Relationship among porcine lncRNA TCONS_00010987, miR-323, and leptin receptor based on dual luciferase reporter gene assays and expression patterns.

Yueyun Ding,Liang Zhang,Jinnan Shang,Yuanlang Wang,Yafei Gao,Xiaodong Zhang,Xudong Wu,Li Qian,Jian Ding,Wei Zhang,Li Wang,Zongjun Yin,Dengtao Li,Chaodong Wu,Chonglong Wang,Min Yang

doi:10.5713/ajas.19.0065

Yueyun Ding, Liang Zhang + Show 14 more

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https://doi.org/10.5713/ajas.19.0065

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Abstract

ObjectiveConsidering 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.MethodsBioinformatics 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.ResultsTarget 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).ConclusionLEPR is a potential target of SSC-miR-323, and TCONS_00010987 might act as a sponge for SSC-miR-323 to regulate LEPR expression.

Highlights

Obesity is a major source of human morbidity and mortality and is becoming more common worldwide [1]
We found that SSC-miR-323 could directly bind lncRNA TCONS_00010987 and leptin receptor (LEPR) mRNA with low free energy of binding
LncRNA TCONS_00010987 interacts with SSCmiR-323 Based on bioinformatics analyses, we identified one putative binding site between lncRNA TCONS_00010987 and SSCmiR-323 (Figure 1a)

Summary

Introduction

Obesity is a major source of human morbidity and mortality and is becoming more common worldwide [1]. Poor expression of LEPR in certain tissues was suggested to lead to leptin resistance, which is commonly associated with obesity [8]. A non-synonymous exonic polymorphism in the LEPR gene has been reported to be strongly associated with fat content in Iberian×Landrace and in Duroc×Landrace/ Large White crossbreds [10]. MiRNAs are small non-coding RNAs that can function widely by modulating multiple physiological processes such as development, immunity, cell differentiation, apoptosis, metabolism, and signal transduction [18]. These molecules regulate fat growth, mainly by acting on transcription factors

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