Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis

Of the over 200 identified mammalian microRNAs (miRNAs), only a few have known biological activity. To gain a better understanding of the role that miRNAs play in specific cellular pathways, we utilized antisense molecules to inhibit miRNA activity. We used miRNA inhibitors targeting miR-23, 21, 15a, 16 and 19a to test efficacy of antisense molecules in reducing miRNA activity on reporter genes bearing miRNA-binding sites. The miRNA inhibitors de-repressed reporter gene activity when a miRNA-binding site was cloned into its 3′-untranslated region. We employed a library of miRNA inhibitors to screen for miRNA involved in cell growth and apoptosis. In HeLa cells, we found that inhibition of miR-95, 124, 125, 133, 134, 144, 150, 152, 187, 190, 191, 192, 193, 204, 211, 218, 220, 296 and 299 caused a decrease in cell growth and that inhibition of miR-21 and miR-24 had a profound increase in cell growth. On the other hand, inhibition of miR-7, 19a, 23, 24, 134, 140, 150, 192 and 193 down-regulated cell growth, and miR-107, 132, 155, 181, 191, 194, 203, 215 and 301 increased cell growth in lung carcinoma cells, A549. We also identified miRNA that when inhibited increased the level of apoptosis (miR-1d, 7, 148, 204, 210, 216 and 296) and one miRNA that decreased apoptosis (miR-214) in HeLa cells. From these screens, we conclude that miRNA-mediated regulation has a complexity of cellular outcomes and that miRNAs can be mediators of regulation of cell growth and apoptosis pathways.

[1]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[2]  G. Ruvkun,et al.  Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans , 1993, Cell.

[3]  B. Reinhart,et al.  The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans , 2000, Nature.

[4]  F. Slack,et al.  The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. , 2000, Molecular cell.

[5]  V. Ambros.  microRNAs Tiny Regulators with Great Potential , 2001, Cell.

[6]  Eric J Wagner,et al.  Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. , 2002, Molecular cell.

[7]  G. Hutvagner,et al.  A microRNA in a Multiple-Turnover RNAi Enzyme Complex , 2002, Science.

[8]  C. Croce,et al.  Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[9]  A. Pasquinelli,et al.  Control of developmental timing by micrornas and their targets. , 2002, Annual review of cell and developmental biology.

[10]  V. Kim,et al.  MicroRNA maturation: stepwise processing and subcellular localization , 2002, The EMBO journal.

[11]  Anton J. Enright,et al.  MicroRNA targets in Drosophila , 2003, Genome Biology.

[12]  Michael Z Michael,et al.  Reduced accumulation of specific microRNAs in colorectal neoplasia. , 2003, Molecular cancer research : MCR.

[13]  Martin Tabler,et al.  Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in Drosophila and the identification of putative target genes. , 2003, Nucleic acids research.

[14]  R. Russell,et al.  bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila , 2003, Cell.

[15]  C. Burge,et al.  Prediction of Mammalian MicroRNA Targets , 2003, Cell.

[16]  B. Cullen,et al.  Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. , 2003, Genes & development.

[17]  Bruce A. Hay,et al.  The Drosophila MicroRNA Mir-14 Suppresses Cell Death and Is Required for Normal Fat Metabolism , 2003, Current Biology.

[18]  B. Cullen,et al.  MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[19]  V. Kim,et al.  The nuclear RNase III Drosha initiates microRNA processing , 2003, Nature.

[20]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[21]  Thomas Tuschl,et al.  Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. , 2004, RNA.

[22]  Sam Griffiths-Jones,et al.  The microRNA Registry , 2004, Nucleic Acids Res..

[23]  Phillip D Zamore,et al.  Sequence-Specific Inhibition of Small RNA Function , 2004, PLoS biology.

[24]  D. Bartel,et al.  MicroRNAs Modulate Hematopoietic Lineage Differentiation , 2004, Science.

[25]  D. Bartel,et al.  MicroRNA-Directed Cleavage of HOXB8 mRNA , 2004, Science.

[26]  Arndt Borkhardt,et al.  High expression of precursor microRNA‐155/BIC RNA in children with Burkitt lymphoma , 2004, Genes, chromosomes & cancer.

[27]  John G Doench,et al.  Specificity of microRNA target selection in translational repression. , 2004, Genes & development.

[28]  Natalie Wilson,et al.  The miRNA Registry , 2004, Nature Reviews Genetics.