The widely used reverse transcriptase inhibitor, zidovudine (AZT), alters micro RNA (miRNA) expression in preimplantation mouse embryos

Abstract

Zidovudine (AZT) is a reverse transcriptase inhibitor used widely to prevent perinatal transmission of HIV. In mice, AZT disrupts early embryo development and prevents telomere elongation. miRNA’s are small non-coding RNA’s that regulate gene expression post-transcriptionally and play important roles during early development, aging and cellular senescence. miRNAs also control the function of telomeres in cancer. While miRNA’s and telomeres are known to play important roles during early development, no studies have evaluated the impact of AZT on miRNAs in mammalian embryos. To further characterize the effects of AZT on early embryos, we profiled miRNA expression, focusing on a number of miRNA’s (let-7a, miR-34a, miR-1247 and miR-762) previously associated with telomere homeostasis. Experimental study. In vivo fertilized mouse zygotes (n=72) were cultured in vitro with three different media: 1. KSOM (n=38), 2. KSOM+AZT 1μM (n=39), and 3. KSOM+AZT 10μM (n=27). The concentrations of AZT were selected to approach those obtained in patients taking AZT to prevent perinatal HIV transmission. After 48 hours in culture, embryo quality was evaluated. miRNAs were extracted from metaphase II mouse oocytes or embryos cultured for 48h in control or AZT-containing media. TaqMan RT-qPCR was used to measure expression of the miRNAs let-7a, miR- 34a, mirR-762 and miR-1247. The effects of AZT 1 μM and AZT 10 μM on miRNA expression in embryos relative to control embryos were measured. All measurements were done in triplicate and multivariable regression analysis determined the association of each miRNAs with AZT treatment, by using the delta delta CT method, to test the difference of differences, using expression of the housekeeping gene, sno 135, as an internal control. Let-7a miRNA expression was significantly downregulated (p<0.05), while miR-1247 (p<0.05) was significantly upregulated by exposure of 1μM AZT to preimplantation embryos. Mir-34a (p<0.05) and miR-762 (p<0.01) were significantly upregulated by AZT 10 μM. We also observed a significant upregulation of miR-1247 (p<0.05) in 1μM AZT treated embryos. However let-7a miRNA expression remained unchanged in 10 μM AZT treated embryos, while miR-34a change was not significant in the 1μM AZT treatment group. AZT, which disrupts early development and inhibits telomere elongation in early embryos, alters expression of a number of miRNAs previously shown to regulate telomere dynamics. Our results also show AZT increases expression of mir34a, which is known to be an inhibitor of totipotency. The relationships among telomere length, miRNA expression, and embryo development need further investigation, both to identify potentially long term effects of prenatal exposure to AZT, and to elucidate the biology of early human development.