Variations In miRNA Binding Sites Research Articles

Abstract A019: Identification and Genomic Analysis of miRNA-mRNA Binding Site SNPs in Hepatitis C Virus-Associated Hepatocellular Carcinoma

Abstract Hepatitis C virus (HCV) infection is a major risk factor for hepatocellular carcinoma (HCC), a leading cause of cancer-related deaths globally. MicroRNAs (miRNAs) play critical roles in gene regulation, and single nucleotide polymorphisms (SNPs) within miRNA binding sites can disrupt this regulation, potentially influencing HCC pathogenesis. The present study aims to identify and characterize SNPs within miRNA binding sites of genes associated with HCV- induced HCC (HCV-HCC). A comprehensive bioinformatics analysis workflow was devised. HCV-specific differentially expressed (DE) miRNAs and mRNAs were extracted from literature and databases. SNPs in the 3' UTRs of HCV-HCC genes were identified, and their impact on miRNA binding was predicted using PoymiRTS and TargetScan. RNAhybrid was used to estimate minimum folding energy (MFE) differences (ΔMFE) between mutant and wild types. Functional enrichment analysis was conducted to elucidate the biological processes affected by these SNPs. Our analysis revealed several significant alterations in miRNA-mRNA binding pairs due to SNPs within genes associated with HCV-HCC. For instance, among the downregulated genes in HCV-HCC, the SNP in CTSB (rs3088245; 193T>C) predicted to increase the stability of the miR-27b - CTSB duplex (ΔMFE: 1.4), suggesting enhanced binding and potentially improved repression of CTSB. Conversely, the SNP in EGR2 (rs61865883; 287 T>A) decreased the stability of the miR-140-5p - EGR2 mRNA interaction (ΔMFE: -1.9), suggesting a weaker binding affinity and potentially reduced repression of EGR2 expression. Considering the tumor suppressor role of EGR2, presence of this SNP is likely to reflect good prognosis. For the upregulated gene oncogene, XIAP, the SNP (rs6648556; 5892 T>C) is predicted to enhance the binding of miR- 200c (ΔMFE: 1.5), likely resulting in increased suppression of XIAP expression in patients carrying this SNP and improved prognosis. Our study emphasizes the importance of considering genetic variations in miRNA binding sites in the context of HCV-HCC. These SNPs may serve as genetic modifiers influencing disease susceptibility and progression. Further experimental studies are warranted to explore their potential as therapeutic agents and predictors of prognosis in HCV-HCC patients. Citation Format: Azeem M Butt, Anum Qamar, Shafiqa Siddique. Identification and Genomic Analysis of miRNA-mRNA Binding Site SNPs in Hepatitis C Virus-Associated Hepatocellular Carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A019.

SNP rs1803622 in hsa-miR-548g binding site at GAPDH alters susceptibility to breast cancer

Breast cancer has been increasing in developing countries; this cancer is becoming a fundamental health problem for these countries. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classical glycolytic enzyme correlated with the hormonal receptor concentrations, cell proliferation, tumor grade and stage, drug resistance, and outcome of breast cancer cells. Furthermore, genetic variations in miRNA binding sites could be played an essential role in breast cancer risk and survival. In this study, the SNP rs1803622 at the 3′-UTR of GAPDH was chosen as a candidate SNP for investigation based on bioinformatics studies. This polymorphism was genotyped in an Iranian population with 123 breast cancer patients and 117 matched healthy controls using the Allele-Specific Primer (ASP)-PCR method. The functional impact of this genetic variation on the post-transcriptional regulation of GAPDH gene were bioinformatically studied. The genotyping data, for the first time, successfully identified frequencies of GG, GT, and TT to be 7%, 61%, and 32% in breast cancer patients, respectively. In particular, the assessed frequencies of allele G was 38% while risk allele T was 62% in these population. Results revealed that compared with the GG genotype of rs1803622, T allele was related to a significantly increased risk of breast cancer in a dominant genetic model Significant difference in allele and genotype distributions in the SNP rs1803622 was found between the high and low expression levels of estrogen, progesterone, and HER2 receptors. Furthermore, allele and genotype frequencies in the SNP rs1803622 were also related to the stage and grade of breast cancer. Surprisingly, Genotype and allele frequencies in this SNP were also significantly different in metastatic and non-metastatic breast cancer samples. Finally, the bioinformatics data suggested that the SNP rs1803622 might be involved in transcriptional regulation of the GAPDH gene by hsa-miR-548g.

Genetics of epigenome might hold the clue for determining the threshold of environmental impact.

Genetics of epigenome might hold the clue for determining the threshold of environmental impact.

MicroRNA-related genetic variants in iron regulatory genes, dietary iron intake, microRNAs and lung cancer risk

Genetic variations in MicroRNA (miRNA) binding sites may alter structural accessibility of miRNA binding sites to modulate risk of cancer. This large-scale integrative multistage study was aimed to evaluate the interplay of genetic variations in miRNA binding sites of iron regulatory pathway, dietary iron intake and lung cancer (LC) risk. The interplay of genetic variant, dietary iron intake and LC risk was assessed in large-scale case-control study. Functional characterization of the validated SNP and analysis of target miRNAs were performed. We found that the miRNA binding site SNP rs1062980 in 3' UTR of Iron-Responsive Element Binding protein 2 gene (IREB2) was associated with a 14% reduced LC risk (P value = 4.9×10 - 9). Comparing to AA genotype, GG genotype was associated with a 27% reduced LC risk. This association was evident in males and ever-smokers but not in females and never-smokers. Higher level of dietary iron intake was significantly associated with 39% reduced LC risk (P value = 2.0×10 - 8). This association was only present in individuals with AG + AA genotypes with a 46% reduced risk (P value = 1.0×10 - 10), but not in GG genotype. The eQTL-analysis showed that rs1062980 significantly alters IREB2 expression level. Rs1062980 is predicted to alter a miR-29 binding site on IREB2 and indeed the expression of miR-29 is inversely correlated with IREB2 expression. Further, we found that higher circulating miR-29a level was significantly associated with 78% increased LC risk. The miRNA binding site SNP rs1062980 in iron regulatory pathway, which may alter the expression of IREB2 potentially through modulating the binding of miR-29a, together with dietary iron intake may modify risk of LC both individually and jointly. These discoveries reveal novel pathway for understanding lung cancer tumorigenesis and risk stratification.

Evaluation of genetic variations in miRNA-binding sites of BRCA1 and BRCA2 genes as risk factors for the development of early-onset and/or familial breast cancer.

Although genetic markers identifying women at an increased risk of developing breast cancer exist, the majority of inherited risk factors remain elusive. Mutations in the BRCA1/BRCA2 gene confer a substantial increase in breast cancer risk, yet routine clinical genetic screening is limited to the coding regions and intron- exon boundaries, precluding the identification of mutations in noncoding and untranslated regions. Because 3' untranslated region (3'UTR) polymorphisms disrupting microRNA (miRNA) binding can be functional and can act as genetic markers of cancer risk, we aimed to determine genetic variation in the 3'UTR of BRCA1/BRCA2 in familial and early-onset breast cancer patients with and without mutations in the coding regions of BRCA1/ BRCA2 and to identify specific 3'UTR variants that may be risk factors for cancer development. The 3'UTRs of the BRCA1 and BRCA2 genes were screened by heteroduplex analysis and DNA sequencing in 100 patients from 46 BRCA1/2 families, 54 non-BRCA1/2 families, and 47 geographically matched controls. Two polymorphisms were identified. SNPs c.*1287C>T (rs12516) (BRCA1) and c.*105A>C (rs15869) (BRCA2) were identified in 27% and 24% of patients, respectively. These 2 variants were also identified in controls with no family history of cancer (23.4% and 23.4%, respectively). In comparison to variations in the 3'UTR region of the BRCA1/2 genes and the BRCA1/2 mutational status in patients, there was a statistically significant relationship between the BRCA1 gene polymorphism c.*1287C>T (rs12516) and BRCA1 mutations (p=0.035) by Fisher's Exact Test. SNP c.*1287C>T (rs12516) of the BRCA1 gene may have potential use as a genetic marker of an increased risk of developing breast cancer and likely represents a non-coding sequence variation in BRCA1 that impacts BRCA1 function and leads to increased early-onset and/or familial breast cancer risk in the Turkish population.

Variation within 3′-UTRs of Base Excision Repair Genes and Response to Therapy in Colorectal Cancer Patients: A Potential Modulation of microRNAs Binding

Colorectal cancer is routinely treated with a 5-fluorouracil (5-FU)-based chemotherapy. 5-FU incorporates into DNA, and the base excision repair (BER) pathway specifically recognizes such damage. We investigated the association of single-nucleotide polymorphisms (SNP) in the 3'-untranslated regions (UTR) of BER genes, and potentially affecting the microRNA (miRNA) binding, on the risk of colorectal cancer, its progression, and prognosis. SNPs in miRNA-binding sites may modulate the posttranscriptional regulation of gene expression operated by miRNAs and explain interindividual variability in BER capacity and response to 5-FU. We tested 12 SNPs in the 3'-UTRs of five BER genes for colorectal cancer susceptibility in a case-control study (1,098 cases and 1,459 healthy controls). Subsequently, we analyzed the role of these SNPs on clinical outcomes of patients (866 in the Training set and 232 in the Replication set). SNPs in the SMUG1 and NEIL2 genes were associated with overall survival. In particular, SMUG1 rs2233921 TT carriers showed increased survival compared with those with GT/GG genotypes [HR, 0.54; 95% confidence interval (CI), 0.36-0.81; P = 0.003] in the Training set and after pooling results from the Replication set. The association was more significant following stratification for 5-FU-based chemotherapy (P = 5.6 × 10(-5)). A reduced expression of the reporter gene for the T allele of rs2233921 was observed when compared with the common G allele by in vitro assay. None of the genotyped BER polymorphisms were associated with colorectal cancer risk. We provide the first evidence that variations in miRNA-binding sites in BER genes 3'-UTR may modulate colorectal cancer prognosis and therapy response.

G‐A variant in miR‐200c binding site of EFNA1 alters susceptibility to gastric cancer

MicroRNAs (miRNAs) post-transcriptionally modulate gene expression by binding to complementary sites at 3'-untranslated regions (3'UTRs) of their target messenger RNAs (mRNAs). Genetic variations in miRNA binding sites may alter individual susceptibilities to many cancers. However, whether miRNA binding site single nucleotide polymorphisms (SNPs) interfere with gastric cancer (GC) susceptibility has not been reported. In this case-control study including 525 GC patients and 501 controls, we selected three miRNA binding site SNPs located in 3'UTRs of genes involved in GC to investigated their associations with GC susceptibility. We identified that rs12904 in ephrin-A1 (EFNA1) gene was significantly associated with risk of GC, with the OR for carrying AG or GG genotype being 0.65 (P = 0.002, OR 0.65; 95% CI, 0.50-0.85) compared with AA genotype. Specifically, we found that rs12904 is in strong linkage disequilibrium (LD) with rs4072037, a susceptibility variant reported by previous genome-wide association study (GWAS). No significant associations were observed for the other two SNPs (rs699517 in TYMS and rs1042542 in BIRC5). Furthermore, luciferase assays indicated EFNA1 as the target of hsa-miR-200c and rs12904 G > A change resulted in altered regulation of luciferase expression. In addition, rs12904 AA genotype was associated with increased expression of EFNA1 mRNA compared with AG or GG genotype in the cancer tissues from 48 patients. Taken together, these findings indicate that the miR-200c binding site SNP (rs12904 G > A) in the 3'UTR of EFNA1 can modulate EFNA1 expression and is associated with GC susceptibility. Larger replication studies are needed to confirm our findings.

Polymorphisms in miRNA-binding sites of nucleotide excision repair genes and colorectal cancer risk

Reduced DNA repair capacity and DNA damage accumulation may lead to cancer development. Regulation of and coordination between genes involved in DNA repair pathways is fundamental for maintaining genome stability, and post-transcriptional gene regulation by microRNAs (miRNAs) may therefore be of particular relevance. In this context, the presence of single nucleotide polymorphisms (SNPs) within the 3'untranslated regions of target DNA repair genes could alter the binding with specific miRNAs, modulating gene expression and ultimately affecting cancer susceptibility. In this study, we investigated the role of genetic variations in miRNA-binding sites of nucleotide excision repair (NER) genes in association with colorectal cancer (CRC) risk. From 28 NER genes, we screened among SNPs residing in their 3'untranslated regions and simultaneously located in miRNA-binding sites, with an in silico approach. Through the calculation of different binding free energy according to both alleles of identified SNPs, and with global binding free energies median providing a threshold, we selected nine NER gene variants. We tested those SNPs in 1098 colorectal cancer cases and 1469 healthy controls from the Czech Republic. Rs7356 in RPA2 and rs4596 in GTF2H1 were associated with colorectal cancer risk. After stratification for tumor location, the association of both SNPs was significant only for rectal cancer (rs7356: OR 1.52, 95% CI 1.02-2.26, P = 0.04 and rs4596: OR 0.69, 95% CI 0.50-0.94, P = 0.02; results not adjusted for multiple testing). Variation in miRNA target binding sites in the 3'untranslated region of NER genes may be important for modulating colorectal cancer risk, with a different relevance according to tumor location.

The single-nucleotide polymorphism T>C rs4353229 in CASP7 gene and mir-224 as new biomarkers of oxaliplatin-induced neurotoxicity in advanced colorectal cancer.

513 Background: Oxaliplatin represents a central component of colorectal (CCR) cancer treatment. It is known that the ways in which oxaliplatin is neurotoxic include not only DNA damage, but also the modulation of specific molecules, such as caspases 3 and 7, involved in regulating the cellular equilibrium between apoptotic death and the cell cycle. MiRNAs are non-coding RNAs that can block mRNA traslation. Recent evidences indicate that variations in miRNA binding sites may contribute to the susceptibility to CCR. However, the effect of these SNPs on miRNA-target interaction remains to be determined. The SNP T>C rs4353229 in CASP7 gene is binding site of miR-224. We hypothesize that miR-224 and the SNP T>C rs4353229 could be new predictive biomarkers of oxaliplatin-induced neurotoxicity in advanced colorectal cancer. Methods: We examined the SNP T>C rs4353229 in CASP7 gene in 62 patients with advanced CCR. All patients had received first line of chemotherapy based in fluoropirimidines plus oxaliplatin. The Oxaliplatin-Specific Neurotoxicity Scale was utilized to assess oxaliplatin-related symptoms. Time to progresion (TTP) and objetive response rate (ORR) were calculated. Genomic DNA was extracted from peripheral blood and genotyped by allelic discrimination. We explored the possible correlation of rs4353229 with neurotoxicity, DCR and TTP using and Chi-cuadrado test and Kaplan-Meier model (log rank) respectively. Results: SNP distribution was 3,2% (ancestral homozygous CC), 46,7% (variant homocygous TT) and 43,7% (heterocygous CT). Median TTP was 10 months (IC95%: 7,4-12,5); regarding CASP7 polymorphism, there were not differences (p=0,41; log rank). ORR was 64,5% and there are not differences between CC, CT and TT patients (p=0,50). Fifteen patients experienced neurotoxicity grade III/IV (24,19%) and it was correlated significantly with ancestral homoygous CC patients (p=0,003). Conclusions: The SNP T>C rs4353229 in CASP7 gene and miR-224 may be new candidates predictor of oxaliplatin-induced neurotoxicity. Further characterization of CASP7 SNPs and miR-224 may open new avenues for the study of cancer and therapeutic interventions.