Evaluation of hsa-miR-2355 MRE Site Sequence Within 3'-UTR of ERCC1 Gene in Breast Cancer Clinical Samples by RFLP Method
Author(s): Aliarash Anoushirvani, Azam Ahmadi*, Mohammad Arjomandzadegan*, Reza Aghabozorgi, Maryam Sahraei, Mona Moghadasi
Aims: The involvement of deregulated miRNAs networks in breast cancer (BC) progression is validated previously. MiRNAs control genes by binding to their regulatory regions. A SNP within a miRNA binding site could change target mRNA level and protein. ERCC1 is involved in the Nucleotide excision repair (NER). This pathway is associated with BC. In present study, we genotyped 3'-UTR ERCC1 in BC patients.
Method: The 3'-UTR of ERCC1 were analysed for MRE sites using bioinformatics software. In present case-control study, we extracted genomic DNA from 34 BC clinical and control samples. These DNAs were used as template in PCR which bind to 3'- UTR ERCC1 gene. The nucleotide sequences of amplicons were evaluated by RFLP using Eco47I restriction endonuclease.
Results: Bioinformatics analysis showed that the restriction sites of Eco47I enzymes in the 2342 bp 3'-UTR of ERCC1 are related to MRE of miR-2355. The frequency of genotypes in present study was, 26.47% TT homozygote, 35.29% TG heterozygote and 38.23% GG mutant homozygote variant in patient group that was different with control group (OR=1.3465, 95% CI=0.7275 to 2.4923, p<0.05). The positive correlation between MRE change of miR-2355 (homozygote TT) with status of HER2 positive, BMI>22 and age>40 years were confirmed statistically (P=0.03).
Conclusion: In present study, we confirmed correlation between MRE nucleotide changes of miR-2355 within 3'-UTR ERCC1 with HER2 positive status. However, there is no association between miR-2355 MRE changes within 3'-UTRERCC1 with tumour-staging. Evaluation the genotype of aforementioned genomics position could be used as a validation test for HER2 status probably.