Andrographolide Inhibits Angiogenesis By Inhibiting The MiR-21/TIMP3 Signaling Pathway

Objective:Recent studies have shown the importance of angiogenesis in tumor growth.As well as anti-angiogenesis is important in the treatment of anti-tumor.Andrographolide(Andro)and its derivatives have extensive pharmacological effects such as anti-inflammatory,anti-infection,anti-tumor.Its role in anti-angiogenesis has gradually attracted attention,however,its direct effect on angiogenesis still needs to be clarified.In this study,we will explore the role of Andro in the inhibition of angiogenesis and vascular endothelial cell migration,and investgate the possible role of miR-21 in the signaling pathways that inhibit angiogenesis.In this work,we have demonstrated the molecular mechanism of Andro inhibition of angiogenesis,which may be used in the clinic to improve outcomes for cancer patients due to the anti-proliferative,anti-metastatic,anti-angiogenic and pro-apoptotic therapeutic potential.Method:Chicken embryo chorioallantoic membrane model(CAM),yolk sac membrane model(YSM)and rat aortic rings in vitro angiogenesis assay were amployed to determine whether Andro has inhibitory effects on angiogenesis.To detecte whether Andro can significantly inhibit the growth of transplanted tumors and reduce the microvessel density of the transplanted tumor tissue,the CAM xenograft breast cancer was used as model.The effects of Andro on cell viability of HUVEC cell was detected by Cell Counting Kit-8.In vitro tube formation assays and transwell migration assays detecte whether Andro has inhibitory effects on tube formation and cell migration in HUVEC cells.Real-time PCR was used to detect the microRNA-21 transcription level in HUVEC cell.Bioinformatics predictions found that 3’UTR ofTIMP3 exists the target binding site for miR-21,and the dual luciferase reporter system verified it.The effect of Andro on TIMP3 and the downstream protein in HUVEC cells were detected by Western Blotting.The experiments of miR-21 overexpression and the interference of TIMP3 on HUVEC cells were used to validate the regulatory network that Andro inhibit angiogenesis.Result:The result of CAM,YSM and rat aortic rings in vitro angiogenesis assay showed that Andro has inhibitory effect on angiogenesis.The CAM xenograft breast cancer assay showed that the tumor volume and microvessel density was significantly reduced with Andro treatment.The activity,angiogenesis and migration of HUVEC were also inhibited by Andro treatment,which were shown by Cell Counting Kit-8,in vitro tube formation assays and transwell migration assays.Result of qRT-PCR showed that Andro reduced the miR-21 expression in HUVEC cells.Bioinformatics prediction found that TIMP3 3′UTR exists the target binding site of miR-21 which was verified by dual luciferase reporter gene system.Western Blotting showed that Andro could upregulate the expression of TIMP3 in HUVEC cells and decreased the expression of matrix metalloprotein 9(MMP9)interacting with it.In addition,the expression of VEGFR2,which was related to the proliferation ability and migration ability of HUVEC cell,was similarly decreased.After Andro treatment the HUVEC cell which have overexpressed miR-21 or suppressed TIMP3,the results of tube formation experiments,transwell migration experiments,and Western Blotting confirmed that Andro inhibits angiogenesis by inhibiting the miR-21/TIMP3/MMP9 pathway.Conclusion:Andro has inhibitory effect on angiogenesis by decreasing the expression of miR-21,and then increase its target protein TIMP3,decrease the expression of MMP9.