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

Abstract

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<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). Conclusion: 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.

Identification of Signatures of Selection by Whole-Genome Resequencing of a Chinese Native Pig

“…In our previous studies, the average backfat thickness and the intramuscular fat content in the longissimus dorsi muscle of ASP at about 100 kg were measured, and the value were 46.01 ± 2.55, 6.54 ± 0.81 (mean ± SD, n = 6) separately (Wang et al, 2020). Based on molecular and multiomics level, we have identified some genes, microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) played an important role in meat quality, lipid metabolism, and fat deposition (Zhang et al, 2015;Hu et al, 2019;Ding et al, 2020;Wang et al, 2020). However, the genetics basis of the characteristics in ASP, particularly at the genomic level, remains largely unknown.…”