| Objective: To investigate the effect of zhiyanxiaojifang(ZYXJF)on the proliferation and apoptosis of SMMC-7721 cells,and to explore the possible mechanism of ZYXJF regulating the biological behavior of tumor cells based on gene chip technology.Methods: Cell Titer GLO assay was used to detect the inhibition rate of ZYXJF(3200,1600,800,400,200,100,50 and 25 μg/m L)on the proliferation of SMMC-7721 cells,and the IC50 was calculated.High concentration of the screened IC50(1500 μg/m L)and low concentration of 1000 μg/m L were used to observe the effect of ZYXJF on the proliferation activity of SMMC-7721 cells at different time through Cell Titer GLO experiment,CCK-8.The effect of ZYXJF on the apoptosis of SMMC-7721 cells was detected by flow cytometry.The total RNA was extracted from SMMC-7721 cells after 1500 μg/m L ZYXJF treatment for 48 h,and then the changes of gene expression profile before and after the intervention were studied by using gene chip technology.|Fold Change| > 1.5 and FDR < 0.05 were used as the screening criteria to screen the differentially expressed genes before and after intervention.IPA-based analysis software was used for disease and function analysis to observe the mainly enriched diseases and functions of differentially expressed genes.The expression of 10 differentially expressed genes with down-regulated expression abundance was verified by RT-PCR,and potential target genes of ZYXJF were screened.The potential target genes screened were analyzed in the GEPIA database for their expression in liver cancer tissues and their relationship with the prognosis of patients,and finally a target gene was selected for follow-up study.After transfected with the DNAJB6-si RNA sequence of SMMC-7721 cells,the1500 μg/m L ZYXJF was used to continue the intervention for 48 h.The effect of DNAJB6 gene silencing on the regulation of ZYXJF on cell proliferation was verified by Cell Titer GLO and CCK-8 assays.Results:(1)The IC50 of ZYXJF on SMMC-7721 cells was 1500μg/m L;(2)The 1000 μg/m L and 1500 μg/m L of ZYXJF could significantly reduce the proliferation activity of SMMC-7721 cells.(3)The 1000 μg/m L and 1500 μg/m L of ZYXJF could significantly promote the apoptosis of SMMC-7721 cells in human liver cancer,and the effect of high concentration group was better than that of low concentration group.(4)The ZYXJF could affect the gene expression profile of the SMMC-7721 cells.A total of 418 differentially expressed genes were screened between the two groups,among which,compared with the blank group,a total of 263 significantly up-regulated genes and 155 significantly down-regulated genes were found in the SMMC-7721 cells of the intervention group.(5)The IPA analysis software showed that the top five diseases and functions associated with the different genes were Tissue damage and abnormal(organismal injury and abnormalities),Cancer,Cell death and survival,Cellular development and Tissue development.(6)RT-q PCR results showed that the the expression of HIST1H2 BK,DNAJB6,ZBED1,and PKDCC in cells was significantly reduced after ZYXJF treatment.(7)GEPIA database analysis showed that DNAJB6 was highly expressed in liver cancer tissues and was associated with poor prognosis of patients.(8)DNAJB6 gene silencing can enhance the effect of ZYXJF on the proliferation of SMMC-7721 cells.Conclusion:(1)ZYXJF inhibited the proliferation and induced apoptosis of SMMC-7721 cells in human liver cancer in concentration and time-dependent manners.(2)DNAJB6 is maybe the key gene for inhibiting SMMC-7721 cells in human liver cancer by ZYXJF.(3)DNAJB6 is highly expressed in hepatocellular carcinoma and is positively correlated with poor prognosis.(4)The mechanism of ZYXJF inhibiting the proliferation of SMMC-7721 cells and promoting apoptosis in human liver cancer may be related to the down-regulation of DNAJB6 gene expression. |
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