| Pigmentation, a physiological process of melanin metabolism, which including melanin synthesis, transfer, redistribution and degradation, contribute to normal human skin color. The skin reacts to numerous external environmental stress and internal physiological factors through all its cellular and molecular components, which form a complicated, sophisticated and highly sensitive signaling network. Pigmentation disorder of skin leads to various pgmentary disrase, such as vitiligo and melasma, the pathogenesis has not yet been fully elucidated.The synthesis of melanin by melanocytes and the transfer of melanosomes to surrounding keratinocytes serve not only as the major determinant of skin color but also as the major source of skin protection against UV irradiation. Melanin transfer, a key step in the process of pigmentation, is the outcome of two important events:(1) Melanosomes move toward the periphery of the cell within the dendrites in melanocytes;(2) Melanosomes are transferred via dendrites to surrounding keratinocytes where they play a critical role in photoprotection.Dendrite, as a crucial morphological feature of melanocytes required for melanin transfer to keratinocytes, plays an extremely important role in pigmentation. It has been found that various stimulus, especially the UVR, could cause significant increase in the number and length of melanocytes dendrites to faciliate melanin tranfer, along with the increase of the melanin synthesis. Abnormal pigmentation attribute to the disorder of melanin metabolism with damaged structure and/or fuction of melanocyte. It was showed that human melanocytes reacted to UVB irradiation in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. Melanosomes can distribute in basal layer and stratum corneum. Our previous work have confirmed that the number and length of melanocytes dendrites were significant increased so that more melanosome transferred to surrounding keratinocytes after UVB irradiation. However, the mechanism underlying is not fully clarified.MicroRNAs (miRNAs) are small,-22nt long, single-stranded molecules which, when complexed with an RNA-induced silencing complex (RISC), are able to form a complementary double-stranded RNA structure by hybridizing to the3’untranslated region of target transcripts, and inhibit translation of their cognate mRNA and/or promote their degradation. miRNAs, as key regulators of gene expression at the post-transcriptional level, play a fundamental role in a wide variety of biological processes through their specific gene regulatory networks. In prior researches, some miRNAs were found with region-specific expression patterns in neural spines development and were showed to play important roles in cell morphology, polarity migration and mobility by regulating actin cytoskeleton pathway. In addition, microRNAs were found differentially expressed after UV irradiation, and regulated cellular responses to UV-induced DNA damage at the post-transcriptional level. Our studies focused on the possible functional role of specific miRNAs associated with melanocyte dendricity, that could improve the basic theory of metabolism of melanin and explore the mechanism of UVR induced skin pigmentation, as well as provide new ideas and theories for pigmentary diseases therapy and cosmetics invention.Methods:Firstly, human menlanocytes PIG1were exposured to100mJ/cm2UVB irradiation, then we analysis the differential expressed microRNA profiles between the treated group (3h,24h postirradiation) and control group (shamirradiation), which were detected by miRNA chip. Next, we used RT-PCR to validate the miRNA expression. In order to perform a functional studies of choosed miRNAs on the melanocytes dendrite formation, specific miRNA mimics and inhibitor were transiently transfected into PIG1cells. We observed the number and length of melanocytes dendrites and the intracellular distribution of F-actin and melanosomes. Bioinformatics softwares were utilized to predict potentially direct target genes for further search. Dual-luciferase reporter gene assay was used to confirm the relationship between miRNA and its target gene.RT-PCR and Western Blot methods were used to validate the possible regulatory role of miRNA in dendrite formation.Results:1. After microRNA profiles analysis, we found that the expression level of many miRNAs chenged after UVB exposure. The results showed there was a large change at3h and24h postirradiation compared with the control group.12miRNAs, including10up-regulated and2down-regulated, were changed at3h (Fold change>2.0) whereas,4miRNAs, including1up-regulated and3down-regulated, were changed at24h.2. RT-PCR results showed that transfection of miR-340mimic and inhibitor can successfully increase or decrease endogenous miR-340expression respectively in PIGl cells. Upregulation of miR-340, the number and length of melanocyte dendrites, were significantly increased compared with the control group (p<0.05). With immunofluorescence, we observed the loss of stress fiber and melanosome gathered at the end of the melanocvte dendrite.3. Biological software predicted that RhoA was a potential target genes of miR-340. The reporter gene assay confirmed that miR-340could down-regulate RhoA expression by directly binding to its3’UTR. Compared with control, RhoA mRNA showed no significant change in PIGl cells transfected with miR-340mimic or inhibitor; but RhoA protein expression changed inversely.Major conclusions:1. UVB could induce a large change of microRNA profile in human melanocyte PIGl, which may be involved in various biological process, including melanocyte dendricity, so that we could select the miRNAs associated with dendrite formation. In additon, the major change of microRNA expression primarily at the early time after UVB irradiation and scarcely any miRNA was detected with significant change at both3h and24h postirradiation, suggesting that some miRNAs were differentially expressed early, but without long-lasting expression.2. miR-340expression was upregulated but not persistent after UVB exposure, miR-340could depolymerize stress fiber, promote dendrite formation and melanosome transport the end of dendrite in melanocytes.3. miR-340may participate in in melanocyte dendricity through the inhibition of RhoA expression.RhoA is a key molecule regulating scaffolding protein F-actin.RhoA, which belongs to Rho family, could promote actin polymerization to form stress fiber, increase cell tension and adhesion,result in dendrite retraction and shorter. It plays a negative role in dendrite formation, we conclude that the positive regulation of miR-340in dendrites formation, was achieved partiy through negatively regulating RhoA. |
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