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. 2016 Sep 27;7(39):63388-63407.
doi: 10.18632/oncotarget.11255.

Identification of Tumorigenesis-Related mRNAs Associated With RNA-binding Protein HuR in Thyroid Cancer Cells

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Free PMC article

Identification of Tumorigenesis-Related mRNAs Associated With RNA-binding Protein HuR in Thyroid Cancer Cells

Federica Baldan et al. Oncotarget. .
Free PMC article

Abstract

RNA binding proteins (RBPs) play a central role in cell physiology and pathology. Among them, HuR is a nuclear RBP, which shuttles to the cytoplasm to allow its RNA targets processing. HuR over-expression and delocalization are often associated to cell transformation. Numerous cancers display increased HuR protein levels and its high cytoplasmic levels has been associated with a worse prognosis.In our study, we first evaluated HuR expression in normal and cancer thyroid tissues and then evaluated its function in thyroid cell lines. HuR is over-expressed in all thyroid tumor tissues; high cytoplasmic levels are detected in all thyroid carcinomas. HuR silencing decreased cell viability and determined apoptotic cell death, in a non-tumorigenic (Nthy-ori-3.1) and a tumorigenic (BCPAP) thyroid cell line. Global transcriptome analysis indicated that HuR silencing, though having similar biological effects, induces distinct gene expression modifications in the two cell lines. By using the RIP-seq approach, the HuR-bound RNA profiles of different thyroid cell lines were evaluated. We show that in distinct cell lines HuR-bound RNA profiles are different. A set of 114 HuR-bound RNAs distinguishing tumorigenic cell lines from the non-tumorigenic one was identified.Altogether, our data indicate that HuR plays a role in thyroid tumorigenesis. Moreover, our findings are a proof of concept that RBP targets differ between cells with the same origin but with distinct biological behavior.

Keywords: HuR; RIP-seq; RNA-binding proteins; RNA-seq.

Conflict of interest statement

CONFLICTS OF INTEREST

Authors have no conflict of interest to report.

Figures

Figure 1
Figure 1. HuR expression in thyroid tissues
Panel A. Immunohistochemical evaluation of HuR expression in normal thyroid tissues (NT), FAs, PTCs, FTCs and ATCs. Two PTC images are shown to highlight variation in HuR cytoplasmic levels. Panel B. Quantification of HuR expression in normal and neoplastic thyroid tissues. It was obtained by using the IHC score, calculated as described in Materials and Methods section. Results are shown as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001 by ANOVA test.
Figure 2
Figure 2. HuR expression in thyroid cell lines
Panel A. Western blot analysis of HuR expression in a non-tumorigenic thyroid cell lines (Nthy-ori-3.1) and in six tumorigenic ones (SW1736, FTC133, WRO, FRO, BCPAP and TPC1). Panel B. Densitometric analysis of HuR protein levels in thyroid cell lines. Panel C. Immunocytochemical staining of Nthy-ori-3.1 and BCPAP cells. The brown signal indicates HuR positivity. Results are shown as mean ± SD. * p < 0.05, ** p < 0.01, **** p < 0.0001 by ANOVA test.
Figure 3
Figure 3. HuR silencing effects on cell viability
Panel A. Nthy-ori-3.1 and BCPAP cells were transfected with non-targeting siRNA (NC, negative control) or three different siRNA sequence specific to HuR (5 nM) and collected after 72 h treatment. HuR protein levels were analyzed by Western Blot analysis, as described in Materials and Methods section. Panel B-C. Nthy-ori-3.1 (B) and BCPAP (C) were transfected to either siRNA1 or siRNA2 or negative control for 72 hours and cell death phenomena was analyzed by Annexin V and PI staining. Panel D. Histogram relative to Annexin V/PI analysis.
Figure 4
Figure 4. HuR silencing effects on gene expression
Panel A. Heat maps showing the hierarchical clustering of RNA targets in Nthy-ori-3.1 and BCPAP cell lines. Cells were treated either with siRNA1 or negative control for 72 hours. Results are showed as siRNA1 compared to control transfected cells. Panel B. Venn diagrams represented the comparison of up-regulated, down-regulated and unmodified genes between Nthy-ori-3.1 and BCPAP cell lines after RNA-seq data analysis. The overlap of Nthy-ori-3.1 and BCPAP circles in Venn diagrams indicated shared modified genes between Nthy-ori-3.1 and BCPAP cell lines.
Figure 5
Figure 5. Validation of HuR silencing effects on gene expression
Relative expression levels of PLAU, ROBO4, SERPINB2, ANGPTL4, ARRB2, LMOD1, ITGB4, PPL, IFIT3, KISS1, ADIRIF and FGFBP1 after HuR silencing. RNA extraction and real time PCR are described in Materials and Method section. For each cell lines, the results were normalized against β actin and expressed in arbitrary unit, calculated as described in Materials and Methods section. Results are shown as mean ± SD of three different experiments. All silenced cell data were statistical significant compared to untreated cells.
Figure 6
Figure 6. HuR-bound RNAs in thyroid cell lines
Panel A. Venn diagrams representing the comparison of HuR RNA target identified in Nthy-ori-3.1 and BCPAP cells by RIP-seq analysis. Panel B. Venn diagrams representing the comparison of HuR RNA target identified in three PTC cell lines: BPCAP, K1 and TPC1 by RIP-seq analysis. Panel C. Venn diagrams representing the comparison of HuR RNA target identified in tumorigenic and non-tumorigenic cell lines.
Figure 7
Figure 7. Localization of HuR-RNAs interaction region
Graphical representation of Nthy-ori-3.1, BCPAP, K1 and TPC1 RIP-seq count distribution across different genomic features (UTR3, intronic, exonic, UTR5, intergenic and downstream). The count distribution was normalized for the number of each region present.
Figure 8
Figure 8. Nthy-ori-3.1 and BCPAP cell lines growth in vitro
Panel A. Colony formation assay of Nthy-ori-3.1, BCPAP and K1. Panel B. Histogram representing the number of colonies per cell line. Results are shown as mean ± SD of three different experiments. *** p < 0.0001 by Student t test. Panel C. Cell viability of Nthy-ori-3.1, BCPAP and FRO cells was determined by MTT assay after 0, 24, 48 and 72 h and expressed as fold change respect t0. All samples were run in quadruplicate.
Figure 9
Figure 9. Functional and interaction HuR target
Venn diagrams representing the comparison between RNA-Seq (Functional) and RIP-Seq (Interaction) HuR-target genes identified in Nthy-ori-3.1 and BCPAP cells. In the circle overlapping area genes that were both Functional and Interaction HuR targets.

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