| Objective: To investigate the effect of transcription factor GCF2 on the proliferation and apoptosis of hepatoma cells,and whether it participates in the proliferation and apoptosis of hepatoma cells through the E2F1/Cyclin A1/CDK2 pathway,which lays the foundation for subsequent experiments.Methods:（1）The expression of GCF2 m RNA in liver cancer tissues（HCC）and in adjacent tissues（ANCT）were analyzed by bioinformatics studies.Immunohistochemical method was used to detect the expression of GCF2 protein in HCC tissues and in ANCT tissues.（2）According to the previous experimental results,to select human hepatoma cell line Hep G2 with high expression of GCF2 for experiments,and the down-regulated GCF2 sequence was transferred into human hepatoma cell line Hep G2 by sirna interference technique.The experiment was divided into three groups: plasmid transfection group（GCF2-si RNA）,negative control group（NC-si RNA）and blank control group（MOCK）.The relative expression of GCF2 gene and protein were detected by RT-PCR and Western blot.（3）The effects of GCF2 on the clone ability and cell proliferation of Hep G2 cells were investigated by cell clone formation test and cck8 cell activity test,respectively.（4）The effects of GCF2 on apoptosis and cell cycle of Hep G2 cells were examined by flow cytometry Annexin V-7AAD/PE double staining and PI single staining respectively.The effects of GCF2 on cell cycle related proteins and apoptosis-related proteins in Hep G2 cells were detected by western blot.Results:（1）Bioinformatics analysis showed that the expression of GCF2 m RNA in HCC tissues was significantly higher than that in adjacent tissues（p<0.01）.The results of immunohistochemistry indicated that GCF2 was expressed highly in HCC tissues,and mainly expressed in cytoplasm.The expression of GCF2 in HCC tissues was significantly higher than that in adjacent tissues（p<0.05）.（2）The expression of GCF2 m RNA and protein were down-regulated effectively in Hep G2 by si RNA targeting GCF2 m RNA（p<0.01）.（3）After down-regulation of GCF2 expression,the results of CCK8 showed that the inhibition rates of the GCF2-si RNA groups were（21.8±0.07）%,（43.6±0.08）%,（24.4±0.05）% at 24 h and 48 h and 72 h,respectively.The NC-si RNA groups were:（15.0±0.08）%,（25.4±0.08）%,（18.8±0.07）%,respectively.The inhibition was most obvious at48 hours after the expression of GCF2 was decreased,and the tran GCF2-si RNA group was significantly higher than the NC-si RNA group.The results of plate cell colony formation experiments showed that the cloning rates of the GCF2-si RNA group,the MOCK group and the NC-si RNA groups were（25.1±2.30）%,（79.6±3.14）%,（65.8±2.43）%,respectively.The clone forming ability of the GCF2-si RNA group was lower than that of the NC-si RNA group and the MOCK group,the differences were statistically significant（p<0.05）.（4）Apoptosis test results show that after decreasing the expression of GCF2 by using the small interfering RNA,the apoptosis rate of the GCF2-si RNA group was（11.64±1.85）% at 48 hours,the MOCK group was（4.3±0.89）%,and the NC-si RNA group was（5.2±0.38）%.The apoptosis rate of the GCF2-si RNA group was significantly increased（p<0.05）.The results of cell cycle test showed that the percentage of S phase was significantly increased（p<0.05）,the G0/G1 phase was significantly decreased,and Cell cycle was blocked in S phase;and the results of western blot showed that compared with the MOCK group and the NC-si RNA group,the expression of Cyclin A1 was significantly increased in the GCF2-si RNA group,and the expression of cyclin-dependent kinase CDK2 and transcription factor E2F1 was significantly decreased.The expression levels of apoptotic proteins p21 and Cleaved Caspase-3 were higher than those of the MOCK group and the NC-si RNA group（p<0.05）.Conclusion:（1）GCF2 is highly expressed in liver cancer tissues and liver cancer cells,suggesting that high expression of GCF2 may be involved in the development of liver cancer.（2）Down-regulation of GCF2 expression can block Hep G2 cell cycle,inhibit cell cloning and proliferation,and promote Hep G2 cell apoptosis,indicating that GCF2 may be involved in the biological behavior of liver cancer development.（3）Down-regulation of GCF2 caused an increase in the expression of cell cycle-associated protein Cyclin A1 and decreased expression of CDK2 and E2F1,suggesting that GCF2 may affect the proliferation and apoptosis of hepatoma cells through E2F1/Cyclin A1/CDK2 pathway,laying a foundation for subsequent research. |
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