The effect of lncRNA UCA1 on PI3K/AKT/mTOR signaling in doxorubicin-resistant ER+ breast cancer

This thesis study aimed to investigate the effect of the long non-coding RNA (lncRNA) UCA1 on PI3K/AKT/mTOR signaling in the development of doxorubicin (Dox) resistance in breast cancer (BC). Initially, doxorubicin resistance was induced in MCF-7 cells, and the resistance level reached 0.64 µM. The development of resistance in resistant cells (MCF-7/Dox) was validated through the examination of cell morphology, the evaluation of multidrug resistance-associated protein 1 (MRP1) gene expression, and the regulation of the PI3K/AKT/mTOR signaling pathway. Resistant cells were observed to adopt a spindle-like shape, exhibit increased length, and contain multiple vesicles in their cytoplasm. Dox resistance was demonstrated by a 4-fold increase in MRP1 gene expression at the transcript level. Additionally, to support the acquisition of mesenchymal features in resistant cells, the expression levels of genes associated with the epithelial-to-mesenchymal transition (EMT) process (E-cadherin, Occludin, Claudin-1, N-cadherin, Twist-1, Vimentin) were analyzed using quantitative PCR (qPCR). In MCF-7/Dox cells, the expression levels of E-cadherin, Occludin, and Claudin-1 genes were decreased, whereas the expression levels of N-cadherin, Twist-1, and Vimentin genes were significantly increased. These findings indicate that resistant cells acquired mesenchymal characteristics. To examine the effect of doxorubicin on cell viability in MCF-7/Dox cells, an MTT assay was performed, and the half-maximal inhibitory concentration (IC50) values of MCF-7/S and MCF-7/Dox cells were determined as 1.65 µM and 128.5 µM, respectively. MCF-7/Dox cells exhibited 78-fold higher resistance to doxorubicin compared to MCF-7/S cells. Furthermore, it was shown that UCA1 gene expression and the PI3K/AKT/mTOR signaling pathway was upregulated in Dox-resistant cells, as evidenced by increased expression levels of genes involved in this pathway, including Akt1, Akt2, mTOR, and PTEN. In the next step of the study, siUCA1 was transfected into the cells using the lipofectamine method. qPCR analyses revealed that UCA1 gene expression was statistically significantly reduced by 50% in transfected cells compared to the control group. siUCA1 transfection significantly decreased IC50 values in both MCF-7/Dox and MCF-7/S cells. This result suggests that UCA1 could be a potential candidate for reversing doxorubicin resistance. Additionally, it was observed that UCA1 silencing suppressed PI3K/AKT/mTOR signaling and significantly reduced the motility of cells. In conclusion, this thesis study is the first in the literature to identify that lncRNA UCA1 may be associated with the reversal of doxorubicin resistance in breast cancer through its regulation of the PI3K/AKT/mTOR signaling pathway.