Effect Of Substrate Stiffness On The Phenotype And Epithelial-mesenchymal Transition Of Breast Cancer Cells MCF-7 Under Hypoxia Microenvironment

Objective: In this study, the microenvironment of human malignant breast cancer tissue was mimicked to explore the effect of hypoxia and substrate stiffness on cell mophology, cell viability and epithelial-mesenchymal transition(EMT) of breast cancer cell MCF-7.Methods: To mimic the stiffness range of malignant breast cancer tissue, polyacrylamide gels with different stiffness(0.5 kPa, 5 kPa and 20 kPa) were prepared. Cytoskeleton staining was applied to detect the change of cells morphology in substrates with different stiffness under normoxia and 1% hypoxia microenvironment. The cells viability was detected by live/dead staining and Hoechst staining. Western blot and immunofluorescence were applied to detect the expression of hypoxia inducible factor(HIF), EMT epithelial marker protein E-cadherin, mesenchymal marker protein Vimentin and transcription factor Snail 1. qPCR was applied to detect the expression of HIF-1a, E-cadherin, Vimentin, Snail 1, matrix metalloproteinase 2(MMP 2) and matrix metalloproteinase 9(MMP 9) genes.Results: Substrates with different stiffness have an important influence on the biological behavior of MCF-7 cells under normoxia and 1% hypoxia microenvironments.(1) The cytoskeleton staining results showed that: under normoxia and 1% hypoxia microenvironment, as the substrate stiffness increased, cells lost their round morphology, and transformed into irregular polygon morphology, while cells spreading area increased and cells circularity decreased.(2) The live/dead staining and Hoechst staining results showed that: substrates with different stiffness had no significant effects on cells viability under normoxia microenvironments. Under 1% hypoxia microenvironment, cells apoptosis was significantly increased on 20 kPa substrate. On 20 kPa substrate, cells apoptosis was significantly higher under 1% hypoxia microenvironment compared with normoxia microenvironment.(3) The results of western blot and immunofluorescence staining showed that: 1% hypoxia microenvironment could promote the expression of HIF-1a protein, reduce the expression of epithelial marker E-cadherin protein, and promote the expression of mesenchymal marker Vimentin protein and transcription factor Snail 1 on the same stiffness substrates in MCF-7 cells. In the same oxygen concentration environment, subtrate stiffness had no significant effects on E-cadherin and Vimentin expression, but promoted the expression of Snail 1 protein.(4) The results of qPCR showed that: on the same subtrate stiffness, 1% hypoxia microenvironment could promote the mRNAs expression of HIF-1a, mesenchymal maker Vimentin, transcription factor Snail 1, MMP 2 and MMP 9, reduce the mRNA expression of epithelial maker E-cadherin. Under 1% hypoxia microenvironment, as the substrate stiffness increased, the mRNA expression of E-cadherin had no significant difference; the increasing substrate stiffness enhanced mRNAs expression of transcripition factor Snail 1, MMP 2 and MMP 9. Conclusion: 1% hypoxia microenvironment could promote EMT of breast cancer cells MCF-7. Stiffer substrate(20 kPa) could induce breast cancer cell MCF-7 apoptosis, promote the loss of epithelial phenotype and obtain of mesenchymal phenotype, lead to occur of cells EMT. Results of this study have important significance to further explore the molecular machanisms of cells EMT on substrates with different stiffness under hypoxia microenvironment.