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. 2021 Jun 23;11(7):1871.
doi: 10.3390/ani11071871.

SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor

Cai Chen  1 , Yao Zheng  1 , Mengli Wang  1 , Eduard Murani  2 , Enrico D'Alessandro  3 , Ali Shoaib Moawad  1 , Xiaoyan Wang  1 , Klaus Wimmers  2 , Chengyi Song  1
Affiliations

SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor

Cai Chen et al. Animals (Basel). .

Abstract

The genetic diversity of the GH/IGF axis genes and their association with the variation of gene expression and phenotypic traits, principally represented by SNPs, have been extensively reported. Nevertheless, the impact of retrotransposon insertion polymorphisms (RIPs) on the GH/IGF axis gene activity has not been reported. In the present study, bioinformatic prediction and PCR verification were performed to screen RIPs in four GH/IGF axis genes (GH, GHR, IGF1 and IGF1R). In total, five RIPs, including one SINE RIP in intron 3 of IGF1, one L1 RIP in intron 7 of GHR, and three SINE RIPs in intron 1, intron 5 and intron 9 of GHR, were confirmed by PCR, displaying polymorphisms in diverse breeds. Dual luciferase reporter assay revealed that the SINE insertion in intron 1 of GHR significantly repressed the GHR promoter activity in PK15, Hela, C2C12 and 3T3-L1 cells. Furthermore, qPCR results confirmed that this SINE insertion was associated with a decreased expression of GHR in the leg muscle and longissimus dorsi, indicating that it may act as a repressor involved in the regulation of GHR expression. In summary, our data revealed that RIPs contribute to the genetic variation of GH/IGF axis genes, whereby one SINE RIP in the intron 1 of GHR may decrease the expression of GHR by acting as a repressor.

Keywords: GH/IGF axis; GHR; SINE; pig; retrotransposon insertion polymorphism; structural variation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PCR detection of RIPs. 1. Bama miniature, 2. Wuzhishan, 3. Congjiangxiang, 4. Jiangkouluobo, 5. Tibetan, 6. Meishan, 7. Sujiang, 8. Ningxiang, 9. Daweizi, 10. Duroc, 11. Yorkshire, 12. Landrace. M: DNA marker DL2000.
Figure 2
Figure 2
Effect of the GHR-RIP10 SINE insertion on GHR promoter activity. (A) The SINE sequence in GHR-RIP10 site with and without SINE insertion and its location on GHR gene. (B) The conservation analysis for the GHR-RIP10 locus. (C) GHR promotor detection results in Hela, PK15, C2C12 and 3T3-L1 cells by dual-luciferase reporter assay. (D) Impact of SINE insertion of GHR-RIP10 on the promoter activity of GHR by dual-luciferase reporter assay. * showed p < 0.05; ** showed p < 0.01.
Figure 3
Figure 3
Association of SINE insertion genotype with the expression of GHR in tissues of 30-day old piglets. (A) Genotype result for 13 Sujiang individuals. (B) RT-qPCR results for GHR expression. * showed p < 0.05; ** showed p < 0.01.
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