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Abstract

BACKGROUND:

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a common malignant tumor associated with EBV infection. Insulin-like growth factor 2 (IGF2) is an imprinted gene and a key protein that regulates growth, especially during normal fetal development. Loss of imprinting (LOI), is a common epigenetic anomaly in a variety of human cancers. However, the promoter methylation, imprinting status and function of IGF2 gene in GC are unclear.

OBJECTIVE:

To explore the role of IGF2 in the occurrence and development of gastric cancer.

METHODS:

The biological function of IGF2 in gastric cancer was investigated by Transwell, wound healing, CCK-8 and flow cytometry assays. IGF2 imprinting status and gene promoter methylation in gastric cancer tissues were detected by PCR-RFLP and BGS.

RESULTS:

The results showed that the expression of IGF2 was higher in GC tissues than adjacent tissues. IGF2 gene promoter methylation and LOI were significantly higher in EBVaGC tissues than in EBV-negative gastric cancer (EBVnGC) tissues. The high expression of IGF2 in gastric cancer can promote the migration and proliferation of gastric cancer cells.

CONCLUSION:

Our data suggest that IGF2 is involved in the occurrence and development of gastric cancer. Targeting IGF2 may be a potential therapeutic target for gastric cancer.

Keywords

Epstein-barr virus insulin-like growth factor 2 genomic imprinting DNA methylation gastric cancer

1. Introduction

Gastric cancer (GC) is often diagnosed at an advanced stage and has a high mortality rate, with 784,000 deaths worldwide in 2018, making it the third most common cause of cancer death worldwide [1]. EBV-associated GC (EBVaGC) is a unique subtype of GC and a common malignancy associated with EBV infection [2]. It has been confirmed that EBV infection induces abnormal methylation of CpG islands and thus causes abnormal changes in host gene expression, which is an important mechanism of EBV involvement in EBVaGC, and also an important mechanism of initiation of malignant transformation of tumor in the early stage of tumor [3]. Therefore, only with a clearer understanding of its tumor biology will it be possible to make substantial progress in the treatment and prevention of gastric cancer [4].

Genomic imprinting, also known as gamete imprinting or parental imprinting, is a special phenomenon that does not follow Mendelian inheritance law and only relies on single parents to pass on some genetic traits. The role of genetic imprinting in human genetic diseases and tumorigenesis has attracted more and more attention. IGF2 is the first confirmed endogenous imprinted gene, and was derived only from the paternal allele [5]. In recent years, with the continuous development of molecular biotechnology, the regulation mechanism of IGF2 imprinting has been further understood, such as enhancer competition theory, chromatin configuration theory, transcriptional cycle theory, etc [6]. IGF2 loss of imprinting (LOI) leads to increased expression of IGF2, and can promote cancer development. For example, IGF2 LOI enhances the pluripotency of colorectal cancer stem cells by promoting tumor autophagy, and IGF2 LOI can promote the development of prostate cancer [7, 8]. Although IGF2 LOI was previously reported to be present in 34.5% of GC tumor tissues [9], the incidence of IGF2 LOI in EBVaGC and its relationship to IGF2 expression have not been studied until now.

In recent years, epigenetic abnormalities, including LOI and DNA methylation, have been identified as important mechanisms in the development of many human cancers. Epigenetic abnormalities of host cells caused by viral infection are the focus of research on oncogenic mechanism of tumor viruses. The main objective of this study was to analyze the promoter methylation and imprinting status of IGF2 in EBVaGC and EBVnGC. And the role of IGF2 gene in the occurrence and development of gastric cancer was elucidated to provide theoretical and experimental basis.

2. Materials and methods

2.1 Cell culture and tissue samples

AGS-EBV, C666-1, Daudi and Raji cell lines were EBV positive cell lines, among which AGS-EBV and C666-1 were donated by Professor Sun Lunquan from Xiangya Medical College, Central South University. SGC7901, HGC-27, BGC823, AGS, MKN-45, CNE and Jurkat cell lines were all EBV negative cell lines, among which SGC7901 and HGC-27 cell lines were preserved in our laboratory. BGC823 and AGS cell lines were donated by Prof. Shao Chunkui, Department of Pathology, the Third Affiliated Hospital of Sun Yat-sen University; MKN-45 cell lines were purchased from Peking Union Cell Bank; CNE cell lines were donated by Prof. Zhu Wei, Department of Pathology, Guangdong Medical College; and Jurkat cell lines were donated by Department of Bioinformatics, Tottori University Health Science Center, Japan. GT38, GT39, Daudi and Raji cell lines (EBV positive) were provided by T. Sairenji (Tottori University, Japan). The SNU719 cell line (EBV positive) was provided by Tao Qian (Chinese University of Hong Kong). MGC803 was provided by Jiang Lei (Peking University). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at 37 ${{}^{\circ}}$ C in a humidified incubator with 5% CO ${}_{2}$ . Paraffin embedded and fresh gastric tissues were collected from the Affiliated Hospital of Qingdao University and Qingdao Municipal Hospital. EBVaGC was determined by in situ hybridization of EBV-encoded small RNA1 (EBER1) [10]. This study received permission from the Medical Ethical Committee of Medical College of Qingdao University.

2.2 Transfection with IGF2 siRNA and plasmid

Specific siRNA against IGF2 and Negative control (NC) were designed and synthesized by GenePharma (Shanghai, China). IGF2 gene was cloned into pcDNA3.1 containing FLAG tag and has identification reports. The sequences are as follows: siIGF2: 5’-GCUC AGAAAUUGGCUUUAATT-3’, 5’-UUAAAGCCAAU UUCUGAGCTT-3’; Negative control (NC) 5’-UUCUC CGAACGUGUCACGUTT-3’, 5’-ACGUGACACGUU CGGAGAATT-3’. Transfection of 50 nM siRNA was performed using Lipofectamine 2000 Reagent (Invitrogen, Thermo Fisher Scientific, Germany). The cells were gathered after 48 h to assay relative gene expression.

2.3 Cell proliferation assay

Cells were seeded at a density of 4 $\times$ 10 ${}^{3}$ per well in 96-well plates overnight. The cells were then transfected with plasmid, siRNA, or NC and incubated for 24, 48, 72, or 96 h. Cell viability was detected using Cell Counting Kit-8 (CCK-8, TargetMol, USA) at the indicated times. Ten microliters of CCK-8 were added to each well, and after 1 h, the absorbance was measured at 450 nm using a microplate reader. We set up a total of 6 repetition holes for each treatment group, and all experiments were triplicated.

2.4 Cell cycle assay

Cells were seeded into six-well plates and incubated overnight. After treatment for 48 h, the cells were digested by trypsin and washed gently with PBS. The collected cells were fixed in 66% ethanol at 4 ${}^{\circ}$ C overnight. Then, the cells were incubated with propidium iodide (PI) and RNase A for 25 min in the dark. The cell cycle was analyzed by using flow cytometry (BD Biosciences).

2.5 Cell apoptosis detection

GC cells were seeded in 6-well plates and were transfected with plasmid, siRNA, or NC for 48h cells and then washed with cold PBS. Apoptosis was detected using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions.

2.6 RNA extraction and quantitative reverse transcription PCR (qRT-PCR)

Total RNA was extracted from the cultured cells using the TRIzol reagent (BioFlux, Beijing) and then reverse transcribed into cDNA according to the manufacturer’s protocol. We tested the absorbance values of the proposed RNAs, all of which were between A260/A280 $=$ 1.9 $\sim$ 2.0, and were retrotranscribed with 1000ng RNA. The products were used as a PCR template in the LightCycler96 SN10700 Sequence Detection System (Roche). All reactions were performed in triplicate, and $\beta$ -actin was used for normalization. The 2 ${}^{-\Delta\Delta\text{Ct}}$ method was used to assess the relative gene expression. The sequences of the specific forward and reverse primers were as follows: for IGF2, 5’-ACACCCTCCAGTTCGTCTGT-3’ and 5’-GGGGTATCTGGGGAAGTTGT-3’; for $\beta$ -actin, 5’-CACCATTGGCAATGAGCGGTT-3’ and 5’-AGGTCTTTGCGGATGTCCACGT-3’.

2.7 Western blot analysis

All cells were lysed using radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor, phenylmethanesulfonyl-fluoride (PMSF), and a phosphatase inhibitor mixture. These samples were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA) using SDS-PAGE gel with a 10% mass concentration, blocked with bovine serum albumin (BSA) with a 5% mass concentration at room temperature for 2 h, incubated with the primary antibody overnight at 4 ${}^{\circ}$ C, cultured with the second antibody the following day, and held at room temperature for 2 h. Following incubation, the membranes were visualized by using an ECL detection kit (Millipore Corporation, Billerica, MA, USA). We used $\beta$ -actin as the loading control. And the protein quantity was measured with a bicinchoninic acid assay kit (CWBIO, China) to ensure the same amount of protein in each lane. The antibodies used include the following: antibodies against $\beta$ -actin (CST, 1:1000); IGF2 (Santa, 1:500); DNMT1 (CST, 1:1000); ZEB1 (Proteintech, 1:1000); Phospho-p44/42 MAPK (Erk1/2) (CST, 1:1000); p44/42 MAPK (Erk1/2) (CST, 1:1000).

2.8 Transwell migration assay

The migration ability of the cells was measured using the transwell inserts with 8 $\mu$ m pores (Corning, New York, USA). 6 $\times$ 10 ${}^{4}$ cells were seeded into the upper chamber with a serum-free medium and the medium was supplemented with 20% FBS in the lower chamber. Cells transfected with siRNA and plasmid were incubated for 48 h. Then the cells in the upper compartment were wiped off, and the cells attached to the bottom of the compartment membrane were fixed and stained for observation under a microscope.

2.9 Wound healing assay

The treated cells were inoculated into the six-well plate, and when the cells had grown to fusion, a high pressure toothpick was used to scrape the fused cell layer to form a linear wound. The cells were washed twice to remove shed cells and debris. Then observe and measure the size of the wound at the specified time.

2.10 Bisufite genomic sequencing (BGS)

BGS specific primers were used to amplify the promoter region of IGF2 coding gene, and TA cloning was combined with DNA sequencing technology to randomly select amplified bands for sequencing. Six clones were selected by sequencing, and the methylation rate was calculated according to the proportion of CG sites in the total CG sites of the six TA clones. The 46 CG sites in the amplified DNA fragments were drawn as dot plots to visually display the methylation state of IGF2, in which the black circle indicated that the CG site was methylated, and the blank circle indicated that the CG site was not methylated.

2.11 PCR-restriction fragment length polymorphism (PCR-RFLP)

Genomic DNA was amplified by polymerase chain reaction (PCR), and Apa I digestion was performed on the amplified products. The forward and reverse specific primers used to amplify exon 9 of IGF2 gene were as follows: 5’-CTTGGACTTTGAGTCAAATTGG-3’ and 5’-GGTCGTGCCAATTACATTTCA-3’. The 25 $\mu$ L PCR reaction was composed of 2 $\mu$ L DNA template, 1.5 $\mu$ L 10 $\times$ reaction buffer, 1.2 $\mu$ L dNTP, 0.5 $\mu$ L of each primer, 0.15 $\mu$ L hot start tap E, and 19.15 $\mu$ L water. Thermal cycling conditions followed an initial denaturation at 94 ${{}^{\circ}}$ C for 5 min and 40 cycles of denaturation at 94 ${{}^{\circ}}$ C for 30 s, annealing at 55 ${{}^{\circ}}$ C for 30 s, and elongation at 72 ${{}^{\circ}}$ C for 60 s. The PCR product was then digested with Apa I at 37 ${{}^{\circ}}$ C for four hours. The products of 231 bp and 61 bp were AA type. Only 292 bp was BB type; 292 bp, 231 bp and 61 bp all appeared as AB type.

Table 1
IGF2 protein expression in GC and normal adjacent tissues of gastric cancer samples

	GC tissues ( $n=$ 77)	Normal adjacent tissues ( $n=$ 59)	$P$ value
1 $+$	37	44
2 $++$	37	15	$P=$ 0.002
3 $+++$	3	0

GC, gastric cancer; GC tissues including EBV-associated gastric cancer and EBV-negative gastric cancer tissues.

2.12 Imprinting status of IGF2

Specimens with DNA genotype AB (heterozygote) were selected for reverse transcription into cDNA. Then PCR was run on cDNA with the same primers used to determine informative status. The RT-PCR products were also digested by Apa I and electrophoresed on 2% agarose gel. Two bands of 160 bp and 38 bp were observed as alleles A, and only 198 bp bands were observed as alleles B, both of which were imprinting maintain. If 198 bp, 160 bp, 38 bp three bands were observed, it was a biallelic expression state (AB type), that is, loss of imprinting.

2.13 Immunohistochemistry

Paraffin embedded and fresh gastric tissues were collected from the Affiliated Hospital of Qingdao University and Qingdao Municipal Hospital. EBVaGC tissues were identified by in situ hybridization for EBV-encoded small RNA1 (EBER1). Sections were incubated for 1h at room temperature with anti-IGF2 antibody (abcam, 1:200). Antigen-antibody complexes were visualized using a DAB chromogenic kit (Zsbio, China). The specimens were randomized, coded, and then analysed by two independent pathologists. The study was conducted in accordance with the ethical standards and the principles of the Declaration of Helsinki and was approved by the Ethical Committee of the Medical College of Qingdao University. Written informed consent was obtained from each participant before the start of the study.

2.14 Statistical analysis

All data are expressed as the means standard error of the mean (SEM). The experimental data were analyzed using Student’s $t$ -test and Chi-square test ( $P<$ 0.05 was considered significant; ns, not significant; ${}^{*}P<$ 0.05; ${}^{**}P<$ 0.01). Statistical analyses were conducted using SPSS 19.0 statistical software (Chicago, IL, USA).

3. Results

Figure 1.

Role of insulin-like growth factor 2 (IGF2) in the development of gastric cancer. A. (A) IGF2 positive expression in EBV-associated gastric cancer (EBVaGC) tissues, the arrow indicates the specific cytoplasmic expression of IGF2 in gastric cancer cells; (B) IGF2 negative expression in gastric tumor tissues. B. The transfection efficiency of IGF2 overexpressing plasmids and siIGF2 in AGS cells. C. Effect of IGF2 and siIGF2 on the migration ability of AGS cells as detected by Transwell assay 48 h after transfection (40x). D. Wound healing assays were used to assess cell migration. E. The apoptosis rate was detected by flow cytometry in AGS cells. F. The cell cycle was observed using flow cytometry in AGS cells. G. After transfection for 24 h, 48 h, 72 h and 96 h, 10 $\mu$ l of CCK-8 solution was added to each well to observe the cell proliferation.

Table 2

IGF2 protein expression in GC and normal gastric tissues of gastric cancer samples

	GC tissues ( $n=$ 77)	Normal gastric tissues ( $n=$ 22)	$P$ value
$-$ /1 $+$	37	21	$P<$ 0.0001
2 $++$ /3 $+++$	40	1

GC, gastric cancer; GC tissues including EBV-associated gastric cancer and EBV-negative gastric cancer tissues.

Figure 2.

Role of insulin-like growth factor 2 (IGF2) in the development of gastric cancer. A. The transfection efficiency of IGF2 overexpressing plasmids in MGC803 cells. B. Effect of IGF2 on the migration ability of MGC803 cells as detected by Transwell assay 48 h after transfection (40x). C. Wound healing assays were used to assess cell migration. D. The apoptosis rate was detected by flow cytometry in MGC803 cells. E. The cell cycle was observed using flow cytometry in MGC803 cells. F. After transfection for 24 h, 48 h, 72 h and 96 h, 10 $\mu$ l of CCK-8 solution was added to each well to observe the cell proliferation.

3.1 The expression of IGF2

Firstly, in order to understand the role of IGF2 in the development of gastric cancer, the expression of IGF2 in gastric cancer tissues and adjacent normal tissues was detected by immunohistochemistry. We found that the expression of IGF2 in gastric cancer tissues was significantly higher than that in adjacent tissues (Fig. 1A) (Table 1). We further examined the expression of IGF2 in normal gastric tissues and found that it was significantly lower than that in gastric cancer tissues (Table 2). Besides, results have shown that there was no significant difference in the expression of IGF2 protein in EBVaGC and EBVnGC tissues (supplementary material, Table S1).

3.2 IGF2 promotes the migration and proliferation of gastric cancer cells

To explore the function of IGF2 in GC cells, we overexpressed IGF2 by transfecting plasmid and suppressed IGF2 expression using siRNA (Fig. 1B). Using Transwell, we found that the overexpression of IGF2 could significantly promote the migration of AGS cells and inhibition of IGF2 could significantly down-regulate the migration ability of AGS cells (Fig. 1C). Wound healing experiments with AGS cells also showed similar results (Fig. 1D). Overexpression and inhibition of IGF2 in AGS cells showed no significant effect on apoptosis and cell cycle by flow cytometry (Fig. 1E and F). Although CCK-8 assays showed that overexpression of IGF2 had no significant effect on cell proliferation, we found that the cell proliferation ability was decreased after interference with IGF2 compared with the control group (Fig. 1G). For further verification, we overexpressed IGF2 in MGC803 cells and obtained similar results as in AGS cells (Fig. 2).

Figure 3.

The molecular mechanism of IGF2 regulation. A. IGF2 overexpression upregulates p-ERK and ZEB1 in AGS cells by Western blot. B. Silencing IGF2 down-regulates p-ERK and ZEB1 in AGS cells through Western blot. C. Western blot showed that IGF2 overexpression upregulates p-ERK and ZEB1 in MGC803 cells.

To further explore the molecular mechanism of IGF2 regulation, we overexpressed IGF2 in AGS cells and MGC803 cells and found that overexpression of IGF2 could activate the ERK pathway and up-regulate ZEB1 (zinc finger E-box binding homeobox 1) (Fig. 3A and C). Down-regulation of p-ERK and ZEB1 was found after inhibiting the expression of IGF2 in AGS cell lines (Fig. 3B).

3.3 IGF2 imprinting status

In order to further investigate the higher expression of IGF2 in gastric cancer tissues than in adjacent tissues, we detected the imprinting status and promoter methylation of IGF2.

Figure 4.

The subtype of insulin-like growth factor 2 (IGF2) gene, imprinting status and methylation status analysis in gastric cancer tissues. A. The subtype of IGF2 gene in gastric cancer tissues. Lane M: DNA marker DL2000; lane 1, 3: homozygotes (231 bp and 61 bp, both alleles with ApaI sites) and lane 2, 4 homozygotes (292 bp, both alleles without ApaI sites); lane 5 and 6: heterozygous specimens (292 bp, 231 bp and 61 bp). B. IGF2 imprinting status in gastric cancer tissues. Lane M: DNA marker DL2000; lane 1 and 6 (198 bp,168 bp and 30 bp): LOI showed both alleles expression; lane 2–4 (198 bp): normal imprinting status showed only one allele expression; lane 5 (168 bp and 30 bp): normal imprinting status showed only one allele expression. C. The methylation status of IGF2 promoter in EBV-associated gastric cancer (EBVaGC) and EBV-negative gastric cancer (EBVnGC) tissues. $\bullet$ indicating methylated CpG site; $\circ$ indicating unmethylated CpG site. D. The methylation status of IGF2 promoter in cell lines. E. IGF2 mRNA expression in EBVaGC cell line (GT38) and EBVnGC cell line (MGC803) were detected by qRT-PCR after treatment with 5-Aza-CdR (7.5 $\mu$ mol/L and 15 $\mu$ mol/L) for 5 days. Expression levels were calculated by qRT-PCR. The $\beta$ -actin gene was used as internal reference and the 2 ${}^{-\Delta\Delta\text{Ct}}$ method was used for relative quantification. F. IGF2 protein expression in EBVaGC cell line (GT38) and EBVnGC cell line (MGC803) were detected by Western blot after treatment with 5-Aza-CdR (7.5 $\mu$ mol/L and 15 $\mu$ mol/L) for 5 days.

3.3.1 IGF2 genotype analysis on DNA level

(1)
DNA IGF2 gene heterozygous screening in gastric cancer tissue

The results of PCR-RFLP showed that among the 80 EBVaGC tissue samples, 40 were heterozygous. Among the 43 EBVaGC adjacent tissue samples, 22 were heterozygous. Among the 92 EBVnGC tissue samples, 56 were heterozygous. Among the 57 EBVnGC adjacent tissue samples, 34 were heterozygous. We can see that there is no significant difference in the proportion of screened heterozygotes in each group. And there was no significant difference in heterozygotes between cancer and adjacent tissues. The classification results are shown in Table 3. The gel imaging results are shown in Fig. 4A.
(2)
DNA IGF2 gene heterozygous screening in cell lines

Table 3
The subtype of IGF2 in gastric cancer and normal adjacent tissues

Tissue type AA $+$ BB AB $P$ value

EBVaGC 40 40 $P=$ 0.902

EBVaGC adjacent 21 22

EBVnGC 36 56 $P=$ 0.882

EBVnGC adjacent 23 34

EBV, Epstein-Barr virus; GC, gastric cancer; EBVaGC, EBV-associated gastric cancer; EBVnGC, EBV-negative gastric cancer.

Table 4
The subtype of IGF2 in cell lines

AA BB AB

EBV-positive cell lines AGS-EBV

C666-1

OB

Raji

Daudi

EBV-negative cell lines AGS HGC-27 Ramos

SGC7901 MKN-45

BGC-823

CNE

Jurkat

EBV, Epstein-Barr virus.

The PCR-RFLP method detected IGF2 genotypes in 5 EBV-positive cell lines and 8 EBV-negative cell lines, and the results showed that the EBV-positive cell lines AGS-EBV, C666-1, OB, Raji, Daudi and EBV-negative cell lines AGS, SGC7901, BGC-823, CNE, Jurkat, HGC-27 and MKN-45 were all homozygous. Only the EBV-negative cell line Ramos is heterozygous. The specific results are shown in Table 4.

Table 5
IGF2 imprinting status in EBVaGC and EBVnGC tissues

Imprinted status EBVaGC ( $n=$ 31) EBVnGC ( $n=$ 45) $P$ value

LOI 19 (61.29%) 15 (33.33%) $P=$ 0.016

Normal imprinting 12 (38.71%) 30 (66.67%)

EBV, Epstein-Barr virus; GC, gastric cancer; EBVaGC, EBV-associated gastric cancer; EBVnGC, EBV-negative gastric cancer; LOI, loss of imprinting.

3.3.2 IGF2 gene imprinting status in gastric cancer tissues

Tissue type	AA $+$ BB	AB	$P$ value
EBVaGC	40	40	$P=$ 0.902
EBVaGC adjacent	21	22
EBVnGC	36	56	$P=$ 0.882
EBVnGC adjacent	23	34

Imprinted status	EBVaGC ( $n=$ 31)	EBVnGC ( $n=$ 45)	$P$ value
LOI	19 (61.29%)	15 (33.33%)	$P=$ 0.016
Normal imprinting	12 (38.71%)	30 (66.67%)

The above-mentioned IGF2 gene heterozygous specimens screened at the DNA level were selected, their RNA was extracted, reverse-transcribed into cDNA, and their imprinting status at the RNA level was detected by PCR-RFLP technology. Among the 40 EBVaGC tissue samples with heterozygous DNA, 31 cases of qRT-PCR amplification were successful. The results of imprinting typing showed that 19 cases were AB biallele expressions, that is, the loss of imprinting. 12 cases were allele type B, that is, the maintain of imprinting. Among the 56 EBVnGC tissue samples with heterozygous DNA, 45 cases of qRT-PCR amplification were successful. The results of imprinting typing showed that 15 cases were AB biallele expression type, that is, the loss of imprinting. 12 cases were allele type A, and 18 cases were allele type B, that is, maintain of imprinting. The results of vertical electrophoresis of some samples are shown in Fig. 4B. Statistical analysis showed that there were significant differences in the distribution of the two imprinting expression states in EBVnGC and EBVaGC ( $\chi^{2}=$ 5.803, $P=$ 0.016), and the LOI in EBVaGC tissues was significantly higher than that in EBVnGC tissues (Table 5).

Table 6
The methylation ratio of IGF2 promoter in gastric cancer tissues

Gastric cancer

tissue type

n

IGF2 promoter methylation rate (

\chi

\pm

P

value

EBVaGC

37.331

\pm

18.692

P=

0.002

EBVnGC

17.022

\pm

12.695

EBV, Epstein-Barr virus; GC, gastric cancer; EBVaGC, EBV-associated gastric cancer; EBVnGC, EBV-negative gastric cancer.

Table 7

The methylation ratio of IGF2 promoter in cell lines

Cell type	$n$	IGF2 promoter methylation rate ( $\chi$ $\pm$ s)	$P$ value
EBV-positive cell lines	3	89.967 $\pm$ 5.454	$P=$ 0.275
EBV-negative cell lines	9	84.978 $\pm$ 6.713

EBV, Epstein-Barr virus.

3.4 IGF2 methylation status analysis

BGS was applied to test the methylation status of IGF2 promoter region. The IGF2 promoter region of DNA samples from 24 EBVnGC and 17 EBVaGC tissues treated with the BGS specific primers (Fig. 4C). The difference between EBVaGC and EBVnGC tissues was statistically significant (Table 6, $P<$ 0.05). And the difference in methylation rate between EBV-positive cell lines and EBV-negative cell lines was no statistically significant (Table 7, $P>$ 0.05). It was found that both EBV-positive cell lines and EBV-negative cell lines were hypermethylated. The average methylation rates of IGF2 promoter in EBV-positive cell lines AGS-EBV, Daudi and C666-1 was about 90%, while in EBV-negative cell lines AGS, HGC-27, MKN-45, BGC823, SGC7901, and MGC803, Jurkat, Ramos and CNE was about 85% (Fig. 4D). Figure 4C and D are methylation dot plots of partial samples.

In order to further explore the role of methylation in IGF2 expression, we observed changes in mRNA and protein levels in EBVaGC cell line GT38 and EBVnGC cell line MGC803 after 5 days of methylation inhibitors 5-Aza-CdR. We found that mRNA levels had significant recovery, and the recovery of EBVaGC cell line was more significant than that of EBVnGC cell line (Fig. 4E). However, at the protein level, we found that IGF2 was significantly down-regulated in EBVaGC cell line under successful demethylation, while IGF2 was not significantly changed in EBVnGC cell line (Fig. 4F). We speculate that the high expression of IGF2 in gastric cancer tissues may be independent of methylation regulation.

4. Discussion

Gastric cancer is one of the most common malignancies and is still one of the leading causes of cancer death worldwide. Due to the lack of early symptoms, most gastric cancer patients are diagnosed with advanced cancer, with metastasis to lymph nodes, distant organs, or both. So far, although physical examination has been effective in reducing the incidence, there is no effective treatment for gastric cancer metastatic disease [11].

Abundant evidence indicates that IGF2 plays a role in promoting cancer [12, 13]. However, the specific role of IGF2 in gastric cancer remains unclear. In our study, the expression of IGF2 in gastric cancer tissues was significantly higher than that in adjacent tissues. Interference with IGF2 significantly inhibited the proliferation and migration of gastric cancer cells. Overexpression of IGF2 promoted migration but had no significant effect on proliferation. Previous studies have shown that IGF2 can be used as a therapeutic target for tumors such as hepatoblastoma [14], adrenocortical adenomas [15], and triple-negative breast cancer [16]. In gastric cancer, Yuasa et al. found that the level of IGF2 DNA methylation of blood leukocytes can be used as an important surrogate marker for the risk of gastric cancer [17]. In our study, we found that inhibition of IGF2 could significantly inhibit the progression of gastric cancer, indicating that IGF2 has a promising potential as a therapeutic target for gastric cancer.

In addition, it was reported that IGF2 could bind to IGF1R to activate PI3K/AKT and MAPK/ERK pathways [18]. ERK pathway is a recognized tumor-related pathway that affects malignant behaviors such as tumor cell proliferation and migration. ZEB1 is one of the most important transcription factors in EMT. In our study, we explored the molecular mechanism of IGF2 promoting the proliferation and migration of gastric cancer cells, and found that IGF2 could activate the ERK pathway and up-regulate the migration-related molecule ZEB1.

IGF2 was the first identified imprinting gene, and LOI is the main reason for its abnormal expression in cancer [6]. Previous studies have shown that IGF2 is imprinted and overexpressed in gastric adenocarcinoma [9, 19]. IGF2 LOI has been found to be highly frequent in Chinese patients with gastric cancer, and is associated with lymph node metastasis and increased risk of gastric cancer [20]. However, the mechanism of IGF2 imprinting in gastric cancer remains unclear. We detected the imprinting status of IGF2 in EBVaGC and EBVnGC tissues and found that IGF2 LOI in EBVaGC tissues was significantly higher than that in EBVnGC tissues at the RNA level. This suggests that EBV infection may be associated with IGF2 imprinting status.

DNA differential methylation, special chromatin structure and antisense transcription products may all play an important role in the production and maintenance of gene imprinting [21, 22]. To further explore whether methylation changes were involved in the expression of IGF2, we used BGS to detect IGF2 methylation in EBVaGC and EBVnGC tissues, and found that IGF2 gene promoter methylation was significantly higher in EBVaGC tissues than in EBVnGC tissues. Studies have shown that a variety of tumor-related genes are hypermethylated in EBVaGC tissues, and the methylation frequency and methylation density of promoter CpG islands in EBVaGC tissues are much higher than those in EBVnGC tissues [23]. However, the IGF2 gene promoter was hypermethylated in both EBV-positive and EBV-negative cell lines. When DNA methylation inhibitor 5-Aza-CdR was added to EBVaGC cell line GT38 and EBVnGC cell line MGC803, qRT-PCR showed that IGF2 mRNA recovered in both GT38 and MGC803, but the recovery of IGF2 in GT38 was more obvious than that in MGC803. At the protein level, after successful inhibition of DNA methyltransferase DNMT1, IGF2 was significantly down-regulated in GT38 but not in MGC803. Studies have shown that AzaC-mediated DMR demethylation of IGF2-H19 locus leads to increased expression of H19 and increased expression of miR-675, which further inhibits the expression of IGF1R and down-regulates the expression of IGF2. IGF2 and IGF1R are down-regulated in a miR-675-dependent manner [24]. IGF2 LOI was previously reported to be associated with decreased H19 expression and H19 promoter hypermethylation [25]. Our previous studies have confirmed that H19 was hypermethylated in EBVaGC cells, and 5-Aza-CdR demethylated treatment could induce the expression of miR-675 [26]. This might be one of the reasons why the mRNA level was up-regulated and the protein level was down-regulated after demethylation of 5-Aza-CdR in GT38 cells. In general, hypermethylation in the promoter region inhibits gene expression, but in EBVaGC, hypermethylation may be associated with LOI and instead induce high protein expression.

In 2014, the Cancer Genome Atlas (TCGA) classified gastric cancer into four subtypes: EBV-positive, microsatellite unstable (MSI), genomic stable (GS), and chromosomal unstable (CIN) tumors[27]. Our study only focused on the differences reflected in EBVaGC, a subtype of gastric cancer, and the remaining EBVnGC. In addition, EBVaGC accounts for less than 10% of gastric cancer, and specimens are difficult to obtain. Therefore, a limited sample was included in this study, which may have an impact on the generalization ability of this research. In the future, more specimens need to be included to explore the differences in imprinted status and IGF2 expression of different gastric cancer subtypes, which will help us to better understand the molecular mechanisms underlying their role in gastric cancer development. In our current study, IGF2 function was mainly investigated in gastric cancer cells. Inherent factors in the genetic background of different cell lines may bias the results. In the future, we will carry out sufficient in vitro experiments to explore the role of IGF2 in the development of gastric cancer.

In conclusion, the expression of IGF2 protein in gastric cancer group was significantly higher than that in adjacent cancer group, and it was found that IGF2 could promote the migration and proliferation of gastric cancer cells. The methylation of IGF2 gene promoter and LOI in EBVaGC tissues were higher than those in EBVnGC tissues, suggesting that EBV infection may be related to the hypermethylation of IGF2 gene promoter and IGF2 imprinting status.

Funding

This study was funded by Natural Science Foundation of Shandong Province [ZR2020MH302; ZR2020MC020].

Author contributions

Conception: Bing Luo.

Interpretation or analysis of data: Jiting Sun.

Preparation of the manuscript: Jiting Sun, Jun Shu, Duo Shi, Wen Liu, Yan Zhang.

Revision for important intellectual content: Jiting Sun, Jun Shu, Duo Shi, Wen Liu, Yan Zhang.

Supervision: Bing Luo.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-230105.

sj-docx-1-cbm-10.3233_CBM-230105.docx - Supplemental material

Supplemental material, sj-docx-1-cbm-10.3233_CBM-230105.docx

Footnotes

Conflict of interest

The authors declare no conflict of interest.

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	AA	BB	AB
EBV-positive cell lines	AGS-EBV
	C666-1
	OB
	Raji
	Daudi
EBV-negative cell lines	AGS	HGC-27	Ramos
	SGC7901	MKN-45
	BGC-823
	CNE
	Jurkat

Effects of methylation and imprinting expression of Insulin-like growth factor 2 gene in gastric cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Cell culture and tissue samples

2.2 Transfection with IGF2 siRNA and plasmid

2.3 Cell proliferation assay

2.4 Cell cycle assay

2.5 Cell apoptosis detection

2.6 RNA extraction and quantitative reverse transcription PCR (qRT-PCR)

2.7 Western blot analysis

2.8 Transwell migration assay

2.9 Wound healing assay

2.10 Bisufite genomic sequencing (BGS)

2.11 PCR-restriction fragment length polymorphism (PCR-RFLP)

Table 1 IGF2 protein expression in GC and normal adjacent tissues of gastric cancer samples

2.13 Immunohistochemistry

2.14 Statistical analysis

3. Results

3.2 IGF2 promotes the migration and proliferation of gastric cancer cells

Table 6 The methylation ratio of IGF2 promoter in gastric cancer tissues

4. Discussion

Funding

Author contributions

Supplementary data

sj-docx-1-cbm-10.3233_CBM-230105.docx - Supplemental material

Footnotes

Conflict of interest

References

Table 1
IGF2 protein expression in GC and normal adjacent tissues of gastric cancer samples

Table 6
The methylation ratio of IGF2 promoter in gastric cancer tissues