title: TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.
abstract: MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.
authors.affiliation: a Department of Cell Biology and of Biochemistry , University of Texas Southwestern Medical Center , Dallas , TX , USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
authors.affiliation: a Department of Cell Biology and of Biochemistry , University of Texas Southwestern Medical Center , Dallas , TX , USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: a Department of Cell Biology and of Biochemistry , University of Texas Southwestern Medical Center , Dallas , TX , USA.
authors.forename: Daliang
authors.initials: D
authors.lastname: Li
authors.affiliation: a Department of Cell Biology and of Biochemistry , University of Texas Southwestern Medical Center , Dallas , TX , USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.affiliation: b Department of Biological Sciences , Center for Systems Biology, The University of Texas at Dallas , Richardson , TX , USA.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Xuan
authors.affiliation: a Department of Cell Biology and of Biochemistry , University of Texas Southwestern Medical Center , Dallas , TX , USA.
authors.forename: Wen-Hong
authors.initials: WH
authors.lastname: Li
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D002460:Cell Line; D015870:Gene Expression; D020869:Gene Expression Profiling; D055786:Gene Knockout Techniques; D017930:Genes, Reporter; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics*
chemical_list: C524940:MIRN21 microRNA, human; D035683:MicroRNAs; D009841:Oligonucleotides; D012333:RNA, Messenger; C477371:locked nucleic acid
keywords: Colorectal cancer; LNA; locked nucleic acid; miR-21; microRNA; microRNA target; target identification; targetlink