Investigation of the Effects of Silencing Mitochondrial Mutant Gene Expressions on Carcinogenesis in a Lung Cancer Cell Model
Project no. 23912 · Status: Kapanmış (Yayın bekleniyor) · 2022
Summary
<p>In this study, we investigated the effects of mitochondrial genome mutations on carcinogenesis in lung cancer, which remains one of the leading causes of cancer incidence and mortality both in Türkiye and worldwide. Specifically, we targeted the <strong>MT-ND2 m.4587T>C (Phe40Leu)</strong> variant, which exists in <strong>a heteroplasmic state in wild-type A549 cells</strong> and <strong>a homoplasmic state in drug-resistant A549 sublines</strong>. Variant-specific siRNA was designed and used to silence the mutation at the mRNA level, and the resulting cellular and mitochondrial impacts were comprehensively evaluated using various functional assays.</p><p>Following siRNA transfection, apoptosis-related assays including Annexin V, Caspase-3 activation, and JC-1 staining were performed via flow cytometry. In A549 cells, significant time-dependent increases in apoptotic signals were observed. Although mitochondrial membrane potential changes were not prominent in healthy T2A cells, Annexin V and Caspase-3 assays revealed significant apoptotic responses, particularly at 72 hours. These findings indicate that silencing this variant may trigger apoptosis in cancer cells.</p><p>Gene expression analysis revealed time-dependent increases in pro-apoptotic genes including <strong>CASP3, CASP7, CASP9</strong>, and <strong>BAX</strong> at 72 hours post-transfection. Notably, upregulation of the tumor suppressor gene <strong>TP53</strong> suggests that silencing of this variant might enhance tumor-suppressive pathways and apoptosis signaling.</p><p>Wound healing assays demonstrated statistically significant time-dependent differences between the treatment and control groups in A549 cells across the 0–120 hour period. In T2A cells, initial differences diminished by 96 hours, indicating cell type–specific responses in migratory behavior.</p><p>Evaluation of mitochondrial function through ROS analysis revealed <strong>no significant changes in A549 cells</strong>, while <strong>T2A cells</strong> showed statistically significant ROS elevation at 72 hours. However, <strong>cell proliferation and clonogenic survival</strong> were not significantly affected by the siRNA treatment.</p><p>In conclusion, our study suggests that siRNA-mediated silencing of the <strong>MT-ND2 m.4587T>C</strong> variant may affect specific cellular processes, particularly apoptosis and migration, in A549 cells. However, the overall impact on carcinogenesis appears to be limited. The lack of similar studies targeting this specific variant via siRNA in the literature adds novelty to our findings. Nevertheless, further studies involving diverse siRNA formulations, dosing strategies, and combination treatments are needed to better understand the therapeutic potential of targeting mtDNA mutations in cancer.</p>