Effects And Mechanism Of MiR-21on Breast Cancer Stem Cell Characteristics And Epithelial-mesenchymal Transition

INTRODUCTIONMiR-21(microRNA-21) is one of the first miRNAs (microRNAs)detected in the human genome, which also is one of the miRNAs known tobe up-regulated in all types of human malignancy. There is abundantevidence suggest that miR-21is aberrantly over-expressed and function asan oncogenic miRNA which involved in all the known processes of breastcancer including cancer cell growth, migration and invasion. However,central molecular mechanisms of miR-21contribute to cancer cellmigration and invasion ability still need to be investigated.Cancer stem cells (CSCs) are predicted to be the cell origin of thetumor and responsible for tumor progression, relapse and metastasis due totheir CSC characteristics (eg. self-renewal capacity and limitlessproliferative potential, as well as invasion and migration capacity).Previously studies have been suggested that breast cancer contains aCSC-compartment, which can be enriched by purifying Aldehyde dehydrogenase1-positive (ALDH1+; ALDHbright) cells or CD44+/CD24-/lowcells by sorting, and also by purifying sphereforming cells (mammospheres)from parental cells.Epithelial-mesenchymal transition (EMT) is also associated withincreased aggressiveness and metastasis in breast cancer, as it allows cellsto migrate and invade surrounding issues and escape into the bloodstream,en route to establishing metastasis. On the molecular level, cellsundergoing EMT towards a more mesenchymal phenotype involves loss orlowered the expression of epithelial markers such as E-cadherin, andincreased the expression of mesenchymal markers such as N-cadherin,Vimentin and α-SMA. There are studies suggested that EMT in breastcancer is tightly linked to the triple negative (ER-negative, PR-negative andHER-2-negative) basal-like phenotype breast cancer subgroup and CSCs.Recently, miR-21has been shown to be associated with CSCcharacteristics and EMT phenotype in the progression, including cellinvasion and migration, of breast cancer. These results strongly suggest thatmiR-21plays a key role in breast cancer progression and metastasis mayinvolve in acquisition of CSC characteristics and EMT process, and theunderlying mechanisms remain unclear. In this study, we demonstrated theeffect and mechanism of miR-21on CSC characteristics and EMT processin the process of migration and invasion, in breast cancer cells. Part I Effects of miR-21on EMT phenotype and CSCcharacteristicsObjective: To explore the effects of miR-21on CSC characteristics andEMT phenotype in breast cancer cells.Methods: We established MCF-7/miR-21cells by transfected hsa-miR-21mimics into breast cancer MCF-7cells, and MDA-MB-231/anti-miR-21cells by transfected hsa-miR-21antagomir into breast cancerMDA-MB-231cells. CSC characteristics were measured using the changesof CSC surface markers (ALDH1and CD44), and the capacity ofsphereforming (mammospheres); EMT was evaluated by the changes ofmesenchymal cell markers (N-cadherin, Vimentin, and α-SMA), epithelialcell marker (E-cadherin), as well as capacities of cell migration andinvasion.Results: Our data showed that re-expression of miR-21induced theacquisition of EMT phenotype by activation of mesenchymal cell markers(N-cadherin, Vimentin, α-SMA) and inhibition of epithelial cell marker(E-cadherin) in MCF-7/miR-21cells, which consistent with increased cellsubpopulation expressing CSC surface markers (ALDH1+andCD44+/CD24-/low) and the capacity of sphereforming (mammospheres). Onthe contrary, antagonism of miR-21reversed EMT and CSC phenotype inMDA-MB-231/anti-miR-21cells. Conclusion: We demonstrated that miR-21is responsible for migrationand invasion by activating the EMT process and enhancing thecharacteristics of CSCs in breast cancer cells, from the pros and cons of thematter.Part II Effects of miR-21on EMT phenotype inthird-sphereforming breast cancer stem cell-like cellsObjective: To culture CSC-like cells, and explore the effects of miR-21onEMT phenotype in CSC-like cells.Methods: Breast cancer mammospheres, including first-sphereforming(1-S), second-sphereforming (2-S), third-sphereforming (3-S), andforth-sphereforming (4-S), were cultured and isolated from MCF-7cells byserum-free suspended culture, and high-purity bCSC-like cell model wasselected. Then the effects of miR-21on the high-purity CSC-like cellbiological features, especially EMT, were evaluated.Results:3-S cells was understood to be a high-purity bCSC-like cell model,showed higher CSC surface markers (ALDH1+and CD44+/CD24-/lowphenotype), sphereforming capacity, as well as migration and invasioncapacities. EMT, which was assessed by over-expression of mesenchymalcell markers (N-cadherin, Vimentin, α-SMA) and suppression of epithelialcell marker (E-cadherin), was induced in3-S CSC-like cells. Moreover, both of HIF-1α and miR-21were up-regulated in the CSC-like cells.Interestingly, antagonism of miR-21by antagomir led to reversal of EMT,down-expression of HIF-1α, as well as suppression of invasion andmigration.Conclusion: Our results demonstrated that the formation of CSC-like cellsundergoing process of EMT-like associated with over-expression ofHIF-1α, both of which are regulated by miR-21.Part III The mechanism of miR-21mediates CSC characteristicsand EMT phenotypeObjective: To explore the underlying mechanism and relative signalingmolecules in miR-21mediating EMT and CSC phenotype.Methods: In this study, MDA-MB-231/anti-miR-21cells were establishedby transfected hsa-miR-21antagomir into basal-like phenotype breastcancer MDA-MB-231cells；down-regulate PTEN by siPTEN; inhibit AKTand ERK1/2pathways by LY294002and U0126, respectively. EMT wasevaluated by the changes of mesenchymal cell markers (N-cadherin,Vimentin, and α-SMA), epithelial cell marker (E-cadherin), as well ascapacities of cell migration and invasion; CSC phenotype was measuredusing the changes of CSC surface markers (ALDH1and CD44), and thecapacity of sphereforming (mammospheres). Results: We found that antagonism of miR-21contributed to PTENup-regulation and AKT/ERK1/2inactivation. Interestingly,down-regulation of PTEN by siPTEN suppressed the effects of miR-21antagomir on EMT and CSC phenotype, confirming that PTEN is a targetof miR-21in reversing EMT and CSC phenotype. Moreover, LY294002(the inhibitors of AKT pathway) and U0126(the inhibitors of ERK1/2pathway), both of which significantly suppressed EMT and CSC phenotype,indicating that AKT and ERK1/2pathways are require for miR-21mediating EMT and CSC phenotype.Conclusion: In conclusion, our results demonstrated that antagonism ofmiR-21reverses EMT and CSC phenotype through targeting PTEN, viainactivation of AKT and ERK1/2pathways, and showed a novelmechanism of which might relieve the maligment biological behavior oftriple negative basal-like phenotype breast cancer.