Changes in microRNA expression profiles in HIV-1-transfected human cells
© Yeung et al; licensee BioMed Central Ltd. 2005
Received: 14 December 2005
Accepted: 28 December 2005
Published: 28 December 2005
MicroRNAs (miRNAs) are small RNAs of 18–25 nucleotides (nt) in length that play important roles in regulating a variety of biological processes. Recent studies suggest that cellular miRNAs may serve to control the replication of viruses in cells. If such is the case, viruses might be expected to evolve the ability to modulate the expression of cellular miRNAs. To ask if expression of HIV-1 genes changes the miRNA profiles in human cells, we employed a high throughput microarray method, termed the RNA-primed Array-based Klenow Enzyme (RAKE) assay. Here, we describe the optimization of this assay to quantify the expression of miRNAs in HIV-1 transfected human cells. We report distinct differences in miRNA profiles in mock-transfected HeLa cells versus HeLa cells transfected with an infectious HIV-1 molecular clone, pNL4-3.
MicroRNAs (miRNAs) are small RNAs of 18–25 nucleotides (nt) in length that are involved in the regulation of a variety of biological processes including developmental timing, signal transduction, apoptosis, cell proliferation and tumorigenesis [1–3]. Recent studies indicate that cellular miRNAs can variably inhibit  or promote  viral replication. Viruses, on the other hand, seem to have developed strategies which include virus-encoded RNAi suppressors [6–12] and/or virus-encoded miRNAs [13–19]. Mechanistically, a current view is that miRNAs function to silence gene expression through imperfect base-pairing with cognate transcripts. Since RNA silencing mediated by miRNA does not require perfect sequence complementarity, one miRNA can target multiply different mRNAs . It is conceivable that viruses may seek to alter cellular miRNA expression in ways that benefit viral replication. Extant findings support such a notion since several viruses have been found to encode RNAi suppressors which could function to influence the cell's overall miRNA milieu [6–12].
The sensitivity of RAKE was evaluated by hybridizing microarray slides with varying amounts of ath-miR-157a. As shown in Fig 2C, RAKE provided robust signals when challenged with as low as 10-7 M of substrate, and offered linear readouts in log2 scale for substrates in the 10-8 to 10-6 M range. We defined our signal as the median of foreground spot fluorescence at 532 nm wavelength minus background (defined by surrounding pixel intensity); negative values were reset as zero.
In conclusion, we describe here a rapid assay that monitors reproducible changes in cells transfected with HIV-1 infectious molecular clone, pNL4-3. We find that a dominant pattern of response in HeLa cells to pNL4-3 transfection is the downregulated expression of many miRNAs. Studies are ongoing to examine changes in miRNA expression patterns in human cells (primary and T cell lines) after infection with HIV-1.
We thank members of the Jeang laboratory and two outside colleagues for their critical reviews of the manuscript. We also thank Dr. Mourelatos for the discussion of RAKE assay technique.
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