Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma

Abstract

Although papillary thyroid carcinoma (PTC) displays strong heritability, no predisposing germ-line mutations have been found. We show that a common G/C polymorphism (rs2910164) within the pre-miR-146a sequence reduced the amount of pre- and mature miR-146a from the C allele 1.9- and 1.8-fold, respectively, compared with the G allele. This is matched by a similar decrease in the amount of each pre-miR generated from the corresponding pri-miR-146a in an in vitro processing reaction. The C allele also interfered with the binding of a nuclear factor to pre-miR-146a. The reduction in miR-146a led to less efficient inhibition of target genes involved in the Toll-like receptor and cytokine signaling pathway (TRAF6, IRAK1), and PTC1 (also known as CCDC6 or H4), a gene frequently rearranged with RET proto-oncogene in PTC. In an association study of 608 PTC patients and 901 controls, we found marked differences in genotype distribution of rs2910164 (P = 0.000002), the GC heterozygous state being associated with an increased risk of acquiring PTC (odds ratio = 1.62, P = 0.000007), and both homozygous states protective with odds ratio = 0.42 for the CC genotype (P = 0.003) and odds ratio = 0.69 for the GG genotype (P = 0.0006). Moreover, 4.7% of tumors had undergone somatic mutations of the SNP sequence. Thus, our data suggest that a common polymorphism in pre-miR-146a affects the miR expression, contributes to the genetic predisposition to PTC, and plays a role in the tumorigenesis through somatic mutation. Preliminary evidence suggests that these effects are mediated through target genes whose expression is affected by the SNP status.

Fig. 1.

Structure, expression, and inhibition of targets of miR-146a. (A) The predicted structure of pre-miR-146a. The location of the G/C SNP is shown by an arrow. The mature miR is shown by full red dots. (B) Northern blot. An oligo DNA complementary to mature miR-146a served as a probe. We loaded 20 μg of RNA from cells transfected with (i) empty vector, (ii) pcDNA3-miR-146a-C, (iii) pcDNA3-miR-146a-G, and (iv) 50% of each pcDNA3-miR-146a-G and pcDNA3-miR-146a-C (C+G). The lane labeled C>G describes an experiment where the expression of mature miR from the pcDNA3-miR-146a-C plasmid was reestablished by introducing the reverse mutation. The control lanes comprise 10 or 50 fmol of RNA oligo of the mature miR-146a (positive control for labeling by the Northern probe), and 50 fmol of RNA oligo complementary to the mature miR-146a (negative control). (C) Dual luciferase assay. The relative luciferase activity of reporter constructs with 3′ UTRs of TRAF6, IRAK1, and PTC1 containing legitimate (studied samples) or mutated (controls) target sites in the presence of miR-146a-C or miR-146a-G.

Fig. 2.

Processing of pri-miR-146a. (A) In vitro processing assay. 1 × 10⁵ dpm of gel-purified pri-miR-146a-C (lane 4) or G (lane 5) or a mixture of 0.5 × 10⁵ cpm of each (lane 6) was incubated with HeLa nuclear extract for 90 min. After treating with proteinase K to digest nuclear proteins, radiolabeled RNAs were separated on a denaturing 12% polyacrylamide/urea gel and visualized by PhosphorImager. Lanes 1 and 2 contain 1 × 10⁴ dpm of each input transcript and lane 3 contains a size ladder of radiolabeled HinfI cut ΦX174 DNA. The bands corresponding to pre-miR-146a are shown enlarged to the right of the autoradiogram, and the relative amount of radioactivity in each band normalized to the product of the G allele is shown beneath. (B) Electrophoretic mobility shift assay. Uniformly labeled transcripts of pre-miR-146a-G (lanes 1–5) or pre-miR-146a-C (lanes 6–10) were incubated for 30 min on ice without (lanes 1 and 6) or with 10 μg of HeLa nuclear extract (filled circles). The mixtures in lanes 3–5 and 8–10 were incubated for an additional 30 min with an equal amount or a 5-fold excess of each unlabeled pre-miR (indicated above the autoradiogram) before electrophoresis on a native 6% polyacrylamide gel. Retarded complexes were visualized by PhosphorImager analysis of the dried gel.

Fig. 3.

Somatic mutations in tumor/normal tissue pairs. (A) An example of chromatographs showing a GG to GC mutation (arrow). (B) Summary of the numbers of cases showing different somatic mutations; arrows indicate direction from germ-line to tumor tissue.