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. 2022 Oct 9;13(10):1828.
doi: 10.3390/genes13101828.

Deciphering the Key Regulatory Roles of KLF6 and Bta-miR-148a on Milk Fat Metabolism in Bovine Mammary Epithelial Cells

Ambreen Iqbal  1 , Haibin Yu  1 , Ping Jiang  1 , Zhihui Zhao  1
Affiliations

Deciphering the Key Regulatory Roles of KLF6 and Bta-miR-148a on Milk Fat Metabolism in Bovine Mammary Epithelial Cells

Ambreen Iqbal et al. Genes (Basel). .

Abstract

MicroRNAs (miRNAs) are non-coding RNAs that regulate the expression of their target genes involved in many cellular functions at the post-transcriptional level. Previously, bta-miR-148a showed significantly high expression in bovine mammary epithelial cells (BMECs) of Chinese Holstein cows producing high milk fat compared to those with low milk fat content. Here, we investigated the role of bta-miR-148a through targeting Krüppel-like factor 6 (KLF6) and further analyzed the role of KLF6 in regulating fat metabolism through targeting PPARA, AMPK/mTOR/PPARG, and other fat marker genes in BMECs of Chinese Holstein. The bioinformatics analysis showed that the 3' UTR of KLF6 mRNA possesses the binding sites for bta-miR-148a, which was further verified through dual-luciferase reporter assay. The BMECs were transfected with bta-miR-148a-mimic, inhibitor, and shNC, and the expression of KLF6 was found to be negatively regulated by bta-miR-148a. Moreover, the contents of triglyceride (TG), and cholesterol (CHO) in BMECs transfected with bta-miR-148a-mimic were significantly lower than the contents in BMECs transfected with bta-miR-148a-shNC. Meanwhile, the TG and CHO contents were significantly increased in BMECs transfected with bta-miR-148a-inhibitor than in BMECs transfected with bta-miR-148a-shNC. In addition, the TG and CHO contents were significantly decreased in BMECs upon the down-regulation of KLF6 through transfection with pb7sk-KLF6-siRNA1 compared to the control group. Contrarily, when KLF6 was overexpressed in BMECs through transfection with pBI-CMV3-KLF6, the TG and CHO contents were significantly increased compared to the control group. Whereas, the qPCR and Western blot evaluation of PPARA, AMPK/mTOR/PPARG, and other fat marker genes revealed that all of the genes were considerably down-regulated in the KLF6-KO-BMECs compared to the normal BMECs. Taking advantage of deploying new molecular markers and regulators for increasing the production of better-quality milk with tailored fat contents would be the hallmark in dairy sector. Hence, bta-miR-148a and KLF6 are potential candidates for increased milk synthesis and the production of valuable milk components in dairy cattle through marker-assisted selection in molecular breeding. Furthermore, this study hints at the extrapolation of a myriad of functions of other KLF family members in milk fat synthesis.

Keywords: KLF6; bovine mammary epithelial cells; bta-miR-148a; milk fat.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Cell morphology (right side) and GFP expression (left side) in BMECs under fluorescence microscope after 24 h of transfection with (A), bta-miR-148a-mimic; (B), bta-miR-148a-inhibitor; and (C), bta-miR-148a-shNC.
Figure 2
Figure 2
Relative expression of bta-miR-148a in BMECs after 24 h of transfection with bta-miR-148-mimic, bta-miR-148a-inhibitor, and bta-miR-148a-shNC. *** p < 0.001.
Figure 3
Figure 3
Relative expression of KLF6 mRNA in BMECs transfected with bta-miR-148a-mimic, bta-miR-148a-inhibitor, and bta-miR-148a-shNC. *** p < 0.001.
Figure 4
Figure 4
Bioinformatics analysis and dual-luciferase reporter assay. (A), Binding site matching in white highlighted area for bta-miR-148a and 3’-UTR KLF6 mRNA. (B), Binding site matching results in 3’-UTR KLF6 mRNA for bta-miR-148a highlighted in red. The yellow highlighted sequence represents the 3’-UTR of KLF6 mRNA. (C); Dual-luciferase reporter assay representing the target site validation. ** p < 0.01, *** p < 0.001.
Figure 5
Figure 5
Relative expression of KLF6 protein in BMECs transfected with bta-miR-148a-mimic, bta-miR-148a-inhibitor, and bta-miR-148a-shNC. (A); Western blot bands for KLF6 protein in different experimental groups. (B); Quantification of KLF6 protein expression. * p < 0.05, ** p < 0.01.
Figure 6
Figure 6
(A) TG’s relative contents in BMECs after 24 h of transfection with bta-miR-148a-mimic, inhibitor, and shNC. (B) Relative contents of CHO in BMECs after 24 h of transfection with bta-miR-148a-mimic, inhibitor, and shNC. ns = non-significant * p < 0.05, ** p < 0.01.
Figure 7
Figure 7
(A) Relative expression of KLF6 mRNA in BMECs transfected with pBI-CMV3-KLF6 and pBI-GFP-Neo-CMV3. (B) Relative expression of KLF6 mRNA in BMECs transfected with pb7SK-KLF6-siRNA1, pb7SK-KLF6-siRNA2, pb7SK-KLF6-siRNA3, and pb7SK-GFP-Neo. **** p < 0.0001.
Figure 8
Figure 8
Cell morphology and GFP expression in BMECs under a fluorescence microscope after 24 h of transfection with; (A) pBI-CMV3-KLF6, (B) pBI-GFP-Neo-CMV3, (C) pb7sk-KLF6-siRNA1, (D) pb7sk-GFP-Neo.
Figure 9
Figure 9
(A) Relative contents of TG in BMECs transfected with pBI-CMV3-KLF6 and pBI-GFP-Neo-CMV3. (B) Relative contents of TG in BMECs transfected with pb7SK-KLF6-siRNA1 and pb7SK-GFP-Neo. (C) Relative contents of TG in BMECs transfected with pBI-CMV3-KLF6 and pb7SK-KLF6-siRNA1. ** p < 0.01.
Figure 10
Figure 10
Cholesterol content in BMECs after KLF6 overexpression and down-regulation (A) Relative contents of cholesterol in BMECs transfected with pBI-CMV3-KLF6 and pBI-GFP-Neo-CMV3. (B) Relative contents of cholesterol in BMECs transfected with pb7SK-KLF6-siRNA1 and pb7SK-GFP-Neo. (C) Relative contents of cholesterol in BMECs transfected with pBI-CMV3-KLF6 and pb7SK-KLF6-siRNA1. ** p < 0.01.
Figure 11
Figure 11
Relative protein expression of KLF6 in BMECs (A) Relative protein expression of KLF6 in BMECs transfected with pBI-KLF6-CMV3, pBI-GFP-Neo-CMV3. (B) Relative protein expression of KLF6 in BMECs transfected with pb7sk-KLF6-siRNA1, pb7sk-GFP-Neo. (C) Relative protein expression of KLF6. (D) Relative protein expression of KLF6. * p < 0.05, ** p < 0.01.
Figure 12
Figure 12
Analysis of lipid metabolism-related genes (A) The KEGG pathway of AMPK/mTOR/PPARG. Arrows point out the lipid metabolism-related pathways (B) The relative mRNA expression of AMPK/mTOR/PPARG in the KLF6-KO-BMECs and normal BMECs. ns = non-significant, **** p < 0.0001.
Figure 13
Figure 13
Protein expression of lipid metabolism-related genes (A,B) The relative protein expression of AMPK/mTOR/PPARG in the KLF6-KO-BMECs and normal BMECs. * p < 0.05, ** p < 0.01.
Figure 14
Figure 14
Analysis of lipid metabolism-related genes (A) The KEGG pathway of PPARA. Arrows points out the PPARA pathways genes associated with lipogenesis and cholesterol metabolism (B) The relative mRNA expression of PPARA and its pathway related genes in the KLF6-KO-BMECs and normal BMECs. ** p < 0.01, *** p < 0.001.
Figure 15
Figure 15
Protein expression of lipid metabolism-related genes (A,B) The relative protein expression of PPARA and their genes are associated with lipogenesis and cholesterol metabolism in the KLF6-KO-BMECs and normal BMECs. ns = non-significant, ** p < 0.01, *** p < 0.001.
Figure 16
Figure 16
(A,B); mRNA expression of marker genes associated with lipogenesis in the KLF6-KO-BMECs and normal BMECs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 17
Figure 17
(A,B) String interaction of different fat metabolism-related genes of Bos Taurus.
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