MicroRNA-200a regulates skin pigmentation by targeting WNT5A and FZD4 in Cashmere goats.

MicroRNAs are small, non-coding RNAs that regulate the expression of target genes. Previous research has demonstrated that microRNA-200a regulates cell apoptosis, tumor progression, and autoimmune disease. Preliminary studies found that microRNA-200a was differently expressed in the skin of Cashmere goats of various coat colors. However, the role of microRNA-200a in skin pigmentation remained poorly understood. In the current study, we investigated the effect of microRNA-200a on pigmentation in Cashmere goats. The expression of target genes was detected by real-time quantitative PCR, western blot, and immunohistochemistry staining both in vivo and in vitro. Luciferase reporter assays were used to demonstrate the relationship between microRNA-200a and its target genes Wnt family member 5A and frizzled class receptor 4 (WNT5A and FZD4) in HEK293T cells. BALB/c mice were injected with antagomiR-200a to detect melanin content and the expression of microRNA-200a and its target genes. The results demonstrated that the expression of microRNA-200a was significantly higher in brown tissue. Luciferase reporter assays confirmed that microRNA-200a targeted WNT5A and FZD4. The expression of WNT5A and FZD4 in the skin of brown Cashmere goats was significantly lower than that in white Cashmere goats by the detection of mRNA and protein levels. Overexpression/inhibition of microRNA-200a in keratinocytes decreased/increased the mRNA and protein expression of WNT5A and FZD4, respectively. In addition, the expression of WNT5A and FZD4 increased in the skin of BALB/c mice injected with antagomiR-200a, but the melanin content decreased. In summary, this study indicated that microRNA-200a regulates skin pigmentation by targeting WNT5A and FZD4 in Cashmere goats.

Differential expression of miR-let7a in hair follicle cycle of Liaoning cashmere goats and identification of its targets.

In order to improve the production and quality of Chinese cashmere, the research of hair follicle development has aroused more and more attention; the regulation mechanism of miRNA in hair follicle development has become a hot spot. A survey of transcriptome profiling screened 10 hair follicle-related miRNAs that were differentially expressed, including miR-let7a. In this study, the expression of miR-let7a was lower in anagen of hair follicle of cashmere goats than that in catagen of hair follicle of cashmere goats (p < 0.01). Results were in accordance with transcriptome data. The expression patterns of miR-let7a target genes (IGF-1R, C-myc, and FGF5) were verified by qRT-PCR, which were consistent with the results of Western blot and showed a downward trend. The dual-luciferase reporter gene system was used to verify the correlation between the expression of miR-let7a and its target genes, and it showed that miR-let7a negatively correlates with C-myc and FGF5. Present study offers new information on miRNAs and their related target genes in the regulation of hair follicle development mechanism.

Differential Expression of miR-202 and Validation of Predicted Target Genes in the Skin Tissue of C57BL/6 Black Mice and BALB/c White Mice.

In recent years, with the need of the fur industry to obtain greater quantities of high-quality fur and the research of hair follicle development attracting greater attention, the role of miRNAs in hair follicle development has become a field of intense interest. miRNAs are short length RNAs that affect gene regulation by binding to the 3'-untranslated region (3'UTR) of the mRNAs of certain target genes. This study predicted and validated the target genes of miR-202 in the skin tissue of C57BL/6 black mice and BALB/c white mice. The expression of miR-202 target genes (wnt5a, kit, and tcf7) was examined by real-time quantitative polymerase chain reaction and western blot in the mouse skin tissue and human embryonic kidney 293T cells (HEK293T cells) which overexpress miR-202. The luciferase reporter gene system was used to verify the correlation between the expression of miR-202 and its putative target genes. We show that the expression of miR-202 was higher in the skin tissue of C57BL/6 black mice than in the skin tissue of BALB/c white mice (p < 0.05). Furthermore, the expression of wnt5a, kit, and tcf7 was observed to be negatively correlated with the expression of miR-202 and to be downregulated by miR-202 in vitro and in vivo. The luciferase reporter gene system indicated that the target sequences of miR-202 are present in 3'UTR of wnt5a, kit, and tcf7 mRNAs. Altogether, this study has shown that the expression of miR-202 was different in the skin tissue of C57BL/6 black mice and BALB/c white mice and that wnt5a, kit, and tcf7 are negatively regulated by miR-202.

Practical synthesis of anti-ß-hydroxy-α-amino acids by Pd(II) -catalyzed sequential C(sp(3) )-H functionalization.

An improved and practical procedure for the stereoselective synthesis of anti-ß-hydroxy-α-amino acids (anti-ßhAAs), by palladium-catalyzed sequential C(sp(3) )-H functionalization directed by 8-aminoquinoline auxiliary, is described. followed by a previously established monoarylation and/or alkylation of the ß-methyl C(sp(3) )-H of alanine derivative, ß-acetoxylation of both alkylic and benzylic methylene C(sp(3) )-H bonds affords various anti-ß-hydroxy-α-amino acid derivatives. As an example, the synthesis of ß-mercapto-α-amino acids, which are highly important to the extension of native chemical ligation chemistry beyond cysteine, is described. The synthetic potential of this protocol is further demonstrated by the synthesis of diverse ß-branched α-amino acids. The observed diastereoselectivities are strongly influenced by electronic effects of aromatic AAs and steric effects of the linear side-chain AAs, which could be explained by the competition of intramolecular C-OAc bond reductive elimination from Pd(IV) intermediates vs. intermolecular attack by an external nucleophile (AcO(-) ) in an SN 2-type process.

The melanocortin 1 receptor (MC1R) inhibits the inflammatory response in Raw 264.7 cells and atopic dermatitis (AD) mouse model.

The alpha melanocyte stimulating hormone receptor (MC1R) is one of five G-protein coupled receptors belonging to the melanocortin subfamily, MC1R gene has been known to play a major role in regulating of fur color in mammals, and α-MSH and ACTH are endogenous nonselective agonists for MC1R. However, we found that MC1R was highly expressed in Raw 264.7 cells which were important inflammatory cells involved in the initiation of inflammatory responses. In addition, Cyclic AMP is not only a key molecule in the MC1R signal transduction pathway, but dampen innate immune-mediated responses. These intriguing biological results triggered the further conformation studies; it suggested that MC1R was very likely to be an important role in immunoregulation. In this study, we were to investigate the immunosuppressive effects of MC1R on inflammation in lipopolysaccharide (LPS) stimulated Raw 264.7 cells and LPS induced vivo 2-chloro-1,3,5-trinitrobenzene (TNCB)-induced atopic dermatitis (AD) model. The effects of the MC1R antagonist psoralen on pro-inflammatory cytokines and signaling pathways were analyzed by enzyme-linked immunosorbent assay, western blot, real-time fluorescence quantitative PCR and Histological analysis. Our results show a consistent and marked effect of high concentrations of MC1R antagonist psoralen increased the level of MC1R mRNA in Raw 264.7 cells by cumulative feedback regulation through preferential binding of MC1R. Moreover, as evidenced by inhibiting the LPS-induced TNF-α, IL-6 and enhancing the expression level of cyclic AMP protein in vitro. In vivo study it was also observed that psoralen promoted on histopathologic changes in the skin tissue of TNCB-induced AD mice. Taken together, our results suggest that MC1R decrease the inflammation in vitro and vivo, and might be a therapeutic signaling pathway to against inflammatory diseases.