The Role Of NF-κB/miR-21 In The Apoptosis Of Hela Cells Induced By Arsenic Via GCLC

ObjectiveAs the fourth most common cancer in women worldwide,chemotherapy is one of the main treatments for cervical cancer.Arsenic is widely used in solid tumor chemotherapy.As the rate-limiting subunit of GSH synthesis,GCLC may be an important target for arsenic to induce apoptosis through mitochondrial apoptotic pathway to exert anti-tumor effect.NF-κB is involved in the regulation of apoptosis and has the function of regulating GCLC.miR-21 plays an important role in the occurrence and development of cervical cancer.It can participate in the regulation of intracellular mitochondrial apoptosis by regulating the level of ROS.NaAsO₂can activate miR-21 by promoting the expression of NF-κB.However,NF-κB/miR-21 can affect GSH synthesis through GCLC and participate in the process of NaAsO₂-mediated oxidative stress-induced mitochondrial apoptosis in cervical cancer cells is rarely reported.Therefore,the purpose of this study was to investigate the role of NF-κB/miR-21 in the apoptosis of Hela cells induced by NaAsO₂through GCLC,and to provide a theoretical basis for exploring the possible mechanism of arsenic in the treatment of cervical cancer.MethodsIn this study,Hela cell line was used as the research object,and different concentrations of NaAsO₂（0,5,10,15,20,25μmol/L）were applied to intervene cells,and then GCLC inhibitor D,L-Buthionine-（SR）-sulfoximine,NF-κB inhibitor BAY 11-7082,miR-21 Inhibitor combined NaAsO₂intervention.Annexin-FITC/PI method,transmission electron microscope,JC-1 dye,kits,CCK-8 method were used to analysis of changes in the apoptosis level,mitochondrial structure,membrane potential changes,glutathione,glutamate,cystine,reactive oxygen species and cell activity.The m RNA and protein expression levels of BAX,Bcl-2,Caspase3,Cleaved-caspase3,NF-κB p65,p-p65,IκBα,p-IκBα,miR-21,Glutamate-cysteine ligase catalytic Subunit（GCLC）,Glutamate-cysteine ligase Modifier Subunit（GCLM）were detected by Western Blot,q RT-PCR,and cellular immunofluorescence.The detection indicators are described in the form of mean±standard deviation,and statistical methods such as ANOVA,Dunnett-t method and independent sample t test are used to analyze the differences of experimental results.A two-sided test was used for the hypothesis test.When P<0.05,the difference was considered to be statistically significant.Results1.The effect of NaAsO₂on apoptosis of Hela cells Compared with the control group,when 15,20and 25μmol/L Na Aa O₂intervened,the cell viability was decreased（P<0.05）,and the apoptosis rate was increased（P<0.05）.When 24,48,and 72 h of 15μmol/L NaAsO₂intervened in the cells,the cell viability was reduced（P<0.05）.2.The effect of NaAsO₂on mitochondrial pathway apoptosis of Hela cells When 15 and 25μmol/L Na Aa O₂intervened,the mitochondrial membrane outside of the cells was ruptured,the connection between the inner membrane and the cristae was interrupted,NaAsO₂decreased the mitochondrial membrane potentialΔΨm and the ratio of Bcl-2/BAX protein expression in cells（P<0.05）,but it increased the protein of Caspase3 and Cleaved-caspase3（P<0.05）.3.The effect of NaAsO₂on the level of oxidative stress in Hela cells When 15,20,25μmol/L NaAsO₂was used to intervene cells for 24 h,the level of ROS was accumulated by NaAsO₂（P<0.05）,GSH levels was decreased（P<0.05）.When 15μmol/L NaAsO₂was used to intervene cells for 24,48,and72 h,the intracellular GSH level was decreased compared with the 0 h group（P<0.05）.4.The effect of NaAsO₂on glutathione synthesis in Hela cells Compared with the control group,after the intervention of 15,20,25μmol/L NaAsO₂,the intracellular Cys content andγ-GCS enzyme activity were decreased.Compared with the control group,the m RNAs of subunit GCLC and GCLM ofγ-GCS were decreased（P<0.05）and the expression level of GCLC protein were decreased（P<0.05）.5.The role of GCLC in NaAsO₂-induced mitochondrial apoptosis by inhibiting GSH synthesis D,L-Buthionine-（SR）-sulfoximine and 15μmol/L NaAsO₂decreased protein of GCLC level and GSH content（P<0.05）,and increased ROS level（P<0.05）.They also down-regulated mitochondrial membrane potentialΔΨm and intracellular Bcl-2/BAX（P<0.05）,the levels of Caspase3 and Cleaved-caspase3 were higher than control（P<0.05）.When used in combination,compared with the 15μmol/L NaAsO₂group,the GCLC protein and GSH level were decreased（P<0.05）,and induced accumulation of ROS（P<0.05）.They dropped mitochondrial membrane potentialΔΨm,Bcl-2/BAX ratio and the cell viability（P<0.05）,but the levels of Caspase3 and Cleaved-caspase3 were higher than NaAsO₂group（P<0.05）.6.The effect of NaAsO₂on the NF-κB signaling pathway in Hela cells 15 and 25μmol/L NaAsO₂groups increased the levels of IκBαm RNA（P<0.05）,and 25μmol/L NaAsO₂group increased the expression of p65 m RNA（P<0.05）.When 15,20 and 25μmol/L NaAsO₂intervened,the expression of IKKβprotein in cells was promoted（P<0.05）,and they increased the protein level of p65 in the nucleus and p-p65/p65,p-IκBα/IκBα（P<0.05）.7.The effect of NF-κB on NaAsO₂induced mitochondrial apoptosis by inhibiting the expression of GCLC When 15μmol/L BAY 11-7082 was used to intervene in cells,the expression levels of GCLC m RNA and protein in cells were increased（P<0.05）.After NaAsO₂combined with BAY 11-7082,it decreased the m RNA expressions of p65,IκBαand the expressions of p-p65/p65,p-IκBα/IκBα（P<0.05）,the expression of GCLC protein,the GCLC and GCLM m RNA were increased（P<0.05）.Moreover,the level of mitochondrial membrane potentialΔΨm,GSH and cell vibility were higher than NaAsO₂group,the level of ROS was inhibited（P<0.05）.8.The role of miR-21 in NaAsO₂-induced mitochondrial apoptosis through NF-κB/GCLC When 15,20,25μmol/L NaAsO₂was used to intervene the cells for 24 h,they increased the expression of miR-21 in the cells（P<0.05）.After using the NF-κB inhibitor BAY 11-7082 in combination with NaAsO₂,the expression level of miR-21 in this group was lower than NaAsO₂group（P<0.05）.After using miR-21Inhibitor to inhibit NaAsO₂activated miR-21,compared with NaAsO₂combined with miR-21 NC group,the expression level of GCLC protein and GSH content in cells increased（P<0.05）,the level of intracellular ROS decreased（P<0.05）,the mitochondrial membrane potential increased（P<0.05）,and the cell activity was increased（P<0.05）.ConclusionNaAsO₂can down-regulate the expression of GCLC,inhibit GSH synthesis and trigger mitochondrial apoptosis in Hela cells,which may be related to NaAsO₂activates the NF-κB signaling pathway to promote the expression of miR-21 and induce the reduction of GCLC expression and results in mitochondrial structural damage and decreased mitochondrial membrane potential.