Qiaoling Zhang

Objective: This study revealed that miRNA-24 downregulates KLF6, regulating melanogenesis in the coat of Cashmere goats.Methods: The correlation between miRNA-24 and coat color was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). GO/KEGG analysis and bioinformatics tools were used to screen for target genes. Targeted interactions were confirmed using the dual luciferase assay. The expressions of miRNA-24 and KLF6 on different colored skins of Cashmere goats, and the KLF6 expression in B16-F10 cells transfected with miRNA-24 mimics/inhibitors/NC were detected by RT-qPCR, Western Blot, and Immunofluorescence. To investigate the pathway that KLF6 influenced, KLF6 was silenced by RNA interference (RNAi), and the melanogenesis pathway’s gene expression was examined. The expression and phosphorylation of STAT3 protein in the pathway were inhibited. RNAi and Stattic were utilized to downregulate its protein expression and phosphorylation for detecting tyrosine expression. BALB/c mice were subcutaneously injected with antagomiRNA-24, a miRNA-24 inhibitor. The influence of miRNA-24 on the downregulation of KLF6 in relation to melanogenesis was examined.Results: MiRNA-24 is related to coat color. KLF6 was chosen as the target gene, and the targeted interaction was validated. The expression trends of miRNA-24 and KLF6 in the skin inversely correlated. The mRNA and protein expression of KLF6 were altered in B16-F10 cells after transfected. These suggested that miRNA-24 downregulates KLF6. Silencing of KLF6 resulted in the downregulation of genes linked to melanogenesis, including WNT1, WNT2, PRKACA, MAPK1, TYR, and STAT3. The suppression of protein and phosphorylation levels of STAT3 decreaseed the mRNA level of TYR, suggesting that KLF6 influences TYR expression by modulation of STAT3 expression and phosphorylation. BALB/c mice injected with antagomiRNA-24 showed increased melanin content and decreased KLF6 expression.Conclusion: MiRNA-24 regulates melanogenesis in the Cashmere goat coat by downregulation of KLF6, thus mediating STAT3 expression and phosphorylation

The rising incidence of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to global public health, highlighting the urgent need for novel therapies and treatments in clinical settings. Caseinolytic protease P (ClpP) serves as a key component of bacterial degradation systems, playing a crucial role in maintaining cellular homeostasis and contributing to pathogenicity. Targeting ClpP function inhibition has demonstrated potential in combating antibiotic resistance and offers a promising therapeutic strategy for treating S. aureus infections. In this study, coniferaldehyde (CA) was identified as a ClpP inhibitor through ClpP peptidase inhibition assay. CA reduced the hemolysis activity, protease hydrolysis and bacterial invasion ability via regulating the transcription of main virulence factors. Furthermore, CA treatment led to a decreased resistance of S. aureus to adverse stimuli, including heat, acidic pH, high osmotic environment, hydrogen peroxide and NaClO stress assays. Notably, CA enhanced the efficacy of the bactericidal antibiotic tigecycline against growing S. aureus in time-killing assays. Molecular simulations and mutagenesis analyses revealed that the amino acids M31 and G33 were critical for the interaction between CA and ClpP. Importantly, CA exhibited excellent protective efficacy against S. aureus pneumonia in murine infection models. Our findings confirm that CA is an effective ClpP inhibitor with potential as a therapeutic agent for S. aureus infections.

Coat colour formation in mammals is influenced by melanogenesis and pigmentation processes regulated by miRNAs, including miRNA-200a. Although miRNA-200a is differentially expressed in the skin of cashmere goats with varying coat colours, its regulatory mechanism remains unclear. In this study, miRNA-200a target genes were predicted using miRBase and TargetScan, identifying GNAI1 and PLCB4 as the target genes through GO and KEGG analyses. Dual-luciferase assays using wild-type and mutant plasmids confirmed a direct interaction between miRNA-200a and the 3’UTR regions of these genes. RT-qPCR and Western blot analyses demonstrated that the expression levels of miRNA-200a and its target genes differed significantly between black and white goat skin. In HaCaT cells, transfection with miRNA-200a mimics or inhibitors altered GNAI1 and PLCB4 expression at both mRNA and protein levels. To validate these findings in vivo, subcutaneous injection of antagomiR-200a into BALB/c mice significantly reduced melanin content (P < 0.01) and increased the expression of GNAI1 and PLCB4. These results indicate that miRNA-200a modulates skin pigmentation by suppressing GNAI1 and PLCB4, thereby influencing coat colour in cashmere goats. This study provides a foundational understanding for leveraging genetic regulation to enhance coat colour diversity and develop naturally pigmented breeds.

Cashmere goats have great economic value because of their highquality cashmere characteristics. The growth and production of Cashmere are affected by the cyclic activity of hair follicles, which includes anagen, catagen and telogen. MiRNAs can regulate gene expression as a key regulatory factor, thereby affecting hair follicle periodical development. Previous studies had found that miRNA-143 differentially expressed at different stages of hair follicle development in Cashmere goats, suggesting that it has a potential role in regulating hair follicle activity, but the specific mechanism is unclear. In order to further analyze the role of miRNA-143 in hair follicle periodical development of Cashmere goats, this study used bioinformatics software to predict the target genes of miRNA-143. Dual luciferase assay, Quantitative reverse transcription polymerase chain reaction, Western Blot and over-expression were performed in vitro and vivo to verify the targeting relationship between miRNA-143 and the target genes and the role of miRNA-143 in hair follicle periodical development of Cashmere goats. The results showed that FGF9 and MAPK1 were targeted by miRNA-143, verified by the Dual-Luciferase Reporter Assay System, miRNA-143 was highly expressed in anagen skin compare to telogen skin of Cashmere goats, while the expression of FGF9 and MAPK1 was reversed. The over-expression of miRNA-143 reduced MAPK1 and FGF9 expression at mRNA and protein levels. In short, this study demonstrated that miRNA-143 might be involved in hair follicle periodical development of Cashmere goats by targeting FGF9 and MAPK1, providing theoretical basis for artificial regulation of hair follicle development and improving cashmere production.

Clostridium perfringens (C. perfringens) is an important zoonotic pathogen. The diseases such as necrotic enteritis (NE), enterotoxemia, gas gangrene and food poisoning caused by its infection seriously threaten the lives of both humans and animals. However, under the severe situation of antibiotic resistance, the development of new antibacterial strategies or drugs deserves great attention. In this study, we selected the virulence factor Type IV pili (TFP) of C. perfringens as the target for drug screening. The gliding motility, biofilm formation, cell adhesion and antibacterial activity of the natural compound isoxanthohumol (IXN) against C. perfringens were determined. Transmission electron microscopy (TEM), TFP gene transcription analysis and Western blot were used to detect the expression of PilA pilin. The therapeutic effect of IXN on C. perfringens infection was demonstrated through a mouse gas gangrene model. It was confirmed that IXN inhibits the function of TFP by down-regulating TFP-encoding genes and two-component regulatory genes. In conclusion, our study shows that IXN has the potential to inhibit the function of TFP in C. perfringens and for anti-infection applications.

Chromaticity is a key indicator of measuring economic worth of Cashmere goats, and gene regulation plays a major role in Cashmere goat color. By targeting the 3^′UTR region of genes, such as miRNA-211, miRNA plays a significant regulatory role in melanogenesis. But the mechanism of how miRNA-211 affects the melanogenesis in Cashmere goats is unclear. As a result, insights into the color regulation by miRNA-211 in Cashmere goats and its brief mechanism are offered in this study. First, the target gene AP1S2 of miRNA-211 was screened by TargetScan and GO/KEGG, and then the targeted relationship was confirmed by the dual-luciferase reporter experiment. In B16-F10 cells, which overexpress miRNA-211 and goat skin, the opposite expression trends between AP1S2 and miRNA-211 were analyzed by reverse transcription–quantitative PCR (RT-qPCR) and western blot. And their opposite expression trends were confirmed in B16-F10 cells by immunofluorescence. The information above showed miRNA-211 downregulated the expression of AP1S2. Afterwards, the impact of miRNA-211 targeting AP1S2 on the melanogenesis of Cashmere goats was confirmed by animal regression studies. Utilizing immunohistochemistry, the expression of AP1S2 in a mouse hair follicle was observed. Following subcutaneous injection of antagomiRNA-211 (chemical modification inhibitor), the expression levels of the AP1S2 gene and protein were enhanced, and the localization in the hair follicle was also increased. Furthermore, reduction in melanin content in the skin was detected. These results showed that miRNA-211 significantly affected the melanogenesis progress via downregulating AP1S2 and advantageously affected the melanin content of Cashmere goat skin.

Increasing Cashmere production can add value because it is the primary product of Cashmere goats. Recent years, peoples find miRNAs are crucial in regulating the development of hair follicle. Following Solexa sequencing, many miRNAs were distinguishingly expressed in telogen skin samples of goats and sheep in earlier study. But the method through which miR-21 controls the growth of hair follicles is still ambiguous. Bioinformatics analysis was used to predict the target genes of miR-21. The mRNA level of miR-21 in telogen Cashmere goat skins was higher than in anagen, according to the results of qRT-PCR, and the target genes expressed similarly with miR-21. Western blot showed similar trend, the protein expression of FGF18 and SMAD7 were lower in anagen samples. The Dual-Luciferase reporter assay confirmed miRNA-21's relationship with its target gene, and the consequences indicated found FGF18 and SMAD7 have positive correlations with miR-21. Western blot and qRT-PCR distinguished the expression of protein and mRNA in miR-21 and its target genes. According to the consequence, we found that target genes expression was increased by miR-21 in HaCaT cells. This study identified that miR-21 might take part in the development of Cashmere goat’s hair follicles by targeting FGF18 and SMAD7.