Ding Yueyun, Qian Li, Wang Li, Wu Chaodong, Li DengTao, Zhang Xiaodong, Yin Zongjun, Wang Yuanlang, Zhang Wei, Wu Xudong, Ding Jian, Yang Min, Zhang Liang, Shang Jinnan, Wang Chonglong, Gao Yafei
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.
Key Laboratory of Pig Molecular Quantitative Genetics of Anhui Academy of Agricultural Sciences, Anhui Provincial Key Laboratory of Livestock and Poultry Product Safety Engineering, Institute of Animal Husbandry and Veterinary Medicine, Anhui Academy of Agricultural Sciences, Hefei, Anhui 230031, China.
Asian-Australas J Anim Sci. 2020 Feb;33(2):219-229. doi: 10.5713/ajas.19.0065. Epub 2019 Jul 1.
Considering the physiological and clinical importance of leptin receptor (LEPR) in regulating obesity and the fact that porcine LEPR expression is not known to be controlled by lncRNAs and miRNAs, we aim to characterize this gene as a potential target of SSC-miR-323 and the lncRNA TCONS_00010987.
Bioinformatics analyses revealed that lncRNA TCONS_00010987 and LEPR have SSC-miR-323-binding sites and that LEPR might be a target of lncRNA TCONS_00010987 based on cis prediction. Wild-type and mutant TCONS_00010987-target sequence fragments and wild-type and mutant LEPR 3'-UTR fragments were generated and cloned into pmiR-RB-REPORTTM-Control vectors to construct respective recombinant plasmids. HEK293T cells were co-transfected with the SSC-miR-323 mimics or a negative control with constructs harboring the corresponding binding sites and relative luciferase activities were determined. Tissue expression patterns of lncRNA TCONS_00010987, SSC-miR-323, and LEPR in Anqing six-end-white (AQ, the obese breed) and Large White (LW, the lean breed) pigs were detected by real-time quantitative polymerase chain reaction; backfat expression of LEPR protein was detected by western blotting.
Target gene fragments were successfully cloned, and the four recombinant vectors were constructed. Compared to the negative control, SSC-miR-323 mimics significantly inhibited luciferase activity from the wild-type TCONS_00010987-target sequence and wild-type LEPR-3'-UTR (p<0.01 for both) but not from the mutant TCONS_00010987-target sequence and mutant LEPR-3'-UTR (p>0.05 for both). Backfat expression levels of TCONS_ 00010987 and LEPR in AQ pigs were significantly higher than those in LW pigs (p<0.01), whereas levels of SSC-miR-323 in AQ pigs were significantly lower than those in LW pigs (p<0.05). LEPR protein levels in the backfat tissues of AQ pigs were markedly higher than those in LW pigs (p<0.01).
LEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.
鉴于瘦素受体（LEPR）在调节肥胖中的生理和临床重要性，且猪LEPR的表达不受长链非编码RNA（lncRNA）和微小RNA（miRNA）调控这一事实，我们旨在将该基因鉴定为SSC-miR-323和lncRNA TCONS_00010987的潜在靶点。
生物信息学分析显示，lncRNA TCONS_00010987和LEPR具有SSC-miR-323结合位点，基于顺式预测，LEPR可能是lncRNA TCONS_00010987的靶点。生成野生型和突变型TCONS_00010987靶序列片段以及野生型和突变型LEPR 3'-UTR片段，并将其克隆到pmiR-RB-REPORTTM-Control载体中以构建各自的重组质粒。将HEK293T细胞与SSC-miR-323模拟物或阴性对照与含有相应结合位点的构建体共转染，并测定相对荧光素酶活性。通过实时定量聚合酶链反应检测安庆六白猪（AQ，肥胖品种）和大白猪（LW，瘦肉品种）中lncRNA TCONS_00010987、SSC-miR-323和LEPR的组织表达模式；通过蛋白质免疫印迹法检测LEPR蛋白在背膘中的表达。
成功克隆了靶基因片段，并构建了四种重组载体。与阴性对照相比，SSC-miR-323模拟物显著抑制了野生型TCONS_00010987靶序列和野生型LEPR-3'-UTR的荧光素酶活性（两者p<0.01），但未抑制突变型TCONS_00010987靶序列和突变型LEPR-3'-UTR的荧光素酶活性（两者p>0.05）。AQ猪中TCONS_00010987和LEPR的背膘表达水平显著高于LW猪（p<0.01），而AQ猪中SSC-miR-323的水平显著低于LW猪（p<0.05）。AQ猪背膘组织中LEPR蛋白水平明显高于LW猪（p<0.01）。
LEPR是SSC-miR-323的潜在靶点，TCONS_00010987可能作为SSC-miR-323的海绵来调节LEPR的表达。
