## 3 Results  
### In Vitro Analysis  
rs12976445 SNP in pri-miR-125a occurring in three genotypes TT, CT, and CC has been associated with cancer and other diseases [32; 33; 34]. We studied the pri-miR-125a amplicon (Figure 1) by sequencing and restriction analysis to determine the genotype frequency and its association with breast cancer. We amplified DNA obtained from 27 breast cancer tumours to assess the frequency of several known SNPs in pri-miR-125a sequence. The sequencing of 27 pri-miR-125a amplicons showed that only nrs12976445 polymorphic site was highly variable in our samples. Subsequently, we estimated the association of rs12976445 with breast cancer using DNA from 304 blood samples, 175 positive (cases, Ca) and 129 negative (controls, Co) samples. We digested the pri-miR-125a amplicon using BeaGI restriction enzyme producing 42 and 205 fragments if C or non-digesting if T variant was present. We performed statistical analysis with SNPStats on-line software. The allele and genotype frequencies analysis are presented in Table 1. We found that rs12976445 in pri-miR-125a followed Hardy-Weinberg equilibrium in breast cancer cases (Ca) set (Table 1). Figure 1: Whole amplicon (247 nt) comprising pre-miR-125a sequence, the fragment of the sequence from Ensembl Gene ID: ENSG00000208008. Lower-case corresponds to pri-miR-125a fragments removed in pre-miR-125a processing; grey upper-case is pre-miR-125a; in bold upper-case we marked mature miR-125a 5p and 3p, respectively; bold underlined lower-case nucleotides are the analyzed U-, C-variants; bold italic lower-case is a 51 nt fragment (51-miR-125a) that was analyzed using computational methods. BaeGI restriction site is located directly before rs12976445 SNP, dividing sequence into 42 nt and 205 nt subsequences. The statistical analysis, comprising odds ratio (OR) at 95% confidence interval (CI), are presented in Table 1. In the co-dominant model (TT vs. TC vs. CC), the heterozygous CT genotype of rs12976445 SNP was slightly more frequent in control (Co) set (50.4%) compared to cases (Ca) (46.3%) with OR Co/Ca = 1.19, 95%, CI = 0.74-1.91 and \(p\) = 0.77 (Table 1). In the dominant model (TT vs. TC + CC), we observed slightly higher level of CT + CC in controls over cases with OR = 1.17, 95% (CI = 0.74-1.85) and \(p\) = 0.5. These calculations indicated the tendency that in breast cancer cases, TT genotype appeared slightly more frequent than CC and CT genotypes. Additionally, we analyzed two other known SNPs, rs143525573 and rs12975333, located in the mature miR-125a coding sequence. The Ddel enzyme was applied to assess these two SNPs. We did not observe the variability of rs143525573 and rs12975333 in the studied group of patients with breast cancer. Since SNP in pri-miR-125a could modulate the level of mature miRNA, we assessed the relationship of rs12976445 variability with the status of human epidermal growth factor receptor 2 (HER2), ER1\(\alpha\) and PR receptors. mRNA encoding receptor HER2 (human epidermal growth factor receptor 2, receptor tyrosine-protein kinase erbB-2, encoded by ERBB2 gene) is a target of miR-125a [33]. HER2 together with ER1\(\alpha\) and PR receptors are used in the diagnosis of breast cancer. Therefore, we decided to perform tests of the relationship of rs12976445 variation with the status of these receptors. HER2 receptor status 0, 1, and 2 without amplification was classified as the negative group (N), while status