The association between IGF1 Gene 3’-UTR polymorphisms and cancer risk

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Introduction
Insulin-like growth factor 1 (IGF1) plays an important role in regulating cellular proliferation and apoptosis. [1] Most circulat-ing IGF1 is bound to insulin-like growth factor binding protein 3 (IGFBP3), which can extend the half-life of IGF1. [2] IGF1 has been implicated in cancer development due to its key role in cell proliferation, differentiation, and apoptosis. [3] Many prospective studies have suggested that elevated IGF1 levels in the circulation can increase cancer risk. [4,5] While nutrition is a key factor that influences IGF1 levels in the circulation, studies of twins have indicated that 40% to 60% of the variation in IGF1 levels in the circulation depends on hereditary factors. [6][7][8][9] Several IGF1 polymorphisms have been identified as risk factors for cancers in genome-wide association studies (GWAS). [10] Three prime untranslated region (3'-UTR) contains important sequences that regulate mRNA transcription, stability, cellular localization, and microRNA binding. [11] Many studies have shown a relationship between IGF1 3'-UTR single nucleotide polymorphisms (SNPs) and cancer susceptibility, but these results are not consistent. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] For example, Jiang et al report that the rs5742714 can increase the risk of gastric cancer, [25] while Ennishi et al maintain that there is no obvious association between rs5742714 and gastric cancer risk. [20] Dong et al report that IGF1 rs6214 can reduce the risk of pancreatic cancer, [31] while Nakao et al's overall analysis indicates that rs6214 does not affect pancreatic cancer risk, but that the polymorphism could increase the risk of pancreatic cancer among patients with body mass indexes (BMIs) of 25 or greater at the age of 20. [21] The inconclusive nature of these results necessitated the present metaanalysis, which will provide a more accurate evaluation of the association between the IGF1 3'-UTR polymorphisms rs5742714, rs6214, and rs6220 and cancer risk.

Ethics statement
As all analyses were based on previously published studies, no ethical approval or patient consent was required.

Search strategy
We performed a literature search for all available articles concerning the association between IGF1 3'-UTR polymorphisms and cancer risk in PubMed, Embase, and Web of Science databases (before April 30, 2018). The following keywords were used: "IGF1 or IGF-1 or insulin-like growth factor 1," "polymorphism or SNP or mutation or variant," and "cancer or carcinoma or tumor." We also identified relevant studies via checking reference lists.

Inclusion and exclusion criteria
The included studies met the following criteria: (1) addressing the relationship between IGF1 polymorphisms and cancer risk, (2) having a case-control or cohort study design, (3) having been published in English, and (4) containing sufficient genotype data.
The exclusion criteria were as follows: (1) not having a case-controlled or cohort study design, (2) being meta-analyses or reviews, and (3) not having sufficient genotype data.

Data extraction and quality score
The 2 authors worked independently to extract information and evaluate the quality of the studies. The following information was extracted: name of first author, publication year, country, ethnicity, type of cancer, genotyping methods, control source, and alleles or genotypes frequency. The quality of the studies was assessed using a quality score form [32] (Supplementary Table 1, http://links.lww.com/MD/C721). Quality scores ranged from 0 to 15. Any disagreement was resolved via discussion.

Statistical analysis
All statistical analyses were performed with the STATA software (Version 12.0, Stata Corporation, College Station, TX). The strength of each association was estimated using ORs and 95% CIs in 5 genetic models: the allele, homozygote, heterozygote, dominant, and recessive models. P values <.05 were considered statistically significant. A Q test and I 2 statistic were used to assess heterogeneity. [33] If the heterogeneity test was P >.1, this would indicate that the heterogeneity was not significant, a fixed-effect model was used to synthesis the OR and 95% CI. [34] Otherwise, the random-effects model was applied. [35] Hardy-Weinberg equilibrium (HWE) for control was calculated via a Chi-squared test. Stratified analyses were conducted by ethnicity, cancer type, control source, and quality score. Sensitivity analyses were carried out to evaluate the stability of the results by omitting a single study each time. Begg test and Egger test were applied to detect potential publication bias. [36,37] 3. Results

Meta-analysis
The associations between IGF1 rs5742714, rs6214, and rs6220 polymorphisms and cancer risk were evaluated using odds ratios (ORs) and their 95% confidence intervals (CIs) under the following 5 genetic models: the allele homozygote, heterozygote, dominant, and recessive models. We also conducted stratified analyses according to ethnicity, cancer type, and quality score.
Only results synthesized from no fewer than 2 studies are shown. In total, our meta-analysis includes 9 studies regarding the rs5742714 polymorphism, which contains 4741 cases and 7267 controls. In overall analysis, no significant association was identified between rs5742714 and cancer risk in any of the 5 models (n = 9, case = 4741, control = 7267, Table 3). In the stratified analysis of ethnicity, no significant association was identified between rs5742714 and cancer risk among the Asian population (n = 7, case = 3395, control = 5863, Table 3). In the stratified analysis of cancer type, no significant association was identified between rs5742714 and the risk of the gastric (n = 2, case = 1283, control = 2135, Table 3) or pancreatic cancer (n = 2, case = 971, control = 2123, Table 3). The results synthesized from studies that scored no less than 12 (n = 7, case = 4213, control = 6730, Table 3) did not display any difference in terms of the results of the overall analysis.
In total, our meta-analysis includes 16 studies regarding the rs6214 polymorphism, which contain 8700 cases and 13,847 controls. In overall analysis, no significant association was identified between rs6214 and cancer risk in any of the 5 models (n = 16, case = 8700, control = 13,847, Table 3). In the stratified analysis of ethnicity, no significant association was identified between rs6214 and cancer risk among the Caucasian (n = 6, case = 4385, control = 6903, Table 3) or Asian (n = 6, case = 2815, control = 5240, Table 3) population. The results of the stratified analysis of cancer type demonstrate that rs6214 reduces the risk of breast cancer under the allele, the heterozygote, and dominant models (n = 4, case = 3550, control = 4617, Table 3 and  Table 3 and Figure 2, AA vs GA + GG: OR, 0.68, 95% CI,0.53-0.87, P = .003). No significant association was identified between rs6214 and the risk of colorectal cancer in this analysis (n = 3, case = 831, control = 2551, Table 3). The results synthesized from studies that scored no less than 12 (n = 12, case = 7302, control = 11,521, Table 3) did not display any difference in terms of the results of the overall analysis.
In total, our meta-analysis includes 9 studies regarding the rs6220 polymorphism, which contain 8127 cases and 10,085controls. In overall analysis, no significant association Table 1 Characteristics of the studies included in the meta-analysis.

First author
Year was identified between rs6220 and cancer risk in any of the 5 models (n = 9, case = 8127, control = 10,085, Table 3). The results of the stratified analysis of ethnicity demonstrate that rs6220 is associated with a significantly increased cancer risk among the Caucasian population under the allele model (n = 4, case = 5524, control = 7077, Table 3, G vs A: OR, 1.06, 95% CI, 1.00-1.13, P = .043). The results of the stratified analysis of cancer type suggest that rs6220 is associated with a significantly increased risk of breast cancer under the homozygote model (n = 3, case = 2859, control = 3765, Table 3, GG vs AA: OR, 1.23, 95% CI, 1.02-1.48, P = .031). The results synthesized from studies that scored no less than 12 showed that rs6220 increased cancer risk under the allele model (n = 7, case = 7614, control = 9569, Table 3, G vs A: OR, 1.06, 95% CI, 1.00-1.11, P = .033). The instability of the rs6220 results in the analyses stratified by score demonstrates that rs6220 tends to increase cancer risk.
The number of studies used in this assessment was limited. Thus, more studies on the associations between the rs5742714, rs6214, and rs6220 polymorphisms and cancer risk are warranted to confirm these conclusions, and the molecular mechanisms via which these polymorphisms function should also be explored.

Sensitivity analysis
Sensitivity was evaluated by deleting each study once at a time. The corresponding ORs were not altered by any single study for rs5742714, rs6214, or rs6220 ( Fig. 3 and Supplementary Table 2, http://links.lww.com/MD/C721), demonstrating that the results were relatively stable in our meta-analysis.

Publication bias
A Begg test and an Egger test were performed to determine the publication biases of the studies. No statistical evidence of publication bias was observed in any of the 5 models for rs5742714, rs6214, or rs6220 (Table 4).

Discussion
The IGF signaling system plays an important role in cell proliferation, differentiation, and apoptosis. [1] IGF1 promotes cell proliferation via the RAS-mitogen-activated protein kinase (MAPK) signaling pathway. [38,39] Moreover, it is also a potent anti-apoptotic molecule that activates the  rs5742714  Total  GG  GC  CC  Total  GG  GC  CC  G  C  G  C  Chia2008  551  449  98  4  683  565  109  9  996  106  1239  127  0.160  15  Suzuki2008  795  629  154  12  721  591  124  6  1412  178  1306  136  0.857  12  Ennishi2011  703  427  238  38  1462  902  479  81  1092  314  2283 Table 3 Meta-analysis of the association between rs5742714, rs6214, and rs6220 polymorphisms and cancer risk. phosphatidylinositol 3-kinase (PI-3K)-AKT pathway. [40,41] Several IGF1 polymorphisms have been found to be associated with elevated IGF1 levels in the circulation, thus increasing the risk of cancer. [12,27] The previous meta-analyses of the relationship between IGF1 polymorphism and cancer focused on studies of IGF1 CA repeat variants. For the first time, we have systematically reviewed and investigated the relationship between the SNPs in the IGF1 gene's 3'-UTR sequences and cancer risk. We have synthesized the results from those groups contained in 2 or more studies. Thus far, for some types of cancer, there is only a single study. We reviewed these studies in the discussion.
In the present meta-analysis, 3 IGF1 polymorphisms were included: rs5742714, rs6214, and rs6220. The criteria for selecting these SNPs were as follows: the SNP should be located in the 3'-UTR region of the IGF1 gene, the SNP should have been reported to have a relationship with cancer risk previously, and the minor allele frequency (MAF) of a selected SNP should be no less than 5% in most of the populations in the 1000 Genomes Project Phase 3 (Supplementary Table 3 The rs5742714 C allele has been reported as a protective mutation in childhood acute lymphoblastic leukemia (ALL) and renal cell carcinoma (RCC). Additionally, the GC and CC genotypes have been reported to reduce the risk of childhood ALL and RCC as compared to the GG genotype. [26,27] Cao et al explored the potential functionality of rs5742714, finding that carriers with the GG genotype had higher levels of IGF1 expression in their renal tissues than carriers with the GC or CC genotype. Further, the rs5742714 C allele was observed to create a microRNA binding site for hsa-mir-580, unlike the G allele. [27] Naoko et al reported that among patients with BMIs 25 or greater at age 20, pancreatic cancer risk was increased with the presence of the C allele for rs5742714. [21] Thus, in the present meta-analysis, we suggest that rs5742714 is not significantly associated with gastric or pancreatic cancer risk. rs6214 is located in the 3'-UTR region of exon 4 in IGF1 and does not cause any amino acid change itself. However, it may have regulatory functions or could be linked with functional alleles at exon 4, leading to a change in the amino acid sequence in the IGF1. [42] Vella et al (2008) tested IGF1 protein levels at birth and at age 7 or 8 years in children who had a different genotype of rs6214. They found that rs6214 polymorphism could increase IGF1 concentrations, but no association was shown between this polymorphism and growth or glucose metabolism. [43] Lu et al reported that rs6214 polymorphism could increase expression of IGF1 mRNA, thus, the difference was not statically significant. [26] Al-Zahrani et al reported that www.md-journal.com that rs6214 was not associated with circulating IGF1 levels among Caucasian women. [12] In fact, the rs6214 polymorphism A allele has been reported to increase childhood ALL risk, [26] as well as increasing esophageal adenocarcinoma (EAC) and head and neck cancer (HNC) risk in women. [23] For the present metaanalysis, it was observed that rs6214 reduced the risk of breast cancer under the allele, the heterozygote, and the dominant models, as well as reducing the risk of pancreatic cancer under the recessive model. It has been reported that the rs6220 G alleles are significantly associated with increasing levels of IGF1, [14] thus increasing prostate cancer risk. Furthermore, rs6220 has been found to reduce the risk of the low-grade gliomas. [16] Interestingly, Al-Zahraniet et al found that there was a statistically significant association between rs6220 and circulating IGF1 levels in females, though, not in males. [12] Moreover, women who have the rs6220 GG genotype had higher IGF1 plasma levels and increased breast density. [29] In the present meta-analysis, it was observed that rs6220 was significantly associated with increasing the risk of breast cancer under the homozygote model. Even if the 3'-UTR sequences cannot translate into proteins, they may contain sequences that are critical for transcriptional regulation, mRNA stability or cellular localization. [17] The biological functions of these polymorphisms in 3'UTR should be explored more in future studies.
The present meta-analysis has several limitations. First, we only included studies published in English. Thus, important studies published in other languages may have been overlooked. Second, the number of studies is relatively small, especially in the stratified analysis. For instance, there is only one study available for rs5742714 regarding ALL, and there is only 1 study available for rs6220 regarding the Asian population, so a pooled study could not be performed for this type of cancer or this ethnicity. Finally, due to the limited information contained in the included articles, we could not analyze adjusted ORs regarding other factors such as gender, age, alcohol intake, and smoking history, which may have influenced the association.
In conclusion, in this study, we systematically reviewed and meta-analyzed the relationship between IGF1 gene 3'-UTR polymorphisms and cancer risk for the first time. We found that rs5742714, rs6214, and rs6220 were not associated with overall cancer risk. In fact, rs6214 reduced the risk of breast and pancreatic cancer, while rs6220 increased the risk of breast cancer. The study also indicated that rs6220 increased overall cancer risk among Caucasian populations. We need welldesigned studies with larger sample sizes to explore the relationship between IGF1 3'-UTR polymorphisms and cancer risk in the future.