Relationship between IGF2BP2 and IGFBP3 polymorphisms and susceptibility to non-small-cell lung cancer: a case–control study in Eastern Chinese Han population

Background IGF2BP2 and IGFBP3 polymorphisms may be associated with cancer risk. Methods With an aim to determine the association of variations in IGF2BP2 and IGFBP3 genes with risk of non-small-cell lung cancer (NSCLC), IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A polymorphisms were selected and genotyped in 521 NSCLC patients and 1,030 controls. Results We found that there was no difference in IGF2BP2 and IGFBP3 genotype distribution among the NSCLC patients and controls. The stratified analyses suggested that IGF2BP2 rs1470579 A>C polymorphism decreased the risk of NSCLC in some subgroups (female subgroup: CC vs AA: adjusted P=0.032 and CC vs AC/AA: adjusted P=0.028; <60 years subgroup: CC vs AA: adjusted P=0.012 and CC vs AC/AA: adjusted P=0.013; and never drinking subgroup: CC vs AA: adjusted P=0.046 and CC vs AC/AA: adjusted P=0.031). The stratified analyses also found that IGF2BP2 rs4402960 G>T polymorphism decreased the risk of NSCLC in some subgroups (female subgroup: TT vs GG: adjusted P=0.031 and TT vs GT/GG: adjusted P=0.026; <60 subgroup: TT vs GG: adjusted P=0.037 and TT vs GT/GG: adjusted P=0.038; and never drinking subgroup: TT vs GT/GG: adjusted P=0.046). Haplotype analysis indicated Ars1470579Crs2270628Grs3110697Grs4402960Ars6953668 haplotype decreased susceptibility of NSCLC (P=0.007). Conclusion Our study suggests that IGF2BP2 rs1470579 A>C, rs4402960 G>T single-nucleotide polymorphisms are candidates for decreased susceptibility to NSCLC among female, <60 years, and never drinking subgroups. In the future, more case–control studies with functional analysis are needed to confirm these preliminary findings.


Introduction
Lung cancer (LC) is the most common malignancy worldwide. It was reported that 1.8 million new LC patients were diagnosed in 2012, which accounted for about 13% of total cancer cases. 1 Because of aging, air pollution, smoking, and exposure to occupational and/or environmental carcinogens, LC constitutes a burden all over the world. Some risk factors mentioned above might contribute to the development of LC; however, other susceptibility factors could also increase the incidence of LC. Nowadays, genetic variants were supported to influence the risk of LC, especially non-small-cell lung cancer (NSCLC), which was a common subtype of LC.
In humans, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is a protein which is encoded by IGF2BP2 gene. 2

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Chen et al factor 2 (IGF2) translation by binding to the 5′ UTR of IGF2 mRNA. 3 Gu et al reported that IGF2BP2 was overexpression in both ovarian cancer and ovarian low malignant potential tumor samples compared to either normal ovary or ovarian adenomas samples. 4 A previous study also found that IGF2BP2/IGF-1/ IGF-1 receptor signaling pathways might involve in cancermediated endothelial recruitment, which was an important feature of metastatic cancer in the tumor microenvironment. 5 In addition, Liu et al found that an lncRNA (IGF2BP2-AS1) was associated with better overall survival in lung squamous cell carcinoma. 6 In view of these previous studies, we thought that IGF2BP2 might influence the development of LC.
IGF family involves IGF ligands, IGF receptors, and IGFbinding proteins (IGFBPs). IGF-1 is a potent mitogen and regulates mitogenesis and antiapoptosis. 7 IGFBP3, a major binding protein of IGF-1, interacts with IGF-1, regulates its biological activity, and may play important roles in antiproliferation and proapoptosis. 8 Papadimitrakopoulou et al reported that IGFBP3 downregulation is an early event during head and neck carcinogenesis. 9 Adenoviral IGFBP3 and farnesyltransferase inhibitor might decrease Akt expression and promote NSCLC cell apoptosis in vitro and in vivo. 10 Results of a previous study highlighted that IGFBP3 could mediate LC progression. In addition, overexpression of IGFBP3 might induce apoptosis of NSCLC cells and promote cisplatin response in vitro. 11 Several case-control studies focused on the association of IGF2BP2 and IGFBP3 polymorphisms with risk to cancer. Results of previous studies indicated that IGF2BP2 rs4402960 G>T single-nucleotide polymorphism (SNP) was associated with the development of breast cancer. 12 Terry et al found that IGFBP3 rs2270628 variants increased IGF1 levels in plasma and was associated with the risk of ovarian cancer. 13 In addition, IGFBP3 rs3110697 variants were significantly associated with IGFBP-3 levels in a multiethnic populations, 14 and IGFBP3 rs3110697 AA genotypes increased the risk of death among Chinese postmenopausal women with breast cancer. 15 However, the relationship between IGF2BP2 and IGFBP3 polymorphisms and NSCLC risk was unclear. With an aim to determine the potential association of genetic variations in IGF2BP2 and IGFBP3 genes with risk of NSCLC in Eastern Chinese Han populations, IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A SNPs were selected and genotyped in 521 NSCLC patients and 1,030 cancer-free controls.

Materials and methods ethics statement
This case-control study conformed to the Helsinki declaration and was approved by the Institutional Review Board of Fujian Medical University. A written consent was obtained from each participant.

subjects
In our study, a total of 521 sporadic NSCLC cases and 1,030 age-and gender-matched controls were enrolled. All participants were recruited from the Department of Thoracic Surgery in Affiliated Union Hospital of Fujian Medical University and Affiliated People's Hospital of Jiangsu University. All NSCLC patients (mean age at 59.76±10.71 years) were diagnosed by pathology. The corresponding information was retrieved from medical files ( Table 1). The cancer-free controls were well-matched to NSCLC patients by age (mean age at 60.34±9.11 years) and sex (P=0.453). Individuals without any history of personal malignancy or autoimmune disorder were included as controls. Both NSCLC cases and controls were hereditarily unrelated and were from Eastern Chinese Han population. Each participant was informed about the study protocols and a written consent was obtained. A body mass index (BMI) ≥24 kg/m 2 was considered as the criterion of Chinese individuals with obesity and overweight. 16,17 The definitions of "ever smokers" and "ever drinkers" were described in our previous study. 18

selection of snPs
To assess the relationship between IGF2BP2 and IGFBP3 SNPs and NSCLC risk, we selected polymorphisms in IGF2BP2 and IGFBP3 gene according to the publications, which were associated with the development of cancer. 12,13,[19][20][21] Dna extraction and genotyping Using a universal Promega DNA kit (Promega Corporation, Fitchburg, WI, USA), genomic DNA was extracted from whole blood sample which was stored with EDTA-anticoagulation tube. IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, 22 rs3110697 G>A and rs6953668 G>A genotypes were determined by a custom-by-design 48-Plex PCR (SNPscan TM kit; Genesky Biotechnologies Inc., Shanghai, China). 23 We used ABI 3730XL sequencer to obtain genotypes. The data were read out by

Baseline characteristics
A total of 521 NSCLC cases were included in this study.  Table 1. The primary information for IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A SNPs was shown in Table 2. The successful ratio was >99.00% for each SNP. Minor allele frequency (MAF) of IGF2BP2 and IGFBP3 SNPs was similar to the data in Chinese database (Table 2). In controls, the genotype frequencies for IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A polymorphisms were in HWE ( Table 2).
association of IGF2BP2 rs1470579 a>C, rs4402960 g>T and IGFBP3 rs2270628 C>T, rs3110697 g>a, and rs6953668 g>a polymorphisms with nsClC Table 3 showed the frequencies of IGF2BP2 and IGFBP3 genotypes in different NSCLC subgroups and control group.
Results of the single locus analyses were summarized in Table 4. We found that there was no difference in IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A genotype distribution among overall NSCLC patients and controls. In addition, similar findings were also identified among different NSCLC subtype and controls.

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Chen et al association of igF2BP2 rs1470579 a>C, rs4402960 g>T and igFBP3 rs2270628 C>T, rs3110697 g>a, and rs6953668 g>a polymorphisms with nsClC in a stratification analysis As shown in Table 5, the stratified analyses suggested that IGF2BP2 rs1470579 A>C polymorphism decreased the risk   However, we found no significant difference in IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A genotype distribution among NSCLC cases and controls (Tables 7-9, respectively).

snP haplotypes
We harnessed an online SHEsis software 31 to establish haplotypes of IGF2BP2 and IGFBP3 gene (Table 10). Finally, 19 haplotypes of IGF2BP2 and IGFBP3 genes were constructed. When A rs1470579 C rs2270628 G rs3110697 G rs4402960 G rs6953668 haplotype was used as reference, A rs1470579 C rs2270628 G rs3110697 G rs4402960 A rs6953668 haplotype decreased susceptibility to NSCLC (P=0.007, Table 10).

Discussion
In this study, we explored the potential relationship of IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A SNPs with susceptibility to NSCLC. We found that IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A polymorphisms might not confer risk to overall NSCLC. However, in stratified analyses, we found significant associations between IGF2BP2 rs1470579 A>C, rs4402960 G>T polymorphisms and decreased risk of NSCLC in female, <60 years, and never drinking subgroups. We also found that A rs1470579 C rs2270628 G rs3110697 G rs4402960 A rs6953668 haplotype decreased susceptibility to NSCLC. To our knowledge, the present study was the first investigation to identify the correlation between IGF2BP2 rs1470579 A>C, rs4402960 G>T polymorphisms and the decreased risk of NSCLC in Asians. Dai et al reported that IGF2BP2 is a tumor promoter which promotes malignancy proliferation through its client mRNAs IGF2 and high mobility group A1. 32 In many human malignancies, the gene encoding IGF2BP2 was found to be amplified and overexpressed. Recently, Barghash et al found that elevated expression of IGF2BP2 was associated with a shorter survival and metastasis in esophageal adenocarcinoma. 33 Several case-control studies reported that IGF2BP2 rs4402960 G>T polymorphism was associated with the risk of T2DM and might affect the therapeutic efficacy of antidiabetic in Chinese population. 34,35 We found that the IGF2BP2 rs4402960 TT genotype was associated with the decreased susceptibility of NSCLC among female,    years, and never drinking patients. Previous report showed that IGF2BP2 rs4402960 G>T polymorphism was associated with the increased risk of breast cancer in a Chinese population. 12 The other case-control study did not find any association between IGF2BP2 rs4402960 G>T polymorphism and colorectal cancer. 36 We identified that IGF2BP2 rs4402960 T allele might probably be a protective factor for NSCLC, which was not consistent with the findings of previous studies. It is believed that there are some LC-related driver genes possessing low frequency variant, which modify the states of chromatin or DNA. 37 In addition, intronic region could bind to some proteins and even directly alter special gene transcription. [37][38][39] rs4402960 G>T polymorphism is located in the intron region of IGF2BP2 gene, which may influence

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Chen et al the post-transcription process. IGF2BP2 rs4402960 G>T polymorphism may accordingly alter the risk of NSCLC through post-transcription process mechanisms, and our study suggested that IGF2BP2 rs4402960 TT genotype and T allele play an important role in lung carcinogenesis. In the future, the function of the IGF2BP2 rs4402960 G>T    40 and Huang et al found that this SNP was a risk factor for T2DM in a Chinese population. 35 In addition, a quantitative assessment demonstrated that this common polymorphism was associated with the development of T2DM. Therefore, whether the A-to-C variant in the intron region of IGF2BP2 gene does influence the expression of IGF2BP2 gene needs to be further studied. Using SHEsis software, 31 we constructed 19 haplotypes to assess the potential inherited patterns of IGF2BP2 and IGFBP3 genes. Compared with A rs1470579 C rs2270628 G rs3110697 G rs-4402960 G rs6953668 haplotype, we found that A rs1470579 C rs2270628 G rs-3110697 G rs4402960 A rs6953668 haplotype significantly decreased the risk of NSCLC (P=0.007, Table 10). To the best of our knowledge, we first identified the relationship of this haplotypes with susceptibility to NSCLC. However, this rare haplotype only influenced a very minor fraction (<1%) of the studied populations. In the future, more studies with a larger sample size and an adequate methodological quality should be performed to confirm or refute these primary findings.
Some limitations in the current study should be acknowledged. First, we selected only some functional polymorphisms in IGF2BP2 and IGFBP3 genes. In the future, a fine-mapping study should be conducted to further study the potential relationship of GF2BP2 and IGFBP3 polymorphisms with risk of NSCLC. Second, in this case-control study, the sample size of NSCLC patients was relatively limited, which might lead to lack of sufficient power to identify true correlation, especially in the subgroup analysis. In the future, more NSCLC cases and controls should be enrolled, and a replicated study should be carried out. Third, this case-control study was hospital-based. The cancer-free controls recruited from local hospitals might not completely represent a general Eastern Chinese Han population. Fourth, the genotype frequencies of IGF2BP2 rs1470579 A>C and rs4402960 G>T polymorphisms were not in HWE, which might lead to bias. Fifth, a functional experimentation was not performed. Finally, because of the lack of the information on survival of NSCLC, we did not further analyze the role of GF2BP2 and IGFBP3 variants on NSCLC prognosis.

Conclusion
Our study suggests that IGF2BP2 rs1470579 A>C, rs4402960 G>T polymorphisms are candidates for decreased susceptibility to NSCLC in Eastern Chinese Han population among female, <60 years, and never drinking subgroups. Compared with A rs1470579 C rs2270628 G rs3110697 G rs4402960 G rs6953668 Cancer Management and Research 2018:10 submit your manuscript | www.dovepress.com

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Chen et al haplotype, A rs1470579 C rs2270628 G rs3110697 G rs4402960 A rs6953668 haplotype significantly decreased risk of NSCLC. In the future, more case-control studies with comprehensive resequencing or SNP functional analysis are needed to confirm these preliminary findings.