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Asian-Australas J Anim Sci > Accepted Articles
DOI: https://doi.org/10.5713/ajas.18.0510    [Accepted] Published online January 3, 2019.
Relationship between porcine miR-20a and its putative target LDLR based on dual luciferase reporter gene assays
Yueyun Ding1, Shujiao Zhu1, Chaodong Wu1, Li Qian1, Dengtao Li1, Li Wang1, Yuanlang Wang1, Wei Zhang1, Min Yang1, Jian Ding1, Xudong Wu1, Xiaodong Zhang1, Yafei Gao2, Zongjun Yin1,*
1Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui 230036, China
2Anhui Haoxiang agriculture and animal husbandry co. LTD, Bozhou, Anhui 236700, China
Correspondence:  Zongjun Yin, Tel: +86-13866191465, Fax: 0551-65786328, Email: yinzongjun@ahau.edu.cn
Received: 6 July 2018   • Revised: 19 September 2018   • Accepted: 13 December 2018
Abstract
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′-UTR fragments were generated by 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 (P < 0.01), but not mutant LDLR-3′-UTR-driven (P > 0.05), 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).

Conclusions

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.
Keywords: SSC-miRNA-20a; LDLR; pGL3-Control Vector; pMR-mCherry Vector; Dual Luciferase Reporter Gene Assay System
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