Expression of microRNA-26b and identification of its target gene EphA2 in pituitary tissues in Yanbian cattle

Abstract

microRNAs (miRNAs/miRs) are a class of single-stranded non-coding RNA molecules of 19–24 nucleotides (nt) in length. They are widely expressed in animals, plants, bacteria and viruses. Via specific mRNA complementary pairing of target genes, miRNAs are able to regulate the expression of mRNA levels or inhibit protein translation following transcription. miRNA expression has a time- and space specificity, and it is involved in cell proliferation and differentiation, apoptosis, development, tumor metastasis occurrence and other biological processes. miR-26b is an miRNA of 22 nt and is important in the regulation of cellular processes. With the advancement of molecular biology techniques in recent years, there have been extensive investigations into miR-26b. Numerous studies have observed that miR-26b is involved in early embryonic development, cell proliferation regulation, pituitary hormone secretion and other physiological activities. miRNAs are associated with the function of propagation. The present study used reverse transcription quantitative polymerase chain reaction to detect the relative expression levels of miR-26b in the pituitary tissue of Yanbian cattle at different developmental stages. The 2 ^−ΔΔCt method was used to calculate the relative gene expression levels. The miRNA target gene database TargetScan and RNA22 were used for prediction of the miR-26b target gene and selective recognition was also performed. The results demonstrated that miR-26b is expressed in the pituitary tissues of Yanbian cattle at 6 and 24 months of age. The relative expression levels of miR-26b in the pituitary tissues of 24-month-old Yanbian cattle were 2.41 times that of those in the six-month-old Yanbian cattle, demonstrating significant differences in the relative expression (P<0.01). The relative expression of the candidate target genes, EphA2 and miR-26b, exhibited the opposite expression pattern. The relative expression levels in the pituitary tissues of six-month-old Yanbian cattle were 3.34 times that of those in 24-month-old Yanbian cattle (P<0.01). There are miR-26b binding sites in the 3′-untranslated region (3′-UTR) of EphA2 in bovine, human, murine and other mammalian mRNAs, suggesting that the EphA2 gene may be a target gene of miR-26b. The results of a Luciferase reporter system assay revealed that miR-26b is able to suppress EphA2 expression at the transcription level. Following the site-directed mutagenesis of plasmid EphA2 3′-UTR pmirGLO-MUT- and miR-26b mimic-transfected HeLa cells, the dual-luciferase reporter gene assay revealed that there were three consecutive nucleotide mutations in the 3′-UTR, binding with the predicted seed region. This may have caused the miR-26b inhibition of luciferase activity to decrease from 60% in the wild-type to 26%, suggesting that miR-26b achieved its function via binding with the TACTTGAA sequence of the 3′-UTR in EphA2. In conclusion, the present study successfully assessed the expression pattern of miR-26b in the pituitary tissue of Yanbian cattle, and also confirmed that EphA2 was a target gene of miR-26b in Yanbian cattle in vitro. The present study provided the theoretical basis to further investigate the role of miR-26b in early embryonic development, pituitary hormone secretion and other reproductive functions.