Regulation And Maintenance Of CAF Activation By Stromal MiR-200s Contribute To ECM Remodeling And Breast Cancer Cell Invasion

Chapter 1 Stromal miR-200 s contribute to breast cancer cell invasion through CAF activation and ECM remodelingObjective: The chapter was to explore how stromal miR-200 s make CAFs become activated and stimulate the dysregulation of ECM proteins,thus influencing cancer cell invasion.Methods: 20 paired primary CAFs and NFs were isolated from breast cancer patients to test miR-200 s expression. NFs and cancer cells were co-cultured to analyze the decreased miR-200 s expression. Luciferase reporter assay was used to detected whether Fli-1 and TCF12 were directly targets of miR-200 s. For CAF activation, miR-200 s and their targets(Fli-1and TCF12) engineered NF or CAF cell lines were constructed to explore the expression of CAF activation markers, migration and invasion, and collagen-remodeling ability. MDA-MB-231 cells were plated on the surface of ECM remodeled by the miR-200 s engineered fibroblasts, the invasion of cancer cells were detected by H&E staining. A total of 2× 106MDA-MB-231 cells mixed with 2× 106 CAF/Ctrl or engineered CAF cells in 200 μl PBS: Matrigel at a 1:1 ratio were subcutaneously were subcutaneously injected into 4-week-old female nude mice, Lung metastasis and ECM remodeling were analyzed by picrosirius red(PR)staining.Results: We found that miR-200 s, which are generally decreased in activated CAFs in breast cancer tissues and normal fibroblasts(NFs)activated by breast cancer cells, played a direct mediator role in the reprograming of NFs into CAFs and in ECM remodeling. NFs with downregulated miR-200 s displayed traits of activated CAFs, including accelerated migration and invasion. Ectopic expression of miR-200 s in CAFs at least partially restored the phenotype and features of NFs. The process of CAF activation may be governed by the targets of miR-200 s,Fli-1 and TCF12, which are responsible for cell development and differentiation and were obviously elevated in CAFs. Furthermore,miR-200 s and their targets influenced the collagen contraction of CAFs.miR-200 s directly or indirectly regulated LOX expression through the target Fli-1, and miR200 s indirectly induced FN expression through TCF12.These phenomena contributed to ECM remodeling, fueling the invasion and metastasis of breast cancer cells both in vitro and vivo.Conclusion: The current studies establish a link between the loss of miR-200 s in breast cancer CAFs and the acquisition of activated CAFsproperties, and provide an important and novel insight into breast CAF activation and ECM remodeling, their induction by miR-200 families and their targets, Fli-1 and TCF12, which triggers tumor cell invasion.Chapter 2 Reciprocal regulation between Dnmt3 b and miR-200 s maintains CAF activationObjective: The chapter was to explore how stromal miR-200 s make CAFs become activated and stimulate the dysregulation of ECM proteins,thus influencing cancer cell invasion.Methods:(1) qRT-PCR was used to detect miR-200 s expression in CAF treated with 5’-aza.(2) Western Blot and IF were used to analyze methyltransferase expression in 3 paired NF and CAF cells.(3) qRT-PCR was used to test miR-200 s expression in Dnmt3 b knockdown CAFs.(4)qRT-PCR 、 Western Blot and Luciferase reporter assay were used to determine whether Dnmt3 b is a target of miR-200 s.(5) Treat NFs with TGF-b1, qRT-PCR was used to test miR-200 s expression, and Western Blot to detect Dnmt3 b, TCF12 and Fli-1 expression.Results: After treatment with 5’-aza, miR-200 s expression was upregulated in CAFs.(2) Western Blot and IF assay showed Dnmt3 b was up-expressed in CAFs.(3) miR-200 s were up-regulated in Dnmt3 b silenced CAFs.(4) Dnmt3 b was a direct target of miR-200b/200 c.(5) miR-200 s were down-regulated in NFs treated with TGF-b1, and kept lower expression in those treated NFs after TGF-b1 was deprived. At the same time, TGF-b1 led to up-regulation of Dnmt3 b, TCF12 and Fli-1 in NFs.Interestingly, TGF-b1 was partially down-regulated in Dnmt3 b silenced CAFs.Conclusion:(1) Dnmt3 b may down-regulate miR-200 s expression through methylation.(2) The reciprocal loop between Dnmt3 b and miR-200 s regulates and maintain CAF activation.(3) TGF-b1 regulates the interaction of Dnmt3 b and miR-200 s.(4) There is a loop between TGF-b1and Dnmt3 b.