BRD4 Inhibitor JQ1 Promotes Melanoma Cell Apoptosis By Regulating Mitochondrial Dynamics

Background: Cutaneous melanoma is a rare skin cancer in which tumors in millimeters are lethal.Compared with other types of skin cancers,cutaneous melanoma has the characteristics of high malignancy,prone to distant metastasis and poor clinical prognosis,which brings endless pain and economic burden to patients.At present,the commonly used treatment methods are mainly surgical resection,radiotherapy and chemotherapy are often used in advanced patients,but the survival rate is low.Therefore,there is an urgent need to find more diverse targets.Bromodomain extraterminal domain proteins inhibitors(BETi)are considered to be one of the promising therapies for melanoma[15].JQ1 is a BET small molecule inhibitor targeting bromodomain protein 4(BRD4),by mimicking acetyl,JQ1 binds to the acetyl-lysine pocket peculiar to the BET family proteins and prevents the transcription of genes downstream of BRD4.JQ1 effects on melanoma are mostly focused on the regulation of apoptosis,cell cycle and other processes.However,the above mechanism does not fully elucidate the therapeutic effect of BETi in cutaneous melanoma.As the center of energy regulation,mitochondria regulates the fate of tumor cells and melanoma cells are more sensitive to changes in glycolysis,which indicates that mitochondrial changes in melanoma cells may play an important role in the occurrence of disease.Mitochondrial dynamics,as a key factor in maintaining mitochondrial function and morphology,is the main regulator of mitochondrial function,and its disruption of fusion and fission balance will damage mitochondria.Therefore,whether BRD4 can play a role in melanoma by regulating mitochondria has become the focus of our research.Objective:In this study,by using BRD4 inhibitor JQ1 to treat melanoma B16 cells,we observed the effect of JQ1 on melanoma cell growth,apoptosis,mitochondrial function,mitochondrial dynamics and energy metabolism and explored the mechanism of action of BRD4 in melanoma.Methods: 1.Databases were used to analyze the expression of BRD4 in melanoma.2.MTT,cell growth counting and plate cloning experiments were used to study the effect of BRD4 inhibitor JQ1 on the viability and proliferation of B16 cells,cell scratch and western blot experiments were used to detect the effect of JQ1 on B16 cell migration.3.Flow cytometry,hoechst staining and western blot experiments were used to detect the effects of JQ1 on B16 cell apoptosis,cell cycle,mitochondrial membrane potential,and ROS content.JC-1 and ROS immunofluorescence experiments further confirmed the changes in mitochondrial membrane potential and ROS content.4.Extract mitochondrial protein and mt DNA,analyze mitochondrial function and mitochondrial quality changes by western blot and q PCR technology.5.Test the glucose content,lactic acid production and lipid synthesis of the cells through biochemical kits to reflect the energy regulation of JQ1 on B16 cells.6.Mito-tracker green mitochondrial staining,transmission electron microscopy,western blot and q PCR technology confirmed that JQ1's regulation of B16 cells is related to mitochondrial dynamics.Results: 1.Database analysis showed that BRD4 was highly expressed in melanoma.Inhibition of BRD4 can inhibit proliferation and migration of melanoma B16 cells.2.Inhibiting BRD4 can promote the apoptosis of B16 cells through the mitochondrial pathway and induce G1 phase arrest.After JQ1 treated melanoma cells,the release of mitochondrial cytochrome C into the cytoplasm increased,the expression of mitochondrial apoptosis-related protein cleaved-caspase 9 increased,and the mitochondrial membrane potential(??m)decreased in a dose-dependent manner.3.Inhibition of BRD4 reduced the activity of the mitochondrial electron transport chain,down-regulated the expression of important subunits of respiratory chain complex NDUFV1,CYC1,COX7 C and ATP5F1 and eventually lead to mitochondrial dysfunction.which may be related to the decrease in mt DNA copy number.4.The inhibition of BRD4 can reduce melanoma glucose uptake and fatty acid synthesis.Treatment with different concentrations of JQ1(125n M or 250 n M)changed the energy metabolism of melanoma cells,resulting in a reduction in glucose uptake,lactate production and fat production.5.The inhibition of BRD4 regulated the apoptosis and metabolism of melanoma by changing the mitochondrial dynamics.After JQ1 treatment,the expression of mitochondrial Dynamin-related protein 1(Drp1)increased,and the expression of mitochondrial fusion proteins(Mfn1 and Mfn2)and optic atrophy 1(OPA1)decreased.Transmission electron microscopy showed mitochondrial division increased in cells treated with JQ1.Further verification of its mechanism revealed that when mitochondrial division inhibitor Mdivi-1(2?M)was added 1 h before JQ1 treatment of melanoma B16 cells,it was observed that apoptosis was significantly reduced,ROS production was reduced,mitochondrial membrane potential increased and B16 cell glucose and lipid metabolism was restored.Conclusion: 1.Inhibiting BRD4 can inhibit the proliferation and migration of melanoma B16 cells,which may be related to the inhibition of c-myc protein.2.Inhibition of BRD4 can cause mitochondrial dysfunction,affect glucose and lipid metabolism and mediate B16 cell apoptosis through the mitochondrial pathway.3.Inhibition of BRD4 can change the mitochondrial dynamics of B16 cells and increase mitochondrial division,which may be related to the increased apoptosis of mitochondrial pathway.