ABSTRACT
According to the Vogelstein's model of colorectal carcinogenesis, genetic variations in highly penetrant genes may be involved in the colorectal cancer (CRC) pathogenesis. Similarly, aberrant function and/or altered expression of microRNAs (miRNAs) often occur in CRC. In this context, polymorphisms in miRNA-binding sites (miRSNPs) may affect miRNA/target gene interaction, resulting in differential mRNA/protein expression and increased susceptibility to common diseases. To explore this phenomenon, we have mined the 3' untranslated regions (3'UTRs) of genes known to be frequently mutated in CRC to search for miRSNPs and tested their association with CRC risk and clinical outcome. Eight miRSNPs (rs1804191, rs397768, rs41116 in APC; rs1137918, s227091, rs4585 in ATM; rs712, rs1137282, rs61764370 in KRAS; rs8674 in PARP1 and rs16950113 in SMAD7) were tested for their association with CRC risk in a case-control study (1111 cases and 1469 healthy controls). The role of these miRSNPs was also investigated in relation to clinical outcome on a subset of patients with complete follow-up. rs8679 within PARP1 was associated with CRC risk and patients' survival. In the dominant model, carriers of at least one C allele were at a decreased risk of cancer (P = 0.05). The CC genotype in rs8679 was also associated with an increased risk of recurrence/progression in patients that received 5-FU-based chemotherapy (log-rank test P = 0.03). Carriers of the homozygous variant genotype TT for rs712 in KRAS gene were associated with a decreased risk of rectal cancer (odds ratio (OR) = 0.65, 95% confidence intervals (CI) 0.43-1.00, P = 0.05) while individuals with colon cancer carrying the heterozygous GT genotype showed a longer overall survival (OS) (P = 0.04). We provide the first evidence that variations in potential miRNA-binding target sites in the 3' UTR of PARP1 gene may modulate CRC risk and prognosis after therapy. Further studies are needed to replicate our finding and assess miRSNPs as predictive biomarkers in independent populations.
Subject(s)
3' Untranslated Regions , Colorectal Neoplasms/metabolism , Genetic Predisposition to Disease , MicroRNAs/metabolism , Poly (ADP-Ribose) Polymerase-1/metabolism , Polymorphism, Single Nucleotide , Adenomatous Polyposis Coli Protein/genetics , Aged , Antimetabolites, Antineoplastic/therapeutic use , Ataxia Telangiectasia Mutated Proteins/genetics , Binding Sites , Case-Control Studies , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/genetics , Female , Fluorouracil/therapeutic use , Humans , Male , Middle Aged , Poly (ADP-Ribose) Polymerase-1/genetics , Prognosis , Proto-Oncogene Proteins p21(ras)/genetics , RNA, Messenger/metabolism , Smad7 Protein/geneticsABSTRACT
Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.
