Gallic Acid Mediates Tumor-suppressive Effects On Osteosarcoma Through The H19-Wnt/β-catenin Regulatory Axis.

Objective1.To investigate the effects of the active molecule gallic acid of traditional Chinese medicine on osteosarcoma cell function and regulation of Wnt/β-catenin signaling pathway through the study of in vitro experiments.2.To clarify the specific effects of the active molecule gallic acid of Chinese medicine on osteosarcoma treatment in the in vivo environment through the study of in vivo experiments.3.To investigate the mechanism of Lnc RNA H19-mediated Wnt/β-catenin signaling pathway occurrence in osteosarcoma treatment with gallic acid through in vitro back-complementation experiments.Methods1.We examined the toxic effects of gallic acid on cells by MTT assay,and screened the best inhibitory concentration for cell viability of osteosarcoma cells by MTT assay;observed the effect of gallic acid on cell proliferation ability by cell cloning assay;examined the effect of gallic acid-induced cell cycle arrest of osteosarcoma cells by flow cytometry;examined the effect of gallic acid-promoted apoptosis of osteosarcoma cells by flow cytometry.The effect of gallic acid on promoting apoptosis in osteosarcoma cells was examined by flow cytometry.2.To explore the ability of gallic acid to inhibit osteosarcoma cell metastasis in vitroThe effect of gallic acid on the migration of osteosarcoma cells was examined by cell scratching assay;the effect of gallic acid on the invasion ability of osteosarcoma cells was examined by cell Transwell-Invasion assay.3.Exploration of gallic acid regulation of osteosarcoma-related RNA expressionThe expression of HOTTIP,H19,linc-ROR,TINCR and HULC were detected by RT-qPCR assay.4.Explore the mechanism of gallic acid-mediated wnt/β-catenin signaling pathway in osteosarcoma cellsTo explore the effect of gallic acid on luciferase activity in osteosarcoma cells by luciferase reporter assay;to explore the specific mechanism of gallic acid-mediated Wnt/β-catenin signaling in osteosarcoma cells by western bloting assay;to explore the expression of related genes downstream of gallic acid-regulated Wnt signaling pathway by RT-qPCR assay5.To explore the mechanism of action of gallic acid in regulating the growth and metastasis of osteosarcoma cells in an in situ animal model of osteosarcoma nude mice.The 143B osteosarcoma cells in logarithmic growth phase were injected into the right tibial bone marrow cavity of 4-week-old male nude mice to construct an in situ model of osteosarcoma,which were treated with gallic acid and saline by gavage.and β-catenin staining,and TUNEL staining to verify the value of gallic acid in treating osteosarcoma in the in vivo setting.6.Explore the molecular mechanism of gallic acid-mediated H19-Wnt/β-catenin signaling pathway in the treatment of osteosarcomaThe cell transfection experiment was used to construct an overexpressed H19 plasmid,and osteosarcoma 143B cells were infected by lentiviral vectors,and pH19 stable transfer cells were obtained after screening.The following experiments were carried out with osteosarcoma cells 143B pH19 as the experimental subject:(1)the effect of overexpression of H19 on the cell viability of osteosarcoma cells intervened with gallic acid was elucidated through MTT experiments;(2)Through luciferase detection experiments,the effect of overexpression of H19 on the inhibition of luciferase activity by gallic acid was explored;(3)Through the western bloting experiment,the effect of overexpression of H19 on the inhibition of gallic acid-induced β-catenin was explored;(4)Through qRT-PCR experiments,the effect of overexpression of H19 on gallic acid inhibiting the expression of related genes downstream of the Wnt signaling pathway was detected.7.To explore the effect of overexpression of H19 on gallic acid in inhibiting the metastasis function of osteosarcoma cellsThe following experiments were carried out with the stable transfer strain of osteosarcoma cells 143B pH19 as the experimental object:(1)the effect of pH19 on gallic acid inhibiting the migration function of osteosarcoma cells by cell scratch experiment;(2)The effect of pH19 on gallic acid inhibiting the invasion function of osteosarcoma cells was detected by cell invasion experiments.Results1.The results of MTT assay showed that gallic acid inhibited the growth and proliferation of human osteosarcoma cells 143B,MG63 and U20S.The results of cell clonal molding showed that gallic acid induced cycle arrest and promoted apoptosis of both human osteosarcoma cells 143B and U20S;2.the results of cell scratch assay and Transwell-Invasion assay showed that gallic acid could significantly inhibit the migration and invasion ability of the cells;3.The results of luciferase reporter assay showed that gallic acid significantly inhibited luciferase activity in osteosarcoma cells.Western bloting results showed that the total expression of β-catenin was reduced by gallic acid in 143B cells and remained consistent in both groups in U20S osteosarcoma cells.The expression of β-catenin intranuclear protein was decreased in both cells.4.The results of in vivo experiments with gallic acid showed that:1)Compared to the control group,the tumor growth rate started to decrease after the administration of gallic acid in the gallic acid group.The tumor size was smaller and the tumor weight was significantly lighter in the gallic acid administration group compared with the control group,which was statistically different;2)The HE staining results of the lung tissues in the control group showed that the mass area of the metastatic tumor lesions in the lung histopathological sections of the gallic acid administration group was smaller than that of the control group,and the number of tumor cells-stained with nuclei was smaller,which was statistically different;3)Compared with the immunohistochemical staining of the control group results,the expression of Ki67 and β-catenin in the in situ tumor tissues of the administered group showed a down-regulation trend.4)Comparing with the control group,TUNEL staining results showed that in the gallic acid administration group,apoptotic cells were significantly increased,and there were statistical differences between the two groups.5.The results of RT-qPCR experiments showed that Lnc RNA H19 was significantly down-regulated by gallic acid achieved in both 143B and U20S osteosarcoma cells.Gallic acid inhibited osteosarcoma cell growth through the H19-Wnt/β-catenin signaling pathway.The results of the molecular mechanism experiments showed that:1)The H19 overexpression plasmid was prepared by cell transfection experiment,and the pH19 stable transfection strain was constructed by lentiviral vector,and pH 19 was upregulated in osteosarcoma cells 143B by qRT-PCR assay.2)The results of the MTT assay showed that the overexpression of H19 played a role in attenuating the inhibition of osteosarcoma cell viability by gallic acid.3)The results of cell clone molding assay showed that overexpression of H19 played a role in attenuating the inhibition of osteosarcoma cell viability by gallic acid;4)Overexpression of H19 reduced the ability of gallic acid to inhibit luciferase activity in osteosarcoma cells.5)Overexpression of H19 partially reversed the down-regulation of Wnt downstream gene activity by gallic acid;6.Using overexpressed H19 osteosarcoma cells 143B as experimental subjects:1)The assay was performed using cell scratch assay,and the results showed that overexpressed H19 partially reversed the ability of gallic acid to inhibit the migration of osteosarcoma cells and overexpressed pH19 cells migrated over a larger area compared to the control group in pBabe.2)The experimental assay was completed using Transwell-Invasion assay,and the results showed that the overexpressed H19 partially reversed the invasive ability of gallic acid to inhibit osteosarcoma cells compared with that in the control pBabe,and more cells were successfully invaded.Conclusion1.Studies from in vitro experiments suggest that gallic acid does have the ability to inhibit cell viability,proliferative function,cell migration and invasion,induce cell cycle arrest and promote apoptosis in human osteosarcoma cells,and induce inactivation of Wnt/β-catenin signaling in osteosarcoma cells,possibly by disrupting the translocation of β-catenin from the cytoplasm to the nucleus,and leading to inhibitory nuclear aggregation for the function of inhibiting osteosarcoma cell growth;2.The in situ tumorigenic animal studies of tibial osteosarcoma in BABL/C nude mice showed that:1)Gallic acid could play a role in inhibiting the growth and metastasis of human osteosarcoma cells 143B;2)The inhibitory effect of gallic acid on the proliferation and metastasis of human osteosarcoma cells was elucidated in the in vivo environment;3)It was clarified that gallic acid in the in vivo environment possesses a role in down-regulating the Wnt/β-catenin signaling pathway and promoting the apoptosis of human The value of gallic acid in down-regulating Wnt/β-catenin signaling pathway and promoting apoptosis in human osteosarcoma cells was clarified.3.Gallic acid can inhibit the growth of osteosarcoma cells through the H19-Wnt/β-catenin signaling axis.Overexpression of H19 partially restored the downstream gene activity inhibited by gallic acid by reversing the expression level of β-catenin in the nucleus,reactivating the Wnt signaling pathway in osteosarcoma cells and thus promoting osteosarcoma cell growth.4.Overexpression of H19 possesses the effect of reversing the function of gallic acid in inhibiting the migration of osteosarcoma cells.