Inhibitory Effect And Molecular Mechanism Of Radix Tetrastingma Hemesleyani Flavone On Hepatocellular Carcinoma Based On MiRNA Regulation

Objective The study investigated the inhibitory effect and mechanism of radix tetrastingma hemesleyani flavone（RTHF）on hepatocellular carcinoma,which provided new ideas for further study on the gene regulation mechanism of RTHF’s anti-hepatoma activity.Methods RTHF were extracted and purified from radix tetrastigma hemsleyani（RTH）produced in Zhejiang Province,and further detected the main active substances.Human hepatoellular carcinomas 2（HepG2）cells were used as target cells to detect the cell proliferation inhibition by CCK-8 assay.The cell cycle and apoptosis were analyzed by flow cytometry,and the cell invasion inhibition rate was observed by Transwell chamber.The kit detects intracellular Ca²⁺ concentration.The miRNA-seq technology was used to detect the differentially expressed miRNA in HepG2 cells.Two target gene prediction software,TargetScan and miRanda,were used to predict the known dysregulated expressed mirRNA online,and the target genes were analyzed by GO and KEGG.qRT-PCR was used to further verify the expression levels of candidate dysregulated expressed miRNA.For a further study,miR-4792 with significant dysregulated expression was selected as the next research target,and transfected cells were divided into four groups,miRNA NC（control group）,miR-4792 mimics（miR-4792 mimics）,RTHF+miRNA NC（miR-4792 was added to RTHF）,RTHF+miR-4792 inhibitor（RTHF was added after miR-4792 was inhibited）.After 48h,the situation of cell proliferation inhibition,cell invasion inhibition,cell cycle arrest and apoptosis,and intracellular Ca²⁺ concentration in different treatment groups were detected by corresponding methods.Then the target gene prediction tools,miRanda,miRTarBase and miRDB,were used to predict the potential target genes of miR-4792,and GO and KEGG analysis were performed on the predicted target genes.Finally,the protein expressions of cells were detected by Western blotting.Results Six monomers namely kaempferol,kaempferol-3-O-neohesperidin,malvacin-3-β-glucoside chloride,myricetin,icariin I and isocaptarin were found by HPLC-MS/MS analysis.The results of CCK-8 assay showed that RTHF concentration of 4μg/mL significantly inhibited the proliferation of HepG2 cells in a dose-time dependent manner.Flow cytometry showed that RTHF at 2-3 μg/mL significantly induced apoptosis and inhibited invasion of HepG2 cells,and the intracellular Ca²⁺ concentration also increased significantly.The miRNA-seq technique was used to sequence the expression of miRNA in HepG2 cells treated with 3 μg/mL RTHF for 48h.The changes of miRNA expression profiles were compared and analyzed.A total of 184 miRNA were found to be dysregulated expressed,and the number of up-regulated and down-regulated mirRNA was 49 and 54.A total of 5526 target genes with known dysregulated expression of miRNA were obtained by prediction,which were enriched in 1371 GO entries and 32 signaling pathways.The expression trend of candidate miRNA expression levels detected by qRT-PCR was similar to that of miRNA-Seq,which verified the reliability of the data.Compared with the control group,the proliferation and invasion ability of miR-4792 mimics group and RTHF+miRNA NC group were significantly decreased,and the proportion of G1 phase,apoptosis level and intracellular Ca²⁺ concentration were significantly increased;Compared with RTHF+miRNA NC group,cell proliferation ability and cell invasion ability of RTHF+miR-4792 inhibitor group were significantly increased,G1 phase ratio,apoptosis level and intracellular Ca²⁺ concentration were significantly decreased.A total of 92 miR-4792 target genes with common intersection were obtained by the three tools,which were enriched in 655 GO entries and 31 signaling pathways.Western blotting results showed that the expressions of FOXC1 and MAPK pathway key proteins in miR-4792 mimics group and RTHF+miRNA-NC group were down-regulated,which were significantly lower than those in miRNA-NC and RTHF+mmiR-4792 inhibitor group.Conclusion Dysregulated mirRNA may play an important role in proliferation inhibition,cycle arrest,invasion inhibition and apoptosis induction in HepG2 cells by regulating the expression of hepatocellular carcinom cell-associated genes.miR-4792,as an inhibitor in HepG2 cell tumors,targets FOXC1 and some apoptosis-related proteins,may be one of the important molecular mechanisms by which RTHF inhibits HepG2 cells.