siRNA-mediated Silencing of OPA1, NME4, and RCC1L: Effects on Proliferation in KP4 Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest and most aggressive malignancies worldwide, characterized by late diagnosis, limited therapeutic options, and extensive metabolic reprogramming. While oncogenic KRAS signaling extensively rewires PDAC metabolism towards glycolysis, pancreatic cancer cells remain actively reliant on mitochondrial oxidative phosphorylation (OXPHOS) and generally on mitochondrial function for survival and proliferation. Mitochondrial dynamics and cristae organization are therefore important processes in the adaptation and progression of cancer cells. This study investigated the contribution of three interconnected mitochondrial proteins, optic atrophy 1 (OPA1), mitochondrial nucleoside diphosphate kinase (NME4), and regulator of chromatin condensation 1 like (RCC1L), to the proliferative capacity of PDAC cells. OPA1 is a key regulator of the inner mitochondrial membrane fusion and cristae architecture, while NME4 and RCC1L are proposed to support OPA1 function through local nucleotide metabolism and guanine nucleotide exchange, respectively. However, their functional contribution to PDAC cell proliferation remains insufficiently characterized. The effects of transient siRNA-mediated silencing of OPA1, NME4, and RCC1L were investigated in the human PDAC cell line KP4 using two different proliferation assays. Moreover, the effect of NME4 silencing on clonogenic capacity was additionally evaluated by colony formation assays using both KP4 and PANC1 cells. The results demonstrated differential effects of the three targets on KP4 proliferation. Both OPA1 and NME4 silencing produced variable effects across independent experiments, While RCC1L silencing displayed the most consistent trend towards reduced proliferation across experiments. However, none of the observed differences between silenced and control conditions reached statistical significance. In contrast, the colony formation assays showed a consistent qualitative increase in crystal violet staining following NME4 knockdown in both KP4 and PANC1 cells, suggesting that NME4 depletion did not impair clonogenic growth under the experimental conditions. Overall, these findings suggest that OPA1, NME4, and RCC1L may have differential contributions to PDAC cell growth, with RCC1L showing the strongest and most consistent trend towards growth inhibition following depletion.