miR-219-5p targets TBXT and inhibits breast cancer cell EMT and cell migration and invasion

Abstract

miR-219-5p has been reported to act as either a tumor suppressor or a tumor promoter in different cancers by targeting different genes. In the present study, we demonstrated that miR-219-5p negatively regulated the expression of TBXT, a known epithelial-mesenchymal transition (EMT) inducer, by directly binding to TBXT 3'-untranslated region. As a result of its inhibition on TBXT expression, miR-219-5p suppressed EMT and cell migration and invasion in breast cancer cells. The re-introduction of TBXT in miR-219-5p overexpressing cells decreased the inhibitory effects of miR-219 on EMT and cell migration and invasion. Moreover, miR-219-5p decreased breast cancer stem cell (CSC) marker genes expression and reduced the mammosphere forming capability of cells. Overall, our study highlighted that TBXT is a novel target of miR-219-5p. By suppressing TBXT, miR-219-5p plays an important role in EMT and cell migration and invasion of breast cancer cells.

Keywords: EMT; TBXT; cell invasion; cell migration; miRNA.

Figure 1. TBXT is a novel target of miR-219-5p

(A) Partial sequence of human TBXT UTR containing miR-219-5p binding site; sequence of mature miR-219-5p; partial sequence of the generated mutant UTR of human TBXT in complementary sites for seed regions in miR-219-5p. (B) Relative luciferase activity of the luciferase reporter gene that a human TBXT 3′-UTR fragment containing the wildtype miR-219-5p binding sequence (WILD) or the mutant miR-219-5p binding sequence (MUTANT) was cloned downstream of it. The luciferase activity was analyzed in 293 T cells that were transfected with either miR-219-5p or the control vector (mock). The data are presented as means ± SD of three independent experiments, and statistical significance was determined by two-tailed, unpaired Student’s t test. (C) The expression level of miR-219-5p in mock cells and miR-219-5p overexpression cells. The expression of TBXT at mRNA level (D), and at protein level (E) in mock cells and in miR-219-5p overexpression cells. The expression of miR-219-5p (F) and TBXT (G) in miR-219-5p knockdown cells. The expression of miR-219-5p (H) and TBXT (I) in mammalian epithelium cells and breast cancer cells.

Figure 2. The inhibition on the breast cancer cell EMT and cell migration and invasion by miR-219-5p is mediated by TBXT

(A) Cell viability of the three cell lines; MDA-MB-231 mock cells, MDA-MB-231 overexpressing miR-219-5p and MDA-MB-231 overexpressing miR-219-5p plus TBXT. (B,C) Immunoblotting of TBXT, E-cadherin, N-cadherin, vimentin in above three cell lines. Representative images and statistical results of Transwell migration assays (D) and Matrigel invasion assays (F) in the three cell lines. (E) Representative images and statistical results of Transwell migration assays and Matrigel invasion assays (G) of miR-219-5p knockdown cells and control cells. The data are presented as means ± SD of three independent experiments, and statistical significance was determined by two-tailed, unpaired Student’s t test. The quantification result shown on each graph represents the statistical analysis of three experiments.

Figure 3. The inhibition of miR-219-5p on breast cancer cell migration andinvasion is not relevant to hormone receptor and Her2 status in cells

(A) Representative images and statistical results of Transwell migration and Matrigel invasion assays of MCF-7 and MCF-7 overexpressing miR-219-5p cells. (B) Representative images and statistical results of Transwell migration and Matrigel invasion assays of BT474 and BT474 overexpressing miR-219-5p cells. The data are presented as means ± SD of three independent experiments, and statistical significance was determined by two-tailed, unpaired Student’s t test. The quantification result shown on each graph represents the statistical analysis of three experiments.

Figure 4. miR-219-5p diminishes the CSC features of MDA-MB-231 cells

(A) qPCR and (B) immunoblotting of Nanog in MDA-MB-231 mock cells, miR-219-5p overexpression MDA-MB-231 cells and miR-219-5p overexpression MDA-MB-231 that was introduced with TBXT. (C) qPCR of Aldh1 in the above three cell lines. (D) Quantification of mammosphere formation by the above three cell lines. The data are presented as means ± SD of three independent experiments, and statistical significance was determined by two-tailed, unpaired Student’s t test. The quantification result shown on each graph represents the statistical analysis of three experiments.