Effects of BCL3 Gene Knockout on Mitochondrial Respiration and ATP Synthesis

Abstract: B-cell CLL/lymphoma 3 (BCL3)， which acts as transcription coregulator by activating or suppressing gene expression via binding transcription factors， is required for cancer cell survival， the underling mechanism is not yet fully known. To investigate the roles of BCL3 in mitochondria function， BCL3 knockout human cervical carcinoma HeLa cells (BCL3-KO cells) were generated by CRISPR/Cas9-mediated genome editing. Real-time quantitative reverse transcription PCR (qRT-PCR) and Western blot were used to validate gene knockout. Using the cell lines， DCFH-DA and JC-1 staining method was performed to test ROS production and mitochondria membrane potential (MMP)， respectively. Cellular energy metabolism was studied using Seahorse XF24 Extracellular Flux Analyzer， and ATP production was also measured. In contrast to wild type HeLa cells， the relative level of ROS was decreased by nearly 50% in BCL3-KO cells; however the decline could be reversed by overexpression of BCL3. Deficiency of BCL3 led to a significant loss of MMP and a reduction in ATP production (P<0.001)， which could be indication of mitochondrial dysfunction. Furthermore， BCL3 knockdown did not affect the rate of basic cellular aerobic respiration， but caused an obvious rise in the rate of carbomyl cyanide 4-(trifluorometyocy) phenylhydrazone (FCCP)-induced cellular aerobic respiration (P<0.001). ATP concentration in BCL3-KO cells was decreased about 40% compared with that of in wild type HeLa cells. Taken together， our results reveal a novel role for BCL3 in regulation of mitochondrial function， which might be one of the reasons for BCL3 to maintain cancer cell survival.

CSTR: 32200.14.cjcb.2018.04.0009