| Object:To investigate the effect of artesunate on angiogenesis and angiogenic mimetic(VM)formation in choroidal melanoma and its effect on the hypoxia-inducible factor 1(HIF-1α)/vascular endothelial growth factor(VEGF)/platelet-derived growth factor(PDGF)pathway,providing new clues for angiogenesis-targeted therapy in choroidal melanoma.Methods:VM formation and positive expression of VEGF and PDGF proteins around the lumen of the VM were detected by immunohistochemical assays in waxed sections of choroidal melanoma tissue.PDGF proteins were positively expressed around the VM lumen.The choroidal melanoma cell line(OCM-1)was used as an in vitro model of tumor proliferation in a hypoxic workstation to investigate the process of neovascularisation and angiogenesis mimicry in tumors under hypoxic conditions and its molecular mechanisms.Firstly,the optimal hypoxia time and hypoxia concentration of OCM-1 cell line were examined by CCK8 assay;the changes of DNA synthesis level in OCM-1 cells were verified by Ed U assay;the changes of HIF-1α/VEGF/PDGF factor expression level in OCM-1 cells under hypoxic conditions were examined by Western blot assay.The changes in the expression levels of HIF-1α/VEGF/PDGF factors in OCM-1 cells under hypoxic conditions were detected by Western blot.The effect of VEGF and PDGF interference on the proliferation and angiogenesis of OCM-1 cells was further verified by silencing VEGF and PDGF genes separately or in combination with RNA interference.HIF-1α expression was induced in hypoxic environment,and its effects on VEGF and PDGF gene expression in VEGF and PDGF interfered and normal OCM-1 cells were investigated,as well as their effects on angiogenesis and angiogenic mimetic formation in OCM-1 cells.Changes in cell function and expression of HIF-1/VEGF/PDGF factors were investigated after artesunate was applied to OCM-1 cells.The effect of artesunate on the expression of HIF-1/VEGF/PDGF factors in each experimental group was investigated in hypoxic culture,and functional and in vitro tumourigenic assays were used to verify the role of artesunate in the growth and angiogenic mimetic formation of choroidal melanoma cells.Results:1.VM formation was observed in paraffin slices of choroidal melanoma tissue with positive expression of VEGF and PDGF in the periphery.2.The optimal hypoxic concentration for OCM-1 cell line to promote tumour proliferation was detected at 1% in CCK8 assay,and the optimal hypoxic time was 6-9h in Edu assay.3.The expression of HIF-1α/VEGF/PDGF in tumour cells after 6-9 h of hypoxia treatment was significantly higher than that in normal control OCM-1 cell lines(P≤0.05),and the expression of VEGF and PDGF was positively correlated with that of HIF-1α,while no HIF-1α was expressed in normoxic cultured cells.4.The tumour cells appeared tubular under hypoxic conditions for 12-24 h,whereas under normoxic conditions the cells only appeared to proliferate irregularly.5.Lentiviral knockdown of VEGF and PDGF gene expression in OCM-1 cell lines was successfully demonstrated by real-time fluorescence quantitative PCR and Western-Blot.6.The knockdown of VEGF and PDGF genes and the combined knockdown group significantly inhibited the scratch healing ability,cell invasion ability and migration ability of OCM-1 cells(p≤0.05).Cells in the combined knockout group migrated7.Matrigel tube formation assay showed that lentiviral knockdown of VEGF and PDGF genes inhibited the tube formation ability of OCM-1 cells under hypoxic conditions.8.Under hypoxic conditions,the number of cells crossing the transwell after 9 h of hypoxia was 80;knockdown of VEGF and PDGF genes also occurred but the number of cells crossing the transwell was minimal,The difference is statistically significant.This indicates that hypoxia can stimulate the proliferation and invasion ability of OCM-1 cells.9.Western blot showed that the protein expression of the three genes HIF-1α/VEGF/PDGF was significantly higher in the si VEGF,si PDGF and si VEGF+PDGF groups under hypoxic conditions compared to normoxic conditions.10.The optimal concentration of artesunate in tumour cells in the CCK8 assay was IC50=40μΜ.Microscopic concentration of 40 μΜ artesunate significantly reduced the proliferation capacity and 3D tumourigenic capacity of the cells.11.The expression of the three factors HIF-1α/VEGF/ PDGF was significantly lower in the artesunate group than in the control group under anoxic conditions,and the difference was statistically significant.Conclusion:1.VEGF and PDGF proteins are highly expressed in choroidal melanoma,and HIF-1α protein expression is elevated under hypoxic conditions and transcriptionally regulates a significant increase in VEGF/PDGF protein expression.2.Silencing of VEGF and PDGF genes reduced both the tube-forming and invasive ability of choroidal melanoma.After hypoxia induction,the silencing effect was attenuated,and VEGF and PDGF expression appeared again with enhanced invasive ability,suggesting that HIF-1α may be the upstream gene that initiates VEGF and PDGF gene expression,where PDGF may be a downstream factor of VEGF,and silencing VEGF and PDGF genes alone may not play a good role in tumor suppression.3.Artesunate acting on choroidal melanoma can inhibit the expression of HIF-1α as well as VEGF and PDGF genes thus inhibiting tumor growth and angiogenic mimicry,which will provide a new direction for the clinical treatment of choroidal melanoma... |
