Function And Mechanism Of RhoA In Hypoxia-induced Angiogenesis Of Breast Cancer

Breast cancer is the most common female malignant tumor. Because of the abnormal proliferation, the metastasis to distant organ and the drug resistance, breast cancer has become the top killer for women health. The early diagnosis, treatment and prognosis of breast cancer have been improved, but the mortality isn't being controlled effectively. As one of cancer biological treatments, antiangiogenic therapy has been concerned. It not only cuts off the nutrition supply for cnacer, but also prevents cancer metastasis by inhibiting angiogenesis. However, nowadays the studies about tumore angiogenesis are few and the mechanisms remain to be unknown. So it is very crucial to discover the mechanism of angiogenesis.As one of the small G protein, RhoA plays the molecular switch on the tumor metastasis, invasion, proliferation, apoptosis and cell cycle. However, the role of RhoA in angiogenesis is still unclear. Hypoxia, a stress factor, anticipates the whole progression of tumor. Hypoxia can promote transcription of tumor-assiociated factors and supply a basic condition for angiogenesis. However, whether RhoA plays an important role in hypoxia-induced angiogenesis and regulates the angiogenic or antiangiogenic factors, we designed this study and investigated to the function and mechanism of RhoA in hypoxia-induced angiogenesis of breast cancer.Methods and results: Firstly, the expression and function of RhoA in the breast cancer cells: (1) The expressions of RhoA in the seven breast cancer cells was investigated by RT-PCR and western blot; (2) After transfection RhoA small interference RNA, the interference efficiency was examined by western blot and RT-PCR. (3) The effect of siRhoA on MCF-7 cells was investigated by MTT assay, cell wound healing assay, cell cycle and apoptosis assay. (4) Suppression of tumor growth and angiogenesis in nude mice model was observed by intratumoral injection siRhoA. Result: The expressions of RhoA in the breast cancer cells have difference which was closely related with metastasis and invasion of cells. siRhoA could reverse the malignant biological behavior of breast cancer cells and intratumoral injection siRhoA also inhibited the tumor growth and VEGF expression.Secondly, the biological funcation of RhoA in hypoxia-induced angiogenesis of breast cancer: (1) under hypoxia the regulation of RhoA on VEGF in the MCF-7 cells was examined by ELISA assay. (2) MCF-7/HUVEC co-culture model was established and under hypoxia the regulation of RhoA in the MCF-7 cells on HUVEC proliferation, migration, and tube and F-actin formations was investigated. Result: under hypoxia active RhoA promoted VEGF secretion of MCF-7 cells, but shRhoA could inhibit it. In the co-culture model, active RhoA in the MCF-7 cells enhanced the HUVEC proliferation, migration, and tube and F-actin formations, but knock down of RhoA could inhibit it. Thirdly, the molecular mechanism of RhoA in hypoxia-induced angiogenesis of breast cancer: (1) The effect of hypoxia on the expressions of RhoA,p53,HIF-1αand VEGF was detected by western blot. (2) Under hypoxia the effect of RhoA on p53 nuclear translocation was observed by laser scanning confocal microscope. (3) The signal pathways of the regulation of RhoA on p53 were explored under hypoxia. Result: Hypoxia could decrease the p53 expression and increase RhoA,HIF-1αand VEGF expressions. Moreover, under hypoxia active RhoA inhibit p53 expression and promote HIF-1αand VEGF expressions, but knock down of RhoA increased the p53 expression and decreased the HIF-1αand VEGF expressions. And under hypoxia active RhoA could enhance the p53 into nuclear, but knock down of RhoA promoted the p53 out of nuclear. At last the molecular mechanism of RhoA in hypoxia-induced angiogenesis may be involved in Akt/MDM2 and ubiquitin-proteasome pathways.Conclusion: (1) The expressions of RhoA in the breast cancer cells have difference which was closely related with metastasis and invasion of cells. (2) The knock down of RhoA could reverse the malignant biological behavior of breast cancer cells and also inhibit the tumor growth and angiogenesis. (3) Under hypoxic condition, RhoA indirectly influenced HUVEC to affect tumor angiogenesis through regulating VEGF level of MCF-7 cells. (4) Under hypoxia RhoA regulated p53 expression to affect HIF-1αand VEGF expressions and anticipate tumor angiogenesis. Importantly, this molecular mechanism may be accomplished through Akt/MDM2 and ubiquitin-proteasome pathways.