Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140

TY - JOUR

T1 - Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140

AU - Nicolas, Francisco Esteban

AU - Pais, Helio

AU - Schwach, Frank

AU - Lindow, Morten

AU - Kauppinen, Sakari

AU - Moulton, Vincent

AU - Dalmay, Tamas

N1 - Keywords: Animals; Cell Line; Chemokine CXCL12; Chickens; Fibroblasts; Mice; MicroRNAs

PY - 2008

Y1 - 2008

N2 - MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. Twenty-one of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay.

AB - MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. Twenty-one of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay.

U2 - 10.1261/rna.1221108

DO - 10.1261/rna.1221108

M3 - Journal article

C2 - 18945805

SN - 1355-8382

VL - 14

SP - 2513

EP - 2520

JO - RNA

JF - RNA

IS - 12

ER -