Investigation of the therapeutic effects of miRNAs targeting CCT3 gene in various cancer cell lines

Molecular chaperone proteins and protein misfolding play a critical role in cancer pathogenesis. CCT3, one of the eight subunits of eukaryotic chaperons, catalyzes the correct folding of proteins involved in cell division, proliferation, and apoptosis pathways. Moreover, CCT3 expression is increased responsively with carcinogenesis. However, how CCT3 drives the cancerous process has not been known. In this study, the mechanistic and functional interactions among CCT3, apoptotic pathways and cell stressors were probed. First, CCT3 expression levels of different 16 cell lines were profiled and CCT3 expressions of CRL-2329 and PC-3 were found to be significantly increased. Then, CCT3 expressions of CRL-2329 and PC-3 lines were suppressed by miR-24-3p, miR-128-3p, and miR-149-5p mimics, and the apoptotic responses of the cell lines to the knockdown of CCT3 were measured by acridine orange/ethidium bromide and Annexin V/PI staining, cell-cycle and mitochondria membrane potential (MMP) analyses, intracellular reactive oxygen species (ROS) measurement and expression levels of the apoptotic gene. After having suppressed CCT3, the cell cycle was arrested in the G0/G1 phase, MMP was impaired, and the intracellular ROS level was increased. These signs of apoptotic flux were corroborated by morphological images, statistically enhanced expression levels of the apoptotic genes (FADD, CAS8, CAS3, P53, BAX, BCL-2, NOXA, CAS9, APAF1, and CYCS), and intracellular free amino acids profile. Strikingly, the suppressed-CCT3 shifted intracellular levels of glutamine, beta-alanine, glycine, serine, asparagine and sarcosine, which are employed in energy metabolism. Consequently, suppression of CCT3 by miRNA mimic transfection spurs apoptosis by unbalancing the homeostasis of intracellular ROS and the profile of free amino acids in energy metabolism. Taken together, it was anticipated that miRNA-mediated inhibition of CCT3 might provide "dual therapeutic strategy" through the conventional ROS-mediated cellular toxicity approach as well as energy withdrawal effect.