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Functional relevance of naturally occurring single nucleotide polymorphisms in bovine leukemia virus-encoded microRNAs.

ABSTRACT: Background: Bovine leukemia virus (BLV) microRNAs (miRNAs) contribute to viral latency and immune evasion. Naturally occurring single-nucleotide polymorphisms (SNPs) within the BLV miRNA cluster have been associated with persistent lymphocytosis in cattle, yet their functional impact remains unclear. Aims: This study aims to (1) evaluate how SNPs in BLV miRNAs affect miRNA biogenesis and mature miRNA levels; (2) determine whether specific SNPs alter miRNA–mRNA interactions and identify affected targets; and (3) characterize transcriptomic changes induced by reference versus SNP-bearing miRNAs. Methods: BLV miRNA loci were PCR-amplified and sequenced from 53 blood samples of infected cattle. Both reference and SNP-containing precursors were cloned into expression vectors and co-transfected with an miRNA-deficient BLV clone into HEK293T cells. Mature miRNA levels were quantified via stem-loop RT-qPCR. Computational target prediction (miRanda) validated changes in mRNA targeting. Gene-expression effects were assessed using Agilent microarrays, with selected findings confirmed by RT-qPCR and subjected to pathway enrichment analysis (IPA). Significance: This work will elucidate how BLV miRNA polymorphisms modify miRNA maturation and target recognition, reshape host gene networks, and contribute to viral persistence and immune modulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE299212 | GEO | 2025/06/15

REPOSITORIES: GEO

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Project description:Background/Objective: Quantitative real-time PCR (RT-qPCR) is widely used in miRNA expression studies on cancer. To compensate for the analytical variability produced by the multiple steps of the method, relative quantification of the measured miRNAs is required, which is based on normalization to endogenous reference genes. A literature search in PubMed revealed that no study has been performed so far on reference miRNAs for normalization of miRNA expression in urothelial carcinoma. The aim of this study was to identify suitable reference genes for miRNA expression studies by RT-qPCR in urothelial carcinoma. Methods: Candidate reference miRNAs were selected from 24 papillary urothelial carcinoma and normal bladder tissue samples by miRNA microarrays. The usefulness of these candidate reference miRNAs together with the commonly for normalization purposes used small nuclear RNAs RNU6B, RNU48, and Z30 were thereafter validated by RT-qPCR in 58 tissue samples and analyzed by the algorithms geNorm, NormFinder, and BestKeeper. Principal Findings: Based on the miRNA microarray data, a total of 16 miRNAs were identified as putative reference genes. After validation by RT-qPCR, miR-101, miR-125a-5p, miR-148b, miR-151-3p, miR-151-5p, miR-181a, miR-181b miR-29c, miR-324-3p, miR-424, miR-874, RNU6B, RNU48, and Z30 were used for geNorm, NormFinder, and BestKeeper analyses that gave different combinations of recommended reference genes for normalization. Conclusions: The present study provided the first systematic analysis for identifying suitable reference miRNAs for miRNA expression studies of urothelial carcinoma by RT-qPCR. Different combinations of reference genes resulted in reliable expression data for both strongly and less strongly altered miRNAs. Notably, RNU6B, which is the most frequently used reference gene for miRNA studies, gave inaccurate normalization. The combination of four (miR-125a-5p, miR-148b, miR-151-3p, and miR-151-5p) or three (miR-148b, miR-874, miR-181b) miRNA reference genes is recommended for normalization. Candidate reference miRNAs (for RT-qPCR analysis) were selected from 24 papillary urothelial carcinoma and normal bladder tissue samples by miRNA microarrays.

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Functional relevance of naturally occurring single nucleotide polymorphisms in bovine leukemia virus-encoded microRNAs.

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