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Gail M. Seigel, Yu-Zhuo Pan, Zihua Hu, Ai-Ming Yu; ABCG2-Regulatory miRNAs And The Stem Cell Phenotype In Human Retinoblastoma Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1595.
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© ARVO (1962-2015); The Authors (2016-present)
Retinoblastoma (RB) is a blinding retinal tumor of early childhood that is often treated with enucleation and/or chemotherapy/radiation therapies. In our previous studies, we have shown that there are small subpopulation(s) of RB cells with stem-like properties that include the expression of ABCG2, a stem cell marker and drug transporter that confers resistance to over 20 chemotherapy drugs. The goal of this study was to compare ABCG2+ and ABCG2- RB cells for the presence of three specific miRNAs that regulate the expression of ABCG2 as a possible target for molecular pharmacological interventions.
We separated human RB143 cells into ABCG2+ and ABCG2- enriched populations by immunomagnetic positive selection and compared the two populations by RT-qPCR and miRNA-selective stem-loop RT qPCR. These methods were used to determine the expression of miRNAs, ABC transporters (MDR1/ABCB1, MRP1/ABCC1 and BCRP/ABCG2), as well as the expression of two additional stem cell markers ALDH1 and CD133.
: ABCG2 (normalized to 18S) was ~51-fold higher in ABCG2+ cells vs. ABCG2- cells, a strong validation of cell enrichment by the immunomagnetic positive selection method. As an additional confirmation of the stem cell phenotype, the stem cell markers ALDH1 and CD133 were ~8- and 10-fold higher in ABCG2+ cells, respectively. When we examined two other ABC transporters, there was no difference in ABCB1 expression between ABCG2- and ABCG2+ cells, while MRP1/ABCC1 was undetectable in both populations. We then analyzed miR-328, miR-519c and miR-520h, three miRNAs that are reported to regulate ABCG2 at the post-transcriptional level. Very interestingly, miR-328 (normalized to U74) was ~9-fold lower and miR-519c was ~15-fold lower in ABCG2+ cells. miR-520h was ~3-fold lower in ABCG2+ cells.
In summary, ABCG2+ enriched cells demonstrate important differences from their ABCG2- counterparts in terms of ABCG2 miRNA regulatory molecules. Additional analysis of functionally relevant miRNAs may lead to new strategies in targeting ABCG2-mediated chemoresistance and metastasis in retinoblastoma, as well as in other malignancies.
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