Relationship among porcine lncRNA TCONS_00010987, miR-323, and leptin receptor based on dual luciferase reporter gene assays and expression patterns
Relationship among porcine lncRNA TCONS_00010987, miR-323, and leptin receptor based on dual luciferase reporter gene assays and expression patterns
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基于双荧光素酶报告基因检测和表达模式的猪 lncRNA TCONS_00010987、miR-323 和瘦素受体之间的关系
DOI：
10.5713/ajas.19.0065
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发表时间：
2020-02-01
期刊：
影响因子：
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通讯作者：
Gao, Yafei
中科院分区：
文献类型：
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作者：
Ding, Yueyun;Qian, Li;Gao, Yafei
目的：考虑到瘦素受体（LEPR）在调节肥胖中的生理学和临床重要性以及猪LEPR表达未知受lncRNA和miRNAs控制的事实，我们旨在将该基因表征为SSC-miR-323和lncRNA TCONS_00010987的潜在靶标。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。产生野生型和突变体TCONS_00010987-靶序列片段以及野生型和突变体LEPR 3 ′-UTR片段，并克隆到pmiR-RB-REPORT（TM）-对照载体中以构建相应的重组质粒。将HEK 293 T细胞与SSC-miR-323模拟物或具有携带相应结合位点的构建体的阴性对照共转染，并测定相对荧光素酶活性。采用实时荧光定量PCR方法检测安庆六端白色猪（AQ）和安庆大白色猪（LW）的lncRNA TCONS_00010987、SSC-miR-323和LEPR的组织表达谱;采用Western blotting方法检测LEPR蛋白在背膘中的表达。与阴性对照相比，SSC-miR-323模拟物显著抑制野生型TCONS_00010987靶序列和野生型LEPR-3 '-UTR的荧光素酶活性（p
Objective: 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.Methods: 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-REPORT (TM)-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.Results: 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 wildtype LEPR-3'-UTR (p