Characterizing the function of EPB41L4A in the predisposition to papillary thyroid carcinoma

Abstract

Papillary thyroid carcinoma (PTC) is the most common histotype of thyroid carcinoma. The heritability of PTC is high compared to other cancers, but its underlying causes are unknown. A recent genome-wide association study revealed the association of a variant at the 5q22 locus, rs73227498, with PTC predisposition. We report that rs17134155, a variant in high linkage disequilibrium with rs73227498, is located in an enhancer region downstream of coding transcripts of EPB41L4A. Rs17134155 showed significant enhancer activity in luciferase assays, and haplotypes containing the protective allele of this variant conferred a significantly lower risk of PTC. While the index SNP, rs73227498, acted as a significant cis-eQTL for expression of EPB41L4A, rs17134155 was a significant cis-sQTL for the alternative splicing of a non-coding transcript of EPB41L4A, called EPB41L4A-203. We also performed knockdown of EPB41L4A followed by microarray analysis. Some of the top differentially-expressed genes were represented among regulators of the WNT/β-catenin signaling pathway. Our results indicate that an enhancer region at 5q22 regulates the expression and splicing of EPB41L4A transcripts. We also provide evidence that EPB41L4A expression is involved in regulating growth and differentiation pathways, suggesting that decreased expression of EPB41L4A is a mechanism in the predisposition to PTC.

Figure 1

PTC GWAS SNP rs73237498 is located downstream of coding EPB41L4A transcripts. (a) Graph of negative log₁₀-transformed P values at the 5q22 locus from Ohio PTC cases and controls in a recent GWAS. (b) Graph of negative log₁₀-transformed P values at the 5q22 locus for expression quantitative trait loci (cis-eQTL) in thyroid tissue.

Figure 2

SNP rs17145155 shows allele-specific expression. (a) Schematic of coding and non-coding transcripts of EPB41L4A and EPB41L4A-AS1. The position of GWAS SNP rs73227498 and rs17134155 are indicated by red arrows. Results of dual luciferase activity in relative fluorescence units (RFU) for downstream EPB41L4A variants in (b) BCPAP and (c) TPC1 cells. Results are shown as mean ± SEM of one experiment with four technical replicates. For each variant, the left allele is the effect allele (EA) and the right is the other allele (OA). Asterisks indicate that the experiment was repeated four times with similar results. Allele-specific expression of EPB41L4A-201, EPB41L4A-203, and EPB41L4A-AS1 in adjacent unaffected thyroid tissue between genotypes for (d) rs73227498 and (e) rs17134155. *P < 0.05, **P < 0.01, ****P < 0.0001 (two-tailed t test).

Figure 3

Knockdown of EPB41L4A shows changes in cell survival and motility. Ingenuity Pathway Analysis depicting the top (a) ‘disease and disorders’ and (b) ‘molecular and cellular functions’ affected on EPB41L4A knockdown. (c) Expression of the candidate genes involved in cellular movement from siRNA knockdown of EPB41L4A is plotted with a heat-map color scale using relative expression fold changes (fold change > 2.0, P < 0.0001, n = 4). (d) Quantitative polymerase chain reaction validations for twenty-seven differentially- expressed genes in BCPAP cells treated with non-specific (siNS) or EPB41L4A (siEPB41L4A) siRNA. **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-tailed t test).

Figure 4

Knockdown of EPB41L4A results in increased proliferation of BCPAP cells. Histogram of propidium iodide-stained BCPAP cells treated with (a) non-specific siRNA (siNS) or (b) EPB41L4A siRNA (siEPB41L4A) for 72 h. The percentage of cells in each phase of the cell cycle (G1, S, G2/M) is shown for each range. (c) The average percentage of BCPAP cells in each phase of the cell cycle is shown. (d) WST-8 proliferation assay of BCPAP cells. The level of metabolic activity is depicted by the absorbance of formazan dye (A = 450). **P < 0.01, ***P < 0.001 (two-tailed t test).

Figure 5

rs17134155 shows changes in alternative splicing of the non-coding EPB41L4A-203 transcript. (a) Schematic of the alternative splicing pattern for EPB41L4A-203. Solid lines represent predominant splice forms for rs17134155 “CC” (top) and “TT” (bottom) genotypes. Dotted lines represent lower-expressed splice isoforms. The position of rs17134155 is depicted by a red arrow. (b) RT-PCR of EPB41L4A-203 minor splice products according to rs17134155 genotype. (c) Graph of percent splicing inclusion (PSI) for exon-included relative to exon-excluded products for major EPB41L4A-203 splice variants. **P < 0.01 (two-tailed t test).