Investigation of miRNA 221/222 cluster profile in exosomes derived from gemcitabine-resistant and gemcitabine-sensitive non-small cell lung cancer stem cells

Objective: The aim of this thesis study was to investigate the expression of miRNA 221/222, MDR1, ABCG2, and Bcl-2 genes in Gemcitabine-resistant cancer stem cells (r-CSCs; CD326+ CD133+ CD44+) and their exosomes (r-CSC-Exo), and to determine the impact of miRNA 221/222 on Gemcitabine resistance by comparing them with Gemcitabine-sensitive cancer stem cells (CSCs; CD326+ CD133+ CD44+) and their exosomes (CSC-Exo). Methods: Gemcitabine resistance was developed in A549 cells using the CFU method (r-A549), followed by isolation of CD326+ CD133+ CD44+ CSCs and their propagation in tumor spheroid form. Subsequently, exosome isolation and characterization were performed from CSC and r-CSC tumor spheroids using flow cytometry, immunofluorescence, qRT-PCR, growth curve, and MTT methods. Finally, the expression of miRNA 221/222, MDR1, ABCG2, and Bcl-2 genes was examined in CSC, CSC-Exo, r-CSC, and r-CSC-Exo using qRT-PCR. Results: Significant and high colony-forming abilities were demonstrated in d-A549 cells through CFU analysis. CD326+ CD133+ CD44+ CSCs isolated from A549 and r-A549 cells exhibited high purity and viability and were expanded in a 3B culture environment. Characterization analysis revealed higher MDR1 and ABCG2 activation, OCT4 gene expression, increased proliferation, and higher IC50 values in d-CSCs compared to CSCs. qRT-PCR analysis of all experimental groups showed significantly higher expression of MDR1 and Bcl-2, as well as miR-221-3p, miR-222-3p, and miR-222-5p in r-CSC-Exo. Conclusion: This thesis study suggests that the expression level of miR-221/222 may be closely associated with Gemcitabine resistance in lung cancer, and miR-222-5p may serve as an early biomarker in Gemcitabine-resistant lung cancer.