Analysis of the functional consequences of five microrna allele variants associated with cancer

Abstract

[Objectives] microRNAs (miRNAs) are crucial post-transcriptional gene regulators involved in a wide range of biological processes. The strong sequence conservatlon of miRNA leads to predict that nucleotide changes in miRNAs may have functional consequences. We aim to study human miRNA genetic varlation and ¡ts possible involvement in disease, particularly ¡n cancer, through a functional approach.

[Methods] SNPs in different regions of 1872 human miRNAs (miRBase Release 20) were retrieved from the 1000 genomes database. The TargetScan algorithm was used for target gene predict¡ons. Comparative expression analysis of different m¡RNA alleles cloned in expression vectors and transfected into HeLa cells was performed by RT-qPCR experiments.

[Results] we ident¡f¡ed 967 out of 1872 miRNAs (5L.7%l carrying 1652 SNPs, 284 of which had a minor allele frequency higher than 0.05, 191 were located in the precursor miRNA region (0.0020 SNP/bp), 58 in the mature miRNA out ofthe seed region (0.0015 SNP/bp) and 35 in the miRNA seed reglon (0.0018 SNP/bp). Five SNPs previously associated with cancer were selected for

further expression and functional studies. Three of them (rs12416605, rs35770269 and rs2910164) were located in the seed region of miR-938, miR- 449c and m¡R-146a, respectively; one (rs11614913) in the mature miR-196a-2 and the last (rs37464441 ¡n the mature miR-499b and seed reg¡on of mlR- 499a. Target gene pred¡ctions using Targetscan revealed that the most frequent alleles ofthe analyzed SNPs had a larger number of potential target genes than the minor alleles with very little overlap between both alleles, in particular, Íor rs3746444 in miR-499a no common target genes between the A and G alleles were found. Furthermore, morphological differences were observed between both alleles of this SNP in miR-499a when the miRNA variants were transfected and expressed in HeLa cells, Moreover, differences in the level of expression between the C and T alleles of the rs11614913 var¡ant, located ¡n the mature miR-196a-2, were detected by RT-qPCR. We are currently investigating potential changes in the regulation of gene networks by the different miRNA alleles associated with cancer through a transcriptome approach.

[Conclusion] Different alleles of common miRNA SNPs associated with cancer could lead to changes In the express¡on of the corresponding miRNAs and affect the spectrum of regulated target genes.