Abstract
Purpose: To discuss the association between adiponectin (ADIPOQ) gene rs2241766 and rs1501299 polymorphisms and the risk of colorectal cancer, and to analyze the role of the interaction between these two loci and environmental factors in colorectal cancer pathogenesis. Methods: The case–control study was performed with a 1:1 match. A self-designed questionnaire was used to perform a face-to-face survey with 600 new primary colorectal cancer cases confirmed by histopathology as well as 600 cases of people receiving a physical examination at the same time. The general information, lifestyle, and diet habits, etc. were collected from two groups of study subjects. Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was used to identify ADIPOQ rs2241766 and rs1501299 genotypes. Results: After adjusting for factors such as colorectal cancer family history, body–mass index (BMI), daily sedentary time, weekly red meat intake frequency, as well regular tea drinking, conditional logistic regression analysis indicated that rs2241766 TG+GG carriers had a higher risk of colorectal cancer than TT carriers (OR=1.433, 95% CI: 1.014–1.985); rs1501299 GT+TT carriers had a lower risk of colorectal cancer than GG carriers (OR=0.723, 95% CI: 0.531–0.902). Generalized multifactor dimensionality reduction analysis showed that ADIPOQ rs2241766 and rs1501299 could have interaction with red meat intake (p=0.001). Conclusion: ADIPOQ rs2241766 and rs1501299 single nucleotide polymorphisms (SNPs) could be associated with colorectal pathogenesis and could have interactions with red meat intake. Both factors impact colorectal cancer occurrence.
Introduction
Adiponectin is a recently discovered specific fat-derived collagen-like protein secreted by fat cells. Many studies have shown that the serum adiponectin concentration negatively correlates with colorectal cancer occurrence risk (Izadi et al., 2012; Joshi et al., 2014). Adiponectin is a product of the ADIPOQ gene, which spans ∼16 kb with three exons on chromosome3q27 and has been linked to a susceptibility locus for metabolic syndrome (Suriyaprom et al., 2014), type 2 diabetes (Ye et al., 2014), cardiovascular disease (Zhou et al., 2014), and cancers (Li et al., 2014a, 2014b).
Previously, ADIPOQ gene exon 2+45T>G (rs2241766) and intron 2+276G>T (rs1501299) polymorphism were reported to be associated with changes in serum adiponectin concentrations (Li et al., 2014c; Pineda-Tenor et al., 2014). Also, several studies have shown that ADIPOQ rs266729 is associated with colorectal cancer occurrence risk (Pechlivanis et al., 2009; Gornick et al., 2011; Pham et al., 2014). However, the association of ADIPOQ genetic polymorphisms with colorectal cancer in the Chinese population remains unclear.
In the present study, we used a 1:1 matching case–control study method to investigate the relationship between ADIPOQ rs2241766 and rs1501299 polymorphisms and colorectal cancer occurrence risk. We also studied the interaction between these two single nucleotide polymorphisms (SNPs) and environmental factors in colorectal cancer pathogenesis.
Materials and Methods
Study subjects
A total of 600 cases of primary and new colorectal cancer patients admitted to the First Affiliated Hospital and the Second Affiliated Hospital of Harbin Medical University from July 2008 to May 2013, confirmed by histopathology, were collected and considered the patient group. The age of the patient group ranged from 28 to 77 years, among which there were 350 male cases, 250 female cases, 402 colorectal cancer patients, and 198 colon cancer patients. The healthy individuals who received physical examinations in the community health center at the same time were chosen as the control group. The control group had no history of tumor and their ages were matched to the patient group. The gender was matched 1:1. All patients and controls were Han Chinese.
Methods
Survey content
A self-designed questionnaire was used, and the face-to-face survey was administered by trained investigators to collect the following information: (1) demographic characteristics, including age, gender, marital status, education, occupation, annual household income (Yuan), height, and health conditions; (2) diet within 10 years before the disease or the survey, including frequency of foods, such as vegetables, fruits, meat (red meat was defined as pork, beef, and lamb; according to the daily 50–75 g standard recommended by the Chinese dietary pagoda, our study uses 7 times/week as the cutoff), bean products, and pickles; (3) personal hobby and life habits, including smoking (regular smoking was defined as ≥1 cigarette per day, continuous or cumulative >6 months), drinking (regular drinking was defined as drinking ≥2 times per week for >6 months), drinking tea (regular drinking tea was defined as drinking tea ≥1 time daily for >3 months), exercise, and daily sedentary time etc.; and (4) tumor family history. The survey followed the principal of informed consent. Vacuum anticoagulant blood collection tubes were used to collect 2 mL of blood from the cubital vein of study subjects, and the blood samples were stored at −20°C for future use. Our study was approved by the Ethics Committee of the First Affiliated Hospital of Harbin Medical University.
Laboratory detection
A total of 0.5 mL of anticoagulant blood was used to extract genomic DNA using a DNA extraction kit (Omega Bio-Tek Company, Guangzhou, China). The DNA was stored at −20°C for future use.
There are 680 SNPs of the ADIPOQ gene listed in the NCBI database (www.ncbi.nlm.nih.gov/snp). Using the Haploview 4.2 software and the HapMap phrase II database, we obtained two tagging SNPs (rs2241766 and rs1501299) for the Han Chinese with the minor allele frequency ≥0.05 and r2≥0.8. The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technology was used to detect ADIPOQ rs2241766 and rs1501299 genotypes. The primer sequences were as follows: rs2241766 forward, 5′-GAA GTA GAC TCT GCT GAG ATGG-3′; rs2241766 reverse, 5′-TAT CAG TGT AGG AGG TCTGTG ATG-3′; rs1501299 forward, 5′-GGC CTC TTT CAT CAC AGA CC-3′; and rs1501299 reverse, 5′-AGA TGC AGC AAA GCC AAA GT-3′. The amplified product of rs2241766 was digested with endonuclease SmaI in a 25°C water bath for 10 h. The amplified product of rs1501299 was digested with endonuclease BsmI in a 65°C water bath for 24 h. The digested products were then analyzed for genotypes using 2.5% agarose gel electrophoresis.
Statistical analysis
The original data were verified and organized. EpiData 3.1 software was used to enter the data, and SPSS 18.0 software was used to perform data analysis. The frequency distribution differences of general demographic characteristics, genotype, and allele between the patient group and the control group, as well as the Hardy–Weinberg equilibrium test of the control group were performed with the χ2 test. The relationship between genes, environmental factors, and colorectal cancer occurrence risk, as well as the interaction between genes and environment were analyzed using a conditional logistic regression model. Multivariate analysis of high-level interaction used the combined method of generalized multifactor dimensionality reduction (GMDR) and logistic regression.
Results
ADIPOQ rs2241766 and rs1501299 PCR amplification
The amplification product of rs2241766 is a 372-bp fragment and has three genotypes after digestion: homozygous wild-type TT (372 bp; one fragment); homozygous mutant GG (219, 153 bp; two fragments); and heterozygous TG (372, 219, 153 bp; three fragments). The amplified rs1501299 product is the 196-bp fragment and has three genotypes after digestion: homozygous wild-type GG (150, 46 bp; two fragments); homozygous mutant TT (196 bp; one fragment); and heterozygous GT (196, 150, 46 bp; three fragments).
The relationship between lifestyle habits, diet, and colorectal cancer occurrence risk
The distribution differences of education, occupation, annual household income, etc. between the patient group and the control group were not significant (p>0.05). Multivariate conditional logistic regression analysis results showed that regular tea drinking was a protective factor for colorectal cancer (OR: 0.455, 95% CI: 0.312–0.843), while daily sedentary time ≥8 h (OR: 1.743, 95% CI: 1.143–2.324), weekly red meat intake >7 times (OR: 1.543, 95% CI: 1.114–2.424), and colorectal cancer family history (OR: 2.122, 95% CI: 1.216–5.939) were risk factors for colorectal cancer. Body–mass index (BMI), drinking, smoking, and annual family income were not associated with colorectal cancer pathogenesis (p>0.05; Table 1).
Table 1.
Logistic Regression Analysis of the Relation Between Risk Factors and Colorectal Cancer
| Variables | OR (95% CI) | p-Value |
|---|---|---|
| BMI (kg/m2) | 0.987 (0.549–1.426) | 0.426 |
| Family income (Yuan) | 0.865 (0.511–1.558) | 0.142 |
| Drinking | 0.869 (0.431–1.433) | 0.554 |
| Smoking | 1.433 (0.994–2.113) | 0.103 |
| Regular tea drinking | 0.455 (0.312–0.843) | 0.003 |
| Daily sedentary time (h) | 1.743 (1.143–2.324) | 0.002 |
| Red meat (time/week) | 1.543 (1.114–2.424) | 0.001 |
| Cancer family history | 2.122 (1.216–5.939) | 0.004 |
BMI, body–mass index.
The relationship between ADIPOQ rs2241766, rs1501299 polymorphisms, and colorectal cancer occurrence risks
The Hardy–Weinberg equilibrium test showed that the ADIPOQ rs2241766 and rs1501299 genotype frequency of the control group complied with the Hardy–Weinberg equilibrium (both p>0.05). The rs2241766 TG+GG genotype frequency in the patient group was higher than the control group (p=0.015) and the rs1501299 GT+TT genotype frequency in the patient group was lower than the control group (p=0.031).
After adjusting for colorectal family history, BMI, daily sedentary time, weekly red meat intake frequency, and regular tea drinking factors, multivariate conditional logistic regression analysis showed that compared with rs2241766 homozygous wild-type TT, rs2241766 TG+GG carriers had a higher risk of colorectal cancer pathogenesis (OR=1.433, 95% CI: 1.014–1.985). Compared with rs1501299 homozygous GG, rs1501299 GT+TT carriers had a lower risk of colorectal cancer pathogenesis (OR=0.723, 95% CI: 0.531–0.902; Table 2).
Table 2.
The Relation Between ADIPOQ rs2241766 and rs1501299 and Colorectal Cancer
| Case group | Control | ||||||
|---|---|---|---|---|---|---|---|
| SNPs | N | (%) | n | (%) | OR (95% CI) | OR (95% CI)a | p-Value |
| rs2241766 | |||||||
| TT | 260 | 43.3 | 308 | 51.3 | 1.00 | 1.00 | |
| TG | 295 | 49.2 | 252 | 42.0 | 1.336 (1.064–1.677) | 1.360 (0.996–1.898) | 0.065 |
| GG | 45 | 7.5 | 40 | 6.7 | 1.135 (0.730–1.765) | 1.433 (0.798–2.324) | 0.024 |
| TG+GG | 340 | 56.7 | 292 | 48.7 | 1.379 (1.099–1.732) | 1.433 (1.014–1.985) | 0.015 |
| rs1501299 | |||||||
| GG | 297 | 49.5 | 255 | 42.5 | 1.000 | 1.00 | |
| GT | 266 | 44.3 | 304 | 50.7 | 0.775 (0.618–0.973) | 0.674 (0.497–0.919) | 0.034 |
| TT | 37 | 6.2 | 41 | 6.8 | 0.896 (0.565–1.418) | 0.799 (0.544–1.211) | 0.140 |
| GT+TT | 303 | 50.5 | 345 | 57.5 | 0.754 (0.600–0.946) | 0.723 (0.531–0.902) | 0.031 |
Adjustment for other risk factors, such as cancer family history, BMI, regular tea drinking, daily sedentary time, and red meat.
SNPs, single nucleotide polymorphisms.
The role of interaction between ADIPOQ rs2241766, rs1501299, and red meat in colorectal cancer pathogenesis
After adjusting for colorectal cancer family history, BMI, daily sedentary time, and regular tea drinking factors, conditional logistic regression results showed that rs2241766 and red meat, rs1501299 and red meat, rs2241766 and rs1501299 two–two interactions were not significant (all p>0.05).
Analysis of higher order interaction among ADIPOQ rs2241766, rs1501299, and red meat using the GMDR method showed that the model with rs2241766, rs1501299, and red meat three factors with the lowest prediction error (39.83%) and the highest cross-validation consistency (10/10) was considered the best model, suggesting that rs2241766, rs1501299, and red meat may have interactions. rs2241766 TG+GG and rs1501299 GG were defined as risk genotypes. Trend χ2 analysis results showed that the carried genotype number had a dose–response relationship with colorectal cancer occurrence risk (p=0.004). Compared with an individual without a risk genotype, the individual carrying 2 risk genotypes had a higher colorectal cancer occurrence risk (OR=1.876, 95% CI: 1.056–2.676). Conditional logistic regression analysis results showed that compared with an individual without a risk genotype and who had red meat intake ≤7 times/week, the individual carrying 2 risk genotypes and had red meat intake >7 times/week had a higher colorectal cancer risk (OR=2.867, 95% CI: 1.123–4.765), suggesting that rs2241766, rs1501299, and red meat could have interactions (Table 3).
Table 3.
Interactions Between ADIPOQ rs2241766 and rs1501299 and Red Meat Intake
| Factor 1 (SNP) | Factor 2 (red meat) | OR (95% CI) | p-Value | OR (95% CI)a | p-Valuea |
|---|---|---|---|---|---|
| rs2241766 | Red meat (times/week) | 1.231 (0.776–2.323) | 0.165 | ||
| TT | ≤7 | 1.00 | |||
| TG+GG | ≤7 | 1.312 (0.781–1.944) | 0.132 | ||
| TT | >7 | 1.561 (0.994–2.421) | 0.076 | ||
| TG+GG | >7 | 2.354 (1.456–3.212) | <0.001 | ||
| rs1501299 | Red meat (times/week) | 1.331 (0.765–2.608) | 0.342 | ||
| GT+TT | ≤7 | 1.000 | |||
| GG | ≤7 | 1.212 (0.756–1.987) | 0.546 | ||
| GT+TT | >7 | 1.543 (0.987–2.955) | 0.121 | ||
| GG | >7 | 2.656 (1.431–4.878) | <0.001 | ||
| rs2241766 | rs1501299 | 0.868 (0.432–1.656) | 0.377 | ||
| TT | GT+TT | 1.000 | |||
| TG+GG | GT+TT | 1.655 (1.121–3.254) | 0.031 | ||
| TT | GG | 1.754 (1.132–2.656) | 0.001 | ||
| TG+GG | GG | 2.754 (1.653–5.527) | 0.004 |
Interaction of two factors.
Discussion
Our results showed that after adjusting for colorectal cancer family history, BMI, daily sedentary time, weekly red meat intake frequency, and regular tea drinking factors, the ADIPOQ rs2241 766 TG+GG carriers had a higher colorectal cancer risk than TT carriers. Furthermore, rs1501299 GT+TT carriers had a lower colorectal cancer risk than GG carriers, suggesting that the rs2241766 T→G mutation was associated with an increased risk of colorectal cancer occurrence, and the rs1501299 G→T mutation was associated with a decreased risk of colorectal cancer occurrence.
The GMDR method analysis results showed that ADIPOQ rs2241766, rs1501299, and red meat may have interactions. The conditional logistic regression method was used to analyze the combined effect between carried risk genotype numbers and red meat intake. The results showed a very good supplement to the above conclusion. It is generally considered that red meat has a large amount of saturated fatty acids as well as animal proteins and is also rich in iron. Currently, international studies all consider red meat a risk factor for colorectal cancer (Ley et al., 2014; Oostindjer et al., 2014). Studies have shown that a large intake of red meat can increase plasma TG and insulin levels (Kucera et al., 2014). A high-fat diet can downregulate adiponectin RNA and protein expression in rats, inducing insulin resistance (IR) occurrence. Gao et al. (2014) also demonstrated that a high-fat diet can induce IR in rats. In addition, Simcox and McClain (2013) showed that an excessive iron overload can directly or indirectly mediate β-cell apoptosis and IR, further affecting diabetes occurrence, and red meat contains a large amount of iron. In summary, the interaction mechanism among ADIPOQ rs2241766, rs1501299, and red meat could together induce IR occurrence, therefore affecting colorectal cancer occurrence.
Currently, there are few studies regarding the association between ADIPOQ rs2241766 and rs1501299 polymorphisms and colorectal cancer occurrence, and the conclusions are not consistent. The studies by Kaklamani et al. (2008) and He et al. (2011) did not find that ADIPOQ rs2241766 and rs1501299 polymorphism were associated with colorectal cancer occurrence; however, one study in Saudi Arabia showed that rs2241766 TG+GG carriers had a lower colorectal cancer occurrence risk (Al-Harithy et al., 2012). The association between ADIPOQ rs2241766 and rs1501299 polymorphisms and colorectal cancer still needs verification through large-sample studies.
Our study collected diet and life habit information of the study subjects from 10 years before the illness/survey; therefore, recall bias may play a role. Only two SNPs, ADIPOQ rs2241766 and rs1501299, were analyzed, and our future study will study additional SNPs of ADIPOQ.
Acknowledgments
We sincerely thank the patients, healthy volunteers, and their families who participated in this study and all the medical personnel involved in specimen collection. This project has been supported by the Department of Education Science and Technology Project of Heilongjiang Province (No.12541303).
Author Disclosure Statement
No competing financial interests exist.
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