6 research outputs found

    Changes in the Expression of miR-381 and miR-495 Are Inversely Associated with the Expression of the MDR1 Gene and Development of Multi-Drug Resistance

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    <div><p>Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3’-UTR of the <i>MDR1</i> gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR. </p> </div

    Generation of K562/ADM cells.

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    <p>(A) K562/ADM cells were established after exposure of K562 cells to ADM. MDR1 expression in parental and drug-treated K562 cells was normalized to the GAPDH housekeeping gene. (B) FACS analysis of K562 and K562/ADM cells stained with P-gp antibody. (C) MTT assay shows survival rate of K562 and K562/ADM cells under VBL treatment. The ratio of OD<sub>490</sub> of VBL-treated cells <i>versus</i> control cells was used as the measure of survival rate. At least three independent experiments were performed and error bars represent standard deviation (S.D.). (D) FACS analysis of Rh123 accumulation in K562 and K562/ADM cells.</p

    Expression of primary transcripts in the miR cluster on chromosome 14q32.31.

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    <p>(A) Schematic representation of known EST sites and selected miRs shown in UCSC genome browser (Human Feb. 2009 (GRCh37/hg19)) at chromosome region 14q32.31. EST primer sets A to E are indicated in triangles below each EST. Transcription start sites sourced from FANTOM CAGE tags are shown as arrows on the top of the 14q32.31 region. (B) Expression of selected miRs (miR-494, miR-495, miR-376c, miR-381 and miR-655) in K562 and K562/ADM cells was validated by stem-loop real-time PCR. The Ct values of these miRs were normalized to that of miR-425 whose level of expression was similar in the two cell lines. The relative expression of miR-494, miR-495, miR-376c, miR-381, miR-655 and miR-16 in K562/ADM cells was calculated. The results are shown as the mean ± SD of three independent experiments. (C) PCR products were resolved on a 2% agarose gel. (D) Expression of the primary transcripts in both K562 and K562/ADM was determined by RT-PCR using primers designed against ESTs A to E. The data were normalized to GAPDH. The results are shown as the mean ± SD of three independent experiments. Quantification of the change of expression level in K562/ADM cells relative to K562 cells is shown on the bottom. (E) Expression of three human RNA nuclease genes, Drosha, DGCR8 and Dicer, in both K562 and K562/ADM cells, was determined by real-time PCR. The expression values were normalized to GAPDH, and are shown as the mean ± SD of three independent experiments.</p

    Validation of mature miR expression.

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    <p>(A) Schematic representation of miRs targeted to the 3'-UTR of the MDR1 mRNA predicted by <a href="http://microrna.org" target="_blank">microRNA.org</a>. MiR-381 and miR-495 are highlighted and their predicted seed binding regions are shown in (B). Numbers at both ends of the fragments represent the relative nucleotide locations in this region. (C) Expression of mature miR-381, miR-495 and miR-16 in K562 and K562/ADM cells was validated by stem-loop real-time PCR. The Ct values of these miRs were normalized to that of miR-425 whose expression was unchanged in both cell types. Next, the relative expression of miR-381, miR-495 and miR-16 in K562 cells and in K562/ADM cells was calculated. The results are shown as the mean ± SD of three independent experiments. (D) The Ct values of miR-381, miR-495 and miR-16 in K562 and K562/VBL cells were normalized to miR-425. The relative expression of miR-381, miR-495 and miR-16 in K562 cells and in K562/VBL cells was calculated. The results are shown as the mean ± SD of three independent experiments.</p

    Manhattan plot of the chromosomal distribution of differentially expressed miRs.

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    <p>The x-axis represents the 23 human chromosomes end-to-end; the y axis shows the expression changes of individual miRs in K562/ADM cells based on miR-seq profiling. Only differentially-regulated miRs showing 2-fold or greater changes in their expression are shown. Grey dots represent miRs on chromosomes 1, 3, 5, 7, and 9 etc. while black dots are miRs on even numbered chromosomes. No miR with a 2-fold or greater fold change mapped to chromosome 21.</p

    Functional analysis of miR-381 and miR-495 in K562/ADM cells.

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    <p>(A) After transfection of K562/ADM cells with mimics of miR-381, miR-495 or negative controls, or transfection with inhibitors in addition to mimics, MDR1 mRNA expression was determined by real-time PCR. Expression values were normalized to GAPDH and are shown as the mean ± SD of three independent experiments. * indicates a significant difference between the two groups of samples with p<0.05 calculated using a two-sample t-test. (B) FACS analysis showed the different levels of P-gp expression in K562 cells and K562/ADM cells without transfection, or transfected with scrambled sequences as negative controls or mimics alone or mimics plus inhibitors of miR-381 or miR-495. (C) Microscopy images of Rh123 accumulation in K562 and K562/ADM cells with different treatments. Each sample had a cell density of approximately 1×10<sup>6</sup> cells/mL. (D) Cell survival rate after transfection with mimics of miRs in the presence of 0, 2 or 10 µg/mL VCR. Data is shown as the mean ± SD of three independent experiments. (E) Relative GFP fluorescence intensity compared to internal control DsRed was analyzed by FACS after co-transfection with mimics of miR-381 or miR-495 or negative control. * indicates a significant difference between the two groups of samples with p<0.05 calculated using a two-sample t-test.</p
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