Genetic Variants In MicroRNA Processing Genes And Binding Sites Modify The Susceptibility Of Breast Cancer

Breast cancer is the most prevalent cancer in both developing and developedcountries. It was reported that the global burden of breast cancer in women wassubstantial and on the increase, with1.38million new cases diagnosed in2008.Although the breast cancer incidence was relative low in China, it had beenincreasing20%to30%in China’s urban registries in the past decade. It is well knownthat the etiology of breast cancer appears to include a complex combination of geneticand environmental. Dietary factors, reproductive patterns, pregnancies, age atmenarche and menopause showed weighty influence on breast cancer risk. However,genetic determinants also play an important role in the etiology of breast cancer,including variants located at both coding and noncoding sequences.MicroRNAs (miRNA) are small, noncoding, double-stranded RNA molecules.Primary miRNA (pri-miRNA) is firstly transcribed from miRNA genes by RNApolymerase and then processed into precursor miRNA (pre-miRNA) by DROSHAwithin the microprocessor complex including DGCR8. The pre-miRNA is exportedinto cytoplasm by Exportin-5and further processed to mature double-strandedmiRNA duplex by Dicer and subsequently one strand of miRNA incorporated intoRNA-induced silencing complex (RISC) that mediated target gene expression. Anumber of studies have highlighted the role of miRNA in various biological processes,even the development of cancers. However, impaired miRNA processing cased a marked change in the transformed phenotype of cancer cells and promotedtumorigenesis. Genetic variants in miRNA processing genes might influence themiRNA maturation. Besides, genetic variants in miRNA binding site might modulatethe miRNA-mRNA interaction through creating or destroying the binding sites andresult in diverse functional consequences. Oncogenes and tumor suppressor genes asmiRNA targets can impact the processing of tumorigeness through miRNAregulation.Although the association between single nucleotide polymorphism (SNPs) andcancer development have been widely studied, evidence for SNPs in miRNAprocessing genes and binding sites with breast cancer susceptibility in Chinesewomen are still lacked. We hypothesized that genetic variation in miRNA processinggenes and binding sites might influence breast cancer risk due to the functionalrelevance, and performed a case-control study to investigate the association above inChinese women.PartⅠEvaluation of genetic variants in microRNA processing genesand risk of breast cancer in Chinese womenMicroRNAs (miRNA) are a class of small, noncoding and double-strandedRNA molecules involved in a diversity of cellular functions. Single nucleotidepolymorphisms (SNPs) in miRNA biosynthesis pathway genes may affect thebiogenesis of miRNAs and the regulatory effect of miRNAs to targeted genes, hencepromoting tumorigenesis. In this study, we systematically selected24functionalSNPs located in8key biosynthesis genes (DROSHA, DGCR8, XPO5, RAN, DICER,AGO2, GEMIN4and HIWI) to investigate the association between those SNPs andbreast cancer risk in Chinese population. All24SNPs were first genotyped in subjectsof stage1(878breast cancer cases and900controls) and three promising SNPs(DROSHA rs2291109, RAN rs7301722and DGCR8rs417309) were further validatedin stage2sample sets (914breast cancer cases and967controls). We found that only one SNP (rs417309) located in3’UTR of DGCR8was associated with a significantlyincreased breast cancer risk in both stage2[odds ratio (OR)=1.59,95%confidenceinterval (CI)=1.09-2.33] and combined sets (OR=1.50,95%CI=1.16-1.93).Furthermore, the bioinformatics prediction suggested that this SNP was located at thebinding sites of miR-106b and miR-579in3’-UTR of DGCR8. To evaluate whetherrs417309variant might affect the binding site of miRNAs, we generated luciferasereporter palsmids of DGCR83’-UTR (containing rs417309G and A allele) that werecotransfected with mir-106b or mir-579in293T, Hela and MCF-7cells. The resultsof vitro experiment showed the variant A allele of rs417309decreased the bindingcapability of miRNAs, which might lead to the up-expression of DGCR8. Overall,these findings suggested that DGCR8rs417309G>A might affect breast cancer riskthrough breaking miRNA binding sites. PartⅡ Genetic variations in a microRNA binding siteassociated with breast cancer susceptibilityGenetic variants may modulate miRNA-mRNA interaction through creating ordestroying miRNA binding sites. Twenty-four single nucleotide polymorphisms(SNPs) that were predicted to affect the binding affinity of reported breast cancerrelated miRNAs to3’-UTR of known genes were genotyped in878breast cancercases and900controls in Chinese women. Three promising SNPs (rs10494836,rs10857748and rs7963551) were further validated in additional914breast cancercases and967controls. The variant allele (C) of rs7963551at3’-UTR of RAD52showed a consistently reduced breast cancer risk in two stages with a combined oddsratio (OR) of0.84[95%confidence interval (CI)=0.75-0.95, P=0.007], which wasmore prominent among women with early age at first live birth (OR=0.71,95%CI=0.58-0.87). A significant interaction between rs7963551and age at first live birth onbreast cancer risk (P for interaction=0.041). Luciferase activity assay showed ahigher expression level for rs7963551C allele as compared with A allele (P =2.95×10-~2, P=1.73×10-~2, P=5.19×10-~3for293T, Hela and MCF-7cell lines,respectively), which may be due to a reduced inhibition from a weakened bindingcapacity of miRNA to3’-UTR of RAD52harboring C allele. These findings indicatethat rs7963551located at hsa-let-7binding site may alter expression of RAD52though modulating miRNA-mRNA interaction and contribute to the development ofbreast cancer in Chinese women.