Pulmonary artery smooth muscle cells (PASMCs) are associated with the development of hypoxic pulmonary hypertension (HPH). Recent studies have implicated a critical role for microRNAs (miRNAs) in HPH; however, their expression and regulation in hypoxia‐mediated phenotypic modulation of PASMCs remains largely unclear. Here, we report that miR‐9 was induced in hypoxia and involved in a hypoxia‐induced phenotypic switch in rat primary PASMCs. Knockdown of miR‐9 followed by hypoxia exposure attenuated PASMCs proliferation and enhanced the expression of contractile genes in vascular smooth muscle cells (VSMCs), while overexpression of miR‐9 in normoxia promoted a proliferative phenotype in PASMCs. The primary transcripts of miR‐9‐1 and miR‐9‐3, but not miR‐9‐2, increased dramatically after hypoxia, whereas silencing of the hypoxia‐associated transcription factor HIF‐1α following hypoxia exposure abolished the enhancement of both primary transcripts in PASMCs. Using in silico analysis, we found three putative HIF‐1α binding motifs on miR‐9‐1 and one motif on miR‐9‐3 located within the 5‐kb region upstream of the transcriptional start sites. Chromatin immunoprecipitation assay revealed that hypoxia enhanced the direct interaction between HIF‐1α and the regulatory elements of miR‐9‐1 and miR‐9‐3. Reporter assays showed that the regulatory regions of miR‐9‐1 and miR‐9‐3 behaved as enhancers in a HIF‐1α‐dependent manner during hypoxia. Taken together, our data uncover a regulatory mechanism involving HIF‐1α‐mediated up‐regulation of miR‐9, which plays a role in the hypoxia‐induced phenotypic switch of PASMCs. J. Cell. Physiol. 229: 1511–1520, 2014. © 2014 Wiley Periodicals, Inc.

Recent clinical study indicated that up-regulation of miR-146b was associated with poor overall survival of patients in esophageal squamous cell carcinoma. However, the underlying mechanism of miR-146b dysregulation remains to be explored. Here we report that miR-146b promotes cell proliferation and inhibits cell apoptosis in esophageal cancer cell lines. Mechanismly, two C/EBPβ binding motifs are located in the miR-146b promoter conserved region. Among the three isoforms of C/EBPβ, C/EBPβ LAP2 positively regulated miR-146b expression and increases miR-146b levels in a dose-dependent manner through transcription activation of miR-146b gene. Together, these results suggest a miR-146b regulatory mechanism involving C/EBPβ, which may contribute to the up-regulation of miR-146b in esophageal squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) is a developmental program, which is associated with esophageal squamous cell carcinoma (ESCC) progression and metastasis. Recently, C/EBPβ is reported to be an EMT inducer in cancer. However, the detailed molecular mechanisms remain unclear. Here we report for the first time, that the truncated C/EBPβ LIP isoform was abnormally overexpressed and correlated with cancer metastasis in clinical specimens of human ESCC. Furthermore, we demonstrate that C/EBPβ LIP mediates epithelial growth factor (EGF) - induced EMT and increases migration and invasion of esophageal cancer cells dependent on a miR-203 inactivation. Finally, we identified miR-203 as a direct target of C/EBPβ LIP. Disruption of C/EBPβ LIP attenuated the EGF-mediated decrease in miR-203, whereas overexpression of C/EBPβ LIP alone markedly suppressed miR-203. In addition, we demonstrated that C/EBPβ LIP inhibited miR-203 transcription by directly interacting with a conserved distal regulatory element upstream of the miR-203 locus, and in doing so, orchestrated chromatin remodeling. In conclusion, our results have revealed a novel regulatory mechanism that involves C/EBPβ LIP-mediated down-regulation of miR-203, which plays a key role in EMT and metastasis.

ABSTRACT Epithelial–mesenchymal transition (EMT) is a developmental program that is associated with esophageal squamous cell carcinoma (ESCC) progression and metastasis. Recently, C/EBP&bgr; has been reported to be an EMT inducer in cancer. However, the detailed molecular mechanisms remain unclear. Here, we report for the first time, that the truncated CCAAT-enhancer-binding protein &bgr; (C/EBP&bgr;) LIP isoform is abnormally overexpressed and correlated with cancer metastasis in clinical specimens of human ESCC. Furthermore, we demonstrate that C/EBP&bgr; LIP mediates epithelial growth factor (EGF)-induced EMT and increases migration and invasion of esophageal cancer cells in a manner that is dependent on miR-203 inactivation. Finally, we identified miR-203 as a direct target of C/EBP&bgr; LIP. Disruption of C/EBP&bgr; LIP attenuated the EGF-mediated decrease in miR-203, whereas overexpression of C/EBP&bgr; LIP alone markedly suppressed miR-203. In addition, we demonstrated that C/EBP&bgr; LIP inhibited miR-203 transcription by directly interacting with a conserved distal regulatory element upstream of the miR-203 locus, and in doing so, orchestrated chromatin remodeling. In conclusion, our results have revealed a new regulatory mechanism that involves C/EBP&bgr;-LIP-mediated downregulation of miR-203, which plays a key role in EMT and metastasis.

Polymorphisms in 3' untranslated region (UTR) of cancer-related genes might affect regulation by microRNA (miRNA) and contribute to carcinogenesis. In this study, we screened several single nucleotide polymorphisms (SNPs) in 3'UTR of cancer-related genes and investigated their effects on the risk of esophageal squamous cell carcinoma (ESCC). First, we used SNaPshot assay to genotype seven 3'UTR SNPs in 537 ESCC cases and 608 normal controls in a Chinese Han population and found that SNP rs6573 in 3'UTR of RAS-related proteins (RAP1A) was significantly associated with ESCC risk [P = 0.02, odds ratio (OR) = 0.43; 95% confidence interval (CI): 0.21-0.91] and pathologic stage (P = 0.03, OR = 1.89; 95% CI: 1.06-3.36). A putative binding site for miRNA-196a (miR-196a) exists in the 3'UTR of RAP1A, and the genetic variant, rs6573 A→C, is present in this binding region. We confirmed that miR-196a regulated the expression of RAP1A by luciferase reporter assay and that the regulation was affected by the RAP1A genotype. SNP rs6573 A to C change interfere in the interaction of miR-196a binding to RAP1A 3'UTR, resulting in higher constitutive expression of RAP1A. Moreover, we observed that RAP1A was overexpressed in the majority of ESCC tissues and correlated with RAP1A genotype and lymph node metastasis. In vitro study indicated RAP1A might function as a promoter for esophageal cancer cell migration and invasion through matrix metalloproteinase 2. Our study highlights RAP1A and SNP rs6573 functioning as potential personal diagnostic and prognosis markers for ESCC.

In most cases, the molecular mechanism underlying the pathogenesis of sporadic Alzheimer's disease (AD) is unknown. Elevated basal cortisol levels in AD patients suggest that glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Amyloid plaques are the hallmark of AD, and they are considered to play an early role in the AD process. However, little is known about how their formation is regulated by stress and GC. Astrocyte accumulation is one of the earliest neuropathological changes in AD. Here, we report that GC elevated amyloid-β (Aβ) production in primary cultures of astrocytes by increasing amyloid precursor protein (APP) and β-site APP-cleaving enzyme 1 gene expression. Notably, GC administered to normal, middle-aged mice promoted the expression of APP and β-site APP-cleaving enzyme 1 in astrocytes, as determined by double immunofluorescence. Additionally, confocal microscopy and ELISA revealed that GC markedly reduced Aβ degradation and clearance by astrocytes in vitro, indicating a decreased neuroprotective capacity of the astrocytes. This may have been due to the decrease of several Aβ-degrading proteases, such as insulin-degrading enzyme and matrix metalloproteinase-9. These effects occurred through the activation of GC receptors. Taken together, our results demonstrate that GC can enhance the production of Aβ, reduce its degradation in astrocytes, and provide a molecular mechanism linking stress factors to AD. Our study suggests that GC can facilitate AD pathogenesis and that reducing GC in the elderly and early AD patients would be beneficial.