cell survival and apoptosis are associated with the cell cycle , which is highly conservative . many cancer genes closely associated with proliferation are closely related to cell apoptosis . with the further understanding of cell apoptosis most cancer chemotherapy aims to induce cell apoptosis in the specific period to kill the tumor cells [ 14 ] . however , there is still little data on the relationship between mitochondrial - mediated u251 cell apoptosis and the cell cycle . we tried to clarify the mechanism of mitochondria - mediated apoptosis through cell cycle , caspase-3 , and caspase-9 . human brain glioma cell line u251 cells ( harbin medical university ) were maintained in dmem medium ( gibco , usa ) containing 10% fetal bovine serum ( gibco , usa ) and penicillin streptomycin at 37c , 5% co2 incubator ( queue systems , the united states ) . the cells were passaged when the density reached more than 410 cells / cm . after removing the medium , when the adherent cells achieved 410 , mitochondrial apoptosis - related gene bax plasmid ( santa cruz , usa ) was applied to stimulate cells for 14 h ; 10 g / ml tpa ( beyotime , shanghai ) was used to stimulate u251 cells , and after adding 12.5 g / ml bax for 24 h , the cells were collected . 10 cells in 100 l fluid after washing were added with annexin v - fitc and pi . after incubating for 15 min away from light , the cells were detected by flow cytometry ( bd , usa ) . the collected cells were added with annexin v  fitc and incubated at room temperature away from light for 30 min . after washing twice with buffer , 1 ml formaldehyde was applied to fix the cells . pi was further used for staining and the cells were detected on flow cytometry after 1 h. cyclins / dna double staining flow cytometry instrument was used to analyze the human brain glioma cell line u251 cell cycle specificity . the cells were fixed by ethanol overnight and treated with tritonx  100 for 5 minutes twice . after washed with pbs , the cells were incubated with bsa diluted antibody at 4c overnight . goat anti - mouse igg were added on the second day at room temperature for 20 min . pi and rnasea were used for dna staining , and the cells were detected by flow cytometry . we extracted 100 mg total protein from each group and separated it by sds - page electrophoresis . after incubation with primary antibody ( 1:500 ) at 4c overnight and hrp - tagged secondary antibody ( 1:1000 ) at 37c for 1 h , the membrane was detected by ecl photochemical method . primer design : caspase-3 : upstream 5-gtactggcattg gcggtatc-3 , downstream 5-gagaatccagggcatccatt-3 ; caspase-9 upstream 5-ggcttggtagtgtttgccat-3 , downstream 5-gggcaaagagtaaacccaca-3 ; 18srna upstream 5-ctgcccctacttgtcatggt -3 , downstream 5-agatgagcctcacagcccta -3 total rna was extracted from the cells . the pcr cycling conditions consisted of an initial , single cycle of 5 min at 95c , followed by 30 cycles of 30 s at 94c , 30 s at 60c , and 60 s at 72c . pcr products were tested by agarose gel electrophoresis and analyzed by software ; 18 s rna was chosen as control . data conformed to normal distribution were presented as means and standard deviation ( s ) . human brain glioma cell line u251 cells ( harbin medical university ) were maintained in dmem medium ( gibco , usa ) containing 10% fetal bovine serum ( gibco , usa ) and penicillin streptomycin at 37c , 5% co2 incubator ( queue systems , the united states ) . the cells were passaged when the density reached more than 410 cells / cm . after removing the medium , when the adherent cells achieved 410 , mitochondrial apoptosis - related gene bax plasmid ( santa cruz , usa ) was applied to stimulate cells for 14 h ; 10 g / ml tpa ( beyotime , shanghai ) was used to stimulate u251 cells , and after adding 12.5 g / ml bax for 24 h , the cells were collected . 10 cells in 100 l fluid after washing were added with annexin v - fitc and pi . after incubating for 15 min away from light , the cells were detected by flow cytometry ( bd , usa ) . the collected cells were added with annexin v  fitc and incubated at room temperature away from light for 30 min . after washing twice with buffer , 1 ml formaldehyde was applied to fix the cells . pi was further used for staining and the cells were detected on flow cytometry after 1 h. cyclins / dna double staining flow cytometry instrument was used to analyze the human brain glioma cell line u251 cell cycle specificity . the cells were fixed by ethanol overnight and treated with tritonx  100 for 5 minutes twice . after washed with pbs , the cells were incubated with bsa diluted antibody at 4c overnight . goat anti - mouse igg were added on the second day at room temperature for 20 min . pi and rnasea were used for dna staining , and the cells were detected by flow cytometry . we extracted 100 mg total protein from each group and separated it by sds - page electrophoresis . after incubation with primary antibody ( 1:500 ) at 4c overnight and hrp - tagged secondary antibody ( 1:1000 ) at 37c for 1 h , the membrane was detected by ecl photochemical method . primer design : caspase-3 : upstream 5-gtactggcattg gcggtatc-3 , downstream 5-gagaatccagggcatccatt-3 ; caspase-9 upstream 5-ggcttggtagtgtttgccat-3 , downstream 5-gggcaaagagtaaacccaca-3 ; 18srna upstream 5-ctgcccctacttgtcatggt -3 , downstream 5-agatgagcctcacagcccta -3 total rna was extracted from the cells . the pcr cycling conditions consisted of an initial , single cycle of 5 min at 95c , followed by 30 cycles of 30 s at 94c , 30 s at 60c , and 60 s at 72c . pcr products were tested by agarose gel electrophoresis and analyzed by software ; 18 s rna was chosen as control . data conformed to normal distribution were presented as means and standard deviation ( s ) . the results showed that after treatment with tpa , u251 cells in the stationary phase obviously entered g2-m phase ( figure 1 ) . g2~m cells in the experimental group reached 31.27% , compared with the control group ( 20.24% ) . ( figure 1 shows the cell cycle of human brain glioma cell line u251 after stimulation by tpa ) . after treated with bax for 24 hours , the apoptotic cell rate increased for 6.8% . after stimulation with tra and bax for 24 h , the cell apoptotic rate up - regulated to 15.2% . tpa stimulation was more apparent in u251 cell apoptosis induction effect ( figure 2 ) . cells in the control group with no tpa stimulation showed no significant cell apoptosis ( figure 3 ) . after stimulation with tpa , cells appeared in g0 , early / late g1 phase , s2 , g2 , and m stages . cyclin e expression decreased after addition of bax and cells were arrested in g1 stage ( figure 4 ) . after being induced by the mitochondrial apoptosis - related gene bax , u251 cells showed obviously elevated caspase-3 and caspase-9 protein expression ( figure 5 ) . after induction by mitochondrial apoptosis - related gene bax , caspase-3 and caspase-9 mrna expression significantly increased ( p<0.05 ) ( table 1 ) . the results showed that after treatment with tpa , u251 cells in the stationary phase obviously entered g2-m phase ( figure 1 ) . g2~m cells in the experimental group reached 31.27% , compared with the control group ( 20.24% ) . ( figure 1 shows the cell cycle of human brain glioma cell line u251 after stimulation by tpa ) . after treated with bax for 24 hours , the apoptotic cell rate increased for 6.8% . after stimulation with tra and bax for 24 h , the cell apoptotic rate up - regulated to 15.2% . tpa stimulation was more apparent in u251 cell apoptosis induction effect ( figure 2 ) . cells in the control group with no tpa stimulation showed no significant cell apoptosis ( figure 3 ) . after stimulation with tpa , cells appeared in g0 , early / late g1 phase , s2 , g2 , and m stages . cyclin e expression decreased after addition of bax and cells were arrested in g1 stage ( figure 4 ) . after being induced by the mitochondrial apoptosis - related gene bax , u251 cells showed obviously elevated caspase-3 and caspase-9 protein expression ( figure 5 ) . after induction by mitochondrial apoptosis - related gene bax , caspase-3 and caspase-9 mrna expression significantly increased ( p<0.05 ) ( table 1 ) . studies   have shown that the cell cycle plays an important role in the process of cell proliferation and apoptosis . the cell cycle is mainly regulated by signaling pathways . signal blockage or strong stimulation can cause cell cycle imbalance and lead to tumor occurrence . a tumor is a kind of disease characterized as abnormal cell proliferation . one kind is the activation of signal transduction pathway , while the other is excessive cell proliferation . signaling pathways includes cdks , cyclins abnormal activation and expression , and gene mutation . however , single - gene locus mutation is unlikely to cause cancer , and multiple genes combined are required to for the occurrence of cancer cells . under the abnormal proliferation condition , cancer treatment is also a process of tumor cell apoptosis initiation , and its core mechanism is the activation of caspase and promoting cells disintegration . phosphorylated akt mutation , bcl-2 , and bax overexpression both can activate apoptosis through mitochondria . there is still lack of related research about the role of mitochondria - mediated apoptosis in human brain glioma cells . this study revealed the relationship between mitochondria - mediated human brain glioma cell apoptosis and the cell cycle through selecting the mitochondria - mediated apoptosis inducer protein bax , applying api technology for cell apoptosis and cell cycle analysis , and exploring the impact of mitochondria - mediated apoptosis on brain glioma cell cycle specificity and apoptosis related proteins caspase-3 and caspase-9 activation . u251 cells proliferate quickly during inflammation or trauma , and its status becomes stable again when the inflammatory lesions fade away [ 1014 ] . when the cells were induced by mitochondrial apoptosis - related gene bax , apoptotic cells were fewer and cells did not enter the cell cycle . on this basis , the tectotype of plasminogen activator tpa stimulus can activate the u251 cells into the cell cycle , promote cell proliferation , and lets cells enter the s and g2/m phases . at the same time , thus , apoptosis can only be detected after the cells enter the metaphase , which is similar with clinical chemoradiotherapy treatment . chemoradiotherapy made the enter the cell cycle , and the cell apoptosis rate is much higher after treatment than in stationary phase . to test whether cell apoptosis has cycle specificity , we tested mitochondria - mediated apoptosis by api method . membrane eversion phenomenon appeared in the early phase of apoptosis , and api method used annexin v combined with this phenomenon to judge apoptosis cycle specificity . the results suggested that mitochondria - mediated apoptosis and cycle specificity apoptosis mainly occurred in the g1 phase . cell cycle protein consists of various types of proteins specifically expressed in the cell cycle that can make cells have a specific cycle manifestation by stimulating the cell cycle - dependent protein kinase . cycline / dna double - stain flow cytometry combined cell place and cycline expression to test the detailed information . our results show that the mitochondria - mediated apoptosis decreased cycline expression in brain glioma cells , and cells were arrested in g1 phase ; this process also complied with apoptosis - related proteins caspase-3 and 9 overexpression and activation . the human brain glioma cells did not start the cycle process in stationary stage and the apoptosis became sensitive only after entering the cell cycle . mitochondria - mediated apoptosis is closely related to the brain glioma cell cycle and occurs in a particular phase . this study investigated mitochondria - mediated apoptosis and has clinical significance in inducing tumor apoptosis and treatment .
backgroundmost mitochondria - mediated apoptosis has some relevance to the cell cycle , but there is still a lack of investigations about u251 cell cycle in human brain glioma cells . in this study , we aimed to clarify the correlation of mitochondria - mediated apoptosis with the u251 cell cycle and its influence on apoptosis , through observing the impact of mitochondria - mediated apoptosis in u251cell specificity cycle arrest and caspase activation.material/methodsannexinv/pi and api were used to label the brain glioma cells for flow cytometry analysis of u251 cell apoptosis and cell cycle . 
 rt - pcr and western blot were performed to detect caspase-3 and caspase-9 activation.resultsperipheral blood in stationary phase is not sensitive to apoptosis induction , but u251 cells have obvious apoptosis . 
 mitochondria - mediated apoptosis mainly occurs in the g1 phase of the cell cycle . 
 caspase-3 and caspase-9 mrnas and proteins expression increased significantly after the cells were treated by mitochondrial apoptosis - related gene bax induction.conclusionsmitochondria-mediated apoptosis is related to the u251 cell cycle with specific g1 stage arrest . 
 caspase activation occurs in the process of cell apoptosis .