Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays

Yueyun Ding; Shujiao Zhu; Chaodong Wu; Li Qian; DengTao Li; Li Wang; Yuanlang Wang; Wei Zhang; Min Yang; Jian Ding; Xudong Wu; Xiaodong Zhang; Yafei Gao; Zongjun Yin

doi:10.5713/ajas.18.0510

Asian-Australasian Journal of Animal Sciences (Jul 2019)

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays

Yueyun Ding,
Shujiao Zhu,
Chaodong Wu,
Li Qian,
DengTao Li,
Li Wang,
Yuanlang Wang,
Wei Zhang,
Min Yang,
Jian Ding,
Xudong Wu,
Xiaodong Zhang,
Yafei Gao,
Zongjun Yin

Affiliations

Yueyun Ding: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Shujiao Zhu: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Chaodong Wu: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Li Qian: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
DengTao Li: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Li Wang: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Yuanlang Wang: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Wei Zhang: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Min Yang: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Jian Ding: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Xudong Wu: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Xiaodong Zhang: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
Yafei Gao: Anhui Haoxiang Agriculture And Animal Husbandry Co. LTD, Bozhou, Anhui 236700, China
Zongjun Yin: Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China

DOI: https://doi.org/10.5713/ajas.18.0510
Journal volume & issue: Vol. 32, no. 7
pp. 922 – 929

Abstract

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Objective Mutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a. Methods Bioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR. Results Gel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = −0.656, p<0.05). Conclusion LDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

Published in Asian-Australasian Journal of Animal Sciences

ISSN: 1011-2367 (Print); 1976-5517 (Online)
Publisher: Asian-Australasian Association of Animal Production Societies
Country of publisher: Korea, Republic of
LCC subjects: Agriculture: Animal culture; Science: Physiology: Animal biochemistry
Website: https://www.ajas.info/

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