EGCG对热应激下巴马香猪背最长肌中lncRNA表达的影响

Effects of EGCG on the expression of lncRNA in the longissimus dorsi muscle of Bama Xiang pig under heat stress

  • 摘要: 目的 探究表没食子酸没食子儿茶素(Epigallocatechin-3-gallate, EGCG)对热应激条件下巴马香猪背最长肌组织中长链非编码RNA (Long non-coding RNA,lncRNA)表达的影响,为揭示热应激条件下EGCG调节巴马香猪肌内脂肪(IMF)沉积的分子机制提供理论依据。方法 选择20头8月龄雄性巴马香猪随机分为4组,对照组(TN)在22 ℃常温环境下饲养,3个高温处理组(HS0、HS250和HS500)均在35 ℃环境下饲养,HS0、HS250和HS500组日粮中分别添加0%、0.025%和0.05%的EGCG。试验结束后屠宰巴马香猪并采集背最长肌组织,提取总RNA并进行转录组测序。使用DESeq筛选差异表达lncRNA,并进行GO功能注释分析、KEGG信号通路富集分析与ceRNA互作网络构建。随机选取5个lncRNA靶基因,利用实时荧光定量PCR验证转录组测序结果的可靠性。结果 TN vs HS0、TN vs HS500和HS0 vs HS500组共筛选出263个差异表达lncRNA,共富集到2819个靶基因,其中lncRNA MSTRG.11234的靶基因PLPP1、ENSSSCT00000068789的靶基因PPARα和MSTRG.26033的靶基因SCD5与脂肪合成密切相关。KEGG信号通路富集分析结果显示,差异表达lncRNA靶基因富集到的信号通路中与脂质代谢相关的信号通路包括过氧化物酶体增殖物激活受体、腺苷酸活化蛋白激酶和磷脂酰肌醇3-激酶-Akt通路等。ceRNA互作网络构建结果显示,部分lncRNA可能通过ceRNA机制参与调控脂质代谢及应激反应。随机选择的5个差异表达lncRNA靶基因,表达趋势与转录组测序结果一致,表明转录组测序结果可靠。结论 筛选出263个差异表达lncRNA,其靶基因显著富集到过氧化物酶体增殖物激活受体、腺苷酸活化蛋白激酶和磷脂酰肌醇3-激酶-Akt通路等脂质代谢相关信号通路,其中PLPP1PPARαSCD5基因与IMF沉积密切相关。热应激可能通过调控lncRNA的表达,影响下游脂质代谢相关基因的转录活性,从而介导IMF沉积过程。

     

    Abstract: Objective This study aimed to investigate the effects of epigallocatechin-3-gallate (EGCG) on the expression of long non-coding RNA (lncRNA) in the longissimus dorsi muscle of Bama Xiang pigs under heat stress, with the goal of providing a theoretical basis for elucidating the molecular mechanism through which EGCG regulated intramuscular fat (IMF) deposition in Bama Xiang pigs under heat stress.Method Twenty male Bama Xiang pigs of 8-month-old were randomly assigned into four groups. The control group (TN) was reared at a normal temperature of 22 ℃, while the three high-temperature treatment groups (HS0, HS250, and HS500) were rear at 35 ℃. In the HS0, HS250, and HS500 groups, 0%, 0.025%, and 0.05% EGCG were added in their diets. At the end of the experiment, the Bama Xiang pigs were slaughtered and the longissimus dorsi muscle tissues were collected, from which the total RNA were extracted and their transcriptomes were sequenced. Differentially expressed lncRNAs were screened using DESeq to perform functional annotation analysis, KEGG signaling pathway enrichment analysis, and ceRNA interaction network construction. Five lncRNA target genes were randomly selected and analyzed using real-time fluorescence quantitative PCR to verify the reliability of the transcriptome sequencing results.Result A total of 263 differentially expressed lncRNAs were screened from the groups of TN vs HS0, TN vs HS500, and HS0 vs HS500, and 2819 target genes were enriched. Among them, the target genes PLPP1 of lncRNA MSTRG.11234, PPARα of ENSSSCT00000068789, and SCD5 of MSTRG.26033 were closely related to fat synthesis. KEGG signaling pathway enrichment analysis showed that, among the signaling pathways enriched by target genes of differentially expressed lncRNAs, peroxisome proliferator-activated receptors, adenosine monophosphate-activated protein kinase, and phosphatidylinositol 3-kinase-Akt pathways and so on were related to lipid metabolism. Results from the ceRNA interaction network revealed that some lncRNAs may regulate lipid metabolism and stress responses through ceRNA mechanisms. Five differentially expressed lncRNA target genes were randomly selected, and their expression trends were consistent with the transcriptome sequencing results, indicating that the transcriptome sequencing results were reliable.Conclusion 263 differentially expressed lncRNAs are screened, and their target genes were significantly enriched in lipid metabolism-related signaling pathways, including the peroxisome proliferator-activated receptor, adenosine monophosphate-activated protein kinase, and phosphatidylinositol 3-kinase-Akt pathways. PLPP1PPARα, and SCD5 genes are closely associated with IMF deposition. Heat stress may mediate IMF deposition by regulating lncRNA expression and affect the transcriptional activity of downstream lipid metabolism-related genes.

     

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