Effects of HuR downregulation on anaplastic thyroid cancer cells

Abstract

Anaplastic thyroid cancer (ATC) constitutes one of the most aggressive types of human solid cancer, and is characterized by the absence of thyroid differentiation features and a marked degree of invasiveness. We have previously demonstrated that the RNA-binding protein Hu antigen R (HuR) is overexpressed in thyroid carcinoma; thus, the biological role of this RNA-binding protein was investigated in the present study using the ATC cell lines SW1736 and 8505C. In both cell lines, HuR protein levels were higher compared with in the non-tumorigenic thyroid cell line Nthy-ori-3.1. HuR silencing by RNA interference in both ATC cell lines decreased cell viability, increased apoptosis rates and reduced the capability to form colonies in soft agar. Thus, HuR plays an important role in the proliferation and aggressiveness of ATC cells. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was able to reduce the viability of ATC cells. The results demonstrated that SAHA was able to decrease HuR expression in SW1736 and 8505C cells. Furthermore, since it is known that the transcription factor nuclear factor (NF)-κB modulates HuR expression, whether SAHA affects the nuclear (active) fraction of NF-κB in ATC cells was investigated. The data suggested that SAHA decreases ATC cell viability by reducing the active form of NF-κB, which, in turn, modulates HuR expression.

Keywords: HuR; RNA-binding proteins; SAHA; anaplastic thyroid cancer; siRNA.

Figure 1.

HuR expression in ATC cell lines. (A) Western blot analysis of HuR expression in a non-tumorigenic thyroid cell lines (Nthy-ori-3.1) and in two ATC-derived cell lines (SW1736 and 8505C). (B) Densitometric analysis of HuR protein levels in thyroid cell lines. For each cell line, the results were normalized against β-actin levels and expressed in arbitrary unit. Results are shown as mean ± standard deviation. *P<0.05 by Student's t-test.

Figure 2.

Biological effects of HuR silencing in ATC cells. (A) SW1736 and (B) 8505C cells were transfected with non-targeting siRNA (CN, negative control) or three different siRNA (1, 2 and 3) sequence specific to HuR (1 nM) and collected after 72 h treatment. HuR protein levels were analyzed by western blot analysis, as described in Materials and methods section. For each cell line, the results were normalized against β-actin levels and expressed in arbitrary unit. (C) SW1736 and 8505C cells were transfected to either siRNA1 (#1.1) or CN for 72 h and cell viability was analyzed by MTT assay. (D) Densitometric analysis of cleaved PARP fraction levels obtained with western blot assay in SW1736 and 8505C cells transfected to either siRNA1 (#1.1) or CN for 72 h. (E) Histogram representing the number of colonies per cell line evaluated by colony formation assay of SW1736 and 8505C transfected to either siRNA1 (#1.1) or CN for 72 h. Results are shown as mean ± standard deviation. *P<0.05 by Student's t-test.

Figure 3.

SAHA effects on HuR expression in ATC cells. (A) Relative expression levels of HuR mRNA after SAHA 3 µM or DMSO treatment for 48 h. RNA extraction and qPCR are described in Materials and method section. For each cell line, the results were normalized against β-actin levels and expressed in arbitrary unit, calculated as described in Materials and Methods section. (B) Western blot analysis of HuR expression in SW1736 and 8505C treated with SAHA 3 µM or DMSO for 72 h. (C) Densitometric analysis of HuR protein levels in ATC cell lines treated with SAHA or DMSO. For each cell line, the results were normalized against β-actin levels and expressed in arbitrary unit. Results are shown as mean ± standard deviation. *P<0.05 by Student's t-test.

Figure 4.

Effects of SAHA treatment on activated NF-κB levels in ATC cell lines. Densitometric analysis of nuclear NF-κB protein levels in SW1736 and 8505C cell lines after DMSO or SAHA 3 µM administration for 24 or 48 h. For each cell line, the results were normalized against LSD1 expression and expressed in arbitrary unit. Results are shown as mean ± standard deviation. *P<0.05 by Student's t-test.