The Study On MiR-488Regulating The Pigment-producing POMC Gene

Coat color is one of the important economic traits of wooler, the study of coat color will help improve the economic value of animal hair and reduce industrial pollution and human damage caused by artificially dyed. However, the formation of animal fur and pigmentation is affected by complex regulatory mechanisms, which including hundreds of regulated genes. POMC is particularly important color-related genes, it plays an important role on the formation, development, pigmentation and other aspects of melanin. POMC could affect the coat color through regulating pigment-producing enzyme changes.MicroRNA (miRNA, miR) are small, non-protein-encoding of endogenous RNAs that founded in eukaryotes, it could post-transcriptionally regulate gene expression via suppression of specific target mRNAs. Predicted by TargetScan, we found a targeting relationship between the POMC and miR-488. Therefore, we assumed that miR-488may be control the formation of coat color and pigmentation by combining with the3’-UTR of POMC. Analysising the Melanin production pathway, we build Dual Fluorescence Report vector of POMC3’-UTR, identified miR-488could post-transcriptionally regulate POMC expression through binding with POMC3’-UTR, then build miR-488eukaryotic expression vector and transfected mouse keratinocytes to make sure the regulatory mechanism at the cellular level. Based on this experiments, it reveals the mechanism of miR-488in the formation of animal pigmentation.1miR-488were found highly homologous in different species through NCBI blast, it can reach94%-100%. By TargetScan (http://www.targetscan.org/) predicted miR-488and POMC targeting relationships, we found there are same binding sites in human, mouse, sheep, etc. As shown above, we guess POMC gene is a target of miR-488.2The primers of miR-488and POMC were designed to identify the endogenous expression in mouse. The result showed both POMC and miR-488were expressed in mouse. 3Different coat color mice RNA were extracted to verify the endogenously express of miR-488and POMC, the results showed that the expression of POMC and miR-488was negatively correlated.4Different coat color mice protein were extracted to verify the express of POMC in protein level by western bolt, the results showed all of the mouse have the POMC expression, and with the different color, the expression of POMC appear differences in protein levels.5The POMC dual fluorescence report vetor and miR-488eukaryotic expression vector were build, and co-transfected into293T cells. The results showed that the firefly luciferase value will decreased in experimental group, indicated miR-488may be affected the expression of the dual fluorescence reporter vector sequence upstream of the firefly luciferase by combining with the3’-UTR of POMC.6We cultured mouse primary keratinocytes and transfected the K14-MCS-mouse pri-miR488eukaryotic expression vector into mouse keratinocytes, then make sure transfection efficiency by observing the expression of GFP fluorescence microscope. By QRT-PCR and western bolt to detecte the expression change in mRNA and protein levels, the result showed POMC mRNA and protein levels had a downward trend,and protein levels change more significant.To sum up:we can determine the POMC gene as a target of miR-488by dual fluorescence reporter, and miR-488affecting the POMC downstream signal transduction by combining with POMC mRNA3’-UTR to achieve the POMC mRNA degradation or to inhibit post-translational regulation of POMC transcription. At the same time,we got miR-488can be involved in pigmentation in cell level. The study not only can rich theoretical knowledge about mammalian coat color formation mechanism, but also provide important genetic resources and experimental basis for color-related microRNAs genetically modified genetic engineering.