MicroRNAs (MIRs) are a novel group of conserved short ∼22 nucleotide-long RNAs with important roles in regulating gene expression. We have established a MIR-specific oligonucleotide microarray system that enables efficient analysis of the expression of the human MIRs identified so far. We show that the 60-mer oligonucleotide probes on the microarrays hybridize with labeled cRNA of MIRs, but not with their precursor hairpin RNAs, derived from amplified, size-fractionated, total RNA of human origin. Signal intensity is related to the location of the MIR sequences within the 60-mer probes, with location at the 5Ј region giving the highest signals, and at the 3Ј end, giving the lowest signals. Accordingly, 60-mer probes harboring one MIR copy at the 5Ј end gave signals of similar intensity to probes containing two or three MIR copies. Mismatch analysis shows that mutations within the MIR sequence significantly reduce or eliminate the signal, suggesting that the observed signals faithfully reflect the abundance of matching MIRs in the labeled cRNA. Expression profiling of 150 MIRs in five human tissues and in HeLa cells revealed a good overall concordance with previously published results, but also with some differences. We present novel data on MIR expression in thymus, testes, and placenta, and have identified MIRs highly enriched in these tissues. Taken together, these results highlight the increased sensitivity of the DNA microarray over other methods for the detection and study of MIRs, and the immense potential in applying such microarrays for the study of MIRs in health and disease.

Protein kinase C encodes a family of enzymes implicated in cellular di erentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCZ in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and a ected the cell cycle at several points. The induced expression of another PKC isoform, PKCd, in MCF-7 cells had opposite e ects and inhibited their growth. PKCZ expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21 WAF1 was also speci®cally elevated in PKCZ expressing cells, but its overall e ects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27 KIP1 were not altered by the induced expression of PKCZ, the cyclin E associated Cdk2 kinase activity was in correlation with the p27 KIP1 bound to the cyclin E complex and not by p21 WAF1 binding. PKCZ expression enhanced the removal of p27 KIP1 from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27 KIP1 to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21 WAF1 correlated with its increased activity in PKCZ-expressing cells. Thus, PKCZ induces altered expression of several cell cycle functions, which may contribute to its ability to a ect cell growth. Oncogene (2001) 20, 6794 ± 6804.

No abstract

No abstract