Rationale:: Endothelial cells have the ability to undergo endothelial–mesenchymal transitions (EndMTs), by which they acquire a mesenchymal phenotype and stem cell–like characteristics. We previously found that EndMTs occurred in the endothelium deficient in matrix γ-carboxyglutamic acid protein enabling endothelial cells to contribute cells to vascular calcification. However, the mechanism responsible for initiating EndMTs is not fully understood. Objective:: To determine the role of specific serine proteases and sex determining region Y-box 2 (Sox2) in the initiation of EndMTs. Methods and Results:: In this study, we used in vivo and in vitro models of vascular calcification to demonstrate that serine proteases and Sox2 are essential for the initiation of EndMTs in matrix γ-carboxyglutamic acid protein–deficient endothelium. We showed that expression of a group of specific serine proteases was highly induced in endothelial cells at sites of vascular calcification in Mgp null aortas. Treatment with serine protease inhibitors decreased both stem cell marker expression and vascular calcification. In human aortic endothelial cells, this group of serine proteases also induced EndMTs, and the activation of proteases was mediated by Sox2. Knockdown of the serine proteases or Sox2 diminished EndMTs and calcification. Endothelial-specific deletion of Sox2 decreased expression of stem cell markers and aortic calcification in matrix γ-carboxyglutamic acid protein–deficient mice. Conclusions:: Our results suggest that Sox2-mediated activation of specific serine proteases is essential for initiating EndMTs, and thus, may provide new therapeutic targets for treating vascular calcification.

Results from recent studies suggest that aberrant microRNA expression is common in numerous cancers. Although miR-338-3p has been implicated in hepatocellular carcinoma, its role in gastric cancer is unknown. To this end, we report that miR-338-3p is downregulated in both gastric cancer tissue and cell lines. Forced expression of miR-338-3p inhibited cell proliferation and clonogenicity and induced a G₁–S arrest as well as apoptosis in gastric cancer cells. Furthermore, P-Rex2a (PREX2) was identified as a direct target of miR-338-3p, and silencing P-Rex2a resulted in the same biologic effects of miR-338-3p expression in gastric cancer cells. Furthermore, both enforced expression of miR-338-3p or silencing of P-Rex2a resulted in activation of PTEN, leading to a decline in AKT phosphorylation. Also, miR-338-3p markedly inhibited the in vivo tumorigenicity in a nude mouse xenograft model system. These results demonstrate that miR-338-3p affects gastric cancer progression through PTEN—AKT signaling by targeting P-Rex2a in gastric cancer cells, which posits miR-338-3p as a novel strategy for gastric cancer treatment. Implications: miR-338-3p acts as a novel tumor suppressor that blocks the growth of gastric cancer cells through PTEN—PI3K signaling by targeting P-Rex2a. Mol Cancer Res; 12(3); 313–21. ©2013 AACR.

Background: MicroRNAs are regulators that can play an essential role in tumorigenesis. Although miR-302 families have been suggested to be tumor repressors in human cancer, the mechanism by which they suppress tumor development remains to be defined. In this study, we discover that miR302b suppresses tumor proliferation may due to directly targeting EGFR in human hepatocellular carcinoma (HCC). Methods: QRT-PCR was used to assess miR-302b and EGFR expression in 27 pairs of clinical hepatocellular carcinoma tissues and their corresponding adjacent nontumorous liver tissues. MTT, colony formation, immunofluorescence staining, and cell cycle assays were used to examine the tumor suppressor role of miR302b in cell proliferation. Luciferase assays were performed to assess the EGFR was a novel target of miR-302b. Western blot assay was used to validate the protein expression level. Results: We demonstrated that miR-302b was frequently down-regulated, whereas EGFR was up-regulated in 27 pairs of clinical HCC and non-tumorous counterparts. The dual-luciferase reporter assays revealed that EGFR was a novel target of miR-302b. Re-expression of miR-302b resulted in the inhibition of proliferation in hepatocellular carcinoma SMMC-7721 cells. The silencing of EGFR by miR-302b or siEGFR led to down-regulation of proliferation-related proteins, such as AKT2, CCND1, and CDK2. Conclusion: miR-302b suppresses HCC growth may due to targeting the EGFR/AKT2/CCND1 pathway.

Cerebral arteriovenous malformations (AVMs) are common vascular malformations, which may result in hemorrhagic strokes and neurological deficits. Bone morphogenetic protein (BMP) and Notch signaling are both involved in the development of cerebral AVMs, but the cross-talk between the two signaling pathways is poorly understood. Here, we show that deficiency of matrix Gla protein (MGP), a BMP inhibitor, causes induction of Notch ligands, dysregulation of endothelial differentiation, and the development of cerebral AVMs in MGP null (Mgp^−/−) mice. Increased BMP activity due to the lack of MGP induces expression of the activin receptor-like kinase 1, a BMP type I receptor, in cerebrovascular endothelium. Subsequent activation of activin receptor-like kinase 1 enhances expression of Notch ligands Jagged 1 and 2, which increases Notch activity and alters the expression of Ephrin B2 and Ephrin receptor B4, arterial and venous endothelial markers, respectively. Reducing the expression of Jagged 1 and 2 in the Mgp^−/− mice by crossing them with Jagged 1 or 2 deficient mice reduces Notch activity, normalizes endothelial differentiation, and prevents cerebral AVMs, but not pulmonary or renal AVMs. Our results suggest that Notch signaling mediates and can modulate changes in BMP signaling that lead to cerebral AVMs. Significance Cerebral arteriovenous malformations (AVMs) are common vascular abnormalities that may lead to strokes. Signaling by bone morphogenetic proteins (BMPs) and Notch play important roles in the formation of cerebral AVMs, but the cross-talk between the pathways is poorly understood. We report that gene deletion of matrix Gla protein (MGP), a BMP inhibitor, causes cerebral AVMs in mice by activating activin receptor-like kinase 1, a BMP receptor. This activation enhances Notch activity and disrupts endothelial cell differentiation by inducing the Notch ligands Jagged 1 and 2. Reducing Jagged 1 and 2 expression prevents the disruption in differentiation and AVM formation. The findings suggest that MGP maintains the balance between BMP and Notch signaling and promotes a normal brain vasculature.

It is well established that transcriptional silencing of critical tumor suppressor genes by DNA methylation is a fundamental process in the initiation of lung cancer. However, the involvement of microRNAs (miRNAs) in restoring abnormal DNA methylation patterns in lung cancer is not well understood. Therefore, and since miRNA-101 is complementary to the 3′-untranslated region of DNA methyltransferase 3A (DNMT3A), we investigated whether miRNA-101 could restore normal DNA methylation patterns in lung cancer cell lines. Bioinformatics has indicated that DNMT3A is a major target of miR-101. In addition, the overexpression of miR-101 downregulates DNMT3A. Using a methylation-specific polymerase chain reaction assay, we demonstrated that methylation of the phosphatase and tensin homolog (PTEN) promoter was reduced in A549 cells transfected with miR-101, but not in the transfected control. Furthermore, overexpression of miR-101 and silencing of DNMT3A suppressed lung cell proliferation and S/G2 transition, and increased apoptosis through the PTEN/AKT pathway in vitro. Furthermore, we observed the opposite phenomenon in A549 cells transfected with a miR-101 inhibitor. Subsequent investigation revealed that overexpression of miR-101 significantly inhibited the tumorigenicity of A549 cells in a nude mouse xenograft model. These results demonstrate that miR-101 affects lung cancer progression through the PTEN/AKT signaling pathway by targeting DNMT3A in lung cells, suggesting that miR-101 may be a novel potential therapeutic strategy in lung cancer treatment.