The Effects Of Angiopoietin On The Proliferation, Invasion And Angiogenesis Of Cervical Cancer

Objective: Angiopoietins and their receptors are mainly expressed in endothelial cells. However, our previous immunohistochemistry(IHC) assays showed that angiopoietins(Ang1 and Ang2) and their receptor Tie2 were also over-expressed in cervical cancer cells. The present study was aimed to investigate the effects of angiopoieteins on the proliferation, invasion and angiogenesis of cervical cancer.Methods:1. Immunocytochemistry and quantitative real-time PCR were used to examine the expression of Ang1, Ang2, Tie1 and Tie2 in cervical cancer cell lines(Si Ha, Hela, and C33A) as well as normal cervical epithelia.2. Human recombinant Ang1(rh Ang1) and Ang2(rh Ang2) were used to elevate angiopoietins in culture supernatent; si Ang1 and si Ang2 were used to down-regulate expression of angiopietins in Hela cells.3. EDU cell proliferation assays, colony forming assays and Ki-67 staining were used to assess the alteration in proliferation ability of cervical cancer cells.4. Scratch tests and transwell assays were used to assess the alteration in migration ability of cervical cancer cells.5. Transwell assays were used to assess the alteration in invasion ability of cervical cancer cells.6. Animal experiments(1) Twenty-four Balb/c nude mice with subcutaneous cervical cancer xenografts were randomly divided into four groups:(1) 5% glucose(control group);(2) 5% glucose plus in vivo-jet PEI(transfection reagent group);(3) in vivo-jet PEI and si Ang1(si Ang1 group);(4) in vivo-jet PEI and si Ang2(si Ang2 group). All agents were intratumorally injected. The tumor diameters were measured every 2 days, then tumor volumes were calculated and tumor growth curves were established.(2) Immunohistochemistry was used to examine the expression of Ang1, Ang2 and Vimentin in the xenografts.(3) The amount and integrity of blood vessels in the xenografts were assessed by immunofluorescence of Endomucin and α-SMA.Results:1. The expression of Ang1, Ang2, Tie1, and Tie2 in cervical cancer cell lines were significantly increased when compared with normal cervical epithelia(P<0.01).2. The migration and invasion but not proliferation ability of cervical cancer cells were enhanced by stimulation with rh Ang1 or rh Ang2(P<0.05).3. The migration and invasion but not proliferation ability of Hela cell were decreased after down-regulation of Ang1(P<0.05); the migration but not invasion and proliferation ability was decreased(P<0.05) after down-regulation of Ang2.4. In vivo, compared with the control group, tumor growth was significantly inhibited by administration of si Ang1 and si Ang2(P<0.01).5. Immunohistochemistry showed a significant decrease in the mesenchymal marker vimentin in the groups transfected with si Ang1 and si Ang2 compared with the control group.6. In the si Ang1 group and the si Ang2 group, the blood vessel counts were decreased compared to the control group, while the difference in α-SMA was not significant.Conclusion: Ang1 and Ang2 promote the migration, invasion and angiogenesis of cervical cancer and might be promising therapeutic targets.