Abstract
Transfection of human oral squamous carcinoma cells (clone E10) with mimics for unexpressed miR-20b or miR-363-5p, encoded by the miR-106a-363 cluster (miR-20b, miR-106a, miR-363-3p, or miR-363-5p), caused 40–50% decrease in proliferation. Transfection with mimics for miR-18a or miR-92a, encoded by the miR-17-92 cluster (all members being expressed in E10 cells), had no effect on proliferation. In contrast, mimic for the sibling miRNA-19a yielded about 20% inhibition of proliferation. To investigate miRNA involvement profiling of miRNA transcriptomes were carried out using deoxyoligonucleotide microarrays. In transfectants for miR-19a, or miR-20b or miR-363-5p most differentially expressed miRNAs exhibited decreased expression, including some miRNAs encoded in paralogous miR-17-92—or miR-106b-25 cluster. Only in cells transfected with miR-19a mimic significantly increased expression of miR-20b observed—about 50-fold as judged by qRT-PCR. Further studies using qRT-PCR showed that transfection of E10 cells with mimic for miRNAs encoded by miR-17-92 - or miR-106a-363 - or the miR-106b-25 cluster confirmed selective effect on expression on sibling miRNAs. We conclude that high levels of miRNAs encoded by the miR-106a-363 cluster may contribute to inhibition of proliferation by decreasing expression of several sibling miRNAs encoded by miR-17-92 or by the miR-106b-25 cluster. The inhibition of proliferation observed in miR-19a-mimic transfectants is likely caused by the miR-19a-dependent increase in the levels of miR-20b and miR-106a. Bioinformatic analysis of differentially expressed miRNAs from miR-106a, miR-20b and miR-363-5p transfectants, but not miR-92a transfectants, yielded significant associations to “Cellular Growth and Proliferation” and “Cell Cycle.” Western blotting results showed that levels of affected proteins to differ between transfectants, suggesting that different anti-proliferative mechanisms may operate in these transfectants.
Highlights
MicroRNAs are associated with regulation of biological phenomena ranging from cancer to cardiac ischemic tolerance (Dharap and Vemuganti, 2010)
In cultures transfected with mimics for miR-20b or miR-363-5p the number of cells were about 55 and 75%, respectively, of that found in scrambled control (Figure 2A3)
Results obtained using qRT-PCR showed that ATF1, KRT14, KRT15, and PSMB6 were differentially expressed in miR-3635p transfectants and AFT1 and PSMB6 in miR-20b transfectants (Table 1)
Summary
MicroRNAs (miRNAs) are associated with regulation of biological phenomena ranging from cancer (for review see Lee and Dutta, 2009) to cardiac ischemic tolerance (Dharap and Vemuganti, 2010). Some miRNAs are transcribed from a single polycistronic transcript, e.g., the miR-17-92 cluster and its two paralogs, the miR-106a-363 and miR-106b-25 clusters (Bartel, 2004; He et al, 2005; Zhang et al, 2009). Fifteen miRNAs encoded by these three clusters can be separated into four families based on their seed sequences (Figure 1). The miR-106a-363 cluster, located on human chromosome X, encodes six miRNAs: miR-106a, miR-18b, miR-20b, miR-19b-2, miR-922, and miR-363. The miR-106b-25 cluster, located on human chromosome 7, encodes three miRNAs: miR-106b, miR-93, and miR-25. While the miR-17-92 and miR-106a-363 clusters reside in introns of non-coding genes, the gene encoding miR-106b-25 is located in intron 13 of MCM7 (Mendell, 2008)
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