The Effects Of DLL4/Notch1Signal Pathway Regulated By FOXC2on Angiogenesis In Breast Cancer

Objective: To explore the effects of transcription factor FOXC2onangiogenesis of breast cancer MDA-MB-231cells and its mechanisms. Toclarify the role of FOXC2in promoting tumor angiogenesis is throughup-regulating of DLL4/Notch1signal pathway.Methods:(1) In vitro experiment: FOXC2-RNAi-LV and emptyvector LV were transfected into breast cancer cell line MDA-MB-231respectively to obtain stable FOXC2-interference MDA-MB-231cells. Theexperiment were devided into non-transfected group, empty-vector groupand FOXC2-interference group. Matrigel assay and Transwell chamber testwere used to observe the change of tube formation and migration ability ofhuman umbilical vein endothelial cells (HUVECs) of MDA-MB-231cellssupernatant in various groups. Western blot method were applied to detectthe expressions of DLL4, Notch1, CD31and MMP2protein.(2) In vivoexperiment: DLL4-RNAi-LV were transfected into breast cancer cell lineMDA-MB-231to obtain stable DLL4-interference MDA-MB-231cells.The experiment were divided into empty-vector group and DLL4- interference group. To establish transplantation modles of DLL4-interference and empty-vector MDA-MB-231cells in nude mice. Toobserve tumor growth, compare the time of tumorgenesis, tumor volumeand tumor weight in two groups and draw the tumor growth curve. Micewere killed after4weeks, The expression of DLL4, Notch1and CD34protein levels were tested by immunohistochemistry.Results:(1) In vitro experiment: RT-PCR and Western blot verifiedthat FOXC2-interference MDA-MB-231cell line was establishedsuccessfully. Matrigel angiogenesis method and Tanswell chamber testshowed that FOXC2interference decreased the number of tube formationand the migration number of HUVECs, compared with non-tranfectedgroup and empty-vector group(P<0.05). Western blot results showed thatthe expression of DLL4, Notch1, CD31and MMP2protein levels weredecreased in FOXC2-interference group(P<0.05).(2) In vivo experiment:The nude mice tumor model is successfully established, the growth oftumors, tumor volume and weight were decreased in DLL4-interferencegroup, compared with empty-vector group(P<0.05). Immunohistochemistryshowed that the expression of DLL4, Notch1and CD34protein levels weredecreased in DLL4-interference group(P<0.05).Conclusion: In vitro experiment showed that FOXC2interference inMDA-MB-231cells can decrease the tube formation ability and migrationability of HUVECs, and also can decrease the expression of DLL4, Notch1, CD31and MMP2protein levels. In vivo experiment verified that themechanism of FOXC2promoting angiogenesis in MDA-MB-231cells maybe related to Notch signaling pathway.