HMGA2 rs968697 T>C Polymorphism is Associated With The Risk of Colorectal Cancer

Background: Recently, a genetic polymorphism (rs968697 T>C) in HMGA2 gene has been reported to be associated with hepatoblastoma risk. However, no studies reported the effect of the polymorphism on the risk of colorectal cancer (CRC). The study aimed to explore whether rs968697 polymorphism had a signicant impact on CRC risk. Methods: A total of 500 CRC patients and 500 age and gender matched healthy individuals were genotyped by Sanger sequencing. Quantitative real-time PCR technology was used to detect the relative expression of HMGA2 gene in 30 pairs of primary CRC and adjacent non-cancerous tissues. Results: HMGA2 rs968697 polymorphism was signicantly associated with CRC risk (CC vs. TT: OR=0.20, 95%CI=0.06-0.70, P=0.01; (CC+CT) vs. TT: OR=0.71, 95%CI=0.53-0.96, P=0.02; CC vs. (CT+TT): OR=0.21, 95%CI=0.06-0.73, P=0.01; C vs. T: OR=0.67, 95%CI=0.51-0.89, P<0.01). The analysis based on tumor stage indicated that HMGA2 rs968697 polymorphism was signicantly associated with CRC tumor stage. In addition, the genotype-tissue expression showed that the rs968697 polymorphism was related to HMGA2 gene expression. The in silico analysis showed that rs968697 polymorphism located in the promoter region of HMGA2 gene could affect the binding of transcription factors. Conclusion: Our study suggested that HMGA2 rs968697 polymorphism was associated with CRC risk and might serve as a reliable biomarker to detect CRC risk.


Introduction
High mobility group AT-hook 2 (HMGA2) gene is located on chromosome12q14.3, and encodes an architectural transcription factor that belongs to the non-histone chromosomal high mobility group protein family. The protein can act as an oncogenic protein closely linked to aggressive tumor behavior and poor clinical outcomes [1][2][3]. Thereinto the promoting role of HMGA2 in the occurrence and development of colorectal cancer (CRC) has been reported [4][5]. For instance, HMGA2 over-expression was associated not only with metastasis but also with reduced survival rates of CRC patients [4]. HMGA2 could promote intestinal tumorigenesis by facilitating MDM2-mediated ubiquitination and degradation of p53 [5]. In recent years, a case-control study also investigated the association of a genetic polymorphism (rs968697 T > C) in the cancer-related gene with cancer risk, and found that the polymorphism was signi cantly associated with hepatoblastoma risk.
In view of the signi cance of rs968697 polymorphism and the critical role of HMGA2 gene in CRC, we speculated that HMGA2 rs968697 polymorphism might be involved in CRC risk. However, no studies reported the effect of the polymorphism on CRC risk. Thus, a case-control study was conducted to assess the strength of the effect. patients was histopathologically con rmed. Healthy individuals were cancer-free individuals living in the same residential area and seeking for routine physical examination. This study protocol was approved by the Institutional Review Board of the Hospital. All enrolled subjects provided written informed consent in the study.

Genotyping
Genomic DNA was extracted from peripheral blood samples by using the TIANamp genomic DNA Kit (Tiangen). The concentration and quality of genomic DNA were determined by using a NanoDrop spectrophotometer (Thermo Fisher Scienti c). Genotyping was performed by Sanger sequencing.

Bioinformatics analysis
RegulomeDB (www.regulomedb.org/regulome-search/) is a database annotating SNPs with known and predicted regulatory elements in the intergenic regions of the human genome [6]. SNPinfo (https://snpinfo.niehs.nih.gov/snpinfo/snpfunc.html) is a web-based tool integrating genome-wide association studies and candidate gene information into functional SNP selection for genetic association studies [7]. In this study, RegulomeDB and SNPinfo were used to explore the latent function of HMGA2 rs968697 polymorphism. For RegulomeDB database, POLR2A ChIP-Seq data from human colon cancer cell (HCT116) was analyzed.

Statistical analysis
In control group, Hardy-Weinberg equilibrium (HWE) of the genotype distribution was analyzed using the chi-square goodness-of-t test. The association between HMGA2 rs968697 polymorphism and CRC risk was evaluated using logistic regression analysis. The association between HMGA2 rs968697 polymorphism and CRC tumor stage was analyzed using the chi-square test. The expression levels of HMGA2 gene were compared among different genotype groups by using the one-way ANOVA. Statistical analyses were performed using SPSS 22.0 software. P value less than 0.05 was considered statistically signi cant.

Results
3.1 HMGA2 rs968697 was signi cantly associated with CRC risk As shown in Table 1, the genotype distribution of HMGA2 rs968697 polymorphism was 371/115/14 (TT/CT/CC) in control group, which conformed to HWE (P = 0.17). In CRC patients, the genotype distribution of HMGA2 rs968697 polymorphism was 400/97/3 (TT/CT/CC). The comparison results showed that HMGA2 rs968697 polymorphism was signi cantly associated with CRC risk. The individuals carrying rs968697 CC genotype had a decreased risk of CRC compared with those carrying the TT genotype (OR = 0.20; 95%CI = 0.06-0.70; P = 0.01). The individuals carrying rs968697 CC and CT genotype had a decreased risk of CRC compared with those carrying the TT genotype (OR = 0.71; 95%CI = 0.53-0.96; P = 0.02). The individuals carrying rs968697 CC genotype had a decreased risk of CRC compared with those carrying the CT and TT genotype (OR = 0.21; 95%CI = 0.06-0.73; P = 0.01). The individuals carrying rs968697 C allele had a decreased risk of CRC compared with those carrying the T allele (OR = 0.67; 95%CI = 0.51-0.89; P < 0.01). Furthermore, the analysis based on tumor stage indicated that rs968697 polymorphism was signi cantly associated with CRC tumor stage ( Table 2).  3.2 Rs968697 polymorphism was related to HMGA2 gene expression As shown in Fig. 1, the genotype-tissue expression showed that rs968697 polymorphism was related to the expression of HMGA2 gene. HMGA2 gene had a higher expression level in CRC and adjacent noncancerous tissues with rs968697 TT genotype than in CRC and adjacent non-cancerous tissues with rs968697 CC genotype.

Rs968697 polymorphism affected the binding of transcription factors
The in silico analysis showed that rs968697 polymorphism located in the promoter region of HMGA2 gene affected the binding of transcription factors, such as DBP, CDPCR3, POLR2A and TAXCREB (Table 3).

Discussion
As one of the most common cancers, CRC was estimated to cause over 1.8 million new cases and 881,000 deaths in 2018 [8]. Although the molecular etiology of CRC is still unknown, its onset is signi cantly associated with genetic variants. Single nueleotide polymorphism (SNP) is one of the most common genetic variants and involves altered cancer risk [9][10][11]. In the current study, we conducted a case-control study investigating the association between HMGA2 rs968697 polymorphism and CRC risk, and found that rs968697 C allele could reduce CRC risk compared with the T allele. To the best of our knowledge, this is the rst epidemiological study exploring the association of HMGA2 rs968697 polymorphism with CRC risk in the Chinese population. Furthermore, we also found that rs968697 polymorphism was related to the expression of HMGA2 gene in CRC and adjacent non-cancerous tissues. Bioinformatics analysis showed that the rs968697 polymorphism had an effect on the binding of transcription factors to the promoter of HMGA2 gene, which provided a possible molecular explanation for the effect of the genetic polymorphism on CRC risk. However, the detailed mechanism still needs to be veri ed by further well-designed experiment. It is worthy of note that the current sample size is relative small, which may not obtain su cient statistical power. Therefore, further replication studies are necessary to fully establish the association between HMGA2 rs968697 polymorphism and CRC risk.

Conclusion
Our molecular epidemiological ndings demonstrated a signi cant association of HMGA2 rs968697 polymorphism with CRC risk. The rs968697 polymorphism may serve as a potential biomarker for CRC risk.

Declarations
Ethical approval and consent to participate The study was approved by the Ethics Committee of Yancheng Teachers' University, and all participants provided written informed consent.
Authors contributions and consent to publish ZL drafted the manuscript. SZ collected clinical samples. JY and XW were responsible for the gures and tables. XG critically revised the manuscript. All authors approved the nal manuscript.