Dysregulation of vitamin D synthesis pathway genes in colorectal cancer: A case‐control study

Abstract Background The cytochromes P450 are a superfamily of enzymes that control the synthesis of the biologically active form of vitamin D, 1,25‐dihydroxyvitamin D3. These enzymes contribute to the formation of 1,25‐dihydroxyvitamin D3, which starts with a 25‐hydroxylation by CYP2R1 and CYP27A1 and a subsequent 1α‐hydroxylation via CYP27B1. Methods By using quantitative real‐time polymerase chain reaction (qRT‐PCR), we analyzed the expression ratio of CYP2R1, CYP27A1 and CYP27B1 genes within the vitamin D metabolic pathway in a total of 75 colorectal cancer (CRC) tissues compared to the adjacent tissues. Furthermore, we evaluated the association of CYP27B1 rs4646536 and CYP2R1 rs12794714 and rs10766196 polymorphisms with CRC risk in a total of 490 subjects, including 245 CRC patients and 245 non‐cancer controls. The genotyping was performed using tetra‐primer amplification refractory mutation system polymerase chain reaction (TP‐ARMS–PCR) method. Results The results indicated 2.3 and 2.7 upregulation of CYP2R1 and CYP27B1 genes in colorectal cancer tissues compared to the adjacent tissues, respectively. Rs12794714 AG genotype increased the risk of CRC (P = .03). Furthermore, a significant association was observed under the dominant inheritance model (P = .039). Conclusion CYP2R1 and CYP27B1 genes were over‐expressed in CRC samples compared to the adjacent control tissues. Furthermore, CYP2R1 rs12794714 variant was associated with the risk of CRC in the studied samples. CYP2R1 rs10766196 and CYP27B1 rs4646536 are not responsible for CYP2R1 and CYP27B1 genes expression alteration, respectively, but CYP2R1 rs12794714 polymorphism may be the reason of CYP2R1 upregulation and increased the risk of CRC.


| INTRODUC TI ON
According to the global cancer project estimation, colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide, accounting for around 1.8 million new cases and 860 000 deaths in 2018. 1 The Iranian National Cancer Registry (IACR) Report shows that the incidence of CRC has increased during the last 25 years. 2 Genetic factors, epigenetic alterations, and environmental factors may be associated with CRC progression. 3,4 Vitamin D is a steroid hormone and is involved in cell proliferation and differentiation. 5 Multiple lines of evidence have shown the vitamin D pathway dysregulation in different types of cancer, for instance, cytochrome P450 (CYP) genes such as CYP2R1, CYP27A1, and CYP27B1. [6][7][8] In summary, vitamin D synthesis is as follows: CYP2R1 and CYP27A1 convert vitamin D to circulating 25(OH)D. Then, CYP27B1 converts 25(OH)D to 1, 25-dihydroxyvitamin D, which is the biologically active form of the vitamin D. 9 Increased concentrations of CYP27A1 had been reported in the nuclei of normal colonic epithelia, aberrant crypt foci (ACF), and adenomatous polyps. 7 The primary site of CYP27B1 expression is in the proximal tubule of the kidney, but it is also expressed in other tissues such as the colon. 10,11 CYP2R1 is expressed in colon; however, liver is the primary site of CYP2R1 expression. 12,13 On the other hand, single-nucleotide polymorphisms (SNPs) could modify the gene expression and, consequently, influence the risk of cancer. 14 Additionally, numerous studies suggested that SNPs in the CYP2R1 and CYP27B1 were found to be associated with CRC risk. 15, 16 Gong et al have shown the association between intronic CYP27B1 rs4646536, located at the long arm of chromosome 12, and CRC risk. However, Dong et al did not find any significant association between this polymorphism and colon cancer. 15,17 Nam et al 18 hypothesized that the CYP2R1 rs12794714 and rs10766196 variants, both located at the CpG island, may modulate the gene transcription and were associated with the risk of developing type 1 diabetes. Furthermore, CYP2R1 rs12794714 was found to significantly associate with CRC and serum level of 25(OH)D3. 16 While several studies have investigated CYP27B1 and CYP2R1 expression and genetic association separately in CRC, only a few of them have simultaneously investigated the expression and genetic association of CYP27B1 and CYP2R1 with CRC. Hence, the current study aimed to investigate the relation between CYP2R1, CYP27B1, and CYP27A1 expressions and CRC. We also intended to assess the contribution of CYP2R1 and CYP27B1 polymorphisms in the pathogenesis of CRC in a sample of Iranian population.

| RNA isolation and reverse transcription
Total RNA was extracted from fresh-frozen CRC tissues and the noncancerous tissues using a GeneAll Hybrid-R™ RNA purification kit (Geneall Biotechnology Co. Ltd) according to the manufacturer's protocol. The quantity and the quality of the total extracted RNA were estimated using NanoDrop ® ND-1000 spectrophotometer (Thermo Fisher Scientific). The RNA purity was evaluated according to the A260/A280 ratio.
The beta-2 microglobulin (β2M) mRNA was used as the reference gene. β2M was selected as the reference gene according to previous research for identification of housekeeping control genes in colorectal cancer. 19 The thermal profile of the reaction was performed using the following conditions: initial denaturation at 95°C for 15 minutes; followed by 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds followed by melting curve stage assessment. The melting curve profile and agarose gel electrophoresis were performed to verify the specificity of primers and the authenticity of the PCR products.

| DNA extraction and genotyping
Genomic DNA was extracted from peripheral blood leukocytes using the standard salting-out procedure and stored at −20°C until further use. 20 The primers used to amplify the target DNA sequences, which were designed by using Primer1 online software available from http://prime r1.soton.ac.uk/prime r1.html, are presented in  genes' expression was significantly upregulated in CRC tissues compared with adjacent tissues (Figure 1A,B). CYP27A1 gene expression did not show any significant expression alteration when comparing the CRC tissues with adjacent tissues ( Figure 1A). The correlations between the expression of CYP2R1, CYP27B1, and CYP27A1 genes and clinicopathological features are shown in Table 3. None of the clinicopathological characteristics were significantly correlated with these genes.   (Table 5). Figure 2 represents 2% agarose gel electrophoresis for identification of CYP2R1 rs12794714 genotypes.

| D ISCUSS I ON
Vitamin D deficiency is inversely associated with the incidence and mortality of colorectal cancer. 21 Under physiological conditions, CYP27B1 and CYP2R1 genes control the vitamin D synthesis, and vitamin D also controls the expression level of these genes. 9 also reported no association between CYP2R1 rs12794714 polymorphism and the risk of breast cancer in Chinese women. In African Americans, CYP2R1 rs12794714 was associated with a decreased risk of CRC. 16 The AAGA haplotype of CYP2R1 rs7936142-rs12794714-rs2060793-rs16930609 was associated with a lower 25(OH)D concentration in the healthy Chinese population. 33 Under the additive and recessive models, the exonic variant CYP2R1 rs12794714 was significantly associated with plasma 25(OH)D concentration in northeastern Han Chinese children and people of Arab origin. 31,34 According to the HaploReg, 35 RegulomeDB, 36

ACK N OWLED G M ENTS
The authors would like to thank the "Gastroenterology and Liver

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.