Mitochondrial Calcium Homeostasis Remodeling Facilitates Colorectal Cancer Growth Via Regulating Mitochondrial Biogenesis

BackgroundMitochondrial calcium homeostasis plays a key role in the pathophysiological process,including energy metabolism,apoptosis and reactive oxygen species(ROS)production.Moreover,mitochondrial calcium homeostasis plays an important role in malignant progression of cancers.However,the functional role of mitochondrial calcium homeostasis in the progression of colorectal cancer is unclear.Our previous study found that the expression level of mitochondrial calcium uniporter MCU is abnormally elevated in colorectal cancer tissues and is associated with poor prognosis in patients with colorectal cancer.Upregulation of MCU expression accelerates cell proliferation and promotes colorectal cancer cell growth.It has been reported that the dephosphorylation state of the mitochondrial transcription factor TFAM can stabilize TFAM protein to promote mitochondrial biogenesis.Based on this,we hypothesise that mitochondrial calcium homeostasis may regulate the phosphorylation state of TFAM via activating mitochondrial enzyme activity,leading to abnormal mitochondrial biogenesis and ultimately promoting malignant progression of colorectal cancer.AimsTo explore that the role and mechanism of MCU-mediated mitochondrial calcium homeostasis facilitates colorectal cancer growth.Methods1.Analysis of the correlation between MCU and the prognosis of patients with colorectal cancer.2.Analysis of the relationship between MCU and mitochondrial biogenesis in colorectal cancer tissues by immunohistochemistry and transmission electron microscopy.3.Analysis of the correlation between MCU and mitochondrial biogenesis using public database in colorectal cancer tissues.4.Using the calcium-binding protein mitopericam targeting the mitochondrial matrix to analyze the effect of MCU on mitochondrial basal calcium levels in colorectal cancer cells.5.Using q PCR,Western Blot,three-dimensional reconstruction,ATP Assay kit and transmission electron microscopy to analyze the role of MCU in mitochondrial biogenesis in colorectal cancer cells.6.Western Blot was used to detect the mitochondrial transcription factor TFAM protein level and its phosphorylation level,and the effect of MCU on mitochondrial transcription factor TFAM protein and its phosphorylation level.7.Using the enzyme activity assay kit to detect mitochondrial PDE2 A enzyme and PKA kinase activity,and analyze the mechanism of mitochondrial calcium promoting mitochondrial biogenesis.8.MTS assay,Ed U assay and Annexin V-FITC/PI apoptosis assay were used to detect the effects of MCU on the growth of colorectal cancer cell.9.Analysis of the effects of MCU on the growth of colorectal cancer cell through animal experiments.ResultsPart Ⅰ: Analysis of expression level of MCU in clinical specimens of colorectal cancer by immunohistochemistry,we found that the expression of MCU was upregulated in colorectal cancer,which was associated with poor prognosis in patients with colorectal cancer.Detecting cell growth by MTS assay and Ed U assay,we found that MCU promotes the proliferation of colorectal cancer cells.Part Ⅱ: Analysis by immunohistochemistry and transmission electron microscopy showed that the expression level of MCU was positively correlated with mitochondrial biogenesis.Through public database GEO analysis,it also showed that the expression level of MCU was positively correlated with mitochondrial biogenesis.Through transfecting calcium-binding protein mitopericam to detect mitochondrial calcium revealed MCU-mediated elevation of mitochondrial calcium.We ultilize q PCR assay,Western Blot,three-dimensional reconstruction assay and ATP assay kit to detect mt DNA copy number,mitochondrial oxidative phosphorylation core subunit protein levels,mitochondrial mass and mitochondrial ATP production,respectively.These results showed that MCU-mediated mitochondrial calcium promotes mitochondrial biogenesis.Part ⅢI: By detecting the mitochondrial transcription factor TFAM protein level and its phosphorylation level,we found that MCU increases the TFAM dephosphorylation level and TFAM protein level of colorectal cancer cells.By constitutively activating TFAM phosphorylation and inhibiting TFAM phosphorylation,we found that MCU-mediated elevation of mitochondrial calcium promotes mitochondrial biogenesis by increasing TFAM dephosphorylation levels.By detecting mitochondrial PDE2 A enzyme and PKA kinase activity,we found that MCU increases the mitochondrial transcription factor TFAM protein and its dephosphorylation level via activating PDE2 A enzyme activity.Part Ⅳ: Detecting cell proliferation and apotosis by MTS assay,Ed U assay and Annexin V-FITC/PI apoptosis assay,we found that MCU-mediated mitochondrial biogenesis promotes the growth of colorectal cancer cells through TFAM.By performing animal experiments confirmed that MCU regulated mitochondrial biogenesis to promote the growth of colorectal cancer.ConclusionsOur study found that MCU-mediated mitochondrial calcium uptake facilitates colorectal cancer growth via activating mitochondrial PDE2 A enzyme activity to increase dephosphorylation of mitochondrial transcription factor TFAM and regulate mitochondrial biogenesis.