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. 2021 Jan 25;11(2):157.
doi: 10.3390/biom11020157.

MicroRNA-193a-5p Regulates the Synthesis of Polyunsaturated Fatty Acids by Targeting Fatty Acid Desaturase 1 (FADS1) in Bovine Mammary Epithelial Cells

Yongliang Fan  1   2 , Abdelaziz Adam Idriss Arbab  1   2 , Huimin Zhang  1   2 , Yi Yang  3 , Xubin Lu  1   2 , Ziyin Han  1   2 , Zhangping Yang  1   2
Affiliations

MicroRNA-193a-5p Regulates the Synthesis of Polyunsaturated Fatty Acids by Targeting Fatty Acid Desaturase 1 (FADS1) in Bovine Mammary Epithelial Cells

Yongliang Fan et al. Biomolecules. .

Abstract

Cardiovascular diseases (CVDs) are seriously threatening to human life and health. Polyunsaturated fatty acids (PUFAs) are known for their role in preventing CVDs. It is beneficial to population health to promote the content of PUFAs in bovine milk. In recent years, limited research based on molecular mechanisms has focused on this field. The biological roles of numerous microRNAs (miRNAs) remain unknown. In this study, a promising and negatively correlated pair of the miRNA (miRNA-193a-5p) and a fatty acid desaturase 1 (FADS1) gene are identified and screened to explore whether they are potential factors of PUFAs' synthesis in bovine milk. The targeted relationship between miRNA-193a-5p and FADS1 in bovine mammary epithelial cells (BMECs) is demonstrated by dual luciferase reporter assays. qRT-PCR and western blot assays indicate that both the expression of mRNA and the protein FADS1 show a negative correlation with miRNA-193a-5p expression in BMECs. Also, miR-193a-5p expression is positively correlated with the expression of genes associated with milk fatty acid metabolism, including ELOVL fatty acid elongase 6 (ELOVL6) and diacylglycerol O-acyltransferase 2 (DGAT2). The expression of fatty acid desaturase 2 (FADS2) is negatively correlated with miR-193a-5p expression in BMECs. The contents of triglycerides (TAG), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) have a significant positive correlation with the expression of FADS1 and a significant negative correlation with the expression of miR-193a-5p in BMECs. For the first time, this study confirms that miRNA-193a-5p regulates PUFAs metabolism in BMECs by targeting FADS1, indicating that miRNA-193a-5p and FADS1 are underlying factors that improve PUFAs content in bovine milk.

Keywords: FADS1; MicroRNA-193a-5p; bovine mammary epithelial cells; bovine milk; polyunsaturated fatty acids.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Principal component analysis of transcriptome sequencing data. (a) Principal component analysis of miRNA sequencing data. (b) Principal component analysis of mRNA sequencing data.
Figure 2
Figure 2
Heat maps of differentially expressed microRNAs (DE miRNAs) and genes (DEGs) in dairy cow mammary glands between early and mid-lactation. (a) Red modules represent upregulated DE miRNAs, and green modules represent downregulated DE miRNAs. (b) Red modules represent upregulated DEGs, and green modules represent downregulated DEGs.
Figure 3
Figure 3
Significant gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs. (a) The top 10 significant GO terms are respectively listed in each catalog, including biological process (BP), cellular component (CC) and molecular function (MF). (b) Circos plots show specific responses and overlapping of DEGs enriched in the top 12 significant GO terms. (c) Circos plots indicate features of DEGs enriched in the top 12 significant GO terms. (d) The top 30 significant KEGG pathways of DEGs. (e) Circos plots show specific responses and overlapping of DEGs enriched in the top 12 significant KEGG pathways. (f) Circos plots indicate the features of DEGs enriched in the top 12 significant KEGG pathways.
Figure 3
Figure 3
Significant gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DEGs. (a) The top 10 significant GO terms are respectively listed in each catalog, including biological process (BP), cellular component (CC) and molecular function (MF). (b) Circos plots show specific responses and overlapping of DEGs enriched in the top 12 significant GO terms. (c) Circos plots indicate features of DEGs enriched in the top 12 significant GO terms. (d) The top 30 significant KEGG pathways of DEGs. (e) Circos plots show specific responses and overlapping of DEGs enriched in the top 12 significant KEGG pathways. (f) Circos plots indicate the features of DEGs enriched in the top 12 significant KEGG pathways.
Figure 4
Figure 4
A network map of the pairs of DE miRNAs and DE mRNAs. The blue triangles represent the downregulated miRNAs, and the red triangles represent the upregulated miRNAs. The blue ellipses represent the downregulated mRNAs, and the red ellipses represent the upregulated mRNAs.
Figure 5
Figure 5
Relative expression of miRNA-193a-5p in BMECs after transfecting the (a) mimic and (b) inhibitor for 48 h, and the relative mRNA expression of FADS1 after transfecting the (c) mimic and (d) inhibitor of miRNA-193a-5p for 48 h (n = 9). NC represents the negative control. (e) Relative expression of the FADS1 protein in BMECs after transfecting the mimic and inhibitor of miRNA-193a-5p for 48 h. (f) Relative luciferase activity in BMECs that transfected and co-transfected with miR-193a-5p mimic and pmirGLO-FADS1-WT/pmirGLO-FADS1-MUT for 48 h (n = 9). Data are presented as mean ± standard error (SE). * represents p < 0.05, indicating significant differences between the groups.
Figure 6
Figure 6
The relative content of triglycerides (TAG) in BMECs after transfecting with the (a) mimic and (b) inhibitor (n = 9). (c) The relative mRNA expression of FADS1 after transfecting siRNA for 48 h. (d) The relative content of TAG in BMECs after co-transfecting with siRNA and the inhibitor of miRNA-193a-5p for 48 h. NC represents the negative control. Data are presented as mean ± standard error (SE). * represents p < 0.05, indicating significant differences between the groups.
Figure 7
Figure 7
Effect of miR-193a-5p mimic and inhibitor on the composition of n-3 fatty acids in BMECs (n = 6). * represents p < 0.05, indicating significant differences between the groups.
Figure 8
Figure 8
Relative expression of genes related to fatty acid metabolism after transfecting with the (a) mimic and (b) inhibitor (n = 9). NC represents the negative control. Data were presented as mean ± standard error (SE). * represents p < 0.05, indicating significant differences between the groups. (c) A protein–protein interaction (PPI) network of FADS1 and genes related to fatty acid metabolism, including FADS2, ELOVL6, DGAT1, DGAT2.
Figure 8
Figure 8
Relative expression of genes related to fatty acid metabolism after transfecting with the (a) mimic and (b) inhibitor (n = 9). NC represents the negative control. Data were presented as mean ± standard error (SE). * represents p < 0.05, indicating significant differences between the groups. (c) A protein–protein interaction (PPI) network of FADS1 and genes related to fatty acid metabolism, including FADS2, ELOVL6, DGAT1, DGAT2.
Figure 9
Figure 9
MiRNA-193a-5p regulated the synthesis of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) by targeting FADS1 in BMECs. After transfecting the miRNA-193a-5p mimics into BMECs, FADS1 expression was downregulated, which led to the upregulation of FADS2 and the downregulation of ELOVL6 and DGAT2.
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