Identification of the most suitable in vitro model to study vimentin-G4 repeats interactions in epithelial-mesenchymal transition

Vimentin (VIM) is an important intermediate filament protein that plays a key role in epithelial-mesenchymal transition (EMT), a process critical for cancer progression and metastasis. While vimentin is predominantly localized in the cytoplasm, recent studies have revealed its presence in the nucleus, where it is able to interact with G-quadruplex (G4) repeat complexes, possibly influencing gene transcription. This study aimed to investigate the nuclear vimentin expression in HGC-27, MDA-MB-231, BT-549, and BT-474 to identify positive and negative models to study the molecular interaction of vimentin with G4 repeats and its implication in cancer metastasis. To analyze the expression and localization of EMT markers, including vimentin, E-cadherin (CDH1), and K+ channel (KCNH1), qRT-PCR, immunofluorescence (IF), immunocytochemistry (ICC), and Western blotting were utilized. Additionally, the BT-474 cell line was treated with StemXVivoTM EMT-inducing Media Supplement to investigate the change in nuclear and cytosolic expression of these markers after the induction of EMT. The results showed that HGC-27, MDA-MB-231, and BT-549 cell lines had high expression of VIM (mesenchymal marker) mRNA, while BT-474 expressed high CDH1 (epithelial marker) mRNA levels. At the protein level, HGC-27, BT549, and MDA-MB-231 showed comparable levels of vimentin in the cytosol, while in the nuclear compartment HGC-27 showed higher expression of this marker. In BT-474, on the other hand, there was no significant difference in the gene and protein expression of vimentin after the induction protocol, but a difference in its subcellular localization (from cytosol to nucleus) probably due to post-translational modifications was observed. Moreover, to investigate a possible mechanism involved in the distribution of vimentin in the subcellular compartments, the KCNH1 K+ channel was evaluated. Interestingly, in BT549 cells, its mRNA expression was high, while in HGC-27 cells, the transporter was not detectable. The other cell lines showed a low KCNH1 mRNA expression, with a slight decrease after the induction treatment in BT-474. In conclusion, these results lead us to hypothesize that the nuclear localization of vimentin may play a role in EMT, potentially through post-translational modifications. KCNH1 could also be a possible actor in vimentin nuclear translocation, but further evaluations are needed.