Breast cancer resistance protein (BCRP/ABCG2) is a molecular determinant of pharmacokinetic properties of many drugs in humans. To understand post-transcriptional regulation of ABCG2 and the role of microRNAs (miRNAs) in drug disposition, we found that microRNA-328 (miR-328) might readily target the 3Ј-untranslated region (3Ј-UTR) of ABCG2 when considering target-site accessibility. We then noted 1) an inverse relation between the levels of miR-328 and ABCG2 in MCF-7 and MCF-7/MX100 breast cancer cells and 2) that miR-328 levels could be rescued in MCF-7/MX100 cells by transfection with miR-328 plasmid. Luciferase reporter assays showed that ABCG2 3Ј-UTR-luciferase activity was decreased more than 50% in MCF-7/MX100 cells after transfection with miR-328 plasmid, the activity was increased over 100% in MCF-7 cells transfected with a miR-328 antagomir, and disruption of miR-328 response element within ABCG2 3Ј-UTR led to a 3-fold increase in luciferase activity. Furthermore, the level of ABCG2 protein was down-regulated when miR-328 was over-expressed, and the level was up-regulated when miR-328 was inhibited by selective antagomir. Altered ABCG2 protein expression was associated with significantly declined or elevated levels of ABCG2 3Ј-UTR and coding sequence mRNAs, suggesting possible involvement of the mechanism of mRNA cleavage. Finally, miR-328-directed down-regulation of ABCG2 expression in MCF-7/MX100 cells resulted in an increased mitoxantrone sensitivity, as manifested by a significantly lower IC 50 value (2.46 Ϯ 1.64 M) compared with the control (151 Ϯ 32 M). Together, these findings suggest that miR-328 targets ABCG2 3Ј-UTR and, consequently, controls ABCG2 protein expression and influences drug disposition in human breast cancer cells.Breast cancer resistance protein BCRP/ABCG2 is an ATPbinding cassette membrane transporter expressed ubiquitously in humans, controlling the absorption, distribution and clearance of numerous xenobiotics, including pharmaceutical agents, dietary carcinogens and conjugated metabolites (Mao and Unadkat, 2005;van Herwaarden and Schinkel, 2006;Vore and Leggas, 2008). In addition, overexpression of ABCG2 and other drug transporters in tumorigenic stem cells represents an important mechanism for multidrug resistance (Dean et al., 2005). Because ABCG2 was discovered in drugresistant human cancer cells (e.g., MCF-7/AdrVp and S1MI80), these cell lines have been widely used for studying the function and regulation of ABCG2 and defining its role in drug disposition and multidrug resistance. In particular, gene amplification (Ross et al., 1999;Knutsen et al., 2000;Volk et al., 2002) has been shown to be an important mechanism for elevated ABCG2 expression in drug-resistant cancer cells. Recent studies have demonstrated that transcriptional factors [i.e., nuclear receptors (Ee et al.,
ABSTRACT:CYP3A4 metabolizes many drugs on the market. Although transcriptional regulation of CYP3A4 is known to be tightly controlled by some nuclear receptors (NR) including vitamin D receptor (VDR/ NR1I1), posttranscriptional regulation of CYP3A4 remains elusive. In this study, we show that noncoding microRNAs (miRNAs) may control posttranscriptional and transcriptional regulation of CYP3A4 by directly targeting the 3-untranslated region (3UTR) of CYP3A4 and indirectly targeting the 3UTR of VDR, respectively. Luciferase reporter assays showed that CYP3A4 3UTR-luciferase activity was significantly decreased in human embryonic kidney 293 cells transfected with plasmid that expressed microRNA-27b (miR-27b) or mouse microRNA-298 (mmu-miR-298), whereas the activity was unchanged in cells transfected with plasmid that expressed microRNA-122a or microRNA-328. Disruption of the corresponding miRNA response element (MRE) within CYP3A4 3UTR led to a 2-to 3-fold increase in luciferase activity. Immunoblot analyses indicated that CYP3A4 protein was down-regulated over 30% by miR-27b and mmu-miR-298 in LS-180 and PANC1 cells. The decrease in CYP3A4 protein expression was associated with significantly decreased CYP3A4 mRNA levels, as determined by quantitative real-time PCR (qPCR) analyses. Likewise, interactions of miR-27b or mmu-miR-298 with VDR 3UTR were supported by luciferase reporter assays. The mmu-miR-298 MRE site is well conserved within the 3UTR of mouse, rat, and human VDR. Downregulation of VDR by the two miRNAs was supported by immunoblot and qPCR analyses. Furthermore, overexpression of miR-27b or mmu-miR-298 in PANC1 cells led to a lower sensitivity to cyclophosphamide. Together, these findings suggest that CYP3A4 gene expression may be regulated by miRNAs at both the transcriptional and posttranscriptional level.
Development of anticancer treatments based on microRNA (miRNA/miR) such as miR-34a replacement therapy is limited to the use of synthetic RNAs with artificial modifications. Herein, we present a new approach to a high-yield and large-scale biosynthesis, in Escherichia coli using transfer RNA (tRNA) scaffold, of chimeric miR-34a agent, which may act as a prodrug for anticancer therapy. The recombinant tRNA fusion pre-miR34a (tRNA/mir-34a) was quickly purified to a high degree of homogeneity (.98%) using anion-exchange fast protein liquid chromatography, whose primary sequence and post-transcriptional modifications were directly characterized by mass spectrometric analyses. Chimeric tRNA/mir-34a showed a favorable cellular stability while it was degradable by several ribonucleases. Deep sequencing and quantitative real-time polymerase chain reaction studies revealed that tRNA-carried pre-miR-34a was precisely processed to mature miR-34a within human carcinoma cells, and the same tRNA fragments were produced from tRNA/ mir-34a and the control tRNA scaffold (tRNA/MSA). Consequently, tRNA/mir-34a inhibited the proliferation of various types of human carcinoma cells in a dose-dependent manner and to a much greater degree than the control tRNA/MSA, which was mechanistically attributable to the reduction of miR-34a target genes. Furthermore, tRNA/mir-34a significantly suppressed the growth of human non-small-cell lung cancer A549 and hepatocarcinoma HepG2 xenograft tumors in mice, compared with the same dose of tRNA/MSA. In addition, recombinant tRNA/mir-34a had no or minimal effect on blood chemistry and interleukin-6 level in mouse models, suggesting that recombinant RNAs were well tolerated. These findings provoke a conversation on producing biologic miRNAs to perform miRNA actions, and point toward a new direction in developing miRNAbased therapies.
Noncoding RNAs (ncRNAs) produced in live cells may better reflect intracellular ncRNAs for research and therapy. Attempts were made to produce biologic ncRNAs, but at low yield or success rate. Here we first report a new ncRNA bioengineering technology using more stable ncRNA carrier (nCAR) containing a pre-miR-34a derivative identified by rational design and experimental validation. This approach offered a remarkable higher level expression (40%-80% of total RNAs) of recombinant ncRNAs in bacteria and gave an 80% success rate (33 of 42 ncRNAs). New FPLC and spin-column based methods were also developed for large- and small-scale purification of milligrams and micrograms of recombinant ncRNAs from half liter and milliliters of bacterial culture, respectively. We then used two bioengineered nCAR/miRNAs to demonstrate the selective release of target miRNAs into human cells, which were revealed to be Dicer dependent (miR-34a-5p) or independent (miR-124a-3p), and subsequent changes of miRNome and transcriptome profiles. miRNA enrichment analyses of altered transcriptome confirmed the specificity of nCAR/miRNAs in target gene regulation. Furthermore, nCAR assembled miR-34a-5p and miR-124-3p were active in suppressing human lung carcinoma cell proliferation through modulation of target gene expression (e.g., cMET and CDK6 for miR-34a-5p; STAT3 and ABCC4 for miR-124-3p). In addition, bioengineered miRNA molecules were effective in controlling metastatic lung xenograft progression, as demonstrated by live animal and ex vivo lung tissue bioluminescent imaging as well as histopathological examination. This novel ncRNA bioengineering platform can be easily adapted to produce various ncRNA molecules, and biologic ncRNAs hold the promise as new cancer therapeutics.
Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3′-untranslated region (3′UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2−) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3′UTR.
BackgroundSingle nucleotide polymorphisms (SNPs) can lead to the susceptibility and onset of diseases through their effects on gene expression at the posttranscriptional level. Recent findings indicate that SNPs could create, destroy, or modify the efficiency of miRNA binding to the 3'UTR of a gene, resulting in gene dysregulation. With the rapidly growing number of published disease-associated SNPs (dSNPs), there is a strong need for resources specifically recording dSNPs on the 3'UTRs and their nucleotide distance from miRNA target sites. We present here miRdSNP, a database incorporating three important areas of dSNPs, miRNA target sites, and diseases.DescriptionmiRdSNP provides a unique database of dSNPs on the 3'UTRs of human genes manually curated from PubMed. The current release includes 786 dSNP-disease associations for 630 unique dSNPs and 204 disease types. miRdSNP annotates genes with experimentally confirmed targeting by miRNAs and indexes miRNA target sites predicted by TargetScan and PicTar as well as potential miRNA target sites newly generated by dSNPs. A robust web interface and search tools are provided for studying the proximity of miRNA binding sites to dSNPs in relation to human diseases. Searches can be dynamically filtered by gene name, miRBase ID, target prediction algorithm, disease, and any nucleotide distance between dSNPs and miRNA target sites. Results can be viewed at the sequence level showing the annotated locations for miRNA target sites and dSNPs on the entire 3'UTR sequences. The integration of dSNPs with the UCSC Genome browser is also supported.ConclusionmiRdSNP provides a comprehensive data source of dSNPs and robust tools for exploring their distance from miRNA target sites on the 3'UTRs of human genes. miRdSNP enables researchers to further explore the molecular mechanism of gene dysregulation for dSNPs at posttranscriptional level. miRdSNP is freely available on the web at http://mirdsnp.ccr.buffalo.edu.
Osteosarcoma (OS) is the most common form of primary malignant bone tumor and prevalent among children and young adults. Recently we have established a novel approach to bioengineering large quantity of microRNA-34a (miR-34a) prodrug for miRNA replacement therapy. This study is to evaluate combination treatment with miR-34a prodrug and doxorubicin, which may synergistically suppress human OS cell growth via RNA interference and DNA intercalation. Synergistic effects were indeed obvious between miR-34a prodrug and doxorubicin for the suppression of OS cell proliferation, as defined by Chou-Talalay method. The strongest antiproliferative synergism was achieved when both agents were administered simultaneously to the cells at early stage, which was associated with much greater degrees of late apoptosis, necrosis, and G2 cell cycle arrest. Alteration of OS cellular processes and invasion capacity was linked to the reduction of protein levels of miR-34a targeted (proto-)oncogenes including SIRT1, c-MET, and CDK6. Moreover, orthotopic OS xenograft tumor growth was repressed to a significantly greater degree in mouse models when miR-34a prodrug and doxorubicin were co-administered intravenously. In addition, multiple doses of miR-34a prodrug and doxorubicin had no or minimal effects on mouse blood chemistry profiles. The results demonstrate that combination of doxorubicin chemotherapy and miR-34a replacement therapy produces synergistic antiproliferative effects and it is more effective than monotherapy in suppressing OS xenograft tumor growth. These findings support the development of mechanism-based combination therapy to combat OS and bioengineered miR-34a prodrug represents a new natural miRNA agent.
Equilibrative nucleoside transporters (ENT) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/Cas9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [ 3 H]uridine uptake in the presence of the ENT specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse transcriptase inhibitors and thirty-seven nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC 50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine if there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, while nevirapine and lexibulin were ENT1 and ENT2 non-transported inhibitors. Bayesian models generated using Assay Central ® machine learning software yielded reasonably high internal validation performance (ROC > 0.7). ENT1 IC 50-based models were generated from ChEMBL; subvalidations using this training dataset correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC 50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 This article has not been copyedited and formatted. The final version may differ from this version.
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