IGFs are key growth-promoting peptides that act in endocrine, autocrine, and paracrine fashion to promote different cellular functions, growth, proliferation, differentiation and survival. IGF signaling is regulated by IGFBPs which can bind IGFs with equal or greater affinity than the IGF1R in both circulation and local tissues[2, 21]. Different studies focused on the potential association of SNPs in IGFBPs gene with growth traits in domestic animals[22, 23, 24]. Few studies concerning retrotransposons insertion polymorphism were reported for the IGFBPs gene. In this study, 12 RIPs, including 7 SINE (ranged from 85bp to 301bp), 3 LINE (ranged from 159bp to 2243bp), and 2 ERV (ranged from 202bp to 327bp), in six pig IGFBPs gene were identified by comparative genomics and PCR. It is known that the repetitive portion of mammal genomes is dominated by SINEs and LINEs, followed by ERV . Our lab also proved this finding for the pig genome. Surveys about the human genome showed that transposable elements are not randomly distributed[25, 26, 27]. In our previous analysis, 2.84%, 0.63%, 35.1%, 1.38% of retrotransposons in the pig genome occupied the 5’ flanking, exon, intron, and 3’ flanking of protein-coding genes. These 12 retrotransposons in IGFBPs were mostly inserted into 5’ or 3’ flanking, only 4 retrotransposons inserted into introns and none into exons. Retrotransposons in introns can affect splicing of genes and form multiple transcripts . Thus, these insertions in 5’ flanks and introns may have function consequences for IGFBPs.
Because of artificial and natural selection, there are major differences in growth and body size among pig breeds that originated in China and Europe[29, 30]. Therefore, we investigated the population distribution of RIPs of IGFBPs in different pig breeds. An interesting distribution was found for IGFBP1-3-RIP, a LINE insertion of 2243bp, which appeared in the Chinese local pig breeds and crossbreeds of Sujiang (Duroc×Jiangquhai×Fengjing), while IGFBP3-3-RIP appeared in the European pig breeds. Selective forces can explain why some transposon elements can reach fixation in certain genomic locations. Maybe these RIPs can be used for tracing and authentication purposes for specific breeds. Some loci (IGFBP4-1-RIP, IGFBP5-1-RIP) are in HW equilibrium, while other loci (IGFBP1-2-RIP, IGFBP1-3-RIP, IGFBP2-1-RIP) for more than three pig breeds deviate from HW disequilibrium, indicating that these loci may have experienced strong selection and play biological roles in gene regulation and phenotype variation. Sujiang and its cross parent Jiangquhai are in genetic disequilibrium in IGFBP1-1-RIP, IGFBP2-1-RIP, IGFBP3-SINE-LINE-RIP, indicating that this locus may have experienced strong purification selection considering the retrotransposon insertion playing positive roles in growth. The insertion alleles were dominated in Chinese local breeds for IGFBP1-1-RIP, IGFBP1-3-RIP, and IGFBP3-2-RIP, while deletion alleles were dominated for IGFBP3-1-RIP and IGFBP3-4-RIP. Whether these RIPs affect the growth between Chinese local breeds and European pig breeds should be studied further.
Previous studies have shown that the variation in the IGFBP3 gene was closely associated with the growth traits of domestic animals. Pigs with the TAT/TAT genotype in intron 2 of IGFBP3 had higher B-point and C-point back-fat thickness than pigs with the GGC/GGC genotype and the allelic frequency of TAT in Chinese breeds was over 50% . SNP in the IGFBP3 gene was connected closely with the energy metabolism of horse and have a significant association with body weight in the Sirohi breed of the goat at different ages . When we investigated the association of IGFBP3-SINE-LINE-RIP in 5’ flanking with growth traits of Large white, we found that the IGFBP3-SINE-LINE-RIP were significantly associated with age at 100 kg body weight, corrected backfat thickness, longissimus area of Large white. There are some important regulatory elements in the 5’ flanking of protein-coding genes and insertion polymorphism by retrotransposons can modify the transcriptional activity of target genes by changing promoter activity, and further influence the biological function of target genes. The IGFBP3 expression was investigated for different genotypes by qPCR in 30-day old Sujiang piglets. The expression of IGFBP3 in SINE+/+/LINE-/- individuals in the liver, backfat was significantly higher compared to SINE-/-/LINE+/+ individuals (P < 0.05). It has been shown previously that IGFBP3 was the most dominant IGFBP in postnatal serum, binding to IGFs with the highest affinity to promote or inhibit growth both in vivo and in vitro. Large amounts of IGFBP3 caused a reduction in the free IGF levels while small amounts of IGFBP3 protected IGFs, intensifying their effects . IGFBP3 knock-down in SMMC-7721 cells can promote cell proliferation rather than affect cell apoptosis. Decreased IGFBP3 expression was related to the over-activated IGF pathway and accelerated HCC cells proliferation. In line with this, homozygotes with the SINE insertion, with a higher level of IGFBP3 in the liver and leg muscle, showed slower growth and a smaller longissimus muscle area compared with homozygote with the LINE insertion, since there was no significant difference between two genotypes for IGFBP3 level in longissimus muscle of 30-day Sujiang piglets. IGFBP3 levels in serum are age-dependent, being low at birth and increasing during childhood to reach a peak during puberty, after which serum concentration of IGFBP3 starts to decrease. The changing pattern was confirmed In EY pigs (F1 crossbreds from Erhualian boars ×Yorkshire sows) that IGFBP3 levels reached their low peak at the perinatal period and high peak value at around puberty respectively . The underlying mechanism for the effect of the spatial-temporal expression of this gene with the SINE insertion on growth traits needs to be revealed by the in-depth study.
Retrotransposons may harbor the primary types of regulatory sequences for their expression and when they are inserted upstream of protein-coding gene, they may contribute enhancer elements in a lineage-specific fashion[38, 39]. Thus, the SINE and LINE in 5’ flanking of pig IGFBP3 were furthered studied here to evaluate whether they acted as an enhancer in gene regulation. The results showed that the SINE insertion could enhance the activity of IGFBP3 promoter in cell lines C2C12, 3T3-L1 and HeLa originated from muscle cells, adipose cells, and cancer cells while LINE showed no significant effect. According to the analysis of the age distribution, the SINEA2 subfamily was one of the youngest SINE families and the sequence harbors a tRNA head which is required for RNA polymerase III dependent transcriptions. L1D14 was a relatively older subfamily compared to the younger L1D1-L1D7. Multiple studies have shown in different ways that SINEs contribute more considerably to regulatory divergence compared to LINEs. This might be the reason that the expression of IGFBP3 of liver and muscle of individuals of SINE+/+/LINE/-/- was higher than that of individuals of SINE-/-/LINE+/+. Therefore, compared with homozygote of LINE insertion, grew faster, had a larger longissimus muscle area and thicker backfat, heterozygote of SINE and LINE insertion had a thinner backfat while a faster growth, a larger longissimus muscle area, which was more adaptive to be selected in commercial breeding.