SINE Insertion in 5’ Flanking of IGFBP3 May Associated With Gene Expression and Phenotypic Variations
Authors: Wang Xiaoyan , An Yalong , Murani Eduard , D'alessandro Enrico , Chi Chengling , Chen Cai , Moawad Ali Shoaib , Li Kui , Wimmers Klaus , Song Chengyi 
Published: July 2021

Abstract
Background: Insulin-like growth factor binding proteins (IGFBPs), specifically binding to IGF1 and IGF2, play an important role in regulating physiological functions of insulin-like growth factors (IGFs). IGFBPs have been considered important candidate genes for economic traits due to their involvement in physiological processes related to growth and development. However, most of the current studies on genetic markers of IGFBPs have focused on SNPs, and large fragment insertion mutations such as retrotransposons have rarely been considered. In this paper, we screened the porcine IGFBP genes (IGFBP1-8) for retrotransposon insertion polymorphisms (RIPs) using bioinformatics prediction combined with the PCR-based amplification. Furthermore, for two linked RIPs their population distribution and impact on promoter activity and phenotype were further evaluated.Results: Screening of IGFBPs identified RIPs in IGFBP1-5 and IGFBP7. In total twelve predicted RIPs were confirmed by PCR. These RIPs were detected in different breeds with an uneven distribution among them. By linkage genetic analysis and PCR verification, IGFBP3-1-RIP and IGFBP3-2-RIP are completely linked, showing only three genotypes, SINE+/+/LINE-/-, SINE-/-/LINE+/+ and SINE+/-/LINE-/+. The age of 100 kg body weight and longissimus muscle thickness of Large white individuals of SINE+/+/LINE-/-genotype were significantly (P<0.05) higher than those of SINE+/-/LINE+/- genotype and SINE-/-/LINE+/+ genotype. However, the longissimus muscle thickness and corrected backfat thickness of SINE+/+/LINE-/- individuals were significantly (P<0.05) thinner than those of SINE+/+/LINE-/- genotype. The expression of the IGFBP3 gene in liver, leg muscles and backfat of 30-day Sujiang piglets with SINE+/+/LINE-/- genotype were significantly higher (P<0.05) than those with SINE-/-/LINE+/+ genotype by the real-time quantitative polymerase chain reaction (qPCR). Further study was conducted to confirm the effect of SINE and LINE insertion on the promoter activity of IGFBP3. First, the core promoter region of the IGFBP3 gene was identified locating within 482bp upstream of ATG by using the dual-luciferase activity assay. Then SINE and LINE were combined with 482bp fragment to construct a recombinant vector respectively based on the PGL3-Promoter-Enhancer. The recombinant vector was transfected into C2C12, 3T3-L1, and Hela cells. The detection of the dual-luciferase reporter gene revealed that only SINE insertion was significantly increased (P<0.05) promoter activity of the IGFBP3 gene, indicating that the SINE may act as an enhancer to regulate the promoter activity of the IGFBP3 gene. Conclusions: Overall, this study identified 12 RIPs in IGFBP gene clusters, which provided useful markers for genetic analysis of pig populations. Furthermore, based on the dual-luciferase activity assay in cells and association analysis, the linked genetic variations generated by SINE and LINE insertions in 5’ flanking of IGFBP3 may associated with variations of gene expression and phenotype.