Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays

  • Ding, Yueyun (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Zhu, Shujiao (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Wu, Chaodong (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Qian, Li (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Li, DengTao (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Wang, Li (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Wan, Yuanlang (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Zhang, Wei (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Yang, Min (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Ding, Jian (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Wu, Xudong (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Zhang, Xiaodong (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University) ;
  • Gao, Yafei (Anhui Haoxiang Agriculture And Animal Husbandry Co. LTD) ;
  • Yin, Zongjun (Anhui Provincial Laboratory of Local Animal Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University)
  • Received : 2018.07.06
  • Accepted : 2018.12.13
  • Published : 2019.07.01
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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'-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 premiR-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 LDLR3'-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). 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.

Keywords

References

  1. Bell DA, Bender R, Hooper AJ, et al. Impact of interpretative commenting on lipid profiles in people at high risk of familial hypercholesterolaemia. Clin Chim Acta 2013;422:21-5. https://doi.org/10.1016/j.cca.2013.03.027
  2. Rodriguez-Perez JM, Posadas-Sanchez R, Blachman-Braun R, et al. HHIPL-1 (rs2895811) gene polymorphism is associated with cardiovascular risk factors and cardiometabolic parameters in Mexicans patients with myocardial infarction. Gene 2018;663:34-40. https://doi.org/10.1016/j.gene.2018.04.030
  3. Vilahur G, Padro T, Badimon L. Atherosclerosis and thrombosis: insights from large animal models. J Biomed Biotechnol 2011;2011:Article ID 907575. http://dx.doi.org/10.1155/2011/907575
  4. Wang Y, Du Y, Shen B, et al. Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA. Sci Rep 2015;5:8256. http://dx.doi.org/10.1038/srep08256
  5. Chen F, Wang Y, Yuan Y, et al. Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting. J Genet Genomics 2015;42:437-44. https://doi.org/10.1016/j.jgg.2015.05.002
  6. Zeng Z, Chen R, Liu C, Yang H, Chen C, Huang L. Evaluation of the causality of the low-density lipoprotein receptor gene (LDLR) for serum lipids in pigs. Anim Genet 2015;45:665-73. https://doi.org/10.1111/age.12183
  7. Goldstein JL, Brown MS. The LDL receptor. Arterioscler Thromb Vasc Biol 2009;29:431-8. https://doi.org/10.1161/ATVBAHA.108.179564
  8. Huang L, Hua Z, Xiao H, et al. CRISPR/Cas9-mediated $ApoE^{-/-}$ and $LDLR^{-/-}$ double gene knockout in pigs elevates serum LDL-C and TC levels. Oncotarget 2017;8:37751-60. https://doi.org/10.18632/oncotarget.17154
  9. Pena RN, Canovas A, Varona L, et al. Nucleotide sequence and association analysis of pig apolipoprotein-B and LDLreceptor genes. Anim Biotechnol 2009;20:110-23. https://doi.org/10.1080/10495390902892518
  10. Brown MS, Goldstein JL. How LDL receptors influence cholesterol and atherosclerosis. Sci Am 1984;251:58-66. https://doi.org/10.1038/scientificamerican1184-58
  11. Momtazi AA, Banach M, Pirro M, Stein EA, Sahebkar A. MicroRNAs: new therapeutic targets for familial hypercholesterolemia? Clin Rev Allergy Immunol 2018;54:224-33. https://doi.org/10.1007/s12016-017-8611-x
  12. Quiat D, Olson EN. MicroRNAs in cardiovascular disease: from pathogenesis to prevention and treatment. J Clin Invest 2013;123:11-8. https://doi.org/10.1172/JCI62876
  13. Goedeke L, Aranda JF, Fernandezhernando C. microRNA regulation of lipoprotein metabolism. Curr Opin Lipidol 2014;25:282-8. https://doi.org/10.1097/MOL.0000000000000094
  14. Goedeke L, Wagschal A, Fernandezhernando C, Naar AM. miRNA regulation of LDL-cholesterol metabolism. Biochim Biophys Acta 2016;1861:2047-52. https://doi.org/10.1016/j.bbalip.2016.03.007
  15. Aryal B, Singh AK, Rotllan N, Price N, Fernandezhernando C. microRNAs and lipid metabolism. Curr Opin Lipidol 2017;28:273-80. https://doi.org/10.1097/MOL.0000000000000420
  16. Zhang XD, Feng YF, Zhang X, et al. Differential expression of miR-145, miR-429 and its target genes in partial reproductive tissues of swine with high and low litter size. Ann Anim Sci 2017;17:671-81. https://doi.org/10.1515/aoas-2016-0082
  17. Soundarya PK, Nirmala P, Ashok KP, Krishna PT. Effect of Lutein in the expression of $PPAR{\alpha}$ and LDLR in hypercholesterolemic male Wistar rats. Int J Basic Clin Pharmacol 2018;7:684-90. https:// doi.org/10.18203/2319-2003.ijbcp20181170
  18. Goedeke L, Rotlan N, Canfran-Duque A, et al. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels. Nat Med 2015;21:1280-9. https://doi.org/10.1038/nm.3949
  19. Jiang H, Zhang J, Du Y, et al. microRNA-185 modulates low density lipoprotein receptor expression as a key posttranscriptional regulator. Atherosclerosis 2015;243:523-32. https://doi.org/10.1016/j.atherosclerosis.2015.10.026
  20. Alvarez ML, Khosroheidari M, Eddy E, Done SC. MicroRNA-27a decreases the level and efficiency of the ldl receptor and contributes to the dysregulation of cholesterol homeostasis. Atherosclerosis 2015;242:595-604. https://doi.org/10.1016/j.atherosclerosis.2015.08.023
  21. Wagschal A, Najafi-Shoushtari SH, Wang L, et al. Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis. Nat Med 2015;21:1290-7. https://doi.org/10.1038/nm.3980
  22. Wang D, Wang Y, Ma J, et al. MicroRNA-20a participates in the aerobic exercise-based prevention of coronary artery disease by targeting PTEN. Biomed Pharmacother 2017;95:756-63. https://doi.org/10.1016/j.biopha.2017.08.086
  23. Liang B, Wang X, Song X, et al. MicroRNA-20a/b regulates cholesterol efflux through post-transcriptional repression of ATP-binding cassette transporter A1. Biochim Biophys Acta Mol Cell Biol Lipids 2017;1862:929-38. https://doi.org/10.1016/j.bbalip.2017.06.002
  24. Brummer A, Hausser J. MicroRNA binding sites in the coding region of mRNAs: extending the repertoire of post-transcriptional gene regulation. Bioessays 2014;36:617-26. https://doi.org/10.1002/bies.201300104

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