The microRNA miR-29a is upregulated in mesenchymal cells and promotes epithelial-to-mesenchymal transition and metastasis. (A) Northern blot analysis using total RNA extracted from confluent epithelial EpRas or mesenchymal RasXT cells was performed to monitor the expression levels of miR-29a. miR-31 and U6-snRNA were used as controls. The numbers indicate normalization by densitometry. (B) EpRas cells were transfected with miR-29a followed by injection into the tail vein of nude mice. Mice were killed after 14 days and lung metastases were counted. (C) EpRas cells were transfected with miR-29a, and after 48 h proliferation was measured. miR, microRNA; miR-scr, microRNA-scrambled; snRNA, small nuclear RNA.

The microRNA miR-29a is overexpressed and tristetraprolin is downregulated in metastatic human breast cancers. (A) Total RNA was extracted from patient samples with invasive breast carcinomas (red) or non-invasive hyperplasias (green). Levels of miR-29a were determined in a padlock assay followed by detection with a ³²P-labelled probe. Results are shown in a box–whisker plot. (B) The level of TTP mRNA was determined by qPCR, and expression of GAPDH was used as normalization control. (C) RT–qPCR of total RNA extracts from MDA-MB-468 cells transfected with LNA-scr or LNA-29a for 72 h was performed to monitor the expression of TTP. LNA, locked nucleic acid; mRNA, messenger RNA; RT–qPCR, reverse transcription–quantitative PCR; scr, scrambled; TTP, tristetraprolin.

The microRNA miR-29a downregulates tristetraprolin. (A) Total RNA was extracted from EpRas and RasXT cells. The levels of TTP as normalized to GAPDH were determined by qPCR. (B) Western blot analysis using whole-cell extracts of EpRas and RasXT cells was performed to monitor the expression of TTP protein. (C) NIH3T3 cells were transfected with miR-scr or miR-29a. Constructs containing the luciferase ORF coupled to the WT and mutated (mut.) versions of the TTP-3′-UTR were used to assess the regulatory impact of miR-29a on TTP. Relative light units (RLUs) are shown. (D) EpRas cells were transfected with miR-scr or miR-29a, and RasXT cells were transfected with LNA-scr or LNA complementary to miR-29a (LNA-29a). Constructs containing the luciferase ORF coupled to the TTP-3′-UTR were used to assess the regulatory impact of endogenous miR-29a on TTP. RLUs are shown. (E) Western blot analysis using whole-cell extracts of EpRas cells and EpRas cells overexpressing miR-29a was performed to monitor the expression of TTP. (F) Western blot analysis using whole-cell extracts of RasXT cells and RasXT cells transfected with LNA-29a was performed to monitor the expression of TTP. LNA, locked nucleic acid; miR-scr, microRNA-scrambled; ORF, open reading frame; qPCR, quantitative PCR; TTP, tristetraprolin; UTR, untranslated region; WT, wild type.

Downregulation of tristetraprolin promotes epithelial-to-mesenchymal transition and metastasis. (A) The levels of miR-29a, TTP, Mcl1, E-cadherin and vimentin were determined by qPCR in EpRas cells stably overexpressing scrambled miRNA or miR-29a (three independent clones are shown), and levels in RasXT cells are shown as a control. (B) Levels of TTP, E-cadherin and vimentin were determined by qPCR in EpRas cells stably overexpressing scrambled siRNA or siTTP (three independent clones are shown). (C) Western blot analysis was performed to monitor the expression of E-cadherin and vimentin using whole-cell extracts of the indicated cell types. vTTP is a TTP variant resistant to siRNA-mediated degradation. (D) EpRas cells stably expressing an shRNA harbouring either miR-scr, miR-29a, siTTP or siTTP+vTTP were cultured on porous supports. Immunofluorescence stainings for E-cadherin, vimentin and F-actin are shown. (E) EpRas cells were transiently transfected with si-scr or siTTP followed by injection into the tail vein of nude mice. Mice were killed after 14 days and lung metastases were counted. qPCR, quantitative PCR; scr, scrambled; shRNA, short hairpin RNA; si, short interfering; TTP, tristetraprolin.