The Function And Mechanism Of MiR-494 And Lysosome Alteration Involved In Snail Induced EMT In Breast Cancer

The conception of EMT is first proposed in the research of development,descripting embryos epithelial cells transformed into mesenchymal cells.Recent study shows process of EMT is also observed in many kinds of cancers.The epithelial-mesenchymal transition(EMT)is considered to be the first step of metastasis,with cell junctions broken,polarity lost,degradation of cell adhesion,cytoskeleton changes,enhanced cell motility and stemness.Snail is the main regulator of EMT,and it can trigger and maintain EMT.Our work are based of the observation in Snail induced EMT.In the first part,we established an EMT cell model MCF-10A-NC/MCF-10A-6SA by stable expression of Snail-6SA in MCF-10 A.After screening from miRNA array and realtime RT-PCR validation,miR-494 is selected for the further functional study in breast cancer.In our study,we find that miR-494 acting as a tumor suppressor,decreased in human breast cancer specimens as well as breast cancer cell lines.Ectopic expression of miR-494 in basallike breast cancer cell lines inhibits cell proliferation,colony formation and migration.Moreover,ectopic expression of miR-494 suppressed neoplasm initiation as well as pulmaonary metastasis in vivo.Furthermore we have identified PAK1,a target gene of miR-494,contributes to the function of miR-494.Remarkably,the expression of PAK1 is inversely correlated with the expression of miR-494 in human breast cancer samples.Furthermore,re-expression of PAK1 partially reverses miR-494 mediated proliferation and clonogenic inhibition as well as migration and invasion suppression in breast cancer cells.Taken together,these findings highlight an important role for miR-494 in the regulation of progression and metastatic potential of breast cancer and suggest a potential application of miR-494 in breast cancer treatment.In the second part,we firstly observed that housekeeping gene GAPDH is upregulated in protein level in Snail overexpressed MCF-10A-6SA cells.And then we find GAPDH degradation organelle lysosome is decreased in MCF-10A-6SA cells.Furthermore,we use TGF-β as well as Snail phosphorylate inhibitor to stabilize the endogenous expression of Snail,and find that endogenous upregulated Snail also induces lysosome alteration.Moreover,we find TFEB,a main regulator of lysosome synthesis,is downregulated in Snail overexpressed cell lines.And promoter luciferase assays show that Snail represses TFEB transcription.