Selection And Function Of MicroRNA Targeting Mepe

Matrix Extracellular Phosphoglycoprotein (MEPE) had been limited to regulation ofbone metabolism, phosphate homeostasis, and dentin. MEPE was first found intumor-induced osteomalacia (the tumor-induced osteomalacia, TIO) by Rowe in2000, inthe same year Petersen and his colleague found the gene which was originally named asOsteoblast/Osteocyte Factor45(OF45) when they screened significant differentiallyexpressed novel gene in a rat differentiated and undifferentiated osteoblasts. MEPE andOF45are homologous genes, Mepe/Of45. Subsequently MEPE/Mepe homologousprotein in mice was also found.microRNAs (miRNAs) represent a class of naturally occurring small noncodingRNA molecules, distinct from but related to siRNAs. Mature miRNAs are19-to25-nucleotide-long molecules cleaved from70-to100-nucleotide hairpin pre-miRNAprecursors. In animals, single-stranded miRNAs bind, through partial sequence homology,to the3'-UTR of target mRNAs and cause a block of translation or, less frequently,mRNA degradation.We previously reported an important function of MEPE/OF45in protecting cellsfrom DNA damage induced killing, which would provide MEPE/OF45as a new targetfor sensitizing tumor cells to radiotherapy or chemotherapy.miRNAs are estimated to target more than30%of animal genes, suggesting thatthey may exert a large combinatorial outcome in biological processes. In this study, wewill focus on selection of miRNAs targeting Mepe and its function in DNA damageresponse, it will be basis to study the signal pathway of MEPE/OF45and its relatedproteins in the cells.Here Human Mepe-3'UTR was searched by NCBI website, and Targetscan was usedto predict the candidate miRNAs targeting Mepe,36candidate miRNAs targeting Mepewere found, and only6predicated miRNAs targeting Mepe were selected by contextscore percentile and consequential pairing and were identified by using dual luciferaseassay. Luciferase analysis showed that the activity of wild type3'UTR reporter wassignificantly suppressed by miR-376a, suppression by miR-376a depends on the wildtype miR-376a complementary sites, and was not longer observed in reporter in whichmiR-376a complementary sites were mutated, so miR-376a directly targets Mepe and miR-376a can repress the translation of Mepe gene in HeLa cells by Western blotting.The results show that introduction of miR-376a expression in human cells resulted indecrease of G2phase arrest following ionizing radiation (IR) by the flow cytometry, andoverexpression of miR-376a sensitized the cells to DNA damage inducers. All theseresults proved that miR-376a sensitized the cells to DNA damage by repressing theexpression of MEPE.In this study, a miRNA targeting Mepe was obtained by bioinformatics andexperimental biology, it will be basis to study potential drugs for radiotherapy orchemotherapy.