| Mitohondrial Ca2+ participates in the control of both cellular life and death, however, a mechanistic understanding of how mitohondrial Ca2+ homeostasis remodeled and its functional roles remains greatly limited in cancers. Here we demonstrated for the first time that mitohondrial Ca2+ uniporter regulator MCUR1 was frequently upregulated in HCC cells to enhance the mitohondrial Ca2+ uptake, which significantly facilitated cell survival by promoting cell proliferation and inhibiting mitohondria-dependent intrinsic apoptosis, and thus contributed to poor prognosis of HCC patients. Mitohondrial Ca2+ homeostasis is the key factor in the regulation of reactive oxygen species ROS, which as an important second messenger, can activate tumor cell proliferation, apoptosis, metastasis and angiogenesis of tumor cells signaling pathways, eventually leading to malignancy. We also found that survival advantage conferred by MCUR1-mediated mitohondrial Ca2+ uptake was majorly caused by elevated mitohondrial ROS production and subsequent AKT/MDM2- mediated P53 degradation. Our study provides evidence supporting a possible tumor-promoting role for MCUR1-mediated mitohondrial Ca2+ uptake and uncovers a mechanism that links remodeling of mitohondrial Ca2+ homeostasis to cancer cell survival. 【Objectives】The study topic to human hepatocellular carcinoma as the research object and aimed to the key mitochondrial Ca2+ uptake regulatory molecule MCUR1. To figrue out whether the mitochondrial calcium homeostasis remodeling mediated by MCUR1 is involved in regulation of the growth of HCC or not, and to explore its mechanisms of MCUR1 mediated mitochondrial calcium homeostasis remodeling regulation on the growth of HCC. 【Methods】1.Immunohistochemistry exams the expression and correlation of MCUR1 and P53 in patients with primary liver cancer of clinical specimens.2.The MTS assay, Colony forming assay, Annexin-V apoptosis detection, Flow cytometry cell cycle assay, EdU incorporation evaluate the effect of MCUR1 on hepatocellular carcinoma cell proliferation, apoptosis and growth.3. Nude Mice Xenograft Model evaluates the effect of MCUR1 on the growth of hepatocellular carcinoma in vivo.4. Rhod-2 and Fluo-4 were used to detect mitochondrial and cellular Ca2+, to analyze the effects of MCUR1 on the level of mitochondrial calcium and the capability of mitochondrial Ca2+ uptake in HCC cells.5. The DCFH-DA and MitoSOX were used to detect cellular and mitochondrial ROS.6. Western Blot analysis the expression of the apoptosis related molecules and proliferation related molecules, to explore its mechanisms of MCUR1 mediated mitochondrial calcium homeostasis remodeling to promote the growth of hepatocellular carcinoma 【Results】Part one: Through the analysing the primary hepatocellular carcinoma clinical specimens found that MCUR1 was frequently up-regulated at both mRNA and protein levels in HCC tissues when compared with paired non-tumor tissues, and HCC patients with higher expression of MCUR1 had significantly poorer overall survival(OS) and recurrence-free survival(RFS). The MTS assay, Colony forming assay, Annexin-V apoptosis detection, Flow cytometry cell cycle assay, EdU incorporation assay found that MCUR1 promotes cell survival in vitro by inhibiting apoptosis and accelerating cell proliferation of HCC Cells. Nude Mice Xenograft Model confirme the functional role of MCUR1 in promoting HCC growth by inducing cell proliferation and inhibiting cell apoptosis.Part two: Rhod-2, Fluo-4 cell staining results found MCUR1 can promote mitochondrial calcium uptake, and enhance the capability of mitochondrial Ca2+ uptake under the state of emergency. Through altering MCU expression, indicated that MCUR1 plays a critical regulatory role in MCU-dependent mitochondrial Ca2+ uptake in HCC cells.Part three: Detecting the cellular ROS and mitochondrial ROS, we found that MCUR1 expression considerably affects the production of ROS in HCC cells. Use the specific scavenger of mitochondrial superoxide MitoTEMPO, and the specific scavenger of mitochondrial calcium PV-Mito-GFP, We found MCUR1-mediated mitochondrial Ca2+ uptake promotes cell growth by increasing ROS production.Part four: Through detecting the expression of the apoptosis related molecules, proliferation related molecules, P53, Akt and MDM2 phosphorylation, we confirmed that MCUR1 mediated mitochondrial calcium homeostasis remodeling promote the growth of HCC through the ROS/AKT/MDM2 pathway to make P53 inactivation. 【Conclusions】In summary, our data demonstrate that MCUR1-mediated mitochondrial Ca2+ uptake plays a critical role in regulation of HCC cell survival through increased mitochondrial ROS production which mediated AKT/MDM2 activation and subsequent regulation of P53 degradation pathway. All of these findings suggest that remodeling of mitochondrial Ca2+ homeostasis is a remarkable cause of malignant transformation in HCC cells. |
