MiR-26b Expression Pattern In The Pitutary Of Yanbian Yellow Cattle And Identification Of EphA2as A Target Gene Of MiR-26b

MicroRNAs （miRNAs） are a class of small RNAs found in animals,plants,DNAviruses and other diverse eukaryotes, which range in size from19to24nucleotides.MiRNAs regulate messenger RNA （mRNA） or protein levels either by promotingmRNA degradation or by attenuating protein translation. As with mRNAs, somemiRNAs are differentially expressed among tissues or developmental stages.MiRNAs play important roles in the regulation of many celluLAR Ⅱ processes,including cell proliferation and differentiation,apoptosis,organism development,tumormetastasis and other biological processes. MiR-26b, a length of22nt miRNA, playsan important role in the regulation of cell biology process. With the development ofmolecuLAR Ⅱ biology techniques, the study of miR-26b is much more specific. Manystudies indicate that miR-26b is involved in early embryo development,cellproliferation regulation,hormone expression regulation in the anterior pituitary andother physiological activities. In one word, miR-26b is essential for the reproductivefunction.This study was designed to detect the relative expression level of the miR-26bbetween the different development stages in the pitutary of YanBian Yellow Cattle byReal-time PCR, and analyze the relative expression with the2^{-△△Ct}method. Thepotential target of miR-26b is predicted using TargetScan and RNA22. EphA2ischosen because it is predicted by the both of two programs. In this study, the relativeexpression level of the EphA2is detected between the different development stages inthe pitutary of YanBian Yellow Cattle by Real-time PCR, the3’-UTR of the EphA2isconfirmed using the pmirGLO Dual-Luciferase miRNA Target Expression vector andsite-directed mutagenesis vector, and EphA2is identified of as a target gene ofmiR-26b.The results indicate that the expression level of miR-26b has extremelysignificant difference in different development stages of YanBian Yellow Cattlepituitary（p<0.01）, the expression of24months of age is2.41fold than6months.Opposite to the miR-26b, the EphA2expression of1months of age is3.34fold than 24months. The binding site for miR-26b is located in3’-UTR of EphA2inbovine,human,mouse and other mammals, suggesting the EphA2gene may be thetarget gene of miR-26b. Moreover, luciferase reporter assays show that miR-26bspecifically binds to the EphA23’-UTR to inhibit the expression of luciferase activity.The inhibition of luciferase activity is also greatly decreased when introducing a3-base mismatch mutation into the seed region of the EphA23’-UTR pmirGLO-MUTvector, show the inhibition to luciferase activity from60%of the wild-type to26%.These data indicate the predicted binding site is critical for the direct and specificbinding of miR-26b to EphA23’-UTR TACTTGAA.In this research, miR-26b expression pattern in the pitutary of YanBian YellowCattle has been successfully detected, and EphA2is identified as a target gene ofmiR-26b in vitro. This research provides the basis for the futher studies of the fuctionof miR-26b in embryonic development, pituitary hormone secretion and otherreproductive process.