Characterization of molecular-level changes due to MicroRNA-125B expression in breast cancer cells by spectroscopic and chemometric analysis techniques

Şimşek Özek, Nihal
MicroRNAs (miRNAs), are 22 nucleotides long, non-coding RNAs that control gene expression post-transcriptionally by binding to their target mRNA’s 3’ UTRs (untranslated regions). Due to their crucial roles in various important regulatory processes and pathways, miRNAs have been implicated in disease mechanisms such as tumorigenesis, metastasis and drug resistance when their expression is deregulated. Moreover, the regulatory roles of these micromanagers have been demonstrated in the metabolism of cancer cells. To date, a significant number of miRNAs and their target messenger RNAs (mRNAs) have been identified and verified. Since one miRNA can target many mRNAs, it is hard to delineate the roles of these molecules in etiopathogenesis and metabolism of cancers. Therefore, to uncover the global roles of these molecules in cancer, both experimental and data analysis methods are required. The current thesis study aimed to examine the alterations in the membrane dynamics, content and structure of molecules especially lipids of miR-125b expressing breast cancer cells compared to controls to obtain a more holistic view of how miRNA expression alters cells. miR-125b expression in breast cancer cell line systems was investigated since this is one the most down-regulated miRNAs in breast cancer. MCF7 and T47D cells stably transfected with miR-125b (MCF7-125b, T47D-125b) and empty vector (MCF7-EV, T47D-EV) were studied. Global molecular changes and specifically the changes in cellular lipids were determined by Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The alterations in membrane dynamics were identified by spin-labelling Electron Spin Resonance (ESR) spectroscopy. In addition to these techniques, to discriminate between EV and miR-125b transfected breast cancer cells, unsupervised chemometric analysis methods including principal component (PCA) and hierarchical cluster analyses (HCA) were applied to the infrared spectra. The spectral results revealed lower saturated and unsaturated lipids, RNA and protein content in both miR-125b transfected breast cancer cells relative to the EV cells. The amount of glycogen, cholesterol ester and triglyceride was found to be decreased in MCF7 cells but opposite results were obtained for T47D cells. The decreased membrane fluidity was acquired in MCF7 cells but for T47D cells an inverse result was obtained. The decline in phospholipid, sphingomyelin, phosphotidyl-choline, sphingolipid and sphingomyelin contents were also observed in both cell lines with miR-125b expression. Based on these alterations, both EV and miR-125b cells lines were discriminated successfully by PCA and HCA methods. Here, a novel approach was proposed to understand the global effects of miRNAs in cells. Potential applications of infrared spectroscopy coupled with chemometric methods are not only limited to research area. This kind of pioneer studies will shed light on the development of future diagnostic tools for deregulated miRNA expression in patient samples.