p53 and miR-34b/c genetic variation and their impact on ulcerative colitis susceptibility

Ulcerative colitis (UC), a chronic inflammatory bowel disease (IBD), is characterized by repeated flare-ups of inflammation that can contribute to the development of colorectal cancer [1]. The pathogenesis of UC remains largely unknown, but involves a complex interaction between immunological, environmental and genetic factors [2]. Among the genetic factors, P53 plays an important role in the development of UC [3]. Of the known TP53 polymorphisms, Arg72Pro (rs1042522) an amino acid substitution of arginine (Arg) to proline(Pro) at position 72 in exon 4, is one of the most widely studied polymorphisms. The Arg72 and Pro72 isoforms have different biochemical and biological properties, such as different binding to components of the transcriptional machinery and different activation of transcription [4]. The Arg72 isoform induces apoptosis faster and suppresses transformation more efficiently than the Pro72. In addition, the Arg form of the p53 protein is more vulnerable than Pro form to binding and degradation by the HPV-E6 oncoprotein [5]. More importantly, the Arg72Pro polymorphism of TP53 influences the p53-mediated inflammatory response. P53 is a transcription factor that regulates a group of target genes such as miRNAs, especially the miR-34 family members (miR-34a, miR-34b and miR-34c) [6]. MicroRNAs (miRNAs) are small, 21–25-nucleotide non-coding RNAs that regulate gene expression by binding to complementary sequences in the 3′-UTR of target mRNAs. A potentially functional common SNP rs4938723 T > C is present in the promoter region of pri-miR-34b/c [7]. Several independent studies suggest that genetic variants in the pri-miRNA sequence of miR-34b/c (rs4938723 T > C) can be used as possible biomarkers of disease development. As p53 physically interacts with miR-34b/c gene, the P53 Arg72Pro and miR34b/c rs4938723 (T > C) polymorphisms were selected to test our hypothesis that these SNPs are associated with the risk of UC. This case-control study comprises 180 UC cases recruited at the Department of Gastroenterology, Ghaem Hospital, Rasht, with disease documented by clinical symptoms, colonoscopic and pathological findings, from June 2013 to July 2015. Endoscopic UC activity at the time of the colonoscopy was categorized according to the Mayo criteria [8]. Histologic scores were graded mild, moderate or severe, and the extent of UC was defined according to Montreal Classification. The frequencies of E1, E2 and E3 were 61.1, 25.6 and 13.3%, respectively. All patients received Mesalazine. Two hundred and fifty healthy volunteers were selected by matching healthy individuals without IBD or known chronic, autoimmune disease based on gender and age. All participants gave written prior informed consent. The genomic DNA was extracted from peripheral blood cells using the GPP solution kit (Gen Pajoohan, Iran) according to the manufacturer’s instruction. Variants of codon72 of P53 were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP) analysis. Amplifications were carried out using specific primers designed by Oligo primer analysis software (Version 7.54, Molecular Biology Insights, USA). Primer sequences were F: 5′-CTTTTCACCCATCTACAGTCCC-3′ and R: 5′-AAACACTGACAGGAAGCCAAA-3′. PCR was performed with a Bio-Rad thermal cycler in 25 μl of PCR mixture containing approximately 30 ng of genomic DNA, 0.1 M deoxyribonucleotide triphosphates (dNPTs), 10 × PCR buffer (50 mM KCl, 10 mM Tris HCl and 0.1%Triton X-100), 1.5 mM MgCl2, 0.5 units of Taq polymerase and 2 pmol of each primer. PCR cycling conditions were implemented with an initial denaturation step for 3 min at 94 °C, followed by 35 cycles for 35 s at 94 °C, 30 s at 57 °C, 30 s at 72 °C and with final extension at 72 °C for 5 min. A