Downregulation of the MicroRNA-200 family induces epithelial to mesenchymal transition.

Epithelial to mesenchymal transition (EMT) is a reversible developmental morphogenesis associated with cancer invasion and metastasis. Essential for the transition is the downregulation of E-cadherin, an adherens junction protein whose transcription is repressed by E-box binding proteins, such as Snail, Slug, Twist, ZEB1, and ZEB2. MicroRNA microarrays showed the miR-200 family and miR-205 are dramatically downregulated during TGF-β- and Pez-induced EMT (Gregory et al. 2008). A search for possible targets of these miRNAs using TargetScan (Grimson et al. 2007) revealed multiple putative miR-200 and miR-205 binding sites in the 3’ untranslated regions (UTRs) of ZEB1 and ZEB2. We hypothesised that ZEB1 and ZEB2 are negatively regulated by miR-200 and miR-205 at the post-transcriptional level. To test this, the ZEB1 and ZEB2 3’UTRs were amplified from human genomic DNA, cloned downstream of a renilla luciferase (RL) reporter then transfected into the Pez-induced model of EMT in Madin-Darby canine kidney (MDCK) cells (Wyatt et al. 2007). Expression of RL-ZEB1/2 was over five-fold lower in epithelial MDCK cells that had high endogenous miR-200 and miR-205 levels compared to mesenchymal MDCK-Pez cells, which were devoid of the miRNAs. Co-transfection of the reporters with synthetic miRNAs (Pre-miR-200a, -200b, -205) into MDCK-Pez cells inhibited RL-ZEB1/2. Conversely, co-transfection of the reporters with antisense inhibitors to the miRNAs (Anti-miR-200a, -200b, -205) into MDCK cells augmented expression of RL-ZEB1/2. Mutagenesis of all miR-200b sites in the ZEB1 and ZEB2 3’UTRs effectively relieved inhibition by endogenous and ectopic miR-200b. These results established miR-200 and miR-205 as negative regulators of ZEB1 and ZEB2. Next, we sought to determine whether miR-200 participates in cancer invasion in vivo. Previous studies reported EMT-like changes, including upregulation of ZEB1 and ZEB2 and loss of E-cadherin, at the invasive front of carcinomas (Aigner et al. 2007a; Brabletz et al. 2001; Hlubek et al. 2007a; Spaderna et al. 2006). Since loss of the miR-200 family is an essential step in the progression of EMT, we predicted these miRNAs would be downregulated at the invasive front of carcinomas where EMT takes place. A method for in situ hybridisation of miRNAs in formalin-fixed paraffin-embedded tissues was developed, and along with immunofluorescence and immunohistochemistry, was used to assess expression of miR-200 and EMT markers in human colorectal adenocarcinomas. Laser capture microdissection and qPCR were used to quantify and compare levels of miR-200 in normal colon epithelium, the tumour core, and the invasive front. Expression of miR-200 was significantly reduced at the invasive front of a subset of colorectal adenocarcinomas displaying degraded basement membranes and markers of EMT in leading edge malignant cells. In contrast, miR-200 expression was maintained across normal epithelium and in carcinomas with intact basement membranes, where no signs of EMT were evident. In regional lymph node and vascular metastases, miR-200 expression was strong, suggesting miR-200 is required to recapitulate the primary tumour phenotype at the metastatic site. These data implicate the miR-200 family in the progression of invasion and metastasis, and add further support to the roles of EMT and MET in the metastasis cascade.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201