Data_Sheet_3_Identification of miRNA in Sheep Intramuscular Fat and the Role of miR-193a-5p in Proliferation and Differentiation of 3T3-L1.XLSX

Intramuscular fat (IMF) is one of the most critical parameters affecting meat quality and mainly affected by genetic factors. MicroRNA as an important regulatory factor, which is still a lack of research in the development of sheep IMF deposition. We used RNA sequencing (RNA-seq) and cell-level validation to explore the role of miRNA in IMF deposition. As for this purpose, longissimus thoracis et lumborum (LTL) samples of 2 month-old (Mth-2) and 12 months-old (Mth-12) Aohan fine-wool sheep (AFWS) were used to identified miRNAs expression. We found 59 differentially expressed miRNAs (DE-miRNA) between these age groups and predicted their 1,796 target genes. KEGG functional enrichment analysis revealed eight pathways involved in lipid metabolism-related processes, including fatty acid elongation and the AMPK signaling pathway. A highly expressed DE-miRNA, miR-193a-5p, was found to serve a function in 3T3-L1 preadipocyte differentiation. Luciferase assay demonstrated that miR-193a-5p directly binds to the 3′-UTR region of ACAA2. By constructing mimics and inhibitor vector transfecting into 3T3-L1 cells to explore the effect of miR-193a-5p on cell proliferation and differentiation, we demonstrated that overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte proliferation, as evidenced by decreased mRNA and protein expression of CDK4 and CyclinB. CCK-8 assay showed that miR-193a-5p significantly inhibited cell proliferation. Similarly, the overexpression of miR-193a-5p inhibited 3T3-L1 preadipocyte differentiation and adipocyte-specific molecular markers’ expression, leading to a decrease in PPARγ and C/EBPα and ACAA2. Inhibition of miR-193a-5p had the opposite effects. Our study lists the miRNAs associated with intramuscular lipid deposition in sheep and their potential targets, striving to improve sheep meat quality.

肌内脂肪（IMF）是影响肉质最为关键的因素之一，其主要受遗传因素影响。作为重要的调控因子，微小RNA（miRNA）在羊肉肌内脂肪沉积发展中的研究尚显不足。本研究采用RNA测序（RNA-seq）及细胞水平验证方法，探究miRNA在肌内脂肪沉积中的作用。为此，选取2月龄（Mth-2）和12月龄（Mth-12）的阿寒细毛羊（AFWS）的长胸腰肌（LTL）样本，以识别miRNA的表达。研究发现，这两个年龄组之间存在59个差异表达miRNA（DE-miRNA），并预测了其1,796个靶基因。KEGG功能富集分析揭示了涉及脂质代谢相关过程的八条通路，包括脂肪酸延长和AMPK信号通路。高度表达的DE-miRNA miR-193a-5p被发现参与3T3-L1前脂肪细胞分化。荧光素酶实验表明，miR-193a-5p直接结合于ACAA2的3′-非翻译区（3′-UTR）。通过构建模拟物和抑制剂载体转染3T3-L1细胞，以探讨miR-193a-5p对细胞增殖和分化的影响，本研究证实了miR-193a-5p的过表达抑制了3T3-L1前脂肪细胞的增殖，这通过CDK4和CyclinB的mRNA及蛋白表达水平的降低得到证实。CCK-8实验显示，miR-193a-5p显著抑制了细胞增殖。类似地，miR-193a-5p的过表达抑制了3T3-L1前脂肪细胞的分化及脂肪细胞特异性分子标志物的表达，导致PPARγ、C/EBPα和ACAA2的表达减少。miR-193a-5p的抑制具有相反的效果。本研究列出了与羊肉肌内脂肪沉积相关的miRNA及其潜在靶基因，旨在提升羊肉品质。