Association of HOTAIR rs2366152 and rs1899663 polymorphisms with colorectal cancer susceptibility in Iranian population: A case–control study

Abstract Background Despite the fact that numerous studies have investigated the association between genetic polymorphisms and colorectal cancer (CRC), more research is required to comprehend the molecular mechanisms of CRC. In the present study, we investigated the association between lncRNA HOTAIR rs2366152 and rs1899663 polymorphisms with CRC susceptibility in the Iranian population. Methods This case–control study consisting of 187 CRC patients and 200 healthy samples. The tetra‐amplification refractory mutation system‐polymerase chain reaction (Tetra‐ARMS‐PCR) technique was used for the genotyping of rs2366152 and rs1899663 polymorphisms. Results The findings showed that the AG genotype of the rs2366152 polymorphism has a protective effect on CRC susceptibility (OR = 0.60, 95% CI: 0.38–0.94, p‐value = 0.023). Furthermore, rs2366152 polymorphism associated with CRC risk in an over dominant inheritance model (p‐value = 0.0089). According to the outcomes of the rs1899663 polymorphism, the GT genotype had protective effects on CRC risk (OR = 0.55, 95% CI: 0.35–0.86, p‐value = 0.008). Moreover, statistical analysis has shown that the rs1899663 polymorphism was associated with CRC risk in dominant (p‐value = 0.013) and overdominant (p‐value = 0.0086) inheritance models in the Iranian population. Conclusion This study confirmed that HOTAIR rs2366152 and rs1899663 polymorphisms associated with CRC risk in different inheritance models. It is indeed necessary to do additional research to verify our findings.


| INTRODUC TI ON
Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in the world. Epidemiological studies have shown that CRC is third in terms of prevalence, with more than 1,900,000 new cases, and second in terms of mortality, with 935,000 deaths. 1 In the Iranian population, CRC is the fourth most widespread cancer in males and the third most prevalent cancer in women. 2 According to the pathophysiology of colon cancer, an accumulation of genetic abnormalities, either somatic or germline, causes the normal colonic epithelium to change into a precancerous lesion and ultimately into an invasive carcinoma. Moreover, genetic alterations can cause cancer cells to undergo apoptotic cell death. 3,4 Numerous risk factors can alter the prevalence of CRC, according to previous research. In addition, there are established strong connections between environmental or genetic factors and the occurrence of cancer. 5,6 Furthermore, dietary and lifestyle risk factors, including high amounts of red and processed meat in the diet, low fiber diet, obesity, low physical activity, alcohol consumption, and smoking, may increase CRC incidence. 7 Researchers have discovered some molecules, such as long noncoding RNA (lncRNA), that play a significant role in the progression of CRC tumorigenesis, despite the fact that the molecular basis of CRC is not entirely known. [8][9][10] LncRNAs are transcribed by RNA polymerase II and have more than 200 nucleotides. 11 They have vital functions in regulating chromatin dynamics, gene expression, cellular growth, cell development, and differentiation. 12 The HOX Transcript Antisense Intergenic RNA (HOTAIR), which is spliced and polyadenylated, is a well-known lncRNA. LncRNA HOTAIR is transcribed from the antisense strand of the HOXC gene cluster and can also alter HOXD gene expression. 13 LncRNA HOTAIR in the combination of polycomb repressive complex 2 (PRC2) and LSD1/REST/ COREST complex, in 5՛ and 3՛ region, respectively, has involved in chromatin regulation and takes part in histone modifications like H3K27 methylation and H3K4 demethylation. 14 Studies have demonstrated the role of HOTAIR in the pathogenesis of multifactorial diseases. For example, variations in the HOTAIR gene increased the risk of recurrent spontaneous abortion (RSA), 15 and bipolar disorder (BD). 16 HOTAIR may also affect type 2 diabetes mellitus (T2DM) susceptibility by modulating various signaling pathways. 17 In addition, studies have shown the HOTAIR gene contributes to tumorigenesis 18  A well-known genetic variant is single nucleotide polymorphism (SNP), which has different roles in cancer susceptibility based on its region. For instance, SNPs in the exons change gene transcription and translation, while SNPs in the introns have roles in RNA splicing, genomic imprinting, and also the function of lncRNAs. 24 The functional SNP rs4759314 A>G in lncRNA HOTAIR is associated with an increased risk of gastric cancer in the Chinese population. LncRNA HOTAIR polymorphism rs4759314 is located in the intronic promoter region, changes promoter activity, and contributes to potential mechanisms for gastric cancer susceptibility by affecting the expression of HOTAIR and HOXC11 genes. 25 Wang et al. 26 discovered that the rs1899663 G>T polymorphism on the lncRNA HOTAIR increases the risk of lung cancer. The Chinese population has been studied for the SNP rs1899663 G>T in the lncRNA HOTAIR. According to the findings, rs1899663 G>T was associated with breast cancer, and the T allele frequency in cases was higher than in controls. More analysis also showed that TT carriers of rs1899663 were associated with an increased risk of breast cancer. 27 Sharma Saha et al. 28 have shown the C allele of rs2366152 is prevalent among cervical cancer cases that lead to low HOTAIR expression. Taken together, according to the meta-analysis study, multiple SNPs are already associated with various kinds of cancer. 29 The association between the lncRNA HOTAIR rs1899663 and rs2366152 and CRC has not been studied in the Iranian population yet. As a result, our case-control study sought to assess the association between the lncRNA HOTAIR rs2366152 and rs1899663 polymorphisms and CRC susceptibility in Iranian population.

| Subjects
In our case-control study, we evaluated 387 participants, comprising 200 healthy controls and 187 colorectal cancer cases which were referred from Taleghani Hospital. First, clinical interviews and medical records, were used to collect some information, such as the age, gender, stage, grade of tumors, family history of cancer, and marital status of participants. Additionally, based on their patients' histories, which included information from clinical evaluations and colonoscopies, the confirmation of every affected person who had been diagnosed with CRC was checked. All participants signed informed consent forms after being informed of the study's goals.
The study protocol was confirmed by the ethics committee of the Shahid Beheshti University of Medical Sciences (IR.SBMU.RETECH. REC.1401.384).

| DNA extraction and genotyping
Peripheral blood samples were collected from all subjects, and genomic DNA was then extracted using the Puregene® Blood Core Kit C (Qiagen). We used the tetra-amplification refractory mutation system-polymerase chain reaction (Tetra-ARMS-PCR) method for genotyping. 30 We used Primer1 online software to design specific primers for each SNP. 31 The Applied Biosystems thermocycler (ABI) was used to perform the Tetra-ARMS-PCR reaction. Each sample had a final volume of 25 μL, contained 2 μL of genomic DNA (100-200 ng), 12.5 μL Taq DNA polymerase 2× master mix Red (Amplicon), 1 μL of each primer (10 pmol), and 6.5 μL of PCR grade water. PCR conditions comprise initial denaturation for 5 min at 95°C, followed by denaturation at 95°C for 1 min and annealing for 50 s at the temperature exhibited in Table 1, and an extension step for 1 min and 20 s at 72°C, throughout the 35-cycle period. The final extension was done for 5 min at 72°C. The primer sequences and annealing temperatures for the rs2366152 and rs1899663 polymorphisms are included in Table 1. Following amplification, PCR products were separated using 2% agarose gel electrophoresis with DNA gel stain in 0.5× tris/borate/EDTA (TBE).

| Bioinformatical analysis
To determine the possible biological role of HOTAIR polymorphisms and to investigate the alteration of the binding affinity of transcription factors at HOTAIR polymorphism locus, we carried out in silico analysis using HaploReg, 32 RegulomeDB, 33 ORegAnno, 34 and PROMO 35 online tools.

| Statistical analysis
In order to calculate allele and genotype frequencies based on the Hardy-Weinberg equilibrium, we used χ 2 test in SNPStats 36 and MEDCALC 37 online software. Statistical analysis evaluated the association of HOTAIR rs2366152 and rs1899663 polymorphisms with CRC in recessive, dominant, overdominant, and log-additive inheritance models. A statistically significant p-value was regarded as being <0.05.

| RE SULTS
Genotypes and allele frequencies of the rs2366152 and rs1899663 SNPs in the cases and healthy groups are shown in Table 2

| DISCUSS ION
According to recent studies, although extensive investigations have been completed about the association between CRC and polymorphisms on lncRNAs having a vital function in growth, metastasis, and treatment, the precise molecular mechanism of CRC is not entirely understood. [38][39][40] Numerous studies have concentrated on the association between SNPs in the lncRNA HOTAIR and cancer susceptibility. 40  polymorphism is associated with low HOTAIR expression in cervical cancer. They also discovered that the C allele of rs2366152 could affect HOTAIR expression levels, particularly in cases of low HOTAIR expression cervical cancer. 28 In the present study, we have also analyzed the association between rs1899663 and CRC risk. Although statistical analysis demonstrated that there were no significant differences in allele frequencies between CRC cases and control groups, it showed that the G/T genotype in the rs1899663 polymorphism is associated with CRC risk (p-value = 0.008) and has protective effects on CRC susceptibility. Furthermore, the rs1899663 polymorphism is asso-

Genomic Research Center, Shahid Beheshti University of Medical
Sciences.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

I N FO R M ED CO N S ENT
The informed consent was taken from all the patients who participated in this study.