MiRNA-24 downregulates KLF6 affecting STAT3 protein expression and phosphorylation regulating melanogenesis in cashmere goat coat
MiRNA-24 downregulates KLF6 affecting STAT3 protein expression and phosphorylation regulating melanogenesis in cashmere goat coat
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Objective: This study revealed that miRNA-24 downregulates <i>KLF6</i>, 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 <i>KLF6</i> on different colored skins of Cashmere goats, and the KLF6 expression in B16-F10 cells transfected with miRNA-24 mimics/inhibitors/<i>NC</i> were detected by RT-qPCR, Western Blot, and Immunofluorescence. To investigate the pathway that <i>KLF6</i> influenced, <i>KLF6</i> 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 <i>KLF6</i> in relation to melanogenesis was examined.Results: MiRNA-24 is related to coat color. <i>KLF6</i> was chosen as the target gene, and the targeted interaction was validated. The expression trends of miRNA-24 and <i>KLF6</i> in the skin inversely correlated. The mRNA and protein expression of <i>KLF6</i> were altered in B16-F10 cells after transfected. These suggested that miRNA-24 downregulates <i>KLF6</i>. Silencing of <i>KLF6</i> resulted in the downregulation of genes linked to melanogenesis, including <i>WNT1</i>, <i>WNT2</i>, <i>PRKACA</i>, <i>MAPK1</i>, <i>TYR</i>, and <i>STAT3</i>. The suppression of protein and phosphorylation levels of <i>STAT3</i> decreaseed the mRNA level of <i>TYR</i>, suggesting that <i>KLF6</i> influences <i>TYR</i> expression by modulation of STAT3 expression and phosphorylation. BALB/c mice injected with antagomiRNA-24 showed increased melanin content and decreased <i>KLF6</i> expression.Conclusion: MiRNA-24 regulates melanogenesis in the Cashmere goat coat by downregulation of <i>KLF6</i>, thus mediating STAT3 expression and phosphorylation
Abstract
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.