Target identification of microRNAs expressed highly in human embryonic stem cells

MicroRNAs (miRNAs) are noncoding RNAs of approximately 22 nucleotides in length that negatively regulate the post- transcriptional expression by translational repression and/ or destabilization of protein-coding mRNAs. The impact of miRNAs on protein output was recently shown that although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. Thus, comparative profilings of miRNAs and mRNAs from the same samples of different cell types may identify the putative targets of miRNAs. In this investigation, both miRNA and mRNA profiles front the undifferentiated human embryonic stem cell line hES-T3 (T3ES), hES-T3 derived embryoid bodies (T3EB), and hES-T3 differentiated fibroblast-like cells (T3 DF) were compared, and 58 genes were found to be targets of four hES cell-specific miRNAs miR-302d, miR-372, miR-200c and/or miR-367 by inverse expression levels (highly negative correlation) of miRNAs to their target mRNAs. Approximately half of these 58 targets are involved in gene transcription . Three common target genes TRPS 1, KLF 13 and MBNL2 of three highly expressed miRNAs miR-302d, miR-372, and miR-200 c were identified, and the target sites of both miR-302d and miR-372 in the 3'UTR of TRPS1, KLF 13, and MBNL2 genes were confirmed by the luciferase assay. The highly expressed mRNAs and miRNA target mRNAs involved in KEGG pathways among DES, T3EB, and T3DF cells were also compared, and the expression levels of target mRNAs predicted by abundantly expressed miRNAs were found to be three- to sixfold lower than those of non-target mRNAs involved in the same signaling pathways.