| Objective: Explain the causes of abnormal proliferation of liver cancer cells;to study the changes of gene expression in different stages of liver cancer;to explore the relationship between CCNA2,CCNB1 and CDK1;to study the effect of Bielong Ruangan Decoction on the proliferation and cell cycle of hepatocellular carcinoma cells.To study the effect of Bielong Ruangan Decoction on the key molecules CCNA2,CCNB1 and CDK1 of liver cancer cells.Methods: GEPIA website was used to analyze and compare the expression differences of key molecules CCNA2,CCNB1 and CDK1 in cell cycle between hepatocellular carcinoma cells and normal liver cells.To explore the key molecules CCNA2 and CCNA2 in different stages of liver cancer.Differences in the expression of CCNB1 and CDK1;the TIMER database was used to explore the relationship between the three key molecules CCNA2,CCNB1 and CDK1,and the proliferation of liver cancer cells and normal liver cells was compared through experiments.The two groups of cells were cultured using the drug-containing plasma containing Bielong Ruangan Decoction and the blank plasma group.The CCK-8 method was used to detect the cell viability,the flow cytometry was used to detect the cell cycle,the real-time fluorescence quantitative PCR was used to detect the m RNA expression of key molecules in the signaling pathway of the two groups of cells,and the Western Blot method was used to detect the expression of key molecules in the signaling pathway of the two groups of cells.SPSS26.0 was used for statistical analysis of the data.Results: 1.In the GEPIA database,TCGA database and GTEx database were used to analyze the expression of CCNA2,CCNB1 and CDK1 genes in liver cancer.CCNA2,CCNB1 and CDK1 m RNA were highly expressed in liver cancer tissues,and the difference was statistically significant(P<0.05).2.In the GEPIA database,the relationship between the expression of CCNA2,CCNB1,CDK1 gene and the clinical stage of liver cancer patients was analyzed,and it was found that the expression of CCNA2,CCNB1 and CDK1 in different tumor clinical stages was statistically significant(P<0.05).3.Analyze the expression correlation of CCNA2,CCNB1 and CDK1 in the TIMER database,and find that the expression correlation of CCNA2,CCNB1 and CDK1 among the three molecules is strong and statistically significant(P<0.05).4.The relative m RNA expression levels of CCNA2,CCNB1 and CDK1 in LX-2 cells were 1.01 ±0.17,1.00 ± 0.06 and 1.00 ± 0.09,respectively.The relative m RNA expression levels of CCNA2,CCNB1 and CDK1 in Hep G2 cells were 1.76±0.39,1.89 ±0.40 and 2.17±0.05,respectively,and the difference between the groups was statistically significant(P< 0.05).5.The relative protein expression levels of CCNA2,CCNB1 and CDK1 in LX-2 cells were 0.06 ±0.02,0.07±0.02 and 0.12±0.03,respectively.The relative protein expression levels of CCNA2,CCNB1 and CDK1 in Hep G2 cells were 0.33±0.02,0.27±0.04 and 0.52±0.04,respectively,and the differences between the groups were statistically significant(P< 0.05).6.The average OD values of LX-2 cells in the blank group,10 % blank plasma control group and 10 % Bielong Ruangan medium dose group were 1.71 ± 0.04,1.64 ± 0.01 and 1.64 ± 0.04,respectively.There was no significant difference between the groups(P > 0.05).The average OD values of Hep G2 cells in the blank group,10 % blank plasma control group and 10 % Bielong Ruangan medium dose group were 1.56 ± 0.01,1.37 ± 0.01 and 1.27 ± 0.01,respectively.The difference between the groups was statistically significant(P < 0.05).7.In LX-2 cells,the proportion of G1 phase cells in the blank plasma group was higher than that in the blank group(P < 0.05);compared with the other two groups,the proportion of cells in the Bielong Ruangan Decoction group increased in G1 phase and G2 phase,and decreased in S phase(P < 0.001).In Hep G2 cells,compared with the other two groups,the proportion of cells in the G1 phase increased(P < 0.05),and the proportion in the S phase decreased(P < 0.01).8.The relative expression levels of CCNA2 m RNA in LX-2 cells in blank group,blank plasma group and Bielong Ruangan Decoction group were 1.00 ± 0.08,1.19 ± 0.01 and 0.91 ± 0.05,respectively.The difference was statistically significant(P < 0.05);the relative expression of CCNB1 m RNA was 1.02 ± 0.24,0.94 ± 0.14 and 1.36 ± 0.06,respectively.The difference was statistically significant(P < 0.05);the relative expression levels of CDK1 m RNA were1.01 ± 0.14,0.80 ± 0.01 * and 1.05 ± 0.04,respectively.The difference was statistically significant(P < 0.05);the relative expression levels of CCNA2 m RNA in Hep G2 cells in blank group,blank plasma group and Bielong Ruangan Decoction group were 1.00 ± 0.11,1.07 ±0.31 and 0.34 ± 0.05,respectively.The difference was statistically significant(P < 0.05);the relative expression of CCNB1 m RNA was 1.01 ± 0.13,1.19 ± 0.10 and 0.38 ± 0.09,respectively.The difference was statistically significant(P < 0.05);the relative expression of CDK1 m RNA was 1.00 ± 0.02,1.09 ± 0.12 and 0.33 ± 0.02,respectively(P <0.05).9.The relative expression of CCNA2 protein in LX-2 cells in blank group,blank plasma group and Bielong Ruangan Decoction group was 0.24 ± 0.02,0.26 ± 0.02 and 0.25 ± 0.02,respectively.The difference was not statistically significant(P > 0.05);the relative expression of CCNB1 protein was 0.34 ± 0.02,0.30 ± 0.02 and 0.27 ± 0.03.The difference was statistically significant(P<0.05).The relative expression levels of CCNA2 protein in Hep G2 cells in blank group,blank plasma control group and Bielong Ruangan Decoction group were0.51 ± 0.06,0.41 ± 0.03 and 0.12 ± 0.02,respectively.The difference was statistically significant(P < 0.05);the relative expression levels of CCNB1 protein were 0.46 ± 0.02,0.39 ± 0.05 and 0.13 ± 0.03,respectively.The difference was statistically significant(P <0.05);the relative expression of CDK1 protein was 0.70 ± 0.03,0.68 ± 0.04 and 0.45 ±0.03,respectively,and the difference was statistically significant(P < 0.05).Conclusion: 1.Bioinformatics analysis showed that the m RNA expression of CCNA2,CCNB1 and CDK1 in normal liver cells and liver cancer cells was different.The expression of the three molecules was related to the clinical stage,and there was a positive correlation between the three molecules.2.The m RNA and protein expression of CCNA2,CCNB1 and CDK1 in LX-2 cells and Hep G2 cells were significantly different,and the cell activity was significantly different.Hep G2 cells had higher cell viability and faster proliferation than LX-2 cells,and the expression of the three molecules in Hep G2 cells was significantly higher than that in LX-2 cells.3.The m RNA and protein expression of CCNA2,CCNB1 and CDK1 in Hep G2 cells after drug intervention of Bielong Ruangan Decoction were significantly different from those in the blank group and the plasma group,and the cell activity was significantly different.The m RNA and protein expression of CCNA2,CCNB1 and CDK1 in Hep G2 cells after the intervention of plasma containing Bielong Ruangan Decoction was significantly lower than that of the blank group and the blank plasma group,and the cell activity was also significantly reduced.Bielong Ruangan Decoction may block the DNA synthesis of cells in G1 phase and inhibit cell division.It is proved that Bielong Ruangan Decoction has the effect of anti-tumor and blocking cell cycle,and may be achieved by affecting the expression of CCNA2,CCNB1 and CDK1 molecules. |
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