Finding And Validation Of MiRNA Targeted To N-acetylglucosaminetransferase

N-acetylglucosaminetransferases (GnTs) catalyze the transfer of N-GlcNAc, which is involved in N-glycans or O-glycans Synthesis and plays very important role in biological process.GnTs could be divided into at least six types, from GnTⅠto GnTⅥ. GnTⅢcould catalyze the GlcNAc of UDP-GlcNAc toβ-Man by the connection ofβ1,4, generating bisecting N-acetylglucosamine whcih will not function as the substrates of GnTⅡ, GnTⅣ, GnTⅤa nd other glycoyltansferases. GnTⅣcould catalyze the GlcNAc of UDP-GlcNAc toβ1,2-Man by the Connection ofβ1,4,participating the N-glycan branching biosynthesis. Altered structure of glycan on glycoprotein are found in tumor. Some glycanstructures,β1,6 N-glycan which is the product of GnTⅤ,have been found to be related with metastasis.microRNA(miRNA) is a kind of new found non-coding small RNA molecules in recent years,which regulates the gene expression in post-transcriptional leve1 by degrading the targeted mRNA or curbing its post. However, little is known about miRNA targeted to N-acetylglucosaminetransferase.This study is to screen and characterize miRNA targeted to N-acetylglucosaminet- ransferase based on the miRNA and bioinformatic analysis. In this study, GnTⅢ/pGL3, GnTⅣ/pGL3 and GnTⅤ/ pGL3 were constructed by subcloning the 3'-UTR fragments of mouse Mgat3,Mgat4a and Mgat5 into pGL3 Luciferase Reporter Vectors, flollowed by co-transfection with pGL3 control or miRNA into mouse Hepa1-6 cells. Analyses of Luciferase in transfected Hepa1-6 cells were performed to screen miRNA candidates targeted to GnTⅢ. Furthermore, real-time PCR and Flowcytometry were used to validate the miRNA transfection on the expression of GnT gene and N-glycan branching biosynthesis. The results showed that Luciferase activity, mRNA levels of Mgat3, the bisecting N-acetylglucosamine level of GnTⅢproduct decreased significantly in Hepa1-6 cells transfected with miR-23. The These results indicate that GnTⅢmight be one of targets of miR-23.