Polymorphisms within micro-RNA-binding sites and risk of sporadic colorectal cancer

Recent evidence indicate that small non-coding RNA molecules,called micro-RNAs (miRNAs), can bind to the 3' untranslatedregions (UTRs) of messenger RNAs and interfere with their translation,thereby regulating cell growth, differentiation, apoptosis andtumorigenesis. Genetic polymorphisms can reside on miRNA-bindingsites. Thus, it is conceivable that the miRNA regulation maybe affected by polymorphisms on the 3' UTRs. Since gene deregulationis one of the key mechanisms by which cells can progress tocancer, we hypothesize that common polymorphisms within miRNA-targetbinding sites could play a role in the individual risk of cancer.In the present study, we selected the 3' UTRs of 104 genes candidatefor colorectal cancer (CRC) and we identified putative miRNA-bindingsites by specialized algorithms (PicTar, DianaMicroT, miRBase,miRanda, TargetScan and microInspector). Fifty-seven single-nucleotidepolymorphisms (SNPs) were identified in miRNA-binding sites.We evaluated the SNPs for their ability to affect the bindingof the miRNA with its target, by assessing the variation ofGibbs free energy between the two alleles of each SNP. We foundeight common polymorphisms that were further investigated bya case–control association studies. The study was carriedout on a series of cases and controls from Czech Republic, apopulation with the highest worldwide incidence of CRC. We foundstatistically significant associations between risk of CRC andvariant alleles of CD86 [odds ratio (OR) = 2.74; 95% confidenceinterval (CI) = 1.24–6.04, for the variant homozygotes]and INSR genes (OR = 1.94; 95% CI = 1.03–3.66, for thevariant homozygotes). These results are the first reportingpositive association between miRNA-binding SNPs sequences andcancer risk. Abbreviations: CRC, colorectal cancer; CI, confidence interval; mRNA, messenger RNA; miRNA, micro-RNA; MAF, minor allele frequency; nt, nucleotide; OR, odds ratio; SNP, single-nucleotide polymorphism; UTR, untranslated region Received October 24, 2007; revised December 6, 2007; accepted December 22, 2007.     

Functional polymorphisms of circadian positive feedback regulation genes and clinical outcome of Chinese patients with resected colorectal cancer

BACKGROUND:

Previous studies have demonstrated that circadian genes play a role in the development and progression of many cancers. This study aims to assess the effects of single nucleotide polymorphisms (SNPs) in circadian genes on recurrence and survival of colorectal cancer (CRC) patients.

METHODS:

Nine functional SNPs in 3 genes (CLOCK, NPAS2, and BMAL1) on the circadian positive feedback loop were selected and genotyped using the Sequenom iPLEX genotyping system in a cohort of 411 resected Chinese CRC patients. Multivariate Cox proportional hazards model and Kaplan‐Meier curve were used for the prognosis analysis.

RESULTS:

The authors identified 2 SNPs in the CLOCK gene to be significantly associated with CRC overall survival. SNP rs3749474 exhibited a significant association with survival of CRC patients in the additive model (hazard ratio [HR], 0.55; 95% confidence interval [CI], 0.37‐0.81; P = .003). In addition, patients carrying the heterozygous variant of rs1801260 had significantly increased overall survival compared with those carrying homozygous wild‐type genotype (HR, 0.31; 95% CI, 0.11‐0.88; P = .03). Findings from functional assay provided further biological support for these significant associations. Stratified analysis found no modifying effect of chemotherapy on the prognostic significance of both SNPs. Moreover, we observed cumulative effects of these 2 SNPs on CRC overall survival (P for trend = .01). Compared with patients carrying no unfavorable genotypes, those carrying 2 unfavorable genotypes had a 2.92‐fold increased risk of death (P = .03).

CONCLUSIONS:

The results suggest for the first time that CLOCK gene polymorphisms may serve as an independent prognostic marker for CRC patients. Cancer 2012;. © 2011 American Cancer Society.     

The PI3K/AKT signaling pathway: Associations of miRNAs with dysregulated gene expression in colorectal cancer

The PI3K/AKT‐signaling pathway is one of the most frequently activated signal‐transduction pathways in cancer. We examined how dysregulated gene expression is associated with miRNA expression in this pathway in colorectal cancer (CRC). We used data from 217 CRC cases to evaluate differential pathway gene expression between paired carcinoma and normal mucosa and identify miRNAs that are associated with these genes. Gene expression data from RNA‐Seq and miRNA expression data from Agilent Human miRNA Microarray V19.0 were analyzed. We focused on genes most associated with CRC (fold change (FC) of >1.5 or <0.67) that were statistically significant after adjustment for multiple comparisons. Of the 304 genes evaluated, 76 had a FC of <0.67, and 57 had a FC of >1.50; 47 of these genes were associated with miRNA differential expression. There were 145 mRNA:miRNA seed‐region matches of which 26 were inversely associated suggesting a greater likelihood of a direct association. Most miRNA:mRNA associations were with factors that stimulated the pathway. For instance, both IL6R and PDGFRA had inverse seed‐region matches with seven miRNAs, suggesting that these miRNAs have a direct effect on these genes and may be key elements in activation of the pathway. Other miRNA:mRNA associations with similar impact on the pathway were miR‐203a with ITGA4, miR‐6071 with ITGAV, and miR‐375 with THBS2, all genes involved in extracellular matrix function that activate PI3Ks. Gene expression in the PI3K/Akt‐signaling pathway is dysregulated in CRC. MiRNAs were associated with many of these dysregulated genes either directly or in an indirect manner.     

Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer

Background:

Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC).

Methods:

Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT–PCR.

Results:

Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001).

Conclusions:

Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.     

Identification of polymorphisms in ultraconserved elements associated with clinical outcomes in locally advanced colorectal adenocarcinoma

BACKGROUND:

Ultraconserved elements (UCEs) are noncoding genomic sequences that completely identical among human, mouse, and rat species and harbor critical biologic functions. The authors hypothesized that single nucleotide polymorphisms (SNPs) within UCEs are associated with clinical outcomes in patients with colorectal cancer (CRC).

METHODS:

Forty‐eight SNPs within UCEs were genotyped in 662 patients with stage I through III CRC. The associations between genotypes and recurrence and survival were analyzed in patients with stage II or III CRC who received fluoropyrimidine‐based adjuvant chemotherapy using a training and validation design. The training set included 115 patients with stage II disease and 170 patients with stage III disease, and the validation set included 88 patients with stage II disease and 112 patients with stage III disease.

RESULTS:

Eight SNPs were associated with clinical outcomes stratified by disease stage. In particular, for patients with stage II CRC who had at least 1 variant allele of reference SNP sequence 7849 (rs7849), a consistent association with increased recurrence risk was observed in the training set (hazard ratio [HR], 2.39; 95% confidence interval [CI], 1.04‐5.52), in the replication set (HR, 3.70; 95% CI, 1.42‐9.64), and in a meta‐analysis (HR, 2.89; 95% CI, 1.54‐5.41). Several other SNPs were significant in the training set but not in the validation set. These included rs2421099, rs16983007, and rs10211390 for recurrence and rs6590611 for survival in patients with stage II disease; and SNPs rs6124509 and rs11195893 for recurrence in patients with stage III disease. In addition, a significant cumulative effect was observed of multiple risk genotypes and potential gene‐gene interactions on recurrence risk.

CONCLUSIONS:

To the authors' knowledge, this is the first study to evaluate the association between SNPs within UCEs and clinical outcome in patients with CRC. The results suggested that SNPs within UCEs may be valuable prognostic biomarkers for patients with locally advanced CRC who receive 5‐fluorouracil–based chemotherapy. Cancer 2012. © 2012 American Cancer Society.     

Interactions between cigarette smoking and selected polymorphisms in xenobiotic metabolizing enzymes in risk for colorectal cancer: A case-only analysis

The metabolism of xenobiotics is complex and involves multiple steps and multiple enzymes. Genetic variation in the genes encoding these enzymes as well as the level of exposure to the substrates of these enzymes could alter metabolism and clearance of potential carcinogens and thus alter cancer susceptibility. This study examined interaction effect between smoking and two single nucleotide polymorphisms (SNPs)—CYP1A1 c.1384A>G (p.Ile462Val) and EPHX1 c.337T>C (p.Tyr113His)—in modulating colorectal cancer (CRC) risk. The SNPs were selected a priori based on functional significance. In a case‐only analysis, unconditional logistic regression was used to examine the associations between smoking and each SNP and between the two SNPs in 786 patients with nonfamilial CRC. There was significant multiplicative interaction for CRC risk between smoking and EPHX1 c.337T>C (odds ratio [OR] = 1.37, 95% confidence interval [CI] = 1.03–1.81, P = 0.03), particularly among smokers with a history of greater than 20 pack‐years of smoking (OR = 1.52, 95% CI = 1.07–2.16, P = 0.02). In addition, there was gene–gene interaction between EPHX1 c.337T>C and CYP1A1 c.1384A>G (OR = 1.61, 95% CI = 1.02–2.55, P = 0.04). Smokers with any variant allele of EPHX1 were at increased risk for CRC, as were individuals with any variant allele of CYP1A1 together with any variant allele of EPHX1. Thus, the study of gene–environment and gene–gene interactions may help to identify high‐risk subgroups that can be targeted for intensive smoking cessation and CRC screening interventions. © 2010 Wiley‐Liss, Inc.     

Evaluation of genetic variants in microRNA biosynthesis genes and risk of breast cancer in Chinese women

MicroRNAs (miRNA) are a class of small, noncoding RNA molecules involved in a diversity of cellular functions. Single nucleotide polymorphisms (SNPs) in miRNA biosynthesis genes may affect the biogenesis of miRNAs and consequently affect the miRNAs regulation. In this study, we systematically selected 24 functional SNPs located in eight key biosynthesis genes of miRNA (DROSHA, DGCR8, RAN, DICER, AGO2, GEMIN3, GEMIN4 and HIWI) and investigated the association between these SNPs and the risk of breast cancer in a Chinese population. All 24 SNPs were firstly genotyped in stage 1 (878 cases and 900 controls) and three promising SNPs (DROSHA rs2291109, RAN rs7301722 and DGCR8 rs417309) were selected for further validation in stage 2 (914 cases and 967 controls). We found that only one SNP (rs417309) located in the 3′‐UTR of DGCR8 was consistently associated with an increased breast cancer risk in two stages with a combined odds ratio (OR) of 1.50 [95% confidence interval (CI) = 1.16–1.93]. Based on the bioinformatics prediction, rs417309 is located at the binding sites of miR‐106b and miR‐579 in the 3′‐UTR of DGCR8. To evaluate whether rs417309 variant affects the binding capacity of miRNAs, we cotransfected luciferase reporter plasmids of DGCR8 3′‐UTR and miR‐106b/miR‐579 in three cell lines. Luciferase activity assay showed a higher expression level with rs417309 A allele compared with G allele in MCF‐7 cell lines (p = 3.31 × 10^–7, 9.29 × 10^–7 for miR‐106b and miR‐579, respectively). Our findings suggested that DGCR8 rs417309 G > A might affect breast cancer risk through the interruption of miRNA binding.     

MicroRNA expression profiling is a potential diagnostic tool for thyroid cancer

BACKGROUND:

Approximately 30% of fine‐needle aspiration (FNA) biopsies of thyroid nodules are indeterminate or nondiagnostic. Recent studies suggest microRNA (miRNA, miR) is differentially expressed in malignant tumors and may have a role in carcinogenesis, including thyroid cancer. The authors therefore tested the hypothesis that miRNA expression analysis would identify putative markers that could distinguish benign from malignant thyroid neoplasms that are often indeterminate on FNA biopsy.

METHODS:

A miRNA array was used to identify differentially expressed genes (5‐fold higher or lower) in pooled normal, malignant, and benign thyroid tissue samples. Real‐time quantitative polymerase chain reaction was used to confirm miRNA array expression data in 104 tissue samples (7 normal thyroid, 14 hyperplastic nodule, 12 follicular variant of papillary thyroid cancer, 8 papillary thyroid cancer, 15 follicular adenoma, 12 follicular carcinoma, 12 Hurthle cell adenoma, 20 Hurthle cell carcinoma, and 4 anaplastic carcinoma cases), and 125 indeterminate clinical FNA samples. The diagnostic accuracy of differentially expressed genes was determined by analyzing receiver operating characteristics.

RESULTS:

Ten miRNAs showed >5‐fold expression difference between benign and malignant thyroid neoplasms on miRNA array analysis. Four of the 10 miRNAs were validated to be significantly differentially expressed between benign and malignant thyroid neoplasms by quantitative polymerase chain reaction (P < .002): miR‐100, miR‐125b, miR‐138, and miR‐768‐3p were overexpressed in malignant samples of follicular origin (P < .001), and in Hurthle cell carcinoma samples alone (P < .01). Only miR‐125b was significantly overexpressed in follicular carcinoma samples (P < .05). The accuracy for distinguishing benign from malignant thyroid neoplasms was 79% overall, 98% for Hurthle cell neoplasms, and 71% for follicular neoplasms. The miR‐138 was overexpressed in the FNA samples (P = .04) that were malignant on final pathology with an accuracy of 75%.

CONCLUSIONS:

MicroRNA expression differs for normal, benign, and malignant thyroid tissue. Expression analysis of differentially expressed miRNA could help distinguish benign from malignant thyroid neoplasms that are indeterminate on thyroid FNA biopsy. Cancer 2011. © 2011 American Cancer Society.     

microRNA-221 and microRNA-18a identification in stool as potential biomarkers for the non-invasive diagnosis of colorectal carcinoma

Background:

The detection of microRNA (miRNA) dysregulation in stool is a novel approach for the diagnosis of colorectal carcinoma (CRC). The aim of this study is to investigate the use of miR-221 and miR-18a in stool samples as non-invasive biomarkers for CRC diagnosis.

Methods:

A miRNA expression array containing 667 miRNAs was performed to identify miRNA dysregulation in CRC tissues. We focused on miR-221 and miR-18a, two significantly upregulated miRNAs which were subsequently verified in 40 pairs of CRC tissues and 595 stool samples (198 CRCs, 199 polyps and 198 normal controls).

Results:

miR-221 and miR-18a were upregulated in the miRNA expression array. miR-221 and miR-18a levels were also significantly higher in 40 CRC tumours compared with their respective adjacent normal tissues. In stool samples, miR-221 and miR-18a showed a significant increasing trend from normal controls to late stages of CRC (P<0.0001). The levels of stool miR-221 and miR-18a were both significantly higher in subjects with stages I+II (miR-221: P<0.0001, miR-18a: P<0.0001) and stages III+IV of CRC (miR-221: P=0.0004, miR-18a: P<0.0001) compared with normal controls. The AUC of stool miR-221 and miR-18a were 0.73 and 0.67 for CRC patients as compared with normal controls, respectively. No significant differences in stool miR-221 and miR-18a levels were found between patients with proximal and distal CRCs. The use of antibiotics did not influence stool miRNA-221 and miRNA-18a levels.

Conclusions:

Stool-based miR-221 can be used as a non-invasive biomarker for the detection of CRC.     

Circulating miR-378 in plasma: a reliable,haemolysis-independent biomarker for colorectal cancer

Background:

Plasma circulating tumour-specific microRNAs (miRNAs) are promising biomarkers of tumour presence and recurrence, especially for diseases whose best chance of successful treatment requires early diagnosis and timely surgery of an already malignant but not yet invasive tumour, such as colorectal cancer (CRC).

Methods:

Expression levels of miRNAs previously found to be differently expressed in tumour vs normal colon tissues were investigated by quantitative real-time PCR in plasma from CRC patients and from healthy donors and confirmed in independent case control series. The validated miRNAs were also measured after surgery. Analyses were repeated on the subsets of haemolysis-free samples.

Results:

We identified four miRNAs differently expressed between the compared groups, two (miR-21 and miR-378) of which were validated. miR-378 expression decreased in non-relapsed patients 4–6 months after surgery and miR-378 ability to discriminate CRC patients from healthy individuals was not influenced by haemolysis levels of plasma samples.

Conclusion:

The miRNA analysis on plasma samples represents a useful non-invasive tool to assess CRC presence as well as tumour-free status at follow-up. Plasma levels of miR-378 could be used to discriminate CRC patients from healthy individuals, irrespective of the level of haemoglobin of plasma samples.     

Haemochromatosis HFE gene polymorphisms as potential modifiers of hereditary nonpolyposis colorectal cancer risk and onset age

Hereditary nonpolyposis colorectal cancer (HNPCC) is characterized by germline mutations in DNA mismatch repair genes; however, variation in disease expression suggests that there are potential modifying factors. Polymorphisms of the HFE gene, which cause the iron overload disorder hereditary haemochromatosis, have been proposed as potential risk factors for the development of colorectal cancer (CRC). To understand the relationship between HNPCC disease phenotype and polymorphisms of the HFE gene, a total of 362 individuals from Australia and Poland with confirmed causative MMR gene mutations were genotyped for the HFE C282Y and H63D polymorphisms. A significantly increased risk of developing CRC was observed for H63D homozygotes when compared with combined wild‐type homozygotes and heterozygotes (hazard ratio = 2.93, p = 0.007). Evidence for earlier CRC onset was also observed in H63D homozygotes with a median age of onset 6 years earlier than wild type or heterozygous participants (44 vs. 50 years of age). This effect was significant by all tests used (log‐rank test p = 0.026, Wilcoxon p = 0.044, Tarone‐Ware p = 0.035). No association was identified for heterozygosity of either polymorphism and limitations on power‐prevented investigation of C282Y homozygosity or compound C282Y/H63D heterozygosity. In the Australian sample only, women had a significantly reduced risk of developing CRC when compared with men (hazard ratio = 0.58, p = 0.012) independent of HFE genotype for either single nucleotide polymorphisms. In conclusion, homozygosity for the HFE H63D polymorphism seems to be a genetic modifier of disease expression in HNPCC. Understanding the mechanisms by which HFE interrelates with colorectal malignancies could lead to reduction of disease risk in HNPCC. © 2009 UICC     

Clinical value of integrated-signature miRNAs in colorectal cancer: miRNA expression profiling analysis and experimental validation

MicroRNA (miRNA) expression profiling of colorectal cancer (CRC) are often inconsistent among different studies. To determine candidate miRNA biomarkers for CRC, we performed an integrative analysis of miRNA expression profiling compared CRC tissues and paired neighboring noncancerous colorectal tissues. Using robust rank aggregation method, we identified a miRNA set of 10 integrated-signature miRNAs. In addition, the qRT-PCR validation demonstrated that 9 miRNAs were consistent dysregulated with the integrative analysis in CRC tissues, 4 miRNAs (miR-21-5p, miR-183-5p, miR-17-5p and miR-20a-5p) were up-regulated expression, and 5 miRNAs (miR-145-5p, miR-195-5p, miR-139-5p, miR-378a-5p and miR-143-3p) were down-regulated expression (all p < 0.05). Consistent with the initial analysis, 7 miRNAs were found to be significantly dysregulated in CRC tissues in TCGA data base, 4 miRNAs (miR-21-5p, miR-183-5p, miR-17-5p and miR-20a-5p) were significantly up-regulated expression, and 3 miRNAs (miR-145-5p, miR-139-5p and miR-378a-5p) were significantly down-regulated expression in CRC tissues (all p < 0.001). Furthermore, miR-17-5p (p = 0.011) and miR-20a-5p (p = 0.003) were up-regulated expression in the III/IV tumor stage, miR-145-5p (p = 0.028) and miR-195-5p (p = 0.001) were significantly increased expression with microscopic vascular invasion in CRC tissues, miR-17-5p (p = 0.037) and miR-145-5p (p = 0.023) were significantly increased expression with lymphovascular invasion. Moreover, Cox regression analysis of CRC patients in TCGA data base showed miR-20a-5p was correlated with survival (hazard ratio: 1.875, 95%CI: 1.088–3.232, p = 0.024). Hence, the finding of current study provides a basic implication of these miRNAs for further clinical application in CRC.     

Meat,vegetables and genetic polymorphisms and the risk of colorectal carcinomas and adenomas

Background  

The risk of sporadic colorectal cancer (CRC) is mainly associated with lifestyle factors, particularly dietary factors. Diets high in red meat and fat and low in fruit and vegetables are associated with an increased risk of CRC. The dietary effects may be modulated by genetic polymorphisms in biotransformation genes. In this study we aimed to evaluate the role of dietary factors in combination with genetic factors in the different stages of colorectal carcinogenesis in a Norwegian population.     

Risk of colorectal cancer in self-reported inflammatory bowel disease and modification of risk by statin and NSAID use

BACKGROUND:

Statins and nonsteroidal anti‐inflammatory drugs (NSAIDs) are associated with reduced risk of colorectal cancer (CRC) in some studies. The objective of this study was to quantify the relative risk of inflammatory bowel disease (IBD) as a risk factor for CRC and to estimate whether this risk may be modified by long‐term use of NSAIDs or statins.

METHODS:

The Molecular Epidemiology of Colorectal Cancer study is a population‐based, case‐control study of incident colorectal cancer in northern Israel and controls matched by age, sex, clinic, and ethnicity. Personal histories of IBD and medication use were measured by structured, in‐person interview. The relative risk of IBD and effect modification by statins and NSAIDs were quantified by conditional and unconditional logistic regression.

RESULTS:

Among 1921 matched pairs of CRC cases and controls, a self‐reported history of IBD was associated with a 1.9‐fold increased risk of CRC (95% confidence interval [CI], 1.12‐3.26). Long‐term statin use was associated with a reduced risk of both IBD‐associated CRC (odds ratio [OR] = 0.07; 95% CI, 0.01‐0.78) and non‐IBD CRC (OR = 0.49; 95% CI, 0.39‐0.62). Stratified analysis suggested that statins may be more protective among those with IBD (ratio of OR = 0.14; 95% CI, 0.01‐1.31; P = .51), although not statistically significant. NSAID use in patients with a history of IBD was suggestive of reduced risk of CRC but did not reach statistical significance (OR = 0.47; 95% CI, 0.12‐1.86).

CONCLUSIONS:

The risk of CRC was elevated 1.9‐fold in patients with IBD. Long‐term statin use was associated with reduced risk of CRC in patients with IBD. Cancer 2011. © 2010 American Cancer Society.