A SNP in the 3′UTR of the porcine IGF-1 gene interacts with miR-new14 to affect IGF-1 expression, proliferation and apoptosis of PK-15 cells

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Highlights

  • One SNP was screened in IGF-1 3′UTR between Bama Xiang pigs and Large White pigs.

  • The SNP downregulated IGF-1 expression by affecting the binding of miR-new14.

  • miR-new14 suppressed cell proliferation and promoted cell apoptosis of PK-15 cells.

Abstract

The kidney of miniature pigs has been considered the most likely potential kidney source for patients needing kidney transplantation. Insulin-like growth factor 1 (IGF-1) is involved in regulating the growth of miniature pigs and inducing growth of kidneys. There are evidences showing that the SNPs in the 3′UTR of a gene may affect the gene expression by affecting the binding to a miRNA target site. In this study, one SNP (rs34142920) was screened in the IGF-1 3′UTR between 2 different body types of porcine breeds, Bama Xiang (BX) pigs, a miniature pig breed, and Large White (LW) pigs by sequencing. The secondary structure of the IGF-1 3′UTR mRNA containing the SNP in BX pigs is different from that of LW pigs. We then verified that there was a porcine miRNA (miR-new14) binding to this SNP in the 3′UTR of IGF-1 via cotransfecting the 3′UTR from the 2 breeds and miR-new14. We further found that the SNP downregulated mRNA and protein levels of IGF-1 by affecting the binding of miR-new14. To understand the function of miR-new14 in porcine kidney (PK-15) cells and its mechanism, cell proliferation and cell apoptosis assays were employed and results showed that proliferation viability of PK-15 cells was weakened and the apoptotic percentage of PK-15 cells was higher in the miR-new14 group. Porcine miRNA reduced the mRNA expression of AKT/ERK and protein levels of p-AKT/p-ERK. These results suggested that the expression of IGF-1 is influenced by this SNP and miR-new14 and that miR-new14 may suppress cell proliferation and promote cell apoptosis in PK-15 cells through regulating AKT and ERK signaling pathways, in which IGF-1 is involved.

Introduction

Kidney transplantation is one of the most effective treatments for patients with end-stage kidney disease. However, the number of patients requiring kidney transplantation is far more than available donor organs [1,2], which has stimulated research into the use of animal organs for transplantation. Recently, miniature pigs were considered as the most likely potential source of donor tissue because their physiological and immune systems are close to those of humans [1,3]. In addition, studies have indicated that larger donor kidney size adjusted to the recipient's body was associated with better long-term graft function [4,5]. Research has suggested that IGF-1, a circulating hormone and tissue growth factor, partly induces growth of the kidney [6,7]. At the same time, IGF-1 plays an irreplaceable role in normal brain development, neuronal growth, and cellular proliferation and differentiation [8,9]. Moreover, IGF-1 is known to be involved in the AKT and ERK signaling pathways [9,10].

MicroRNAs (miRNAs), a class of small noncoding RNAs (∼22 nucleotides), are known to regulate gene expression at a post-transcriptional level and play a critical role in a variety of cellular functions and physiological activities by targeting genes [11,12]. In addition, binding can be affected by SNPs at the miRNA target site [13,14]. Moreover, there was a study showing that a SNP suppressed the expression of TAP2 by interacting with hsa-miR-1270 [15]. We found that miR-new14 was a new potential porcine miRNA that bound to the 3′UTR of the IGF-1 gene, which includes a SNP in miniature pigs [16].

Therefore, the aim of this study was to verify whether miR-new14 targeted the 3′UTR of the IGF-1 gene and to clarify the effect of the miR-new14 on IGF-1 expression, proliferation, and apoptosis of PK-15 cells. Furthermore, the potential effects on proliferation and apoptosis were investigated.

Section snippets

Experimental animals and sample collection

All animal experiments were performed in accordance with the rules and regulations of the Animal Care and Experimentation Committee of Jilin University (Changchun, China). DNA samples were obtained from ear marginal tissues of 46 Bama Xiang pigs (BX) and 114 Large White pigs (LW); all the pigs were randomly selected and had no common ancestor within 3 generations. The RNA samples were obtained from the kidneys of 6 pigs for each breed. Porcine genomic DNA and RNA were both extracted according

SNP in 3′UTR of IGF-1 affected gene expression

A SNP (rs34142920) was screened at position c.674C > T in the 3′UTR of IGF-1 for LW and BX pigs (Fig. 1A). The genotype of all of LW pigs was TT homozygous, whereas the genotype of BX pigs was CT heterozygous, and the dominant allele for this SNP in BX pigs was the C allele (the frequency of the C allele was about 70%). As shown in Figure 1B, we found that the psiCHECK-2-3′UTR-B construct had significantly lower luciferase activity compared with the psiCHECK-2-3′UTR-L construct in PK-15 cells (P

Discussion

IGF-I, at least in part, mediates kidney growth in healthy infants, and IGF-I could be involved in a pathway which programs the renal system [7]. Moreover, there are many studies indicating that SNPs in the 3′UTR of a gene could affect the gene's expression; for example, a SNP in the 3′UTR of the bovine HMGB1 gene led to significant differences in the relative expression of HMGB1 mRNA in cows with the genotype GG and the genotype AA [18]. Another study demonstrated that a 3′UTR SNP affected

CRediT authorship contribution statement

Jie Song: Methodology, Data curation, Writing - original draft, Writing - review & editing. Linlin Hao: Methodology, Data curation, Writing - original draft. Wenzhen Wei: Supervision, Validation. Rui Yang: Supervision, Validation. Chunli Wang: Supervision, Validation. Hongwei Geng: Resources. Haoyang Li: Resources. Siyao Wang: Investigation. Guanhong Lu: Investigation. Tianqi Feng: Investigation. Xiaotong Sun: Investigation. Songcai Liu: Methodology. Gang Wang: Methodology, Data curation.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (31672514), China, Technological Development Plan of Jilin Province (20170101024JC), China, and Project funded by China Postdoctoral Science Foundation (2018M643106), China.

Authors' contributions: J.S., L.H., Y.C., G.W., and S.L. designed experiments; J.S. and L.H. carried out experiments; J.S., L.H., and G.W. analyzed experimental results; H.L. and H.G. collected all of animal samples; T.F., S.W., G.L., and

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