Establishment of twist-1 and TGFBR2 as direct targets of microRNA-20a in mesenchymal to epithelial transition of breast cancer cell-line MDA-MB-231.

Messenchymal to epithelial transition (MET) is a significant physiological phenomenon involved in embryogenesis and cancer. This study aims at investigating the mechanism of microRNA-20a (miR-20a) mediated regulation of mesenchymal to epithelial transition and identification of its direct target genes in breast cancer cell-line, MDA-MB-231. Reduced migratory and invasive property, altered cellular morphology along with reduced capability for attachment to basement membrane was acquired by over-expression of miR-20a in invasive MDA-MB-231 cell-line initially expressing low level of this micro-RNA, indicating direct correlation between abundance of miR-20a and metastatic property. The switch from mesenchymal to epithelial cells mediated by miR-20a involved post-transcriptional down-regulation of twist1, which in turn controls downstream epithelial markers like E-cadherin, claudin and mesenchymal markers like N-cadherin, fibronectin, the crucial players of mesenchymal to epithelial transition (MET). Furthermore, another key component, TGF-β and one of its receptors (TGFBR2) were found to be down-regulated by miR-20a. Additionally, reporter assay established that post-transcriptional down-regulation of TGFBR2 occurred through direct binding of miR-20a to its 3'UTR, thus abrogating the TGF-β signaling pathway resulting in inhibition of MET. Delineating the underlying molecular mechanism of miR-20a-mediated MET and defining the target genes will help us to introduce a miRNA-mediated effective therapeutic strategy against breast cancer.

De S ，Das S ，Mukherjee S ，Das S ，Sengupta Bandyopadhyay S ... -  《-》

Molecular mechanism of miR-153 inhibiting migration, invasion and epithelial-mesenchymal transition of breast cancer by regulating transforming growth factor beta (TGF-β) signaling pathway.

To investigate the role and mechanism of action of miR-153 in the migration, invasion, and epithelial-mesenchymal transition (EMT) of breast cancer cells. Quantitative real time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-153 and transforming growth factor beta receptor 2 (TGFBR2) in tissue specimens and cells. miR-153 overexpression in breast cancer cells was achieved by miR-153 mimic transfection. Mobility and invasiveness of breast cancer cells were evaluated by transwell assay. EMT was evaluated by Western blot detecting the protein level of E-cadherin and Vimentin. Interaction of miR-153 and 3'-untranslated region (UTR) of TGFBR2 messenger RNA (mRNA) was investigated by luciferase reporter assay. The expression of miR-153 in breast cancer tissue specimens and MDA-MB-231 cells was significantly lower than that in nonmalignant counterparts, inversely correlating with that of TGFBR2 mRNA. Transfection with miR-153 mimic significantly increased miR-153 level in MDA-MB-231 cells while inhibiting its migration, invasion, and EMT in vitro, which could be mimicked by TGFBR2 knockdown. Luciferase reporter assay confirmed two targets of miR-153 on the 3'-UTR of TGFBR2 mRNA. Restoring TGFBR2 protein level by transient overexpression largely rescued migration, invasion, and EMT of MDA-MB-231 cells that were repressed by miR-153 mimic transfection. miR-153 inhibits breast cancer cell migration, invasion, and EMT by targeting TGFBR2.

Wang J ，Liang S ，Duan X 《-》

miR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer.

Yu J ，Xie F ，Bao X ，Chen W ，Xu Q ... -  《Molecular Cancer》

FGF2 inhibits endothelial-mesenchymal transition through microRNA-20a-mediated repression of canonical TGF-β signaling.

Endothelial-to-mesenchymal transition (EndMT) is characterized by the loss of endothelial cell markers and functions, and coincides with de novo expression of mesenchymal markers. EndMT is induced by TGFβ1 and changes endothelial microRNA expression. We found that miR-20a is decreased during EndMT, and that ectopic expression of miR-20a inhibits EndMT induction. TGFβ1 induces cellular hypertrophy in human umbilical vein endothelial cells and abrogates VE-cadherin expression, reduces endothelial sprouting capacity and induces the expression of the mesenchymal marker SM22α (also known as TAGLN). We identified ALK5 (also known as TGFBR1), TGFBR2 and SARA (also known as ZFYVE9) as direct miR-20a targets. Expression of miR-20a mimics abrogate the endothelial responsiveness to TGFβ1, by decreasing ALK5, TGFBR2 and SARA, and inhibit EndMT, as indicated by the maintenance of VE-cadherin expression, the ability of the cells to sprout and the absence of SM22α expression. FGF2 increases miR-20a expression and inhibits EndMT in TGFβ1-stimulated endothelial cells. In summary, FGF2 controls endothelial TGFβ1 signaling by regulating ALK5, TGFBR2 and SARA expression through miR-20a. Loss of FGF2 signaling combined with a TGFβ1 challenge reduces miR-20a levels and increases endothelial responsiveness to TGFβ1 through elevated receptor complex levels and activation of Smad2 and Smad3, which culminates in EndMT.

Correia AC ，Moonen JR ，Brinker MG ，Krenning G ... -  《-》

Sensitization of Gastric Cancer Cells to 5-FU by MicroRNA-204 Through Targeting the TGFBR2-Mediated Epithelial to Mesenchymal Transition.

Gastric cancer (GC) is the most common gastrointestinal malignancy, causing cancer-related deaths in East Asia. MicroRNAs (miRNAs) are small non-coding RNAs aberrantly expressed in human tumors. In this study, we aim to investigate the roles of miR-204 in the epithelial to mesenchymal transition (EMT)-associated chemosensitivity. The expression of miR-204 was detected in clinical tumor samples and GC cell lines by real time PCR. Tumor cell's growth, invasion, and migration were measured by MTT assay, wound healing assay, and transwell invasion assay, respectively. Western blot method was used to detect the protein levels of indicated genes. Luciferase reporter assay was performed to validate the target gene of miR-204. The in vivo role of miR-204 was measured using a xenograft mouse model of GC. By comparing the expressions of miR-204 in human gastric tumors and their adjacent normal tissues, it was disclosed that miR-204 was significantly downregulated in gastric tumors. Moreover, miR-204 was downregulated in multiple GC cell lines compared with normal gastric epithelial cells. Overexpression of miR-204 suppressed GC cells' proliferation, invasion, and migration. It is noteworthy that 5-FU treatments induced miR-204 expression and suppressed TGF-β pathway. By establishment of 5-FU resistant GC cell line, it was revealed that miR-204 was significantly downregulated in 5-FU resistant GC cells, representing mesenchymal features with downregulation of epithelial marker, while mesenchymal markers were upregulated. We identified TGFBR2 as a direct target of miR-204 by Western blot method and luciferase assay in GC cells and tumor samples as well. In addition, overexpression of miR-204 sensitized GC cells to 5-FU in vitro. Xenograft experiments demonstrated that the combination of miR-204 and 5-FU efficiently inhibited tumor growth and improved survival rate of mice as well. Eventually, we illustrated the restoration of TGFBR2 in miR-204 overexpression GC cells, which recovered resistance to 5-FU treatments compared with miR-204 overexpression GC cells. This study describes a miRNA-based therapeutic strategy against 5-FU resistance in GC, contributing to the development of anti-chemoresistance therapeutic agents.

Li LQ ，Pan D ，Chen Q ，Zhang SW ，Xie DY ，Zheng XL ，Chen H ... -  《-》