Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma Krystian Jazdzewski*, Elizabeth L. Murray†, Kaarle Franssila‡, Barbara Jarzab§, Daniel R. Schoenberg†, and Albert de la Chapelle*¶ *Human Cancer Genetics Program, Comprehensive Cancer Center, Ohio State University, 850 Biomedical Research Tower, 460 West 12th Avenue, Columbus, OH 43210; †Department of Molecular and Cellular Biochemistry, Ohio State University, 366 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210; ‡Department of Pathology, Helsinki University Central Hospital, FIN-00029, Helsinki, Finland; and §Department of Nuclear Medicine and Endocrine Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Wybrzeze Armii Krajowej 15, Gliwice 44-101, Poland Contributed by Albert de la Chapelle, March 17, 2008 (sent for review February 12, 2008) Although papillary thyroid carcinoma (PTC) displays strong heri- microRNAs) that are processed to form mature microRNA tability, no predisposing germ-line mutations have been found. We duplexes that are Ϸ22 nucleotides long. A protein complex show that a common G/C polymorphism (rs2910164) within the (called RNA-induced silencing complex; RISC) facilitates the pre-miR-146a sequence reduced the amount of pre- and mature coupling of one strand of the microRNA duplex with matching miR-146a from the C allele 1.9- and 1.8-fold, respectively, compared mRNA sequences in the 3Ј untranslated regions of target genes; with the G allele. This is matched by a similar decrease in the the other strand of the duplex is degraded. The binding of the amount of each pre-miR generated from the corresponding pri- microRNA to mRNA leads to inhibition of the translation of the miR-146a in an in vitro processing reaction. The C allele also latter, thus disrupting the expression of the protein. MicroRNAs interfered with the binding of a nuclear factor to pre-miR-146a. The regulate such major processes as development, apoptosis, cell reduction in miR-146a led to less efficient inhibition of target genes proliferation, and hematopoiesis; they may act as tumor sup- involved in the Toll-like receptor and cytokine signaling pathway pressor genes and oncomirs (12–14). The expression of miRNAs (TRAF6, IRAK1), and PTC1 (also known as CCDC6 or H4), a gene varies between cancer and normal cells and varies among frequently rearranged with RET proto-oncogene in PTC. In an different types of cancer (15, 16). association study of 608 PTC patients and 901 controls, we found We reasoned that the previous failure to identify genes predisposing or contributing to PTC might be because these ؍ marked differences in genotype distribution of rs2910164 (P 0.000002), the GC heterozygous state being associated with an genes show low penetrance. The mechanisms may require the interaction of two or more genes; thus, regulatory, rather than ,(0.000007 ؍ P ,1.62 ؍ increased risk of acquiring PTC (odds ratio for protein-encoding, genes might be involved. MicroRNAs fulfill 0.42 ؍ and both homozygous states protective with odds ratio .for the GG these criteria 0.69 ؍ and odds ratio (0.003 ؍ the CC genotype (P Moreover, 4.7% of tumors had undergone We and others have described several microRNAs showing .(0.0006 ؍ genotype (P somatic mutations of the SNP sequence. Thus, our data suggest transcriptional up-regulation in PTC tumors, the most striking that a common polymorphism in pre-miR-146a affects the miR being a 19-fold increase in the quantity of miR-146 (17, 18). expression, contributes to the genetic predisposition to PTC, and Human miR-146 occurs in two distinct forms: miR-146a encoded plays a role in the tumorigenesis through somatic mutation. on chromosome 5q33 and miR-146b encoded on chromosome Preliminary evidence suggests that these effects are mediated 10q24. Because the mature 22-mers differ by only 2 nt, many of through target genes whose expression is affected by the SNP the predicted target genes are common to both miRs, whereas, status. in addition, each is predicted to have specific targets unique to that miR. The two related miR-146 are differentially regulated, genetic predisposition ͉ microRNA processing ͉ polymorphism ͉ miR-146 ͉ with miR-146a (but not miR-146b) strongly induced by lipopoly- thyroid cancer saccharide. It is proposed that miR-146 has a role in Toll-like receptor and cytokine signaling and thus in the immune re- apillary thyroid carcinoma (PTC) is the most common sponse, and there is evidence that miR-146a is regulated by Pthyroid malignancy, accounting for Ϸ80% of all thyroid NF-kappa B (19). cancers. The incidence of PTC in the United States has increased In an effort to elucidate the putative role of miR-146 in PTC we sequenced the pri-miR-146a and pri-miR-146b in the genomic in recent years and has reached Ϸ26,500 cases annually (1). The DNA of those 15 patients whose samples had been analyzed for pathogenesis of PTC involves alterations in the RET/PTC-RAS- global miR expression (17). No previously undescribed sequence BRAF signaling pathway. Activating mutations in BRAF and changes were detected. However, in the pre-miR-146a we noted RET/PTC gene rearrangements are frequent somatic changes in a common G/C polymorphism designated rs2910164. It resides PTC tumors (2–6). A strong inherited genetic predisposition is at position ϩ60 relative to the first nucleotide of pre-miR-146a, suggested by case-control studies showing a 3- to 8-fold increase placing it in the passenger strand (Fig. 1A). The rarer C allele in risk in first-degree relatives, one of the highest risks of all causes mispairing within the hairpin and a lowering of the cancers (7, 8). Despite unequivocal evidence of an inherited predisposition, large families displaying Mendelian inheritance of PTC are rare, and no predisposing genetic factors have been Author contributions: K.J., D.R.S., and A.d.l.C. designed research; K.J. and E.L.M. performed convincingly described, even though several putative loci have research; K.J., K.F., and B.J. contributed new reagents/analytic tools; K.J., E.L.M., D.R.S., and been identified by linkage analysis (9, 10). A.d.l.C. analyzed data; and K.J., D.R.S., and A.d.l.C. wrote the paper. MicroRNAs (miRs) are small noncoding RNA molecules that The authors declare no conflict of interest. function as negative regulators of the expression of other genes; ¶To whom correspondence should be addressed. E-mail: [email protected]. miRs inactivate specific mRNAs and interfere with the transla- This article contains supporting information online at www.pnas.org/cgi/content/full/ tion of target proteins (11). MicroRNAs are transcribed from 0802682105/DCSupplemental. MEDICAL SCIENCES endogenous DNA and form hairpin structures (called pre- © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0802682105 PNAS ͉ May 20, 2008 ͉ vol. 105 ͉ no. 20 ͉ 7269–7274 Downloaded by guest on September 24, 2021 A an even smaller variation of loading amount (cv ϭ 6.8%). Pre-miR-146a and the mature miR were expressed from both pre-miR146a+60G alleles; however, the amount of each differed between the alleles. The expression of pre-miR-146a from the C allele was 1.9-fold lower than from the G allele, and the amount of mature miR-146a was 1.8-fold lower from the C allele compared to the pre-miR146a+60C G allele [Fig. 1B and supporting information (SI) Table S1]. Reduced expression of pre- and mature miR-146a was reversed B tRNA by mutating the C back to G (Fig. 1B), thus indicating that this single nucleotide difference significantly alters the amount of lacZ miR-146a produced from the C allele. Importantly, the ratio of pre-miR precursor-to-mature form was unchanged (2.2 and 2.1 for the G and C alleles, respectively), which makes it unlikely that this results from a block in Dicer processing. We conclude that the miR-146a GϾC substitution in pre-miR-146a results in reduced amounts of pcDNA3 C G C+G C>G c +rt tc r+ c rt - 10 50 50 mature miR-146a. fm lo fmol fmol Processing of pri-miR-146a. To determine whether the differences 1 4. in Fig. 1 resulted from allele-specific differences in nuclear C control miR-146a G miR-146a C 1 2. processing we examined the production of pre-miR-146a from 0.1 the pri-miR in HeLa nuclear extract (23) (Fig. 2A). Phospho- rImager analysis of products separated on a 12% denaturing 8.0 polyacrylamide/urea gel showed that twice as much pre-miR- 0.6 146a was generated from the G allele compared with the C allele, 4.0 and a 1:1 mixture generated the anticipated mean of these two 0 2. values. These results suggest that the 2-fold difference in miR- Relative Luciferase Activity 0 0. 146a in Fig. 1 results from differences in nuclear processing. TRAF6 IRAK1 PTC1 RNA electrophoretic mobility shift assay (EMSA) of nuclear protein binding to pre-miR-146a provided further evidence for Fig. 1. Structure, expression, and inhibition of targets of miR-146a. (A) The differences between the two alleles (Fig. 2B). A retarded 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 complex was formed when each of the pre-miRs was incubated DNA complementary to mature miR-146a served as a probe. We loaded 20 ␮g on ice with HeLa nuclear extract (lanes 2 and 7); however, the of RNA from cells transfected with (i) empty vector, (ii) pcDNA3-miR-146a-C, complex assembled on pre-miR-146a-G was shifted further when (iii) pcDNA3-miR-146a-G, and (iv) 50% of each pcDNA3-miR-146a-G and an equal amount of the unlabeled pre-miR was added (compare pcDNA3-miR-146a-C (CϩG).