The Effects of ANRIL Polymorphisms on Colorectal Cancer, Tumor Stage, and Tumor Grade among Iranian Population

doi:10.21203/rs.3.rs-3615539/v1

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Research Article

The Effects of ANRIL Polymorphisms on Colorectal Cancer, Tumor Stage, and Tumor Grade among Iranian Population

https://doi.org/10.21203/rs.3.rs-3615539/v1

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published 05 Apr, 2024

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Background: Colorectal cancer (CRC) is a type of neoplasm, developing in the colon or rectum. The exact etiology of CRC is not well known, but the role of genetic, epigenetic, and environmental factors are established in its pathogenesis. Therefore, the aim of this research was to explore the effects of ANRIL polymorphisms on the CRC and its clinical findings.

Methods and Results: The peripheral blood specimens were collected from 142 CRC patients and 225 controls referred to Milad Hospital, Tehran, Iran. PCR- RFLP method was used to analyze ANRIL rs1333040 rs10757274, rs10757274 and rs1333048 polymorphisms. The ANRIL rs1333040 polymorphism was related to a higher risk of CRC in the co-dominant, dominant, and log-additive models. ANRIL rs10757274, rs10757274, and rs1333048 polymorphisms showed no effect on CRC susceptibility. Haplotype analysis showed a higher risk of CRC in individuals carrying the CGAA haplotype of ANRIL rs1333040/ rs10757274/ rs4977574/rs1333048 polymorphisms and a lower risk of TGGA haplotype compared to TGGC haplotype. The rs1333040 polymorphism was associated with higher TNM stages (III and IV). ANRIL rs10757274 polymorphism was lower in CRC patients over the age of 50 years only in the dominant model. In addition, the rs10757274 was associated with well differentiation in CRC patients.

Conclusion, The ANRIL rs1333040 polymorphism was associated with a higher risk of CRC and higher TNM stages. ANRIL rs10757274 polymorphism was associated with the well-differentiated tumor in CRC.

ANRIL

Colorectal Cancer

Polymorphism

Tumor grade

Tumor stage

Colorectal cancer (CRC) is a kind of neoplasm, developing in the colon or rectum. CRC is the third leading reason of cancer death in the world, and its prevalence is rising in developing countries [1]. The death rate of CRC has been reduced because the screening techniques and treatment have improved. However, distant metastasis, especially in the liver, and the cell stemness properties of cancerous cells could affect the prognosis and high fatality rate [2]. Evidence showed the effect of environmental and genetic factors on CRC pathogenesis [3]. Smoking, nutrition, obesity, insufficient physical exercise, and aging can be considered as other risk factors for CRC. Evidence showed that both genetic and epigenetic alterations, that change normal glandular epithelium in the invasive adenocarcinomas, are known as the main factors contributing to CRC improvement[4].

Recently, the role of long non-coding RNAs(lncRNAs) in the pathogenesis of various cancers, including CRC is known[5]. The lncRNAs represent a set of non-coding RNAs that have more than 200 nucleotides in length and are not translated into proteins. These molecules can have various effects on numerous biological events, including the control of transcription and post-transcription processes, differentiation, development, epigenetic alteration, apoptosis, and cell cycle control [6]. There are several lncRNAs, including HOTAIR[7, 8], MALAT1[9], H19[10] and ANRIL[11], that could affect the various diseases like cancers.

ANRIL is a lncRNA which its gene (ANRIL, CDKN2B antisense RNA 1) is located on the small arm of chromosome 9(9p21). ANRIL gene contains more than 21 exons, and many of them consist of SINE, LINE, and Alu repetitive elements [12].

Various cellular processes are described, such as tumorigenesis, genotoxic stress, and inflammation that affect the ANRIL expression[12]. The transcription of ANRIL is activated by E2F1 following the genotoxic stress of DNA damage in an ATM-dependent manner. ANRIL is believed to initiate cell growth by blocking the INK family inhibitors after DNA repair ends and permitting it to enter the cell cycle again [13]. Indeed, evidence showed that TET2, as a tumor suppressor, controls ANRIL expression when binds to its promoter in human gastric cancer[14]. It is indicated that the regulation of ANRIL transcription affects not only oncogenesis and DNA damage but also various processes, including inflammation and cell senescence[12].

Evidence showed that the homozygous deletion or the silencing of the INK4b-ARF-INK4a gene cluster is observed in about 40% of human cancers which ANRIL is one of the most commonly changed lncRNAs in both the development and the progression of cancers [15]. In normal cells, an increase in ANRIL transcription by E2F1 is necessary for suppressive genes, namely p14ARF, p15INK4b, and p16INK4a, at the late phase of response to DNA damage so as to return to basal cellular levels after completing the DNA repair. However, in the neoplasmic conditions, the abnormal ANRIL expression would result in the inhibition of the control in DNA damage response, leading to genomic instability, and consequently, tumor improvement [13, 16].

Evidence showed that various suspected transcription factor binding sites are disrupted by different polymorphisms in the 9p21 locus. Therefore, the locus expression should be controlled by various signaling pathways. The response to at least two of these signaling pathways (STAT1 and RAS) is mediated by ANRIL. Moreover, different polymorphisms in those regions can be controlled independently by the expression of ANRIL and CDKN2A/B in association with various diseases. Different variants identified in the ANRIL locus have been shown a significant association with tumor development[16].

Previous studies have investigated the effects of ANRIL polymorphisms on cancer susceptibility; however, there are no published reports on the association between ANRIL variants and CRC. Therefore, this study aimed to explore the probable effects of ANRIL polymorphisms on CRC and its clinical findings.

Subjects

This case-control study was conducted on patients and controls referred to Milad Hospital, Tehran, Iran, from October 2018 to November 2020. The ethical committee of Zahedan University of Medical Sciences approved the study protocol and the written informed consent form was signed by all the participants.

The peripheral blood specimens were collected from 142 patients with CRC who underwent surgical removal of colorectal cancer in the Milad Hospital Surgery Ward. 225 controls who were referred to check up at the same hospital were included in the study.

The diagnosis of colorectal adenocarcinoma was based on the criteria of histological confirmation. The CRC patients had no preoperative chemotherapy or radiotherapy. The clinical characteristics and pathological reports were also obtained for patients. The control group consisted of healthy individuals having no history of cancer and other serious diseases who were matched for age, sex and BMI and CRC patients.

Sample collection

The peripheral blood specimens from all participants were collected in an EDTA-containing tube. The DNA was extracted from peripheral blood leukocytes by the salting-out method, using the DNA extraction kit according to manufacturer protocol (Sinacolon, Iran).

The quality and quantity of DNA were assayed by electrophoresis on 1% agarose gel and NanoDrop spectrophotometer, respectively.

Genetic analysis

To detect ANRIL rs1333040 rs10757274, rs10757274, and rs1333048 polymorphisms, the polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method was used as described previously [11]. The PCR amplification was conducted in a final reaction mixture of 18 µL, containing, 1 µL each primer (10 µM), 9 µl of 2X master mix, 6 µL deionized water, and 1 µL template DNA (100 ng/µL). The cycling program was performed as follows: the first denaturation at 95°C for 6 min, followed by 35 cycles of 30 sec at 95°C, 30 sec at optimal annealing temperature for each polymorphism (52, 59, 50, and 51°C respectively), extension at 72°C for 30 sec, and final extension step of 72°C for 6 min. The PCR products were then digested with the (BsmI, BsmAI, Hin6I, and Dra I restriction enzymes respectively).[17, 18] The digested fragments were separated by 2% agarose gel electrophoresis which stained with Syber green.

Statistical analysis

SPSS version 23 was used for statistical analysis. The descriptive statistics were examined by Fisher's exact test or T-test. Mann-Whitney test was used instead of T-test if the data were abnormal. Differences between the genotypic and allelic distributions of the study groups and subgroups were calculated using SNPStats (http://bioinfo.iconcologia.net/snpstats/start.htm) in various genetic models by obtaining the odds ratio (OR) and their CI of 95% (95% CI ). To examine the Hardy-Weinberg equilibrium (HWE), the Chi-squared (χ²)-test was employed.

The demographic and clinical data of both groups are presented in Table1. Due to matching the groups, no significant variation was observed between the two groups with respect to the mean age (58.5±13.4 and 56.4±9.3 in CRC patients and controls, respectively, P>0.05). No significant difference was observed between the two groups in terms of the frequency of male and female participants.

Table 2 presents the frequency of ANRIL polymorphisms in CRC patients and the control group. All loci were not deviated from hardy Weinberg equilibrium. The frequencies of rs1333040 CT and TT genotypes in the CRC group ( 49 and 43%, respectively) were significantly higher than those in the control group(46.2 and 33.4%, respectively) and these genotypes were associated with a higher risk of CRC in the co-dominant model[OR = 2.81(1.36-5.81), OR= 3.40 (1.62-7.13), p = 0.002, respectively). In addition, rs1333040 polymorphism was associated with 3.06- fold increased risk of CRC in the dominant model [OR= 3.06 (1.53-6.13), P=0.0001] and 1.62- fold increased risk of CRC in the log-additive model [OR=1.62 (1.18-2.21), P=0.002]. No relationship was observed between this variant and CRC in the recessive and over-dominant models. The ANRIL rs10757274, rs10757274, and rs1333048 polymorphisms showed no association with CRC in all genetic models.

Haplotype analysis (Table 3) showed a 6.65-fold increased risk of CRC in individuals carrying the CGAA haplotype of ANRIL rs1333040/ rs10757274/ rs4977574/rs1333048 polymorphisms compared to the TGGC haplotype [OR= 6.65 (2.02 - 21.89), p = 0.002]. However, the TGGA haplotype was associated with 11-fold lower risk of CRC [OR= 0.09(0.02 - 0.50), p=0.006)]

The possible effects of ANRIL rs1333048, rs4977574, rs10757274, and rs1333040 polymorphisms on tumor location, tumor grade, tumor size, and TNM stage were assayed in the co-dominant, dominant, recessive, and over-dominant models. The significant results are presented in Tables 4 and 5. The frequencies of rs1333040 CT and TT genotypes were higher than that in CC genotype in CRC patients with higher TNM stages (III and IV), leading to the 4.66-fold increased risk of higher stages in CRC patients in the dominant model [OR= 4.66 (1-22.46), P=0.031]. However, this variant had no effect on other tumor parameters and was not related to age and sex.

The effect of ANRIL rs10757274 polymorphism was lower in CRC patients over the age of 50 years only in the dominant model [OR=0.22((0.05-0.99), p=0.019]. In addition, the frequencies of ANRIL rs10757274 AG and GG genotypes were higher in CRC patients with the well-differentiated tumor, and this variant was associated with a 3.08-fold increased risk of the differentiated CRC tumors compared to poor or moderate one in the dominant model[OR= 3.08(1.13-8.40), p=0.024).

The present study showed a higher frequency of rs1333040 CT and TT genotypes in the CRC group and also this variant was associated with a higher risk of CRC in the co-dominant, dominant, and log-additive models. However, the ANRIL rs10757274, rs10757274, and rs1333048 polymorphisms showed no association with CRC in all genetic models. The CGAA haplotype of ANRIL rs1333040/ rs10757274/ rs4977574/rs1333048 polymorphisms was associated with the TGGC haplotype and lower risk of CRC. In addition, the rs1333040 polymorphism (CT and TT genotypes) was associated with higher TNM stages (III and IV in CRC patients in the dominant model. The ANRIL rs10757274 polymorphism was lower in CRC patients over the age of 50 years only in the dominant model. The ANRIL rs10757274 variant was associated with an increased risk of differentiated CRC tumors compared to poor or moderate in the dominant model.

ANRIL is a well-known lncRNA (also called CDKN2BAS) and encodes a large antisense non–coding RNA. ANRIL affects several biological processes and regulates the processes that can play key roles in tumor promotion and progression. Therefore, it is a potential candidate in various diseases like cancers. GWAS has introduced several genetic variants in INK4b-ARF-INK4a (such as ANRIL SNPs) and proposed the impacts of these variants on the susceptibility to the development of several diseases like cancers[12].

Important roles of ANRIL in the invasion and metastasis of TC cells were described by Zhao et al. They indicated that ANRIL could reduce the expression of p15INK4b tumor suppressor by blocking the TGF-β /Smad signaling pathway [19]. The results obtained from Liu et al.’s study indicated the up-regulation of ANRIL in association with poor prognosis and tumorigenesis of oral squamous cell carcinoma (OSCC)[20]. In Vitro declined proliferation, migration, and invasion of osteosarcoma cells in knockdown of ANRIL expression condition were found by Cheng et al.’s study demonstrating its role in cellular biology and oncogenesis [21]. Likewise, higher expression of ANRIL in hepatocellular carcinoma is reported by Huang et al. [22]. In many studies, ANRIL was introduced as an oncogene involved in several cancers, such as gastric cancer[23], breast cancer[24], lung cancer[25] and thyroid cancer[11].

Regarding the effects of genetic factors on cancer pathogenesis, several studies investigated the effects of genetic variants in different genes on the promotion and progression of cancer. Long non-coding RNAs like ANRIL were among the candidate genes [9, 11].

In a study on genome-wide association performed by Bahrami et al, the common genetic variants in a region on chromosome 9p21 were introduced as a risk factor for CRC. The genes in this locus were CDKN2B, CDKN2A, and 3' end of CDKN2BAS (ANRIL), and several genetic polymorphisms were recognized in this region. ANRIL showed post-transcriptional modulatory activity and disturbed the expression of other genes near itself [26]. In the large meta-analysis performed by Timofeeva et al., a novel locus for squamous cell carcinoma at 9p21, including the ANRIL rs1333040 variant was also introduced [27].

The effects of ANRIL polymorphism on various diseases have been studied and the relation between these variants and several cancers has been indicated. However, there is no published report on the possible effects of ANRIL variants and colorectal cancer. Kang et al. (2015) showed no association between ANRIL rs2151280 T/C polymorphism and esophageal cancer risk [28]. In their study, Deng et al. (2019) found that the ANRIL rs2151280 variant genotype (A > G) was related to the susceptibility to glioma [29]. In another study, Zhou et al. indicated that the ANRIL rs1537373 variant might affect transcription factor binding and control CDKN2B expression, associated with the predisposition to pancreatic cancer (PC)[30].

Omrani et al. performed the random forest (RF) algorithm to predict prostate cancer risk and showed that ANRIL rs1333048 AA and rs10757278 GG could predict the risk of prostate cancer in the Iranian population [31]. In addition, Gong et al showed no relation between ANRIL rs10120688 and rs1333049 polymorphisms and lung susceptibility; however, they indicated that ANRIL rs10120688 polymorphism was associated with response to platinum-based chemotherapy [32].

A meta-analysis was performed by Huang et al. (2018) on 17 studies investigating the effects of long non-coding RNA polymorphisms on cancer risk. The ANRIL rs4977574, rs1333048, and rs10757278 polymorphisms but not rs1333045 were accompanied by overall cancer risk[33]. The ANRIL rs17694493 GG genotype was associated with 4.93- 4.93-fold increased risk of gastric cancer as compared with the CC genotype in Petkevicius et al’s study [23]. Chen et al. indicated the ANRIL rs1333049 as a biomarker for better prognosis for lung cancer patients with platinum-based chemotherapy [25]. The possible effects of ANRIL rs1333045, rs4977574, rs1333048, and rs10757278 genotypes on prostate cancer were examined by Taheri et al.’s study performed in Iran and despite the results of the present study, they indicated the relation between rs4977574, rs1333048 and rs10757278 polymorphisms but not rs1333045 and PC risk [34]. In their study, Yao et al. evaluated the relation between ANRIL rs1333045, rs4977574, rs1333048, and rs10757278 polymorphisms and cervical cancer (CC) and they showed only the effect of rs4977574A allele and lower risk of CC[35]. Khorshidi et al. studied the relation between ANRIL rs1333045, rs1333048, rs4977574, and rs10757278 variants and breast cancer (BC) and found that there was a relation between rs1333045TT genotype and BC before multiple-testing correction. In addition, they showed a higher frequency of TCGA haplotype in BC patients[24].

In another study, Maruei-Milan et al. found no association between none of the ANRIL rs11333048, rs4977574, rs1333040, and rs10757274 SNPs and papillary thyroid cancer (PTC). However, the AAAC haplotype showed a decreased risk of PTC, and the CAAC, and also CAGT haplotypes were associated with an increased risk of cancer. In this study, the rs1333048 CC genotype was higher in patients with larger tumors in the recessive model. The rs4977574 AC genotype was associated with smaller tumor size in the over-dominant model. rs10757274 AA and rs1333040 CC genotypes were associated with a decreased risk of III-IV cancer stages[11].

In the present study, for the first time, we studied the effects of ANRIL polymorphisms on colorectal cancer. However, our study had some limitations, first, a small sample size, particularly the analysis of subgroups might affect the accuracy of the present results. Second, the results would have been more valuable if the study had been performed on cancerous tissue and healthy lateral tissue about ANRIL expression. Further studies are needed to assay these aspects

The current study indicated the role of the rs1333040 variant and the higher risk of CRC in the co-dominant, dominant, and log additive models. The CGAA haplotype of ANRIL rs1333040/ rs10757274/ rs4977574/rs1333048 polymorphisms was associated with the TGGC haplotype with a lower risk of CRC. In addition, the rs1333040 polymorphism was related to higher TNM stages (III and IV) in the CRC group. The ANRIL rs10757274 polymorphism was lower in CRC patients over the age of 50 years. The ANRIL rs10757274 variant was associated with an increased risk of differentiated CRC tumors compared to poor or moderate.

CRC: Colorectal cancer

ANRIL: Antisense Noncoding RNA in the INK4 Locus

PCR-RFLP: Polymerase Chain Reaction- Restriction Fragment Length polymorphism

lncRNAs: long non-coding RNAs

HOTAIR: HOX antisense intergenic RNA

MALAT1: metastasis associated in lung adenocarcinoma transcript 1

H19: H19 Imprinted Maternally Expressed Transcript)

SINE: short-interspersed nuclear element

LINE: long-interspersed nuclear element

ATM: Ataxia telangiectasia mutated

E2F1: E2 promoter binding factor 1

STAT1: Signal transducer and activator of transcription 1

RAS: Rat sarcoma

BMI: Body Mass Index

TNM: Tumour, node, and metastasis

Acknowledgments

The authors would like to express their sincere gratitude to the Research Department of Zahedan University of Medical Sciences, for their assistance (project No. 9291). In addition, the authors would like to thank Milad Hospital Surgery Department and the individuals who helped us in this work.

Author Contributions

All authors contributed to the study's conception and design. Material preparation, data collection and analysis were performed by [Mina Asadi-Tarani], [Ahmadreza Darashti], [Mohammadsaeid Javadi] and [Saeedeh Salimi]. The first draft of the manuscript was written by [Saeedeh Salimi] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

All authors declare no conflict of interest to disclosure.

Ethical approval

This study was performed in line with the principles of the Declaration of Helsinki. The study protocol was approved by the ethics committee of Zahedan University of Medical Sciences (IR.ZAUMS.REC.1399.349).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, upon reasonable request.

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Table1: Demographic and clinical characteristics of Colorectal Cancer (CRC) patients and controls.

Clinical characteristics

CRC

n=142

Control

n=225

Age(years)

58.5±13.4

56.4±9.3

Sex

Male

Female

91(64.1)

51(35.9)

130(57.8)

95(42.2)

Location

Rectum

Colon

44(31)

98(69)

Grade

Poor differentiated

Moderate differentiated

Well differentiated

unknown

11(8.5)

44(34.1)

74(57.4)

Tumor Size

<4cm

≥4cm

unknown

59(45.4)

71(54.6)

TNM stage

I- II

III- IV

Unknown

67(51.9)

58(47.5)

N stage

Unknown

53(41.7)

74(58.3)

Table2. Allelic and genotypic frequency of ANRIL polymorphisms in Colorectal Cancer patients and controls

		n (%)		*P-value/* CI (95%)
	AA	Aa	Aa	Codominant Aa vs AA aa vs AA	Dominant Aa+ aa vs AA	Recessive aa vs Aa+ AA		Over dominant Aa vs AA+ aa	Log additive
*rs1333040 C/T*	CC	CT	TT
Control	46(20.4)	104(46.2)	75(33.4)	0.002 2.81 (1.36-5.81) 3.40 (1.62-7.13)	0.0001 3.06 (1.53-6.13)	0.064 1.51 (0.98-2.32)	0.57 1.13 (0.74-1.72)		0.002 1.62 (1.18-2.21)
Case	11(8)	70(49)	61(43)	0.48 1.42(0.80-2.53) 1.27 (0.67-2.39)
*rs10757274 A/G*	AA	AG	GG
Control	47(21)	112(50)	66(29)		0.26 1.37 (0.79-2.37)	0.92 0.98 (0.62-1.55)		0.34 1.23 (0.81-1.87)	0.57 1.09 (0.81-1.49)
Case	23(16)	78(55)	41(29)
*rs10757274 A/G*	AA	AG	GG
Control	68(30.3)	100(44.4)	57(25.3)	0.75 0.96 (0.58-1.58) 1.16 (0.67-2.03)	0.46 1.03 (0.65-1.63)	0.90 1.20 (0.75-1.92)		0.59 0.89 (0.58-1.36)	0.60 1.08 (0.81-1.42)
Case	42(29.5)	59(41.6)	41(28.9)	0.75 0.96 (0.58-1.58) 1.16 (0.67-2.03)
*rs1333048 A/C*	AA	AC	CC
Control	38(17)	118(52)	69(31)	0.44 0.72 (0.41-1.28) 0.91 (0.50-1.67)	0.40 0.79 (0.46-1.35)	0.53 1.15 (0.74-1.81)	0.21 0.77 (0.50-1.17)		0.96 0.99 (0.73-1.34)
Case	29(20)	65(46)	48(34)

Table 3: Haplotypes frequency of ANRIL polymorphisms in Colorectal Cancer (CRC) patients and controls

	rs1333040/ rs10757274/ rs4977574/rs1333048	CRC	control	P-value

1	TGGC	0.145	0.149	-
2	CGAA	0.020	0.163	0.002	6.65 (2.02 - 21.89)
3	TAGC	0.063	0.152	0.23	1.79 (0.69 - 4.63)
4	TAAA	0.124	0.056	0.20	0.57 (0.24 - 1.34)
5	TGAC	0.127	0.044	0.085	0.42 (0.16 - 1.12)
6	TGAA	0.049	0.080	0.37	1.59 (0.58 - 4.40)
7	CAAC	0.042	0.062	0.48	1.52 (0.47 - 4.90)
8	CAGC	0.068	0.049	0.52	1.34 (0.55 - 3.25)
9	TAGA	0.047	0.034	0.21	2.52 (0.60 - 10.54)
10	CGAC	0,077	0.035	0.31	0.53 (0.16 - 1.80)
11	TAAC	0.037	0.036	0.62	1.42 (0.36 - 5.62)
12	TGGA	0.086	0.014	0.006	0.09 (0.02 - 0.50)
13	CAAA	0.029	0.049	0.50	1.54 (0.44 - 5.36)
14	CGGC	0.009	0.043	0.17	3.31 (0.61 - 17.98)
15	CAGA	0.027	0.021	0.30	0.44 (0.09 - 2.11)
16	CGGA	0.052	0.015	0.56	0.62 (0.13 - 3.07)

Table4: Association of rs1333040 C/T polymorphism with clinical characteristics in Colorectal Cancer. patients.
*Characteristics*				P-value OR (95% CI)
	CC	CT	TT	Codominant1	Codominant2	Dominant	Recessive	Overdominant	Log-additive
Age, years
<50	3(8)	18(47)	17(45)
≥50	8(8)	52(50)	44(42)	0.96 1.08 (0.26-4.53)	0.97 (0.23-4.10)	0.97 1.03 (0.26-4.10)	0.80 0.91 (0.43-1.91)	0.78 1.11 (0.53-2.34)	0.85 0.94 (0.52-1.72)
Gender
Male	8(9)	44(49)	38(42)
Female	3(6)	26(50)	23(44)	0.79 1.58 (0.38-6.47)	1.61 (0.39-6.71)	0.49 1.59 (0.40-6.29)	0.82 1.09 (0.55-2.16)	0.90 1.05 (0.53-2.07)	0.63 1.14 (0.66-1.99)
Tumor Location
Rectum	4(9)	22(50)	18(41)
Colon	7(7)	48(49)	43(44)	0.9 1.25 (0.33-4.71)	1.37 (0.36-5.25)	0.69 1.30 (0.36-4.69)	0.74 1.13 (0.55-2.32)	0.91 0.96 (0.47-1.96)	0.66 1.14 (0.64-2.01)
Grade
Poor/Moderate differentiated	6(12)	20(39)	25(49)
Well differentiated	5(7)	37(53)	28(40)	0.30 2.22 (0.60-8.19)	1.34 (0.36-4.95)	0.39 1.73 (0.50-6.03)	0.32 0.69 (0.33-1.44)	0.14 1.74 (0.84-3.61)	0.71 0.90 (0.51-1.58)
Tumor size
<4 cm	3(5)	30(51)	26(44)
≥4 cm	8 (11)	35(50)	28(39)	0.43 0.44 (0.11-1.80)	0.40 (0.10-1.69)	0.20 0.42 (0.11-1.67)	0.59 0.83 (0.41-1.67)	0.86 0.94 (0.47-1.87)	0.32 0.76 (0.43-1.32)
TNM stage
I-II	9(13)	30(45)	28(42)
III-IV	2 (3)	32(52)	28(45)	0.096 4.80 (0.96-24.03)	4.50 (0.89-22.72)	0.031 4.66(1-22.46)	0.07 1.15 (0.57-2.30)	0.44 1.32 (0.66-2.63)	0.22 1.41 (0.81-2.46)
N stage
N0	9(12)	34(46)	31(42)
N1	2(4)	27(51)	24(45)	0.22 3.57 (0.71-17.93)	3.48 (0.69-17.64	0.82 3.53 (0.73-17.06)	0.70 1.15 (0.56-2.34)	0.58 1.22 (0.60-2.48)	0.30 1.35 (0.76-2.38

Table 5: Association of rs10757274 A/G polymorphism with clinical characteristics of Colorectal Cancer.
*Characteristics*				P-value OR (95% CI)
	AA	AG	GG	Codominant1	Codominant2	Dominant	Recessive	Overdominant	Log-additive
Age, years
<50	2(5)	25(66)	11(29)
≥50	21(20)	53(51)	30(29)	0.054 0.20 (0.04-0.93)	0.26 (0.05-1.29)	0.019 0.22 (0.05-0.99)	0.99 1.00 (0.44-2.26)	0.11 0.54 (0.25-1.17)	0.23 0.70 (0.40-1.25)
Gender
Male	14(16)	50(56)	26(29)
Female	9(17)	28(54)	15(29)	0.96 0.87 (0.33-2.27 )	0.90 (0.31-2.57 )	0.79 0.88 (0.35-2.20 )	1 1.00 (0.47-2.12 )	0.84 0.93 (0.47-1.85 )	0.88 0.96 (0.57-1.61 )
Tumor Location
Rectum	7(16)	21(48)	16(36)
Colon	16(16)	57(58)	25(25)	0.4 1.19 (0.43-3.29)	0.68 (0.23-2.03)	0.95 0.97 (0.37-2.56)	0.19 0.60 (0.28-1.29)	0.25 1.52 (0.74-3.11)	0.34 0.77 (0.45-1.33)
Grade
Poor/Moderate differentiated	13(25)	25(50)	13(25)
Well differentiated	7(10)	41(59)	22(31)	0.078 3.05 (1.07-8.66)	3.14 (1.00-9.89)	0.024 3.08 (1.13-8.40)	0.48 1.34 (0.60-3.00)	0.30 1.47 (0.71-3.04)	0.078 1.64 (0.94-2.87)
Tumor size
<4 cm	7(12)	35(59)	17(29)
≥4 cm	16(23)	36(50)	19(27)	0.27 0.45 (0.17-1.23)	0.49 (0.16-1.47)	0.11 0.46 (0.18-1.22)	0.79 0.90 (0.42-1.95)	0.33 0.71 (0.35-1.4)	0.28 0.75 (0.44-1.27)



TNM stage
I-II	11(16)	38(57)	18(27)
III-IV	9(15)	35(56)	18(29)	0.94 1.13(0.42-3.04)	1.22 (0.41-3.66	0.77 1.16 (0.44-3.01)	0.78 1.11 (0.52-2.40)	0.98 0.99 (0.49-1.99)	0.72 1.10 (0.65-1.88)
N stage
N0	6(11)	30(57)	17(32)
N1	15(21)	41(55)	18(24)	0.33 1.83 (0.64-5.27)	2.36 (0.74-7.50	0.17 1.99 (0.72-5.53)	0.34 1.47 (0.67-3.22)	0.89 1.05 (0.52-2.14)	0.15 1.48 (0.86-2.57)

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The Effects of ANRIL Polymorphisms on Colorectal Cancer, Tumor Stage, and Tumor Grade among Iranian Population

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