We investigated the role of
mitochondrial DNA (
mtDNA) copy number alteration in human
renal cell carcinoma (RCC). The
mtDNA copy numbers of paired
cancer and non-
cancer parts from five resected RCC kidneys after radical
nephrectomy were determined by quantitative polymerase chain reaction (Q-PCR). An RCC cell line, 786-O, was infected by lentiviral particles to knock down mitochondrial transcriptional
factor A (TFAM). Null target (NT) and TFAM-knockdown (TFAM-KD) represented the control and knockdown 786-O clones, respectively.
Protein or
mRNA expression levels of TFAM;
mtDNA-encoded
NADH dehydrogenase subunit 1 (ND1), ND6 and
cytochrome c oxidase subunit 2 (COX-2); nuclear
DNA (nDNA)-encoded
succinate dehydrogenase subunit A (SDHA); v-akt murine
thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT and v-myc myelocytomatosis viral oncogene homolog gene (c-MYC)-encoded MYC; glycolytic
enzymes including
hexokinase II (HK-II),
glucose 6-phosphate
isomerase (GPI),
phosphofructokinase (PFK), and
lactate dehydrogenase subunit A (LDHA); and
hypoxia-inducible factors the HIF-1α and HIF-2α,
pyruvate dehydrogenase kinase 1 (PDK1), and
pyruvate dehydrogenase E1 component α subunit (PDHA1) were analyzed by Western blot or Q-PCR. Bioenergetic parameters of cellular metabolism, basal mitochondrial oxygen consumption rate (mOCRB) and basal extracellular acidification rate (ECARB), were measured by a Seahorse XF(e)-24 analyzer. Cell invasiveness was evaluated by a trans-well migration assay and
vimentin expression.
Doxorubicin was used as a chemotherapeutic agent. The results showed a decrease of
mtDNA copy numbers in resected RCC tissues (p = 0.043). The TFAM-KD clone expressed lower
mtDNA copy number (p = 0.034), lower
mRNA levels of TFAM (p = 0.008), ND1 (p = 0.007), and ND6 (p = 0.017), and lower
protein levels of TFAM and COX-2 than did the NT clone. By contrast, the
protein levels of HIF-2α, HK-II, PFK, LDHA, AKT, MYC and
vimentin; trans-well migration activity (p = 0.007); and drug resistance to
doxorubicin (p = 0.008) of the TFAM-KD clone were significantly higher than those of the NT clone. Bioenergetically, the TFAM-KD clone expressed lower mOCRB (p = 0.009) but higher ECARB (p = 0.037) than did the NT clone. We conclude that a reduction of
mtDNA copy number and decrease of respiratory function of mitochondria in RCC might be compensated for by an increase of
enzymes and factors that are involved in the upregulation of glycolysis to confer RCC more invasive and a
drug-resistant phenotype in vitro.