Abstract
Background: Kras mutation is frequently detected in pancreatic cancers and leads to altered energy metabolite. Here we investigated molecule markers related with Kras mutation, which could be used as developing new target for Kras mutant driven cancer. Methods: A knockin BxPC-3/ Kras G12D cell line was constructed by CRISPR/Cas9 system. Proliferation and metabolite characterization in BxPC-3/ Kras G12D was compared with wild type BxPC-3 by using colony formation assay and mitochondrial dyes. The differential genes were screened using mitochondrial metabolite-related genes PCR array. The expression of COX6C was confirmed by real time polymerase chain reaction (RT-PCR) and western blot. COX6C expression in 30 pairs of tissue microarray of pancreatic carcinoma and matched adjacent tissues was analyzed by immunohistochemistry. ATP production stimulated by metabolites substrate assay was carried out to investigate whether the decreased COX6C by siRNA transfection affected the metabolite characterization of pancreatic cancer cells. Results: The BxPC-3/ Kras G12D displayed faster proliferation, increased mitochondrial mass and ATP, elevated mitochondrial membrane potential than BxPC-3. Using mitochondrial metabolite-related genes array, we identified COX6C was an up-regulated gene driven by Kras G12D . There was a notable difference of COX enzyme activity between BxPC-3 and BxPC-3/ Kras G12D . The overexpression of COX6C was found in pancreatic cancer tissues. Using COX6C siRNA-mediated knockdown, the cell viability, COX enzyme activity and ATP production of BxPC-3/ Kras G12D cells significantly decreased. Conclusions: These results suggested that activation of Kras G12D in pancreatic cancer cells increased the COX activity and ATP production of mitochondrial via up-regulation of COX6C. This regulatory subunit of COX may have utility as a Ras effector target for development of anti-pancreatic cancer therapeutics.
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