Breast Cancer

lincRNA-ROR[LncRNA]

miR-205[miRNA]

ZEB1[mRNA]

Mcf7, Bt474, Mda-Mb-435, Mda-Mb-231, Bt549 And Mda-Mb-436

Mus musculus (mouse)

Cell Death Dis 2014 Jun 12 5, e1287 doi:10.1038/cddis.2014.249 PMID:24922071

LincRNA-ROR induces epithelial-to-mesenchymal transition and contributes to breast cancer tumorigenesis and metastasis

Western blot;qRT-PCR;luciferase reporter assays

we used a psiCHECK2 reporter plasmid to test the relationship between linc-ROR and miRNAs. We cloned the linc-ROR full-length transcript or ZEB2 3'-UTR (untranslated region) into the 3'-UTR of Renilla luciferase (Rluc) gene of psiCHECK2. The psiCHECK2 plasmid also contains a downstream constitutively expressed firefly luciferase gene as an internal control for normalization. We then showed that transient co-transfection of mir-205-5p mimics into HEK293T cells inhibited the Rluc activity in a dose-dependent manner , while the mir-34a-5p and let-7a-5p mimics had little influence on Rluc activity of psiCHECK2-ROR . To determine whether linc-ROR acts as a sponge to mir-205, we transfected psiCHECK2-ZEB2 into HEK293T cells together with the mir-205-5p mimics and increasing amounts of linc-ROR, and we detected that the Rluc activity increased in response to linc-ROR in a dose-dependent manner . Together, these results strongly point to a role of linc-ROR as a molecular sponge for mir-205, and presumably linc-ROR may regulate EMT program through preventing degradation of mir-205 targets (ZEB1 and ZEB2). This was in accordance with the ZEB1 and ZEB2 expression levels after linc-ROR/mir-205 overexpression or silencing. Our data also showed that several ZEB1/2-related miRNAs such as mir-205 and mir-200 family members were downregulated in linc-ROR-overexpressing cells. Thus, we conclude that lin-ROR functions as a molecular sponge for mir-205 to affect mir-205 targets in the process of EMT.