Function and Mechanism of RhoA in Hypoxia-induced Angiogenesis of Breast Cancer
|School||Fourth Military Medical University|
|Keywords||RhoA Hypoxia Breast cancer cells Endothelial cells Neovascularization p53 HIF-1α VEGF|
Breast cancer is the highest incidence of malignant tumors of women around the world. Due to the abnormal proliferation of breast cancer cells, the distant organ transfer ability, as well as resistance to existing drugs, making it the number one killer of serious harm to the health of women. Although the early diagnosis of breast cancer, treatment and prognosis detection level is rising, but the mortality rate of patients with breast cancer is still no effective control. Anti-angiogenic therapy as a tumor biological treatment means more and more attention, it can not only cut off the nutrient supply of primary tumor, and can control distant metastasis by inhibiting angiogenesis. However, tumor angiogenesis research is relatively small, its molecular mechanism is not fully understood, to find significant angiogenesis pathway upstream switch molecules. RhoA as a small G protein family, involved in tumor metastasis, invasion, proliferation, apoptosis and cell cycle regulation, and other aspects, and plays the role of molecular switches in many signaling pathways. However, the mechanism of action of RhoA in tumor angiogenesis is no clear conclusion. Hypoxia as a stress factor throughout the entire process of tumor development. Hypoxia can affect not only the transcriptional expression of tumor-related factors, and provide a \Well, RhoA whether hypoxia-induced tumor angiogenesis play a role in it? Our ability RhoA to start regulating angiogenesis signaling pathway? With this question, we conducted a RhoA in the hypoxia-induced breast cancer freshmen function and its mechanism of angiogenesis research. Research Methods and Results: 1, RhoA expression analysis in breast cancer cell lines and malignant behavior of tumor cells: (1) seven RhoA expression in breast cancer cell lines were detected by RT-PCR and western blot ; (2) transfection against human RhoA small interfering RNA interference efficiency, western blot and RT-PCR validation; (3) by the MTT assay, monolayer cells scratches experiments, cell cycle and apoptosis detection experiments the observation siRhoA on MCF-7 cell proliferation, migration, cell cycle and apoptosis; (4) nude mice injected siRhoA experimental and immune histochemical analysis to observe siRhoA tumor growth and VEGF expression. Results: RhoA differences in breast cancer cell lines express this expression differences in cell invasion and metastasis; siRhoA can effectively reverse the malignant behavior of tumor cells, and in vitro tumor injection can play the same inhibitory and inhibiting vascular role. 2, RhoA biological functions in the formation of hypoxia-induced breast cancer neovascularization analysis: (1) ELISA detection hypoxic conditions, MCF-7 cells, RhoA VEGF secretion levels; (2) the establishment of MCF-7 / HUVEC co-culture model, observed in the hypoxic stimulation of RhoA activity changes in MCF-7 cells HUVEC proliferation, migration, the impact of the lumen and the formation of F-actin. Results: Under hypoxic conditions, the activity of RhoA can promote MCF-7 cell secretion of VEGF silencing RhoA can inhibit the secretion of VEGF; in the co-culture model, when activated RhoA in MCF-7 cells, can promote HUVEC proliferation, migration, lumen and the formation of F-actin, RhoA silencing in MCF-7 cells, the biological behavior of HUVEC was inhibited. 3, RhoA neovascularization in hypoxia-induced breast cancer molecular mechanisms: (1) detected by western blot regulation of hypoxia on RhoA, p53, HIF-1α and VEGF protein levels and of RhoA these three molecules role; (2) confocal microscope RhoA on p53 nuclear translocation in the absence of oxygen; (3) western blot and immune coprecipitation detection RhoA regulation of p53 signaling pathway. Results: Hypoxia can inhibit the expression of p53, promoting of RhoA, HIF-1α and VEGF expression; able to inhibit the expression of p53, promote HIF-1α and VEGF expression of RhoA activation and RhoA silencing p53 expression is upregulated down HIF-1α and VEGF expression; under hypoxic conditions, activation of RhoA can promote p53 into the nucleus, silence RhoA p53 into nuclear significantly weakened; Akt/MDM2 and ubiquitin - proteasome pathway may be involved in RhoA on the molecular mechanisms of hypoxia-induced tumor angiogenesis. Conclusions: (1) RhoA differentially expressed in seven breast cancer cell lines, which may transfer and cell invasion ability; (2) siRhoA can reverse the malignant behavior of tumor cells transplanted tumor injection can inhibit tumor growth and VEGF expression; (3) under hypoxia, RhoA possibly through the regulation of the expression of VEGF and indirectly affect HUVEC cell proliferation, migration, lumen and F-actin forming ability, which participate in the formation of tumor angiogenesis process; (4) under hypoxic conditions, RhoA vascular suppressor p53 regulation affecting angiogenic factors HIF-1α and VEGF expression involved in tumor angiogenesis. This mechanism may be realized through Akt/MDM2 ubiquitin - proteasome pathway.