Novel alternative splice variant of IGF-1R and its mRNA expression patterns in BaMa and Landrace pigs

Sus scrofa, especially the miniature breeds, has widely been used as biomedical model for human disease researches because of the similarity in physiological characteristics, as well as in anatomy and organ constructions with those of human (Swindle et al., 2012). The Chinese local breed, BaMa pigs, are highly inbred, genetically stable and with mini-body size (adult mean body weight, 40kg) (Wu et al., 2001; Liu et al., 2010). In this study, BaMa pigs were chosen as the representative model of the miniature pigs and Landrace pigs (adult mean body weight, 250 kg) as large pig breeds. The two pig breeds were opposite in many growth characters but they were all genetically stable and their genetic backgrounds were clear (Cheng et al., 2016).
 
Insulin-like growth factor 1 receptor (IGF-1R) is an important member of the IGFs (Insulin-like growth factors) system which is important to animal growth and development (Baker et al., 1993). LeRoith et al., (1995) indicated that IGF-1R was a widely existing cell-surface receptor and expressed in liver, muscle, bone and other tissues. Guntur et al., (2013) showed that IGF-1R played a role in many crucial biological processes including cell proliferation, differentiation and survival.
 
Alternative splicing (AS) is a common event in eukaryotes (Li et al., 2012) and there are many alternatively spliced protein-coding genes in multicellular organisms (Ast et al., 2004). AS leads to the emergence of many different mature transcripts from the same primary RNA sequence (Sammeth et al., 2008). Yang et al., (2015) found that many genes had alternative splicing variants in livestock. Zhou et al., (2014) indicated that alternative splicing events played an important role in the development of gluconeogenesis. Some studies indicated that a single-nucleotide polymorphism (SNP) site may result in aberrant transcript expression. For example, the intron 18 of MST1R (RON) gene had a C to A SNP, which resulted in alternatively spliced cDNA lacking exon 19 of MST1R (Angeloni et al., 2003).
 

The animal experimental protocol was approved by the Use and Care Ethics Committee of Jilin University (Changchun, China). Liver, cartilage and muscle tissues were collected over a seven-day period following the birth of BaMa (n=5) and Landrace (n=5) pigs respectively. All tissues were immediately frozen in liquid nitrogen after slaughter.
 
RNA extraction, Synthesis of cDNA first strand and RT-PCR amplification
 
Total RNA was extracted from the three tissues using Trizol (Invitrogen Corp, Carlsbad, CA) following the manufacturer’s protocol. The amount of 2.5 μg of RNA was reverse transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher). To detect IGF-1R splicing variants, 4 pairs of primers (Primers-1 to Primers-4, Table 1 and Fig 1) were synthesized and used in the RT-PCR amplification. RT-PCR amplification was carried out in a total volume of 20 μL with 50 ng cDNA template, 10 μL PrimeSTAR HS (Premix) (Takara), 10 pmol of each primer. After pre-denaturation for 5 min at 95°C, 30 cycles of a denaturation step at 95°C for 30 s, an annealing step at 58°C for 30 s and an elongation step at 72°C for 30 s were followed with a final extension of 5 min at 72°C. All RT-PCR products were electrophoresed on 2% agarose gels (BLOWEST, Beijing, China). The purified products obtained were sent to the Genewiz (Beijing, China) for sequencing.
 
 

 

 
Measurement of IGF-1R ISO01 and IGF-1R AS02 mRNA levels using qRT-PCR
 
qRT-PCR (quantitative real-time PCR) was performed in triplicate for each sample with the Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific, MA, USA) in an ABI PRISM 7900HT thermocycler (Applied Biosystems, USA). We chose the ACTB (GenBank: XM_003124280.5) as control, the primer (Primers-ACTB) was listed in (Table 1). A 20μL qRT-PCR reaction mixture contained 50ng cDNA, 10 pmol of each primer, 10ìL 2× SYBR Green PCR Master Mix. The qRT-PCR reaction conditions were as follows: denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s. Expression levels of each sample were recorded as threshold cycle (Ct). Data analysis was performed using the comparative Ct (2 -DDCt) method (Meenakshi et al., 2017; Singh et al., 2017).
 
DNA preparation and PCR amplification
 
Ear notch samples were collected over a seven-day period following the birth of BaMa (n=100) and Landrace (n=100) pigs respectively. All individuals were selected randomly, equal numbers of males and females were obtained. Phenol-chloroform method (Aneet Kour et al., 2017) with minor modifications was used for DNA isolation from ear notch samples of pigs. A DNA pool was prepared to detect the SNP located in the IGF-1R (GenBank: NM_ 214172.1) intron 19, which was near the splice site. The pool contained mixtures of equal amounts of the individual genomic DNA for each pig. PCR amplification was carried out in a total volume of 50 μL with 100 ng DNA template, 25 μL PrimeSTAR HS (Premix)(Takara), 20 pmol of each primer. PCR conditions included initial heating at 95°C for 5 min, 30 cycles of 30 s for denaturation at 95°C, 30 s for annealing at 59°C, and 30 s for extension at 72°C, followed by a 5 min extension at 72°C. The primer (Primers-SNP) was shown in (Table 1).
 
Statistical analysis
 
The results are expressed as the mean ± standard deviation of at least three separate experiments performed in triplicate. Relative quantification of IGF-1R gene mRNA expression was done using the standard curve method for relative real-time PCR (Ast et al., 2004). Values are expressed as mean ± standard deviation (SEM) and the statistical significance between groups was determined by multiple T-tests (GraphPad Prism 6). P <0.05 (*) were considered statistically significant. Genotypes of known SNP were determined by sequencing and the individual genotypes and allele frequencies were calculated and analyzed separately. The Chi-square test (c²) was performed using SPSS software (Version 17.0, SPSS, Inc. USA) and P <0.05 was defined as statistically significant.

Identification of alternative splicing variants of the porcine IGF-1R
 
Four isoforms of IGF-1R gene for pigs were predicted by the Ensembl genome browser (http://may2017.archive. ensembl.org/Sus_scrofa/Gene/Summary?db=core; g=ENSSSCG00000004812), which we refer to as IGF-1R ISO01 (ENSSSCT00000005311.3), IGF-1R AS02 (ENSSS CT00000033584.1), IGF-1R AS03 (ENSSSCT000000 35767.1) and IGF-1R AS04 (ENSSSCT00000034979.1). According to the Ensembl genome browser, the alternative splicing patterns of the IGF-1R gene in pigs were shown in (Fig 1) (Zhang et al., 2015). In this study, only two of the four IGF-1R gene isoforms, IGF-1R ISO01 and IGF-1R AS02, were found in the liver, muscle and cartilage tissues of pigs, between which IGF-1R AS02 was a novel IGF-1R splice variant. Using primers Primers-1 and Primers-2, the reaction resulted in the amplification of 146bp and 263bp RT-PCR products, respectively (Fig 2). The sequence analysis of 263bp RT-PCR product was coincident with the porcine IGF-1R AS02 mRNA which was displayed in the Ensembl genome browser database. Alignment of multiple sequences of IGF-1R ISO01 and IGF-1R AS02 mRNA was shown in (Fig 3). Corresponding PCR products of IGF-1R AS03 and IGF-1R AS04 were not detected in these tissues (data not shown), which may indicate that they did not exist in these tissues or their expression was too low to be detected. Therefore, we only quantitatively detected the expression of IGF-1R ISO01 and IGF-1R AS02 using qRT-PCR. According to the data predicted through the Ensembl genome browser, IGF-1R AS02 splice variant did not code for protein. IGF-1R AS03 and IGF-1R AS04 splice variants encoded 103aa and 70aa respectively, but the biotype of IGF-1R AS04 was nonsense-mediated decay (Brogna and Wen 2009). All of the proteins encoded by the IGF-1R AS03 and IGF-1R AS04 splice variants lost the conserved domain of the IGF-1R gene.
 

 

 
Expression of IGF-1R ISO01 and IGF-1R AS02 in different pig breeds and tissues
 
To confirm tissue expression profiles of IGF-1R ISO01 and IGF-1R AS02, qRT-PCR was performed in the collected tissues (liver, muscle and cartilage) from BaMa and Landrace pigs (Fig 4). Both IGF-1R ISO01 and IGF-1R AS02 were expressed in the liver, muscle and cartilage tissues from two pig breeds and IGF-1R ISO01 had higher expression levels than IGF-1R AS02 in all of these tissues. In cartilage, the expression level of IGF-1R ISO01 mRNA in BaMa was significantly higher than that in Landrace (P<0.05), whereas the expression level of IGF-1R AS02 mRNA in BaMa was much lower than that in Landrace (P<0.05). For the expression of IGF-1R ISO01, both BaMa and Landrace pigs showed no significantly different in liver and muscle (P<0.05), but it was significantly higher in the liver and muscle than in the cartilage (P<0.05) (Fig 4B). In BaMa pigs, IGF-1R ISO01 was 1.86-fold (P<0.05) more expressed in the liver than in cartilage and 1.76-fold (P<0.05) more abundantly in muscle than in cartilage. In Landrace pigs, the expression levels of IGF-1R ISO01 in liver and muscle was 3.29-fold (P<0.05) and 3.04-fold (P<0.05) higher than that in cartilage respectively. Surprisingly, there was no significantly different in the mRNA expression level of the IGF-1R AS02 in liver and muscle tissues among the two pig breeds, but it was differently expressed in cartilage tissue among the two pig breeds (P<0.05) (Fig 4C). In BaMa pigs, the expression level of IGF-1R AS02 in cartilage was 6.28-fold (P<0.05) higher than that in muscle and 23.89-fold (P<0.05) higher than that in liver. In Landrace pigs, the expression of IGF-1R AS02 in cartilage was 3.83-fold (P<0.05) and 37.38-fold (P<0.05) higher than that in muscle and liver respectively. In addition, we found that the expressions of IGF-1R AS02 were negatively correlated with that of IGF-1R ISO01 in the three tissues.
 

 
 
In our study, it showed that the expression levels of IGF-1R ISO01 were negatively correlated with that of IGF-1R AS02 in pigs, which meant the expression level of IGF-1R AS02 may directly affect the expression of the IGF-1R ISO01. Moreover, the postnatal increase of muscle mass depended on the interaction between IGF-1 and its receptor, IGF-1R, which mediated the transduction of metabolic signal in the GH/IGF pathways to adjust bone growth and protein synthesis (Wang et al., 2008; Delafontaine et al., 2004). Liu et al., (1993) demonstrated that mice lacking functional IGF-1R were born with the weight less than half the normal and died invariably at birth. Therefore, the differential expression of IGF-1R caused by the AS in pigs could influence muscle growth.
 
Of the cartilage tissue, it was noteworthy that the expression level of IGF-1R ISO01 in BaMa pig was significantly higher than that in Landrace pig (P<0.05). This may indicate that IGF-1R ISO01 mainly affected the growth of BaMa cartilage among newborns. In other words, the expression level in cartilage may be one of the factors which affected the porcine growth. We found that the expression levels of IGF-1R ISO01 and IGF-1R AS02 in muscle and liver were significantly different from those in cartilage of BaMa and Landrace pigs, and there was a tissue-specific expression pattern of IGF-1R ISO01 and IGF-1R AS02 in pigs. Some studies had shown that ASs formed by primary RNA splicing may have different functions (Delafontaine et al., 2004; Charge et al., 2004), and sometimes the expression of these ASs was also negatively correlated. That is to say, the gene may change its transcriptional level through alternative splicing, thereby affecting its function. In our previous studies, IGF-1R protein in Landrace pig was significantly higher than that in BaMa pig at birth (Cheng et al., 2016). However, in this study, the mRNA expression level of IGF-1R in Landrace and BaMa pigs showed no significantly different. The expression of IGF-1R protein and its mRNA was partially inconsistent, which may be the result of later modifications of the process of translation.
 
SNP validation and genotype frequency estimation
 
Some studies indicated that the SNP site may result in increased transcript expression. Ma et al., (2014) revealed that splicing resulted in extra transcript expression. Other study also found that the SNP in intron 24 of PCLO significantly reduced the splicing efficiency (Seo et al., 2013). In this study a SNP: g.5 A> G (Variant ID: rs329600317) was found in the intron 19 of IGF-1R gene of BaMa and Landrace pigs, which was near the splice site according to the Ensembl genome browser. The frequencies of genotype of the SNP were significantly different between Landrace and BaMa pigs (P<0.0001) (Table 2). Our study initially found that there was no significant association between the SNP and splice variants we studied. Interestingly, we found that the predominant allele of Landrace and BaMa pig breeds were G and A, respectively. Whether the SNP could affect other alternative splicing needs further research and exploration.
 

In this study, we identified IGF-1R AS02 as a novel splice variant of IGF-1R gene in pig breeds. In cartilage tissue, the expression level of IGF-1R ISO01 was higher in BaMa pig than in Landrace pig, while the expression levels of IGF-1R AS02 was negatively correlated to IGF-1R ISO01 (P<0.05). For g.5bp A>G in the intron 19 of IGF-1R gene, the predominant alleles of Landrace and BaMa pigs were G and A, respectively. Whether the formation of splice variants between pig breeds was due to the SNP in intron 19 of IGF-1R gene needs further research.

This work was supported by the National Natural Science Foundation of China (31772699 and 31672514) and the Jilin Scientific and Technological Development Program (20170101024JC).