The Research Of Molecular Mechanism Of Hepatocellular Carcinoma Cells Appotosis Induced By Mitofusin-2

At present, liver cancer is one of common malignant tumor and the second cause of death of malignant tumors in our country. Liver cancer was mostly detected at middle-late stage for early HCC with no specific symptoms and signs. Besides the hepatectomy, there are still lacks of other effective treatments for liver cancer. The developing trends and hot issues of treatments for HCC focus on intervening the activation and proliferation of tumor cells. Cancer formation is associated with uncontrolled cell hyperplasia. To develop and maintain tissue homeostasis in multicellular organisms, apoptosis plays a key role during such pathological hyperplasia.Hyperplasia suppressor gene (HSG), a novel gene associated with hypertension, was also called mitofusin2(Mfn2) for its relation to mitochondrion fusion. Mfn2had been confirmed to suppress cells proliferation mediated by inhibiting the activation of Ras-Erk passageway, up-regulating P27and P21Cipl, down-regulating PCNA and arresting the cells at G1/G0phase, which implied that Mfn2was closely related to tumors development.Part I Mfn2induces HCC cells apoptosis via the mitochondrial apoptotic pathway. Aims:Mfn2is a novel gene which remarkably suppresses the injury-mediated proliferation of vascular smooth muscle cells (VSMCs) and has a potential apoptotic effect. We aim to evaluate whether Mfn2can induce HCC cells apoptosis in vitro and investigate molecular mechanism of apoptosis.Methods:The Mfn2expression of tumor tissues and adjacent normal tissues in mRNA and protein levels were analyzed by QRT-PCR and immunohistochemistry, irrespectively. The overexpression Mfn2cell model was established by exogenously adding recombinant Mfn2protein to HepG2and Huh7cell lines. The impact of Mfn2on cells apoptosis, mitochondrial membrane potential (△Ψm) and intracellular reactive oxygen species (ROS) was analysed by using flow cytometry. Western blot was used to detect the release of cytochrome c, Bax mitochondrial translocation and the cleaved form of caspase3.Results:QRT-PCR and immunohistochemistry showed downregulation of Mfn2expression at the mRNA and protein levels in tumour tissues, compared withadjacent non-cancer tissues. Furthermore, according to immunohistochemistry results, Mfn2immunostaining was very weak in HCC tissue (P<0.05) and was significantly associated with tumour size and TNM stage (P=0.038and0.040, respectively), and Kaplan-Meier survival curves showed that patients with HCC with lower Mfn2expression had a poorer prognosis. Overexpression of Mfn2in HepG2and Huh7cells induced apoptosis, reduced the mitochondrial membrane potential (△Ψm) and elevated intracellular reactive oxygen species (ROS). Conclusion:Mfn2was low expression in HCC tissue and was significantly associated with tumour size, TNM stage and patient’s prognosis. We confirmed that Mfn2induced HCC cells apoptosis via mitochondrial apoptotic pathway. Part Ⅱ Mitofusin-2triggers mitochondria Ca2+influx from the endoplasmic reticulum to induce apoptosis in hepatocellular carcinoma cellsAims:In previous studies, we confirmed that mitofusin-2(Mfn2) induced apoptosis in hepatocellular carcinoma (HCC) cells. Mfn2, anchored on the outer mitochondrial membrane, participates in mitochondrial fusion and contributes to the maintenance of the mitochondrial network. Moreover, Mfn2builds a bridge between endoplasmic reticulum and mitochondrion. We aim to investigate the molecular mechanism of apoptosis of the HCC cells induced by Mfn2via analysis Ca2+flow direction in ER and mitochondrion.Methods:The overexpression Mfn2cell model was established by exogenously adding recombinant Mfn2protein to HepG2cell line. We quantified the ER and mitochondrial Ca2+concentrations by using calcium ion tracer. Moreover when HepG2cells were overexpressed with Mfn2, the drugs of heparin and RU360were used to figure out the Ca2+flow direction in ER and mitochondrion, and cell apoptosis, mitochondrial membrane potential (△Ψm), intracellular reactive oxygen species (ROS), the release of cytochrome c and mitochondrial permeability transition pore (MPTP) permeability were evaluated.Results:Overexpression of Mfn2induced HepG2cells apoptosis, reduced the mitochondrial membrane potential (△Ψm) and endoplasmic reticulum (ER) calcium ion (Ca2+) concentrations, and elevated intracellular reactive oxygen species (ROS) and mitochondrial Ca2+concentrations. However, when HepG2cells overexpressing Mfn2were treated with both heparin, which inhibits inositol1,4,5-trisphosphate (InsP3)-activated Ca2+channel release from the ER, and RU360, which specifically blocks Ca2+uptake into the mitochondria, there was no induction of apoptosis, decline in△Ψm or ER Ca2+, or increase in intracellular ROS or mitochondrial Ca2+. We also found downregulation in the expression of mitochondrial calcium uptakel and2(MICU1and MICU2),’gatekeepers’ of mitochondrial calcium influx, in cells transfected with Adv-Mfn2.Conclusion:We confirmed that Mfn2induced apoptosis in HCC cells by triggering influx of Ca2+into the mitochondria from the ER, and we found that the increased accumulation of mitochondrial Ca2+was caused by downregulation of MICUs regulated by Mfn2overexpression.