Ding Yueyun, Zhu Shujiao, Wu Chaodong, Qian Li, Li DengTao, Wang Li, Wang Yuanlang, Zhang Wei, Yang Min, Ding Jian, Wu Xudong, Zhang Xiaodong, Gao Yafei, Yin Zongjun
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.
Anhui Haoxiang Agriculture And Animal Husbandry Co. LTD, Bozhou, Anhui 236700, China.
Asian-Australas J Anim Sci. 2019 Jul;32(7):922-929. doi: 10.5713/ajas.18.0510. Epub 2019 Jan 3.
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.
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.
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).
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.
低密度脂蛋白受体（LDLR）的突变参与了心脏代谢疾病，如猪的家族性高胆固醇血症，该受体编码一种对哺乳动物胆固醇稳态和脂质代谢至关重要的蛋白质。尽管微小RNA（miRNA）可以控制LDLR的调节，但其在猪的心脏代谢疾病中对循环胆固醇和脂质水平的影响尚不清楚。我们旨在鉴定和分析LDLR作为SSC-miR-20a的潜在靶基因。
生物信息学分析预测猪LDLR是SSC-miR-20a的靶标。通过聚合酶链反应（PCR）生成野生型和突变型LDLR 3'-非翻译区（UTR）片段，并分别克隆到pGL3-Control载体中，构建pGL3 Control LDLR野生型-3'-UTR和pGL3 Control LDLR突变型-3'-UTR重组质粒。通过将猪pre-miR-20a编码序列插入pMR-mCherry的HindIII和BamHI位点之间构建miR-20a表达质粒，并通过测序确认构建体。将miR-20a表达或pMR-mCherry对照质粒与携带相应3'-UTR的构建体共转染HEK293T细胞，并测定相对荧光素酶活性。使用逆转录定量PCR分析猪肝脏组织中miR-20a和LDLR mRNA的相对表达水平及其表达水平的相关性。
凝胶电泳和测序表明靶基因片段成功克隆，并且成功构建了三种重组载体。与pMR-mCherry相比，miR-20a表达载体显著抑制野生型LDLR-3'-UTR驱动的（p<0.01），但不抑制突变型LDLR-3'-UTR驱动的（p>0.05）荧光素酶报告活性。此外，miR-20a和LDLR在猪肝脏组织中表达水平相对较高。Pearson相关性分析显示猪肝脏miR-20a和LDLR水平呈显著负相关（r = -0.656，p<0.05）。
LDLR是miR-20a的潜在靶标，miR-20a可能直接结合LDLR 3'-UTR以转录后抑制表达。这些结果对理解猪心血管疾病的发病机制和进展具有重要意义。
