We previously reported a gradual increase of relative mitochondrial DNA (mtDNA) copy number during the progression of esophageal squamous cell carcinoma (ESCC). Because mitochondria are the intracellular organelles responsible for ATP production, we investigated the associations among mtDNA copy number, mitochondrial bioenergetic function, tumor invasion and the expression levels of epithelial mesenchymal transition (EMT) markers in a series of seven ESCC cell lines, including 48T, 81T, 146T, TE1, TE2, TE6 and TE9. Among them, TE1 had the highest relative mtDNA copy number of 240.7%. The mRNA of mtDNA-encoded ND1 gene (2.80), succinate-supported oxygen consumption rate (11.21 nmol/min/10⁶ cells), ATP content (10.7 fmol/cell), and the protein level of mitochondrial transcription factor A (TFAM) were the highest and the lactate concentration in the culture medium (3.34 mM) was the lowest in TE1. These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria. Furthermore, TE1 showed the highest trans-well migration activity of 223.0 cells/field, the highest vimentin but the lowest E-cadherin protein expression levels, which suggest that TE1 had the highest invasion capability. We then conducted a knockdown study using pLKO.1-based lentiviral particles to infect TE1 cells to suppress the expression of TFAM. Molecular analyses of the parental TE1, control TE1-NT and TFAM knockdown TE1-sh-TFAM(97) cells were performed. Interestingly, as compared to the control TE1-NT, TE1-sh-TFAM(97) exhibited lower levels of the relative mtDNA copy number (p = 0.001), mRNA of mtDNA-encoded ND1 gene (p = 0.050), succinate-supported oxygen consumption rate (p = 0.065), and ATP content (p = 0.007), but had a higher lactate concentration in the culture medium (p = 0.010) and higher protein level of lactate dehydrogenase. A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97). Significantly, compared to the control TE1-NT, TE1-sh-TFAM(97) had a lower trans-well migration activity (p < 0.001), a higher E-cadherin level but a lower vimentin protein level, which indicates a decrease of invasiveness. Taken together, we suggest that high relative mtDNA copy number and bioenergetic function of mitochondria may confer an advantage for tumor invasion of ESCC.

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 (mOCR_B) and basal extracellular acidification rate (ECAR_B), 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 mOCR_B (p = 0.009) but higher ECAR_B (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.

The role of mitochondrial DNA (mtDNA) alterations in the pathophysiology of systemic lupus erythematosus (SLE) remains unclear. We investigated sequence variations in the D310 region and copy number change of mtDNA in 85 SLE patients and 45 normal subjects. Leukocyte DNA and RNA were extracted from leukocytes of the peripheral venous blood. The D310 sequence variations and copy number of mtDNA, and mRNA expression levels of mtDNA-encoded genes in leukocytes were determined by quantitative real-time polymerase chain reaction (Q-PCR) and PCR-based direct sequencing, respectively. We found that leukocyte mtDNA in SLE patients exhibited higher frequency of D310 heteroplasmy (69.4% vs. 48.9%, p = 0.022) and more D310 variants (2.2 vs. 1.7, p = 0.014) than those found in controls. Among normal controls and patients with low, medium or high SLE disease activity index (SLEDAI), an ever-increasing frequency of D310 heteroplasmy was observed (p = 0.021). Leukocyte mtDNA copy number tended to be low in patients of high SLEDAI group (p = 0.068), especially in those harboring mtDNA with D310 heteroplasmy (p = 0.020). Moreover, the mtDNA copy number was positively correlated with the mRNA level of mtDNA-encoded ND1 (NADH dehydrogenase subunit 1) (p = 0.041) and ATPase 6 (ATP synthase subunit 6) (p = 0.030) genes. Patients with more D310 variants were more susceptible to lupus nephritis (p = 0.035). Taken together, our findings suggest that decrease in the mtDNA copy number and increase in D310 heteroplasmy of mtDNA are related to the development and progression of SLE, and that the patients harboring more D310 variants of mtDNA are more susceptible to lupus nephritis.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease with heterogeneous clinical manifestations. A diverse innate and adaptive immune dysregulation is involved in the immunopathogenesis of SLE. The dysregulation of immune-related cells may derive from the intricate interactions among genetic, epigenetic, environmental, and immunological factors. Of these contributing factors, non-coding RNAs (ncRNAs), including microRNAs (miRNAs, miRs), and long non-coding RNAs (lncRNAs) play critical roles in the post-transcriptional mRNA expression of cytokines, chemokines, and growth factors, which are essential for immune modulation. In the present review, we emphasize the roles of ncRNA expression in the immune-related cells and cell-free plasma, urine, and tissues contributing to the immunopathogenesis and tissue damage in SLE. In addition, the circular RNAs (circRNA) and their post-translational regulation of protein synthesis in SLE are also briefly described. We wish these critical reviews would be useful in the search for biomarkers/biosignatures and novel therapeutic strategies for SLE patients in the future.

Oxidative stress elicited by reactive oxygen species (ROS) and chronic inflammation are involved both in deterring and the generation/progression of human cancers. Exogenous ROS can injure mitochondria and induce them to generate more endogenous mitochondrial ROS to further perpetuate the deteriorating condition in the affected cells. Dysfunction of these cancer mitochondria may possibly be offset by the Warburg effect, which is characterized by amplified glycolysis and metabolic reprogramming. ROS from neutrophil extracellular traps (NETs) are an essential element for neutrophils to defend against invading pathogens or to kill cancer cells. A chronic inflammation typically includes consecutive NET activation and tissue damage, as well as tissue repair, and together with NETs, ROS would participate in both the destruction and progression of cancers. This review discusses human mitochondrial plasticity and the glucose metabolic reprogramming of cancer cells confronting oxidative stress by the means of chronic inflammation and neutrophil extracellular traps (NETs).

We used gastric cancer cell line AGS and clinical samples to investigate the roles of mitochondrial DNA (mtDNA) alterations and mitochondrial respiratory dysfunction in gastric adenocarcinoma (GAC). A total of 131 clinical samples, including 17 normal gastric mucosa (N-GM) from overweight patients who had received sleeve gastrectomy and 57 paired non-cancerous gastric mucosae (NC-GM) and GAC from GAC patients who had undergone partial/subtotal/total gastrectomy, were recruited to examine the copy number and D310 sequences of mtDNA. The gastric cancer cell line AGS was used with knockdown (KD) mitochondrial transcription factor A (TFAM) to achieve mitochondrial dysfunction through a decrease of mtDNA copy number. Parental (PT), null-target (NT), and TFAM-KD-(A/B/C) represented the parental, control, and TFAM knocked-down AGS cells, respectively. These cells were used to compare the parameters reflecting mitochondrial biogenesis, glycolysis, and cell migration activity. The median mtDNA copy numbers of 17 N-GM, 57 NC-GM, and 57 GAC were 0.058, 0.055, and 0.045, respectively. The trend of decrease was significant (p = 0.030). In addition, GAC had a lower mean mtDNA copy number of 0.055 as compared with the paired NC-GM of 0.078 (p < 0.001). The mean mtDNA copy number ratio (mtDNA copy number of GAC/mtDNA copy number of paired NC-GM) was 0.891. A total of 35 (61.4%) GAC samples had an mtDNA copy number ratio ≤0.804 (p = 0.017) and 27 (47.4%) harbored a D310 mutation (p = 0.047), and these patients had shorter survival time and poorer prognosis. After effective knockdown of TFAM, TFAM-KD-B/C cells expressed higher levels of hexokinase II (HK-II) and v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT, but lower levels of phosphorylated pyruvate dehydrogenase (p-PDH) than did the NT/PT AGS cells. Except for a higher level of p-PDH, the expression levels of these proteins remained unchanged in TFAM-KD-A, which had a mild knockdown of TFAM. Compared to those of NT, TFAM-KD-C had not only a lower mtDNA copy number (p = 0.050), but also lower oxygen consumption rates (OCR), including basal respiration (OCR_BR), ATP-coupled respiration (OCR_ATP), reserve capacity (OCR_RC), and proton leak (OCR_PL)(all with p = 0.050). In contrast, TFAM-KD-C expressed a higher extracellular acidification rate (ECAR)/OCR_BR ratio (p = 0.050) and a faster wound healing migration at 6, 12, and 18 h, respectively (all with p = 0.050). Beyond a threshold, the decrease in mtDNA copy number, the mtDNA D310 mutation, and mitochondrial dysfunction were involved in the carcinogenesis and progression of GACs. Activation of PDH might be considered as compensation for the mitochondrial dysfunction in response to glucose metabolic reprogramming or to adjust mitochondrial plasticity in GAC.