Foreseeing Seed Extracts Regulate The Expression Of Ribosomal Protein-related Genes In Liver Cancer Cells And Inhibit Their Malignant Proliferation

OBJECIITVETo observe the effects of proliferation inhibition and ribosome-related genes expression of SMMC-7721 and Hep G2 hepatoma cells after treatment on extracts of Chinese herbal medicine Akebiae fructus(Yuzhizi,YZZ),and screen some possible key genes to lucubrate their roles for illustrating the target and mechanism of YZZ on inhibiting the malignant proliferation of hepatoma cells.METHODS1.The effect of YZZ on ribosome-related genes in hepatocellular carcinoma cells.PCR experiments were performed to confirm the previous study results of gene chips on some typical ribosome-related gene expression change after YZZ treatment on SMMC-7721 and Hep G2 cells.Identify their change and compare the change to normal liver cells,sceening 3?5 possible key gene to lucubrate.2.The roles of some ribosome-related genes in hepatoma cells.The gene silencing and overexpression of the selected genes were carried out by sh RNA and c DNA technology.The gene silencing and overexpression effects were verified by PCR and Western blot.The effect of gene silencing and overexpression on cell growth viability was observed by MTT.Cell cytometry was used to observe the regulation of gene silencing and overexpression on cell cycle and apoptosis;Western blot was used to detect the expression of other selected gene proteins expression after gene silencing.3.The mechanism of YZZ inhibiting malignant proliferation of hepatoma cells over ribosome-related genes.Key proteins in Mdm2-p53 pathway and MAPK signaling pathway refer to the above ribosomal proteins were detected by Western blot,key cell cycle protein were also detected and compared to cell cycle result via cytometry to identify partly approach and mechanism of YZZ.RESULTS1.After high concentration of YZZ(750?g/ml)treatment to 7721 and Hep G2 hepatoma cells,and the viability of cells was significantly inhibited.RTq PCR results showed that the m RNA level of RPS3 A,RPS8,RPS13,RPS16,RPS29,RPSA,RPL6,RPL10,RPL15,RPL17,RPL19 and RPLP0 was significantly down-regulated in both cell.There are exceptions in RPS26 and RPS6KA3.In Hep G2,RPS26 expression increased after treatment.RPS6KA3 expression increased in 7721 cells with dose increasing and decreased with dose increasing in Hep G2.RPSA and RPS26 were consistently down-regulated in 7721 and Hep G2 cells compared to L-02 normal liver cells.Therefore,we speculate that RPS6KA3,RPS26 and RPSA may play important roles in inhibiting the malignant proliferation of liver cancer cells.2.We silenced and overexpressed three RPS6KA3,RPS26 and RPSA genes in the two cells.MTT results showed that in 7721 cells,silencing RPS6KA3 inhibited cell proliferation,and its overexpression promoted cell proliferation;silencing RPS26 promoted cell proliferation,and its overexpression inhibited cell proliferation;RPSA overexpression promoted cell proliferation,while in Hep G2 cells silence RPSA promotes cell proliferation.No significant changes in cell cycle arrest and apoptosis were observed in each group.Although the MTT results showed a significant change in cell proliferation,but the range of the change was limited(<10%)and was inconsistent with the trend of YZZ treatment,we believe that the three genes RPS6KA3,RPS26 and RPSA are not direct targets of YZZ in inhibiting hepatoma cells proliferation.3.After YZZ treatment,while RPS26 and RPSA proteins were significantly downregulated,Mdm2 protein was also significantly down-regulated.In Hep G2 cells,p53 expression was activated and cells were induced to S phase arrest.In 7721 cells,p53 m RNA levels were significantly reduced and cells were induced to G2/M cycle arrest.Decrease in CCND2 and CREB protein expression was observed in both cells.CONCLUSIONS1.While YZZ inhibit the proliferation of hepatoma cells,the m RNA levels of ribosomal proteins including RPS3 A,RPS8,RPS13,RPS16,RPS29,RPSA,RPL6,RPL10,RPL15,RPL17,RPL19 and RPLP0 are decreased.2.Although RPS6KA3 promoted 7721 cell proliferation,RPS26 inhibited 7721 cell proliferation,and RPSA promoted 7721 cell proliferation inhibited Hep G2 cell proliferation,RPS6KA3,RPS26 and RPSA were not direct targets for YZZ inhibiting hepatoma cell proliferation.2.YZZ may regulate the m RNA and protein expression of ribosomal proteins and inhibit 7721 cells proliferation by inhibiting CREB and CCND2 expression,inhibiting Mdm2 and p53 protein expression,and inducing cell cycle arrest in G2 phase.3.YZZ may regulate the m RNA and protein expression of ribosomal proteins and inhibit Hep G2 cells proliferation by inhibiting CREB and CCND2 expression,inhibiting Mdm2 and activating p53,and inducing cells cycle arrest in S phase.