| Summary: | The mechanisms of modulating milk production traits remain largely unknown. Based on our previous RNA-seq, <i>DDIT3</i> was presumed as a novel, promising candidate gene for regulating milk protein and fat traits in dairy cattle. To further detect the genetic effect of <i>DDIT3</i> and its potential molecular mechanisms in regulating milk production traits in dairy cattle, here, we performed a genotype-phenotype association study. Two SNPs, g.-1194 C>T and g.-128 C>T, were significantly associated with MY (<i>p</i> = 0.0063), FY (<i>p</i> = 0.0001) and PY (<i>p</i> = 0.0216), respectively. A luciferase assay demonstrated that the allele T of g.-128 C>T increased the promoter activity by binding the HSF2, while allele C did not. To further reveal the molecular regulatory mechanisms, the <i>DDIT3</i>-knockdown MAC-T cells were established. It was observed that <i>DDIT3</i> silencing could induce apoptosis and increase the number of PI-positive cells. Meanwhile, <i>DDIT3</i> silencing led to increased expression of inflammatory markers, such as <i>IL-6</i>, <i>IL6R</i>, <i>IL1B</i>, <i>IL7R</i>, <i>IL1RL2</i>, <i>IL1A</i>, <i>STAT1-5</i>, <i>MYC</i>, <i>IGFBP4,</i> and <i>IGFBP5</i>, and especially for <i>IL-6</i> (log<sub>2</sub>FC = 4.22; <i>p</i> = 3.49 × 10<sup>−112</sup>). Additionally, compared with the control group, increased lipid accumulation was found in the <i>DDIT3</i>-knockdown MAC-T cells. Thus, our results proved that lower expression of <i>DDIT3</i> could result in increased lipid accumulation and apoptosis via up-regulating the expression of <i>IL-6.</i> These findings provided clues about the regulatory mechanisms of milk production traits in dairy cattle.
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