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Mitochondria is one of the important organelles, which is composed of outer mitochondrial membrane and inner mitochondrial membrane. Mitochondrial structure and function are regulated by mitochondrial dynamics. Mitochondrial fusion- and fission-related proteins may participate in the process of mitochondrial fusion and fission, mediate mitochondrial dynamics, thereby regulating cell structure, function and energy metabolism. Mitofusin (MFN) 2, a protein located on the outer mitochondrial membrane of mammalian, has guanosine triphosphatase activity, which may mediate mitochondrial fusion, participate in mitophagy, formation of mitochondria-associated endoplasmic reticulum membrane and apoptosis, and significantly affect the incidence and development of ischemia-reperfusion injury (IRI). In this article, the structure, regulation, function of MFN2 and its role in IRI were reviewed, and the relationship between MFN2 and IRI and underlying mechanism were investigated, aiming to provide novel targets and ideas for the prevention and treatment of IRI.
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Objective:To explore the mechanism of energy changes in the three stages of the formation of coronary heart disease due to blood stasis in rat model from the perspective of mitochondrial fusion-fission dynamic changes. Method:Thirty healthy male rats were divided into the blank control group （<italic>n</italic>=6） and model group （<italic>n</italic>=24） using SPSS 21.0 simple random sampling method. The rats in the blank control group were fed an ordinary diet， while those in the model group a high-fat diet. After seven days of adaptive feeding， the rats were treated with intragastric administration of vitamin D<sub>3</sub> （VitD<sub>3</sub>） at 300 000 U·kg<sup>-1</sup> and then at 200 000 U·kg<sup>-1</sup> 14 d later. The high-fat diet continued for 21 d， and six rats were randomly selected as samples for the pre-stage blood stasis syndrome group， followed by model verification and sampling. The remaining rats continued to receive the high-fat diet for 30 d， and six were randomly selected and categorized into the sub-stage blood stasis syndrome group， followed by model verification and sampling. The rest of rats were classified into the heart blood stasis syndrome group. While continuing the high-fat diet， they were also treated with multipoint subcutaneous injection of isoproterenol （ISO，5 mg·kg<sup>-1</sup>） for three consecutive days. One week later， the electrocardiogram （ECG） was recorded for determining whether the modeling was successful and the samples were taken at the same time. The changes in mitochondrial morphology and quantity were observed under a transmission electron microscope. The expression of mitochondrial dynamics-related proteins was measured by Western blot and the cellular localization of related proteins by immunofluorescence assay. Result:The levels of total cholesterol and low-density lipoprotein cholesterol in the pre-stage and sub-stage blood stasis syndrome groups were significantly increased as compared with those in the blank control group （<italic>P</italic><0.05）. The blood rheology index in the pre-stage blood stasis syndrome group was significantly elevated in contrast to that in the blank control group （<italic>P</italic><0.05）. The three-layered membrane of the aorta in the blank group was intact. However， the tunica media of the pre-stage blood stasis syndrome group began to show obvious calcification， with a small number of inflammatory cells adhering to the intima. The subintima and media smooth muscles in the sub-stage blood stasis syndrome group exhibited cavity structures. The three-layered structure of the arterial wall in the heart blood stasis syndrome group was severely damaged. The ECG of the blank control group revealed the regular appearance of P wave，regular QRS waveform （no broadening or deformity）， and no obvious ST-segment depression or elevation. The ECG of the pre-stage blood stasis syndrome group showed no obvious abnormalities as compared with that of the blank control group. In the sub-stage blood stasis syndrome group， the ECG showed an upward trend of the J point and slight ST-segment elevation， with the elevation≤0.1 mV. The ECG in the heart blood stasis syndrome group displayed significant ST-segment depression （>0.1 mV） and J point depression >0.1 mV. The mitochondria in the blank control group were normal in size and morphology， with clear and dense cristae， whereas those in the pre-stage blood stasis syndrome group were fusiform with sparse cristae. Some mitochondria in the sub-stage blood stasis syndrome group were significantly elongated， and even vacuole-like changes were present. In the heart blood stasis syndrome group， the mitochondria were ruptured. As demonstrated by comparison with the blank control group， the expression levels of mitofusin 2 （Mfn2）， dynamin-related protein 1 （Drp1）， and fission protein 1 （Fis1） in the model group were significantly up-regulated （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the pre-stage blood stasis syndrome group， the heart blood stasis syndrome group exhibited down-regulated Mfn2 （<italic>P<</italic>0.05）. Compared with the blank control group and the pre-stage blood stasis syndrome group， the sub-stage blood stasis syndrome group and the heart blood stasis syndrome group displayed down-regulated optic atrophy 1（OPA1） （<italic>P</italic><0.05，<italic>P</italic><0.01）. The Drp1 and Fis1 protein expression declined significantly in the sub-stage blood stasis syndrome group in comparison with that in the pre-stage blood stasis syndrome group （<italic>P</italic><0.05，<italic>P</italic><0.01）. The expression levels of Mfn2 and Drp1 in the heart blood stasis syndrome group were lower than those in the sub-stage blood stasis syndrome group （<italic>P<</italic>0.01）. The comparison with the blank control group showed that Mfn2 and OPA1 were extensively accumulated in mitochondria of both the pre-stage and sub-stage blood stasis syndrome groups， while the red-stained Mfn2 was significantly reduced in the heart blood stasis syndrome group. The Drp1/Fis1 fluorescence was weak in the blank group and the pre-stage blood stasis syndrome group but strong in the sub-stage blood stasis syndrome group and heart blood stasis syndrome group. Conclusion:The cardiomyocyte mitochondria dynamics changes with the change in energy demand of cardiomyocytes. Mfn2 is dominated by fusion effect in the early stage of the formation of coronary heart disease due to blood stasis. With the gradual development of this disease， Mfn2 begins to mediate mitochondrial autophagy. OPA1 plays a role in intimal fusion and cristae integrity. The decreased OPA1 expression is closely related to the accelerated progression of coronary heart disease differentiated into blood stasis syndrome. The process by which Drp1 and Fis1 separate damaged mitochondria to prepare for mitochondrial autophagy contributes to alleviating the imbalance between the energy demand and supply of human body.
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Objective To investigate the protective effect and mechanism of curcumin on acute lung injury in septic mice.Methods Totally 120 clean BALB/c male mice were randomly (random number) divided into 8 groups:sham group,sepsis group,curcumin control group,curcumin intervention group,negative virus-sepsis group,negative virus-curcumin intervention group,Mfn2 interference-sepsis group,and Mfn2 interference-curcumin intervention group,15 rats in each group.Mice in the sepsis and the curcumin groups were given the cecal ligation and puncture (CLP) mice in the curcumin intervention and curcumin control groups were given curcumin 200 mg/(kg·d) for 1 week,and mice in the negative virus-sepsis group and negative virus-curcumin intervention groups were established by injection of a negative adeno-associated virus in the tail vein.The Mfn2 interference-sepsis and Mfn2 interference-curcumin intervention groups were established by injecting an adeno-associated virus carrying the Mfn2 interference sequence through the tail vein.Mice were sacrificed after 24 h in each group.The degree of lung injury was examined by lung wet-to-dry weight ratio and pathological examination.The inflammatory factors of alveolar lavage fluid including TNF-α and IL-6 were detected by ELISA,the activation of caspase-3,a key molecule for apoptosis,was detected by Western blot,and apoptosis was detected by TUNEL.The data were analyzed by SPSS 22.0 software,the count data was analyzed by x2 test,and the comparison of measurement data between groups was analyzed by one-way ANOVA.Results Compared with the sham group,the wet-to-dry weight ratio of lung tissue in the sepsis group was significantly increased (71.11 ± 3.78 vs 31.11 ± 5.61,P=0.002),the histopathological score was significantly higher (P=0.006),the inflammatory factors TNF-α (P=0.001) and IL-6 (P=0.012) were dramatically increased,and the apoptosis of lung tissue and the expression of caspase-3 cleaved were also significantly increased (P=0.001).Compared with the sepsis group,the wet-to-dry weight ratio and the histopathological score of lung tissue in the curcumin-treated group was significantly lower (32.84 ± 6.15 vs 71.11 ± 3.78,P=0.004),and the inflammatory factors TNF-α(P=0.013) and IL-6 (P=0.003) were obviously decreased,and apoptosis and apoptosis-related protein caspase-3 cleaved expression were also dramatically decreased (P=0.012).After Mfn2 was down-regulated,Mfn2 interference-curcumin intervention group interfered with Mfn2.Compared with the sepsis group,the dry-to-wet weight ratio and the histopathological score of the lung tissue of the mice was not significantly decreased.Further studies found that after down-regulating Mfn2,compared with the Mfn2 interfere-sepsis group,Mfn2 interfere-curcumin intervention group had no such performance.The inflammatory factors TNF-α and IL-6 were not significantly decreased,and the apoptosis of lung tissue and the expression of apoptosis-related protein caspase-3 cleaved were not significantly reduced.Conclusion Curcumin may attenuate acute lung injury in sepsis by up-regulating the expression of Mfn2.
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Mitochondria homeostasis is essential to cell activities,while mitochondrial dysfunction is involved in the pathogenesis of many muscular diseases,such as congenital myopathy and muscular dystrophy.However,its detailed mechanism has not been clarified.Mitofusin-2 (Mfn2) is located in the outer mitochondrial membranes and endoplasmic reticulum(ER) mitochondrial contact sites.Recently,researchers have found that in addition to its participation in tethering mitochondiia to mitochondria and ER to mitochondria,Mfn2 regulates mitochondrial dynamics and multiple cellular processes including oxidative stress,Ca2 + signaling pathways,energy metabolism,and PINK1/Parkin induced mitophagy,playing an important role in maintaining the mitochondrial quality control.And abnormal expression of Mfn2 also related to many kinds of pathological states.Here we review its molecular structure and recent advances in the role of Mfn2 in maintaining mitochondrial homeostasis in skeletal muscle.
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Objective To investigate the effects of ghrelin on proliferation of vascular smooth muscle cells(VSMC)and the expression of mitochondrial fusion 2(Mfn-2)in cultured human aortic smooth muscle cells(HASMCs).Methods HASMCs were cultured in vitro,treated with different concentrations(10-9,10-8,10-7,10-6,10-5 mol/L)ghrelin or 10-6 mol/L ghrelin for different time(0,6,12,18,24 h).Subconfluent HASMCs at passage 4-6 were used in experiments.MTT essay was used to investigate the effect on proliferation of HASMCs.RT-PCR and Western blot were used to analyse the expression of Mfn-2.Results 10-7-10-5 mol/L ghrelin inhibited the proliferation of HASMCs,and the inhibitory effect of concentration of 10-6 mol/L was the most obvious(P<0.01).Ghrelin inhibited the proliferation of HASMCs in 6-24 h,and it reached the peak at 24 h(P<0.01).10-6 mol/L ghrelin significantly increased the expression of Mfn-2 mRNA and protein(P<0.01).The up-regulation of 10-6 mol/L ghrelin on Mfn-2 mRNA and protein expression reached the peak at 18 h(P<0.01).Conclusion Ghrelin might inhibit the proliferation of HASMC by up-regulating the expression of Mfn-2.
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Objective: To investigate the dynamic changes of mitofusin-2 (Mfn2) expression in rat cerebral arteries after experimental subarachnoid hemorrhage (SAH) and to reveal the relationship between Mfn2 and cerebral vasospasm (CVS). Methods: A SAH model was induced by endovascular perforation of the intracranial portion of the internal carotid artery. One hundred and forty six male SD rats were randomly divided into six groups; sham group and SAH groups which were sacrificed at different time points (24 hours, 48 hours, 72 hours, 7days and 14days). Mortality, neurobehavioral score and brain water content were measured. Histology was conducted to observe the morphological changes. Western blotting and RT-PCR were performed to measure the Mfn2 protein and mRNA changes of the major cerebral arteries at different time points after SAH. Results: Blood clot surrounded the basilar artery gradually dissipated after SAH. HE staining showed that the most severe morphological vasospasm in basilar arteries was observed at the 24th hour after SAH. No positive immunohistochemical staining of Mfn2 was shown in the media layer of basilar artery at the 7th day after SAH. Western blotting showed that Mfn2 protein was remarkably increased at the 48th hour and the 72th hour after SAH compared to sham groups (P < 0. 05) and significantly decreased at the 7th day after SAH (p < 0. 05). The protein level at the 14th day after SAH was almost the same level with the sham and SAH 24 hours groups. The mRNA level changed in the same tendency as the protein level. Conclusion: This study indicate that Mfn2 plays essential roles in both acute and delayed CVS which may provide a theoretical basis for understanding of the mechanism of the CVS after SAH.
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Objective To investigate the inhibitory effect of testosterone on oxidized low-density lipoproteins ( ox-LDL)-stimulated phenotypic transition and proliferation of vascular smooth muscle cells ( VSMCs) in vitro, and to explore its possible mechanisms. Methods Rat VSMCs cultured in vitro were divided into control group, ox-LDL group(50μg/mlox-LDL),fetalbovineserum(FBS)group(10% FBS),andtestosteronegroups(5×10-8 or5×10-7 mol/L testosterone plus 50μg/ml ox-LDL) . The effect of testosterone on ox-LDL-induced proliferation of VSMCs was explored by WST-1 assay. The cell cycle distribution was determined using flow cytometry. Western blotting was used todetecttheexpressionsofmitofusin2(Mfn2),phosphorylatedextracellularsignal-regulatedkinases1/2(p-ERK1/2) , proliferating cell nuclear antigen ( PCNA) ,α-smooth muscle actin (α-SMA) ,and osteopontin ( OPN) . Results Compared with control group, the proliferation of VSMCs was promoted by ox-LDL, the number of VSMCs decreased in G0/G1 phase and increased in S phase significantly, the expression levels of Mfn2 and α-SMA were significantly reduced, and the expression levels of p-ERK1/2, PCNA, and OPN were significantly raised in ox-LDL group. Compared with ox-LDL group, the proliferation of VSMCs was inhibited, the number of VSMCs increased in G0/G1 phase and decreased in S phase in two testosterone groups, along with the increased expressions of Mfn2 andα-SMA, and the descended expressions of p-ERK1/2, PCNA, and OPN. Conclusions Testosterone inhibits phenotypic transition and proliferation of VSMCs induced by ox-LDL in vitro, which may be related to the up-regulated expression of Mfn 2 and the suppression of ERK1/2 pathway.
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Objective To investigate the effect and underlying mechanism of nocodazole on the inhibition of rVSMCs proliferation. Methods rVSMCs were divided into four groups, group A (normal culture), group B (serum-free culture for 24 h) , group C ( 18 h normal culture after 48 h of serum-free culture ) , and group D ( nocodazole treatment for 12 h after thymidine treatment for 12 h) . Flow cytometry, transmission electron microscopy, and metabolism measurements were performed and mitofusin-2 ( Mfn-2 ) expression was detected. Results Flow cytometry analysis showed rVSMCs of group B、C、D were arrested to G0/G1 , S and G2/M phases, respectively. Less and smaller mitochondria were observed in group D by transmission electron microscopy in nocodazole-treated rVSMCs. Compared with groups A and C, there were significant decreases in glucose and L-amino acid metabolism, levels of ATP, and marked increase in NADH in group D(P<0. 05). Western Blot showed that G2/M cell cycle arrest and nocodazole could induce up-regulation of Mfn-2 in rVSMCs(P<0. 05). Conclusion Nocodazole can block the energy metabolism and proliferation in rVSMCs, which is probably associated with the role of Mfn-2 on anti-atherosclerosis.
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PURPOSE: Charcot-Marie-Tooth disease type 2A (CMT2A) is caused by mutations in the mitofusin 2 (MFN2) genes associated with variable central nervous system (CNS) involvement. The authors report a case of a middle-aged woman with genetically confirmed CMT type 2 (CMT2), combined with delayed-onset bilateral optic neuropathy. CASE SUMMARY: A 47-year-old woman presented with complaints of subacute decrease of visual acuity in both eyes. Her corrected visual acuity was 20/200 in the right eye and 20/320 in the left eye. Fundus photographs revealed bilateral disc pallor and diffuse retinal nerve fiber layer defects. No papillomacular bundle defect was observed. Goldmann perimetry showed central scotoma in both eyes. She had suffered from muscle wasting of the legs and foot deformities such as high arches and hammer toes since childhood and required a wheelchair for ambulation. A series of CMT gene mutation tests revealed an MFN2 gene mutation, c.617C>T (p.Thr206Ile), and the patient was diagnosed with CMT2A. CONCLUSIONS: Charcot-Marie-Tooth disease is a common inherited neuromuscular disorder and CMT2A, an axonal CMT neuropathy, is associated with bilateral optic neuropathy. Therefore, suspecting CMT and testing for gene mutations as part of the work-up in patients with subacute bilateral optic neuropathy associated with peripheral neuropathy is critical.
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Female , Humans , Middle Aged , Axons , Central Nervous System , Charcot-Marie-Tooth Disease , Foot Deformities , Hammer Toe Syndrome , Leg , Nerve Fibers , Optic Nerve Diseases , Pallor , Peripheral Nervous System Diseases , Retinaldehyde , Scotoma , Visual Acuity , Visual Field Tests , Walking , WheelchairsABSTRACT
PURPOSE: Mitofusin2 gene (Mfn2, also named Hyperplasia suppressive gene, HSG) is very important in the origin and development of hypertension. However, the mechanism of Mfn2/HSG expression regulation was not uncovered. This study was designed to explore the association of a novel 5'-uncoding region (UCR) -1248 A>G variation of HSG/Mfn2 gene and hypertension. MATERIALS AND METHODS: 472 healthy, normotensive subjects [normotension (NT) group], 454 prehypertensive subjects [prehypertension (PH) group] and 978 hypertensive patients [essential hypertension (EH) group] were screened for an association study between 5'-UCR -1248 A>G of Mfn2/HSG and hypertension by polymerase chain reaction and DNA sequencing after venous blood was drawn and DNA was extracted. RESULTS: When comparing the A and G frequency in EH, PH and NT groups, in total, NT group significantly had higher A frequency than in PH group [odds ratio (OR)=1.605, confidence interval (CI) 95%=1.063-2.242, p=0.025] and EH group (OR=5.395, CI 95%=3.783-7.695, pG variation was significantly related with blood pressure level (B=-1.271, Wald=40.914, CI 95%=-1.660 - -0.881, pG variation of Mfn2/HSG gene was a novel variation and may be associated with hypertension in Chinese.
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Female , Humans , Male , China , GTP Phosphohydrolases/genetics , Gene Expression Regulation , Genetic Association Studies , Genotype , Hypertension/genetics , Logistic Models , Mitochondrial Proteins/genetics , Polymorphism, Single Nucleotide , Sequence Analysis, DNAABSTRACT
Angiotensin Ⅱ (ANG Ⅱ) plays an important role in the pathogenesis of atherosclerosis by inducing proliferation of vascular smooth muscle cells (VSMCs).In our study,we observed the effects of valsartan on proliferation of cultured VSMCs treated with or without ANG Ⅱ by cell count ing and methyl thiazolyl tetrazolium (MTT) assay,and detected the expression of mitofusin 2 (Mfn2),a newly discovered cell proliferation inhibitor and a related cell proliferation signaling pathway protein by Western blotting.ANGⅡ at a concentration of 10-6 mol/L significantly stimulated VSMCs proliferation,down-regulated the expression of Mfn2 and up-regulated the expression of Raf and ERK1/2.Valsartan inhibited such effects of ANG Ⅱ at concentrations of 10-5 and 10-6 mol/L,but not at 10-7 mol/L.Valsartan had no significant effect on the proliferation of untreated VSMCs.These results suggest that valsartan inhibits ANGⅡ-induced proliferation of VSMCs in vitro via Mfn2-Ras-Raf-ERK/MAPK signaling pathway.
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Objective To study the effects of protein kinase A (PKA) phosphorylation site' s mutation of mitofusin-2 (Mfn2) on intimal proliferation after balloon injury of carotid artery of rats. Method Rat model of carotid artery balloon injury was established and infected with Adv-LacZ, Adv-Mfn2, AdvMfn2-S442A or Adv-Mfn2-S442D from the peri-arterial sheathes of vessels, while phosphate buffered solution (PBS) used instead of above infectious adenovirus as uninfected group and sham operation as control group. The rats were sacrificed 14 days after balloon injury of carotid artery in order to measure the level of Mfn2 protein and the prol9iferation cell nuclear antigen (PCNA) with immunohistochemistry staining. The morphology of vessels was observed with HE staining. All data were statistically analyzed with ANOVA and Dunnett-t test. Results Fourteen days after surgery, the levels of Mfn2 protein significantly increased in arteries infected with Adv-Mfn2, Adv-Mfn2-S4442A and Adv-Mfn2-S442D compared with those in control group, sham operation group and Adv-LacZ infected group. The ratio of intimal area/medial area (I/M) and percentage of PCNA positive cells in both Adv-Mfn2 and Adv-Mfn2-S442A groups markedly decreased compared with control group (P ＜0. 01 ) . Compared with the Adv-Mfn2 group, the I/M and the percentage of PCNA positive cells reduced more significantly in Adv-S442A group (P ＜ 0. 01 ) . However,the I/M and the percentage of PCNA positive cells in Adv-LacZ and Adv-S442D groups were not significantly different from those found in the control group. Conclusions The over-expression of Mfn2 gene may effectively inhibit intimal proliferation after balloon injury of carotid artery of rats. The inhibitory effects of Mfn2-S442A are more obvious than those of Mfn2. However, the Mfn2-S442D is out of the inhibitory effect on neo-intimal proliferation.
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Objective To study the effects of RNA interference(RNAi)-mediated silencing of mitofusin-2 (Mfn2) gene on glycornetabolism and insulin resistance in BALB/c mice. Methods Mfn2 short hairpin RNA (shRNA) and negative control green fluorescent protein(GFP) -expressed plasmid vectors were constructed. 44 mice were randomly divided into transfection group (Mfn2) and negative control group (HK). 1.5 ml GFP-expressed plasmid(negative control or Mfn2 shRNA,75 μg for each mouse)was injected into the mice in 5 seconds through vena caudalis. Five days later, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test(IPITT)were performed to evaluate glycometabolism and insulin sensitivity. D-3-[3~H]-glucose in PBS buffer were injected via the tail vein. Blood samples were taken at specific time points. Radioactivity was measured in all samples with liquid scintillation counter. The rates of hepatic glucose production in vivo were calculated. Mfn2 protein expression in hepatic tissue was detected by Western blot. Results Compared with HK mice, the Mfn2 expressions of Mfn2 mice decreased markedly(8.05±0.15 vs 8.56±0.01 ,P<0.05). The fasted blood glucose leves [(6.95±0. 83 vs 4.68±0. 29) mmol/L,P<0. 05] in Mfn2 mice were higher than those in HK mice. The insulin sensitivity of Mfn2 mice decreased markedly compared with HK mice. The rate of hepatic glucose production was significantly elevated in Mfn2 mice [(49.53±16.31)μmol·kg~(-1)·min~(-1)],compared with negative control mice[(24.91±4.07)μmol·kg~(-1)·min~(-1),P<0.05].Conclusion The down-regulatd expression of Mfn2 induces glycometabolic disorder and insulin resistance in BALB/c mice. Mfn2 plays an important role in maintaining glucose homeostasis in vivo.
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Objective To investigate the effect of tMfn2 gene on inhibiting the proliferation of vascular smooth muscle cells (VSMCs) and related mechanism. Method VSMCs were transfected with adenovirus vector encoding tMfn2 or Mfn2 (Adv-tMfn2, Adv-Mfn2). The abundance of tMfn2 protein and Mfn2 protein were deter-mined by Western blot analysis using Mfn2 N-term antibody. The effect of tMfn2 on the proliferation of VSMCs was explored by cell counting and MTT assay. The cell cycle was analyzed using flow cytometry. Western blot were used to detect the expression of p-ERK1/2 and p-Raf-1. Results The results of cell counting and MTT both indi-cated that tMfn2 gene displayed more remarkable effect on inhibiting the proliferation of VSMCs than Mfn2 (P <0.01). Flow cytometry showed that most of the cells infected with Adv-tMfn2 or Adv-Mfn2 were blocked in the stage of G0/G1 and few entered into the S phase. Western blot indicated that overexpression of tMfn2 gene resulted in downregulation of phosphorylated ERK1/2 and Raf-1 protein (P < 0.01). These results demonstrated tMfn2 had stronger effect than Mfn2 (P < 0.01). Conclusions tMfn2 gene is superior to Mfn2 gene in attenuating the proliferation of VSMCs via the Ras-Raf-ERK1/2 signaling pathway.
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Mitofusin-2 (Mfn2) gene expression is positively correlated with insulin sensitivity in patients with type 2 diabetes. However,it is unclear if Mfn2 is involved in carbohydrate metabolism and lipid homeostasis. In order to investigate the specific functions of Mfn2 in glycometabolism and lipid homeostasis in BALB/c mice,a RNA interference technique-mediated hydrodynamic injection was developed,in which short hairpin RNAs (shRNAs) were used to inhibit the Mfn2 expression in vivo. Seventy-two mice were randomly divided into two groups:the Mfn2 reduction group (Mfn2/shRNA) and the negative control group (NC). Intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests were used to evaluate glycometabolism and insulin sensitivity.D-(3-3H) glucose or 3H2O was injected into the tail vein or intraperitoneally to facilitate the calculation of the rate of hepatic glucose production and fatty acid synthesis in vivo. The results showed that,in Mfn2/shRNA mice,the liver Mfn2 protein was significantly decreased,and fasting blood glucose concentrations were increased by approximately 48%,when compared with the NC mice. In parallel with the changes in fasting glucose levels,hepatic glucose production was significantly elevated in Mfn2/shRNA mice. When insulin was administrated,these mice exhibited impaired insulin tolerance.It was also found that the reduction of Mfn2 markedly decreased the rate of fatty acid synthesis in the liver,and the Mfn2/shRNA mice exhibited hypertriglyceridema. Taken together,our results indicate that Mfn2 plays an important role in maintaining glucose and lipid homeostasis,and in the development of insulin resistance in vivo.
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Objective:To investigate the expressions of Mfn2(mitofusin 2) in non-small-cell lung cancer (NSCLC) tissues and non-cancerous lung tissues,and analyze its relationship with clinicopathological characteristics of lung carcinomas.Methods:The expressions of Mfn2 mRNA and protein in 92 cases of NSCLC tissues and 27 cases of non-cancerous lung tissues were detected by in situ hybridization and immunohistochemical methods. Results:The positive rates of Mfn2 mRNA and protein in pulmonary squamous cell carcinoma were higher than those in adenocarcinoma (83.33% and 89.58% vs 56.82% and 65.91%), respectively. The positive rates of Mfn2 mRNA and protein in NSCLC were higher than those in the non-cancerous lung tissues (25.93% and 29.63%) . The difference was statistically significant among the three groups (P0.05). The expression of Mfn2 mRNA was consistent with that of Mfn2 protein in NSCLC.Conclusion:Mfn2 was involved in the initiation and progression of lung cancer, and the expression of Mfn2 was related to the histological types of lung cancer and its differentiation degree.
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Objective To investigate the expression of hyperplasia suppressor gene(HSG)/mitofusin 2 (Mfn2)and P21 WAF1 in non-small cell lung cancer(NSCLC).Methods The expression of(HSG/Mfn2)and P21 WAF1 was detected in 92 cases of NSCLC samples by immunohistochemistry(SP).Results The absorptance value of the expression of HSG/Mfn2 in squamous cell carcinoma,adenocarcinoma,and non-cancer tissue were 15.06±2.73,12.21±2.96 and 10.36±3.60,respectively(P<0.05),and they were associated with tumor differentiation.The absorptanee valtue of the expression of P21 WAF1 in squamous cell carcinoma,adenocarcinoma,and non-cancer tissue were 3.16±0.98,3.44±0.22,0.06±0.32.The expression of P21 WAF1 in squamous cell carcinoma and adenocarcinoma Was higher than that in non-cancer tissue(P<0.05),and was closely associated with tumor differ-entiation and lymph node metastasis.Conclusion HSG/Mfn2,P21WAF1 is closely related to the pathogenesis and development of lung cancer.There is positive correlation between the HSG/Mfn2 and P2l WAF1 in lung cancer.
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In order to evaluate the effect of mitofusin-2 gene (mfn2) on proliferation and chemotherapy sensitivity of human breast carcinoma cell line MCF-7 in vitro, pEGFPmfn2 plasmid carrying full length of mitofusin-2 gene was transfected, by using sofast, into MCF-7 cells. Mitofusin-2 gene expression in MCF-7 cells transfected by sofast after 48 h was detected by PCR and Western blotting, and the stable expression of GFP protein in MCF-7 cells by Western blot analysis. The proliferation of MCF-7 cells was assayed by MTT and cell counting. By using PI method, the effects of mfn2 on the cell cycle distribution of MCF-7 were measured. Annexin-V/PI double labeling method was em- ployed to detect the changes in apoptosis induced by chemotherapeutics before and after transfection. The results showed that the MCF-7 cells transfected with mfn2 gene could stably and highly express GFP protein. MTT assay revealed that after transfection of mfn2 eDNA, the proliferation of MCF-7 cells was significantly inhibited. DNA histogram showed that cells arrested in S phase, and the per- centage of S phase cells was 42.7, 17.2 and 19.6 in mfn2 cDNA transfection group, blank plasmid transfection group and blank control group, respectively (P<0.05). The apoptosis ratio of the cells transfected with mfn2 gene was increased from 3.56% to 15.95%, that of the cells treated with camptothecin (CAMP) followed by mfn2 gene transfection was 69.6%, and that in blank plasmid transfection group and blank control group was 31.0% and 23.4% respectively (P<0.05). It was suggested that transfection of mfn2 gene could significantly inhibit the proliferation of MCF-7 cells and pro- mote their sensitivity to CAMP with a synergic effect.
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BACKGROUND: Mutations in mitofusin2 (MFN2) are a major underlying cause of axonal Charcot-Marie-Tooth neuropathy (CMT). It has been reported that patients with an early age of onset ( or =10 years, LO) in CMT2A with MFN2 mutations. There are few studies about CMT patients with MRI studies and we performed leg MRIs for better understanding of CMT2A. METHODS: We identified 19 patients (EO=10; LO=9) with MFN2 mutations. We used functional disability scales and CMT neuropathy scales for the grading of disability. Nerve conduction studies and MRIs of the lower leg were performed in all patients. RESULTS: We confirmed that EO had more severe leg muscle involvement than LO by leg MRI. In 7 out of 9 in LO, there were some degree of asymmetric leg muscle weakness and MRI findings explained the nature of asymmetry, that is, asymmetric cross-sectional areas or fatty infiltration. MRI of EO showed marked fatty infiltration on all three compartments whereas that of LO showed rather selective involvement of the posterior compartment. These results were well correlated with clinical findings that in LO, five patients could not do toe walking whereas only one could not do heel walking. CONCLUSIONS: MRI of the leg may be a useful tool for evaluating axonal CMT neuropathy, and asymmetric leg muscle weakness may be the characteristics of an axonal CMT. In addition, more prominent involvement of the posterior leg in LO is a very interesting phenomenon, which is in contrast to the length-dependent involvement in congenital demyelinating neuropathy.
Subject(s)
Humans , Age of Onset , Axons , Charcot-Marie-Tooth Disease , Heel , Leg , Magnetic Resonance Imaging , Muscle Weakness , Neural Conduction , Phenotype , Toes , Walking , Weights and MeasuresABSTRACT
BACKGROUND: Mitofusin 2 (MFN2) is a membrane protein and is an essential component of mitochondrial fusion machinery. Mitochondrial fusion is essential for various biological functions in mammalian cells. Thus mutations in MFN2 are the underlying cause of Charcot-Marie-Tooth neuropathy type 2A (CMT2A). However, there has been no reports investigating the MFN2 genes in Korean CMT patients. Therefore, we investigated to find the clinical and genetic characteristics in Korean patients with the MFN2 gene mutation. METHODS: We examined the mutations of the MFN2 gene in 137 Korean CMT families. According to criteria from the European CMT consortium, CMT2 was 45 families. Mutations were confirmed by both strands sequencing. Nerve conduction studies were carried out in CMT patients having each mutation. RESULTS: Eight pathogenic mutations were found in 10 families. Six mutations (Leu92Pro, Gly127Asp, His165Arg, Ser263Pro, Arg364Trp, Met376Thr) were determined to be novel, and those were not detected in the 100 healthy controls. A de novo missense mutation was found in three CMT families (30%). The frequency of the MFN2 mutation was 22.2%, which was higher than those found in the Cx32 mutation. In CMT2A, the frequencies with early age at onset (<10 years) and flat feet were 46.2%. CONCLUSIONS: We found MFN2 mutations in patients with sporadic or dominantly inherited CMT. In the majority of cases with CMT type 2, the axonal neuropathy, may be due to MFN2 mutations.