Fig. 2g and Fig. 2h).
We also assessed the effect of NCOA3 down-regulation on the growth of xenograft tumors in nude mice. As shown in Fig. 3a, compared with xenograft tumors derived from si-NC transfected cells, the ones derived from si-NCOA3 transfected cells grew more slowly. The volume and mean weight xenograft tumors derived from si-NCOA3 transfected cells was signifcantly smaller as compared with si-NC transfected ones (P = 0.003) (Fig. 3b). Tumor sections were stained for Ki-67 expression to assess the proliferation index. As shown in Fig. 3c, the percentage of Ki-67 positive cells of si-NCOA3 was signifcantly decreased. The data were presented as mean ± SEM. Statistically significant differences were indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
NCOA3 down-regulation inhibits thyroid cancer cell migration and invasion
The effect of NCOA3 knockdown on thyroid cancer cell migration and invasion was also assesed. As shown in Fig. 4a and 4b, there was significantly smaller amount of migrated and invaded cells in the si-NCOA3-transfected cells than si-NC. To the opposite, As shown in Fig. 4c (left panel), we also found that ectopic expression of NCOA3 significantly promoted the ability of cell migration (the upper pannel) and invasion (the lower pannel). Quantitative analysis of colony numbers was shown in the right panel. The data were presented as mean ± SEM. Statistically significant differences were indicated: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
To explore the mechanism of the relationship between NCOA3 and metastatic phenotypes of thyroid cancer cells, we investigated the effect of NCOA3 on the marker of epithelial mesenchymal transition (EMT), which is critical during tumor metastasis including thyroid cancer[16]. Immunofluorescence (Fig. 5a) and western blot (Fig. 5b) assay indicated that knocking down NCOA3 in K1 and FTC133 cells substantially increased the expression of E-cadherin (a epithelial cell marker), and also reduced the expression of vimentin (a mesenchymal marker). These results indicated that the EMT process activated by NCOA3 may contribute to metastatic of thyroid cancer. Besides, we observed that NCOA3 knockdown signifcantly decreased the expression of β-catenin protein (Fig. 5a and b). β-catenin is a key component of the canonical Wnt signaling pathway, and its imbalance in structure can cause metastasis[17, 18]. These observation suggests the involvement of NCOA3 in the regulation of Wnt/β-catenin pathway, further contributing to thyroid cancer cell growth and metastasis.
NCOA3 modulates major signaling pathways in thyroid cancer
Thyroid cancer had a greater mutation burden of PI3K/AKT pathway effectors, BRAF and RAS, which were the predominant drivers for proliferation and distant metastases, especilly for PDTC and ATC[4]. ErbB receptors was demonstrated to form a positive feedback loop with MAPK pathways and lead to the activation of MAPK/ERK and PI3K/AKT pathways[19]. Previous study has demonstrated that ERK1/2 is responsible for Rb phosphorylation[20], which is critical for the regulation of mammalian cell cycle entry[21]. We thus want to determine whether oncogenic role of NCOA3 in thyroid cancer is associated with the activation of these pathways by western blot analysis. As shown in Fig. 6a-c, NCOA3 knockdown dramatically decreased the expression of ErbB receptors, phosphorylation of ERK (p-ERK), AKT (p-AKTS473 and p-AKTT308) and Rb (p-Rb) in K1 and FTC133 cell lines as compared with controls. Collectively, these findings suggest that NCOA3 functions as a critical oncogene in thyroid cancer through modulating major signaling pathways.