Prediction And Validation Of The MiRNAs Involved In The Biosynthesis And Proliferation Of Peroxisome

MiRNAs are small non-coding RNAs with 17-25 nucleotides in length and believed to be involved in a broad range of biological function, including cell proliferation, cell differentiation, cell apoptosis, development, responses to diseases and signal transduction, by base-pairing with complementary sites of target transcript to negatively regulate gene expression at the posttranscriptional level. Peroxisomes are ubiquitous eukaryotic organelles involved in various oxidative reactions. The biogenesis and proliferation of peroxisome are regulated by Peroxisome Proliferator-Activated Receptor(PPAR) family and pex (encoding peroxin) gene family. Mutant or aberrant expression of these genes will lead to disorder of peroxisome biogenesis and various serious diseases. The biogenesis and proliferation of peroxisome are regulated in various ways, but it is still a question that whether miRNAs involved in.In this study, we used a bioinformatics approach to identify conserved miRNAs which can complement with 3'untranslation region of target transcripts involved in peroxisome biogenesis and proliferation, these miRNAs were validated by Dual-Luciferase Reporter system. The following is the summarization of our result:1,After using four kinds of miRNA target predict software to analyze 11 members of pex family(PEX1,PEX2,PEX3,PEX5,PEX6,PEX7,PEX10,PEX12,PEX13,PEX16,PEX19) and 3 members of ppar family(PPARA,PPARG,PPARB), we found numerous miRNA:mRNA basepairing in the 3'UTR of these genes:137 miRNAs was predicted by TargetScan,141 miRNAs was predicted by MicroCosm Targets,306 miRNAs was predicted by miRanda,44 miRNAs was predicted by Pictar. Among these results,129 base-pairing of miRNAs and their targets predicted by two softwares,26 base-pairing of miRNAs and their targets predicted by three softwares,4 base-pairing of miRNAs and their targets predicted by four softwares. In our resultes,4 base-pairing was found in pexl,18 in pex3,22 in pex5,13 in pex6,31 in pex7,3 in pexl2,7 in pexl3,4 in pexl9,13 in ppara,14 in pparg. No base-pairing detected in transcripts of PEX 1,PEX2, PEX10,PEX16 and PPARB.2,Using miRBase to analyze the conservatism of 26 miRNAs predicted by three software, we found that most of them are conserved, such as mir-19a/b,mir-15a/b,mir-16,mir-29a/b are conserved in 20 species of metazoan.3,Based on literature mining, we found mir-15a,mir-16,mir-19a,mir-27a,mir-29a,mir-195,mir-301 etc were positive correlated with their target mRNA, and these miRNAs were identified experimentally.4,After co-transfecting of miRNAs expression constructs and psiCHECK-2-target 3'UTR constructs to meNPC, the activity of Renilla luciferase was measured by GloMaxTM-Multi+and firefly luciferase was used as internal control to normalize Renilla luciferase data.The resultes showed that:mir-19a can down regulate ppara; mir-15a and mir-16-1 can down regulate pex13; mir-29a can down regulate pex5; mir-27a can down regulate pparg, suggesting that miRNAs are involved in the biogenesis and proliferation of peroxisome.Our works laid the foundation of uncovering of the function and mechanism about miRNA in the biogenesis and proliferation of peroxisome, and make a new thinking about the development of therapy of Peroxisome-Biogenesis Disorders.