PTCSC3 Is Involved in Papillary Thyroid Carcinoma Development by Modulating S100A4 Gene Expression

Abstract

Context: We previously showed that a long noncoding RNA gene, PTCSC3, located close to the variant rs944289 that predisposes to papillary thyroid carcinoma (PTC) might target the S100A4 gene.

Objective: The aim was to investigate the impact of PTCSC3 on S100A4 expression and its role in cancer development.

Design: S100A4 abundance was analyzed by quantitative PCR (qPCR) in unaffected and tumor tissue from n = 73 PTC patients. The expression of PTCSC3 and S100A4 was studied in BCPAP and TPC-1 cell lines with forced expression of PTCSC3 by qPCR. Expression of S100A4 target genes (VEGF and MMP-9) was studied in the BCPAP cell line with forced expression of PTCSC3 by qPCR, reverse transcriptase PCR, and Western blot. The impact of PTCSC3 on BCPAP motility and invasiveness was analyzed by the Transwell and Matrigel assays, respectively.

Setting: This was a laboratory-based study using cells from clinical samples and thyroid cancer cell lines.

Main outcome and measure: We aimed to find evidence for a link between the expression of PTCSC3 and thyroid carcinogenesis.

Results: Expression data from PTC cell lines pinpointed S100A4 as the most significantly downregulated gene in the presence of PTCSC3. S100A4 was upregulated in tumor tissue (P = 9.33 × 10(-7)) while PTCSC3 was strongly downregulated (P = 2.2 × 10(-16)). S100A4 transcription was moderately correlated with PTCSC3 expression in unaffected thyroid tissue (r = 0.429, P = .0001), and strongly in unaffected tissue of patients with the risk allele of rs944289 (r = 0.685, P = 7.88 × 10(-5)). S100A4, VEGF, and MMP-9 were suppressed in the presence of PTCSC3 (P = .0051, P = .0090, and P =.0037, respectively). PTC cells expressing PTCSC3 showed reduction in motility and invasiveness (P = 4.52 × 10(-5) and P = 1.0 × 10(-4), respectively).

Conclusions: PTCSC3 downregulates S100A4, leading to a reduction in cell motility and invasiveness. We propose that PTCSC3 impacts PTC predisposition and carcinogenesis through the S100A4 pathway.

Figure 1.

PTCSC3 and S100A4 expressions assessed by qPCR in unaffected thyroid tissue and PTC tumor tissue in 73 patients. The results are expressed as relative expression in tumor tissue normalized to paired unaffected tissue (T/N fold change). S100A4 showed overexpression (relative expression in tumor > 1.0) (P = 9.33 × 10⁻⁷) and PTCSC3 showed underexpression (relative expression in tumor < 1.0) (P = 2.2 × 10⁻¹⁶). x-axis: genes; y-axis: relative expression in tumor.

Figure 2.

The results of motility and invasion assays. A, Representative images (10× magnification) of slides from the Transwell assay of control cells (BCPAP-C) and BCPAP-P cells expressing PTCSC3 showing suppression of motility in BCPAP-P when compared to BCPAP-C (left). The effect on motility potential in BCPAP cells generated by the restoration of PTCSC3 expression calculated as a ratio of the average cell number in BCPAP-P divided by the average cell number in BCPAP-C. The thyroid cancer cells with restored PTCSC3 expression showed significant reduction in cell motility (P = 4.52 × 10⁻⁵) (right). B, Representative images (10× magnification) of slides from the Matrigel assay of control cells (BCPAP-C) and BCPAP-P cells with stable expression of PTCSC3 showing suppression of BCPAP-P invasiveness when compared to BCPAP-C (left). The BCPAP-P/BCPAP-C ratio calculated in the same way as for the motility assay showed significant reduction in invasiveness in BCPAP cells with restored expression of PTCSC3 (P = 1.0 × 10⁻⁴, respectively) (right). ***, P < .0001.

Figure 3.

PTCSC3 and S100A4 expression analyzed in control cells (BCPAP-C) and cells with stable expression of PTCSC3 (BCPAP-P). A, PTCSC3 and S100A4 expression analyzed by RT-PCR showed restoration of PTCSC3 expression and suppression of S100A4 in BCPAP-P. GAPDH served as loading control. B, PTCSC3 shows strong abundance in stable clones (fold change 30, P = 2.32 × 10⁻⁶). The data are presented as relative expression (BCPAP-P/BCPAP-C ratio). C, S100A4 expression analyzed by qPCR showed significant suppression in BCPAP cells with restored PTCSC3 expression (P = .0051). The data are presented as relative expression (BCPAP-P/BCPAP-C ratio). D, S100A4 expression analyzed by Western blot showed suppression at the protein level in BCPAP-P. hsp90 served as loading control. **, P = .01–.001.

Figure 4.

Expression of VEGF and MMP-9 in control cells (BCPAP-C) and BCPAP-P cells expressing PTCSC3 assessed by qPCR and expressed as relative BCPAP-P/BCPAP-C expression ratios. A, Expression of VEGF at messenger RNA level shows significant suppression in BCPAP-P cells (P = .0090). B, Expression of MMP-9 at messenger RNA level shows significant suppression in BCPAP-P cells (P = .0037). *, P < .05; **, P = .01–.001.

Figure 5.

PTCSC3 and S100A4 expression analyzed in control cells (TPC1-C) and cells with stable expression of PTCSC3 (TPC1-P). A, PTCSC3 shows strong abundance in stable clones (fold change 91, P = 1.23 × 10⁻⁸). The data are presented as relative expression (TPC1-P/TPC1-C ratio). B, S100A4 expression analyzed by qPCR showed significant suppression in TPC-1 cells with restored PTCSC3 expression (fold change 0.53, P = .0137). The data are presented as relative expression (TPC1-P/TPC1-C ratio). *, P = .05–.01; ***, P < .001.