EVALUATION OF HMGA2 AND SMARCA5 GENES EXPRESSION IN 4T1 CELLS EXPOSED TO METHOTREXATE: BIOINFORMATIC AND EXPERIMENTAL STUDY

Breast cancer is a threatening disease in females and is the second common cancer
among women after lung cancer. The aim of this research is to bioinformatically and experimentally
evaluate the effect of methotrexate (MTX) on the expression of HMGA2 and SMARCA5 genes in the
MTX treated 4T1 cancer cell line.
Materials & Methods: To perform this study, initially microarray data were collected from Gene
Expression Omnibus (GEO) then they were analyzed using Probabilistic neural networks (PNN) in
MATLAB 2018a software as a bioinformatics tool. In the next step, the specific primers were designed
for HMGA2 and SMARCA5 genes. MTX cytotoxicity was assessed on 4T1 cancer cells using MTT
assay. Finally, Real-time PCR was used for evaluating the effect of MTX on the rate of HMGA2 and
SMARCA5 genes expression.
Results: The bioinformatic analysis showed that HMGA2 and SMARCA5 expression levels notably
reduced and increased respectively in MTX treated group in comparison with the control (untreated)
group. MTT assay indicated that MTX decreased the 4T1 cell viability in a concentration-dependent
manner and the IC50 value of MTX was estimated to be 208 µg/ml in 48 hours. Real-time PCR data
showed that the expression level of HMGA2 decreased significantly (p ≤ 0.05), while the SMARCA5
gene was significantly (p ≤ 0.05) upregulated which is consistent with bioinformatic analysis results.
Conclusion: The obtained results suggest that HMGA2 and SMARCA5 genes may be used as MTX
target genes in breast cancer therapy. However, further studies need to be carried outto confirm the
results