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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.45 no.4 Santiago  2012

http://dx.doi.org/10.4067/S0716-97602012000400004 

Biol Res 45: 351-356, 2012

RESEARCH ARTICLES

 

IGF2 expression in blood is not associated with its imprinting status in healthy pregnant Chinese women

 

Ru-Fei Gao1, Xue-Qing Liu1, Ying-Xiong Wang1, Xue-Mei Chen1, Yu-Bin Ding1*, Jun-Lin He1

1Department of Reproductive Biology, Chongqing Medical University, Chongqing, China.


ABSTRACT

Loss of Imprinting (LOI) of IGF2 and over-expressed IGF2 are associated with tumorigenesis. Our previous epidemiological study found a relatively high frequency of IGF2 LOI in healthy mid-gestation pregnant women. The aim of this study is to determine whether the expression of IGF2 is associated with its imprinting status in healthy Chinese pregnant women. The IGF2 imprinting status of 300 pregnant women was analyzed. 20 cases of IGF2 LOI and 20 cases of IGF2 retention of imprinting (ROI) were selected randomly for IGF2 expression analysis. The expression pattern of IGF2 between the group with IGF2 ROI and group with IGF2 LOI in healthy Chinese pregnant women was evaluated by real time PCR and western blot. The result showed no significant differences between IGF2 ROI and LOI groups in mRNA and protein levels. These results imply that IGF2 imprinting status has no obvious impact on its expression. There may be some unknown important factors other than imprinting status driving IGF2 expression.

Key words: Insulin-like growth factor 2; loss of imprinting; retention of imprinting; pregnancy.


 

INTRODUCTION

Genomic imprinting is a common mechanism by which a gene is preferentially expressed based on the parental origin of the allele (Byun et al. 2007). Insulin-like growth factor 2 (IGF2), located at 11p15.5, has been known as a maternally imprinted and paternally expressed gene in most human tissues (Vu & Hoffman 1994). It plays a fundamental role in embryo growth, differentiation and placental development (Birnbacher et al. 1998; Constancia et al. 2002; Naeve et al. 2000; Wang et al. 1994). Studies have demonstrated that the IGF2 LOI gene is associated with the tumorigenesis of different types of cancers (Byun et al. 2007; Cui et al. 2002; Fu et al. 2008; Honda et al. 2008). 67-77% of IGF2 LOI was observed in sporadic Wilms' tumor cases (Ogawa et al. 1993), 47% in adult lung cancer (Suzuki et al. 1994), 86% in rhabdomyosarcoma (Zhan et al. 1994) and high incidence of LOI of IGF2 in other tumors including testicular germ cell tumors (van Gurp et al. 1994), choriocarcinoma (Hashimoto et al. 1995), bladder cancer (Byun et al. 2007) and colorectal tumors (Cui et al. 2003).

However, recent studies revealed that IGF2 LOI was not a phenomenon specific to cancer, it may occur in normal tissues such as normal cervical tissues adjacent to cervical cancers (Douc-Rasy et al. 1996) and noncancerous liver specimens around hepatocellular carcinomas (Takeda et al. 1996). In the human fetal liver IGF2 appeared to be imprinted, while relaxing its imprinting in the second half of the 1st year of postnatal life (Davies 1994); LOI remained in the adult liver (Kalscheuer et al. 1993). An epidemiological study also showed that IGF2 LOI is found in the cord blood of newborns (Dai et al. 2007).

In theory, IGF2 LOI would lead to a doubling of IGF2 mRNA abundance and a 2-fold increase in IGF2 production (Vu et al.). However, mixed results were shown on the relationship between IGF2 expression and its imprinting status. Wu et al. (1997) found that LOI had strong association with IGF2 overexpression and played an important role in carcinogenesis of cancers. IGF2 LOI also resulted in overexpression of IGF2 in childhood tumors (Muller et al. 2000; Ogawa et al. 1993; Roy et al. 2000). Micha et al. (1999) observed that none of the pancreatic tissues with IGF2 LOI showed concomitant overexpression of IGF2 transcripts. The LOI of IGF2 may induce both increased or decrease expression, which could initiate the onset of Wilms' tumor (Yun 1998). Wang et al. (1996) reported that increased expression of the IGF2 gene in Wilms' tumor did not depend on loss of genomic imprinting or loss of heterozygosity. In prostate cancer, IGF2 imprinting correlated with IGF2 expression in tissues associated with the tumor, but not within the tumor foci (Bhusari et al.). In addition, no association of IGF2 LOI with increased expression of IGF2 mRNA was observed in Ewing's sarcoma (Zhan et al. 1995). Waterland et al. (2006) revealed that a folate-deficient diet caused significant IGF2 LOI in mice offspring and IGF2 expression was significantly reduced in the treatment group. And LOI patterns have no direct influence on altered expression levels of imprinted genes on growth in adult mouse hybrids (Shi et al. 2005). These studies indicated that LOI may not be always involved in the regulation of IGF2 expression and some other unknown factors may be driving IGF2 expression.

To obtain a better understanding of the relationship between IGF2 expression and the imprinting status of IGF2, peripheral blood of healthy pregnant women was evaluated by screening the expression and imprinting status of IGF2. Here we examine the correlation of LOI and IGF2 expression in pregnant women.

MATERIALS AND METHODS

Sample Collection

All the peripheral blood samples (n=300) chosen for our study were obtained from healthy pregnant women aged 20 to 30 years who visited the First and the Second Affiliated Hospital of Chongqing Medical University in the period from February 2008 to August 2009. The research was approved by Local Ethics Committee and informed written consent was obtained from all participants. With clinical examination and ultrasound examination, the gestation (20.6±1.2 weeks) was calculated. The subjects who had a history of gastrointestinal disease or major pregnancy complications were excluded. The pregnancy process and pregnancy outcome were followed up till the children were born.

With standard procedures, fasting blood samples were obtained into EDTA-containing vacuum tubes and 0.5 ml peripheral blood was frozen at -20°C for DNA and protein extraction. The separated lymphocytes were used for RNA extraction.

DNA extraction

Genomic DNA was extracted using a TIANamp Blood DNA Kit (TIANGEN Biotechnology Co. Ltd, Beijing, China) according to the manufacturer's instructions and the extracted DNA was stored at -20 °C for genotyping analysis.

cDNA synthesis

Total RNA extraction was performed using Trizol reagent (Invitrogen Corporation, USA). Quantification and purity were assessed by optical density (OD) measurement at 260nm and 280nm. Integrity of the total RNA was examined by agarose gel electrophoresis and stored at -20° C. The reverse transcription of cDNA was performed with 0.5 µg of total RNA in a 10µl reaction using the First Strand synthesis for RT-PCR kit (TAKARA Biotechnology Co. Ltd, Dalian, China). cDNA was stored at -80 °C for analysis of IGF2 LOI and ROI and Realtime PCR.

Analysis of IGF2 LOI and ROI

To identify heterozygous cases for imprinting analysis, polymorphic pattern of genomic DNA was examined by ApaI polymorphism of axon 9 for IGF2. Cases heterozygous for IGF2 were further analyzed with PCR products of the first stand cDNA as described and for the analysis of imprinting status. The 236bp RT-PCR products (Fig. 1 A) of the IGF2 gene were digested with ApaI endonuclease, followed by electrophoresis on 3% Biowest agarose gel. The digestion products showing both 236bp and 173bp fragments are indicative of LOI and retention of IGF2 imprinting (ROI) (Fig. 1 B). All the protocols were described in our previous study (Gao et al. 2011).


Real-time PCR of IGF2 mRNA expression between LOI and ROI

All Real-time PCR were performed using SYBR Premix Ex Taq (TAKARA Biotechnology Co. Ltd, Dalian, China) and a Light Cycler (Bio-Rad iQTM5 Muliticolor Realtime PCR detection system). Briefly, 2µ l of aliquots containing cDNA were amplified in a total volume of 20µl containing 4µl of a 5xSYBR PreMix Ex Taq and 0.2µM of each primer. For internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also amplified. The following specific primers for IGF2 and GAPDH were used: IGF2 (forward: 5'-CACGCCAAACACTGAAT-3', reverse, 5'-GCAAGCGTTACAAGGTTA-3', product size: 142bp), GAPDH (forward: 5'-CTCTCTGCTCCTCCTGTTCGACAG-3', reverse, 5'-GTGGAATCATATTGGAACATGT-3', product size:

230bp) (Sangon Biological Engineering Technology & Services Co. Ltd, Shanghai, China). All samples were run in triplicate. For appropriate negative controls, the RNA template was replaced with nuclease-free water in each run. Melting curves of the products were obtained after cycling with a stepwise increase of temperature from 55 °C to 95 °C. At the end of 40 cycles, reaction products were separated electrophoretically on an agarose gel and stained with ethidium bromide for visual confirmation of the PCR products. Relative gene expression levels were calculated with the 2־AACT method.

Western blotting analysis

The total protein concentration of plasma samples was evaluated using the Bradford assay. Equal amounts (30µg per lane) of diluted plasma samples were incubated in SDS-PAGE sample buffer, subjected to SDS-PAGE analysis with 12% acrylamide gel, and then electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA). The membrane was subsequently blocked in 5% skim milk powder in 0.1% Tris-buffered saline/Tween-20 and then incubated with rabbit polyclonal to IGF2 primary antibody (Abcam, Cambridge, MA, USA) followed by incubation with the appropriate secondary antibody of peroxidase-conjugated goat anti-rabbit IgG (Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China). Immuno-reactive proteins were visualized by a chemiluminescence reaction using the ECL Western blocking detection system (Amersham).

Bisulfite genomic sequencing for methylation analysis

The IGF2 DMR0 region (GenBank nucleotides 631-859, accession No. Y13633) has been previously reported to be hypomethylated in colorectal cancers with LOI at IGF2 (Cui et al. 2002). In this study, the methylation status of this region was quantified in normal pregnancy via BSP. Genomic DNA extracted from peripheral blood was treated with sodium bisulfite using EZ DNA Methylation-Gold kit according to the manufacturer's instructions. Each PCR mix reaction contained 10 pmol of the forward primer(5'-TAGGGTGGTGTTTGTGGGGA-3'), 10 pmol of the reverse primer (5'- AAAACTTCCCRATACTAATAAAAAAACC -3'), 3 µl of bisulfite-modified DNA, 1 µl of 10 pmol/ µl primers, 1 µl of 10 mmol/L dNTP, 5 µl of 10x PCR buffer, 0.8 µl of HotStart Taq DNA polymerase (Takara). PCR amplification conditions were as follows: initial denaturation at 98 °C for 5 min; 40 cycles of 45 sec at 94 °C, 45 sec at 56 °C, and 1 min at 72 °C; followed by a final extension at 72 °C for 10 min. Products purified with the Universal DNA Purification kit (Tiangen, Beijing, China) were cloned into pUC18-T and transformed into chemically induced competent E. coli. Five insert-positive clones of each sample were chosen for sequencing by the Sangon company.

Statistical analysis

All statistical analyses were carried out using SAS for WINDOWS software (version 8.2; SAS Institute Inc, Cary, NC).

Data are presented as the means ±S.D. Differences in IGF2 mRNA and protein levels between IGF2 LOI and ROI were compared using Student's t test. The relationship between the expression and the imprinting status of IGF2 was examined with logistic regression models. P < 0.05 was considered to indicate statistical significance.

RESULTS

For the ApaI polymorphism site in exon 9 of IGF2, the unrestricted PCR product (AA genotype) has a size of 236 bp, and complete restriction (GG genotype) generates bands of 173bp and 63bp. Samples with 236 bp and 173 bp fragments were heterozygotes (AG genotype), which made them informative for further analysis of imprinting status (Figure.1). Of the 300 women analyzed, 164 (54.67%) cases were heterozygous with the AG genotype and informative for imprinting status analysis of IGF2. Of the 164 cases analyzed for imprinting status, 44 (26.83%) were IGF2 LOI cases while 120 (73.17%) were IGF2 ROI. The IGF2 DMR0 region has been previously reported to be hypomethylated in colorectal cancers with LOI at IGF2 (Cui et al. 2002). Our BSP results showed that, except for CpG 2, all the CpG sits in this region were also hypomethylated in blood lymphocytes in normal pregnancy with IGF2 LOI (Figure. 2).


20 cases of IGF2 LOI and 20 cases of IGF2 ROI were selected randomly for further analysis. The general characteristics of subjects with IGF2 LOI and IGF2 ROI are presented in Table 1. The mean age was 25.3 ±4.2 years in the group with ROI and 24.2±2.8 years in the group with LOI. The mean gestational age at enrollment was 20.9±1.5 weeks in the group with ROI and 20.6±1.7 weeks in the group with LOI. 5.7% of the group with ROI and 6.1% of the group with LOI took antibiotic medicine for a cold during pregnancy. 3.9% of the group with ROI and 4.0% of the group with LOI had bad habits such as smoking and drinking alcohol which could be harmful to embryo development. None of the subjects had complications related to pregnancy. No obvious difference was found in the general characteristics between LOI and ROI.


Expression of IGF2 mRNA was detected by Real-time PCR. The amplification curve and melt curve for Real-time PCR are shown in Fig. 3A and Fig. 3B, respectively. The relative amounts of IGF2 mRNA (IGF2/GAPDH mRNA) in blood lymphocytes in normal pregnancy are shown in Fig. 3C. The IGF2 mRNA level in the group with LOI was a little lower than in the group with ROI, but the difference between the group with ROI and LOI was not significant (P=0.281).


The protein level of IGF2 in different groups with ROI and LOI in plasma was detected by western blot (Figure 4). The IGF2 protein expression was associated with mRNA expression. Densitometric analysis revealed that there was no difference of IGF2 protein expression between the two groups (P=0.275). This showed that there was not a close correlation between the mRNA (OR=1.401, P=0.290) and protein (OR=1.418, P=0.284) expression and the imprinting status of IGF2. Pregnancy outcome was followed up, and no birth defects were found in the two groups.


DISCUSSION

The frequency of IGF2 LOI, which ranges from 12% to 100% in various tumors, suggested that aberrant imprinting is an important epigenetic cause of human carcinogenesis. However, a relatively high frequency of IGF2 LOI was also found in normal human tissue including human fetal liver in the second half of the 1st year of postnatal life (Davies 1994) and the cord blood of newborns (Dai et al. 2007). In our previous epidemiological study, 14.67% (44/300) of pregnant women showed IGF2 LOI. These results indicated that IGF2 LOI is not tumor specific and may be required in some normal biological processes. The association of IGF2 LOI and IGF2 expression in tumor tissues has been demonstrated in several clinical and animal studies, although the results were mixed. Some studies showed that IGF2 overexpression in mRNA was associated with IGF2 LOI in various tumors (Hajdu et al.; Martin-Trujillo et al.; Xu et al. 2006). However, some other studies also showed that IGF2 overexpression was not dependent on loss of genomic imprinting or loss of heterozygosity (Bhusari et al. ; Wang et al. 1996; Zhan et al. 1995). Some researchers demonstrated that IGF2 LOI was associated with decreased expression of IGF2 (Shi et al. 2005; Waterland et al. 2006).

In our study, the IGF2 expression was evaluated in order to find out whether LOI had effects on IGF2 expression in mid-gestation pregnant women. We found that the IGF2 mRNA level in the group with LOI was a little lower than in the group with ROI in peripheral blood lymphocyte of normal pregnancy. But there was no significant difference between the group with ROI and LOI. These results suggest that some unknown factors other than LOI might drive the expression of IGF2.

Although the precise effect of IGF2 LOI on biological processes is not clear, LOI has been demonstrated to be associated with increased cellular proliferation and therefore plays a role in tumorogenesis (Byun et al. 2007; Cui et al. 2003; Hashimoto et al. 1995; Kaneda et al. 2007; Suzuki et al. 1994; van Gurp et al. 1994; Zhan et al. 1994). Little is known of the importance of IGF2 in adults, except the brain and liver, where there is biallelic expression of IGF2. In this study, the follow-up analysis of pregnant outcome showed no obvious harmful effects on offspring in the IFG2 LOI group (data not shown). The imprinting status of IGF2 in fetus and placenta would be more important to produce an effect on pregnancy and offspring. It may indicate that IGF2 LOI might be a common phenomenon in mid-gestation pregnant women, or it might be a salvage pathway for some unknown bad effects of pregnancy. This may also give us a clue to explain why frequent IGF2 LOI is found in some persons who do not suffer from a disease such as cancer.

ACKNOWLEDGEMENTS

This research was supported by the National Natural Science foundation of China (No.30700898); Chongqing Population and Family Planning Commission (No.2007-1-002); Chongqing Education Commission (No. kj090313); Natural Science Foundation Project of CQ CSTC (No.cstc 2009BA5082, 2009BB5271, 2011JJA10085); Excellent Doctoral Dissertation of Chongqing Medical University.

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*Corresponding author: Yu-Bin Ding and Jun-Lin He, Chongqing Medical University, No.1 Yixueyuan Road, Yuzhong Street, Chongqing 400016, China. Tel: +86-23-68485001. Fax: +86-2368485001. E-mail: dingyb@gmail.com and hejunlin_11@yahoo.com.cn.

Received: December 12, 2011. In Revised form: July 9, 2012. Accepted: July 24, 2012.

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