MicroRNA-221 Mediates the Effects of PDGF-BB on Migration,Proliferation, and the Epithelial-Mesenchymal Transition in Pancreatic Cancer Cells

The platelet-derived growth factor (PDGF) signaling pathway has been found to play important roles in the development and progression of human cancers by regulating the processes of cell proliferation, apoptosis, migration, invasion, metastasis, and the acquisition of the epithelial-mesenchymal transition (EMT) phenotype. Moreover, PDGF signaling has also been found to alter the expression profile of miRNAs, leading to the reversal of EMT phenotype. Although the role of miRNAs in cancer has been documented, there are very few studies documenting the cellular consequences of targeted re-expression of specific miRNAs. Therefore, we investigated whether the treatment of human pancreatic cancer cells with PDGF could alter the expression profile of miRNAs, and we also assessed the cellular consequences. Our study demonstrates that miR-221 is essential for the PDGF-mediated EMT phenotype, migration, and growth of pancreatic cancer cells. Down-regulation of TRPS1 by miR-221 is critical for PDGF-mediated acquisition of the EMT phenotype. Additionally, the PDGF-dependent increase in cell proliferation appears to be mediated by inhibition of a specific target of miR-221 and down-regulation of p27Kip1.     

MicroRNA-30c contributes to the development of hypoxia pulmonary hypertension by inhibiting platelet-derived growth factor receptor β expression

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). MicroRNAs have been implicated in the regulation of cell proliferation and might be implicated in the etiology of PAH. Data from in vivo and in vitro cell culture models showed that hypoxia inhibits microRNA-30c (miR-30c) expression in PASMCs. Inhibition of miR-30c by either hypoxia or AMO-30c results in PASMC proliferation (cell viability, 5-bromo-2-deoxyuridine (BrdU) incorporation, proliferating cell nuclear antigen, Ki67, and tubulin polymerization) and the inhibition of apoptosis (cell cycle progression, Cyclin A and Cyclin D, and TUNEL staining). Moreover, down-regulation of miR-30c also results in the phenotype switch from contractile to synthetic PASMC (SM22α and Calponin, osteopontin expression, and wound healing assay). In contrast, these effects were reversed by the application of an miR-30c mimetic under hypoxic conditions. Mechanically, miR-30c inhibited the platelet-derived growth factor receptor β (PDGFRβ) expression by directly binding to the 3′ untranslated region of PDGFRβ mRNA (luciferase reporter assays, and PDGFRβ-masking antisense oligodeoxynucleotides). Pharmacological inhibition of PDGFR by AG-1296 displayed similar effects to the miR-30c mimetic. These data suggest that the down-regulation of miR-30c accounts for the up-regulation of PDGFRβ expression, and subsequent activation of PDGF signaling results in the hypoxia-induced PASMC proliferation and phenotype switching. Therefore, increasing miR-30c expression levels could be explored as a potential new therapy for hypoxia-induced PAH.     

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.     

MicroRNA-124 upregulation inhibits proliferation and invasion of osteosarcoma cells by targeting sphingosine kinase 1

Increasing evidence has confirmed that the dysregulation of microRNAs (miRNAs) contributes to the proliferation and invasion of human cancers. Previous studies have shown that the dysregulation of miR-124 is in numerous cancers. However, the roles of miR-124 in human osteosarcoma (OS) have not been well clarified. Therefore, this study was to investigate the biological functions and molecular mechanisms of miR-124 in OS cell lines, discussing whether it could be a therapeutic biomarker of OS in the future. In this study, our results demonstrated that miR-124 was down-regulated in OS cell lines and tissues. Furthermore, the low level of miR-124 was associated with increased expression of Sphingosine kinase 1 (SPHK1) in OS cells and tissues. Up-regulation of miR-124 significantly inhibited cell proliferation, invasion, and MMP-2 and -9 expressions of OS cells. Bioinformatics analysis predicted that the SPHK1 was a potential target of miR-124. Further study by luciferase reporter assay demonstrated that miR-124 could directly target SPHK1. Overexpression of SPHK1 in OS cells transfected with miR-124 mimic partially reversed the inhibitory of miR-124. In conclusion, miR-124 inhibited cell proliferation and invasion in OS cells by downregulation of SPHK1, and that downregulation of SPHK1 was essential for the miR-124-inhibited cell invasion and in OS cells.     

miR-124和miR-520b下调Eps8表达并抑制HeLa细胞增殖

Eps8是一个多功能的信号分子,参与肌动蛋白重排、受体内吞和肿瘤的发生发展. 为了寻找靶向Eps8的microRNAs (miRNAs)并研究其在宫颈癌中的调控作用,本文采用软件预测获得4个可能调控Eps8表达的miRNAs. 利用双荧光素酶报告系统和Western印迹研究发现,miR-124 和miR-520b结合到人EPS8 mRNA的3′非翻译区(untranslated region, UTR)并有效抑制Eps8蛋白的表达. 进一步细胞存活检测、MTT法和克隆形成实验分析显示,miR-124和miR-520b过表达显著抑制HeLa细胞的生长和增殖.而且,miRNA调节的Eps8下调能提高HeLa细胞对化疗药物顺铂的敏感性. 同时证明,miR-124和miR-520b激活肿瘤抑制基因p53与下游基因p21报告基因转录活性,也相应地上调了p53与p21的蛋白表达. 这些结果提示,miR-124和miR-520b下调癌基因EPS8表达,从而抑制HeLa细胞增殖,负调控宫颈癌细胞生长.     

Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

Background

Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_i in PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺ channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺ influx and increase in [Ca²⁺]_i is involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_i leads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.

Methods

Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_i was measured with a dynamic digital Ca²⁺ imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.

Results

Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_i and Ca²⁺ entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400"}}SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.

Conclusion

The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.     

LncRNA NRON alleviates atrial fibrosis via promoting NFATc3 phosphorylation

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a dominant role in the development of pulmonary arterial hypertension (PAH). Some studies and our previous work found that disturbance of fatty acid metabolism existed in PAH. However, the mechanistic link between fatty acid catabolism and cell proliferation remains elusive. Here, we identified an essential role and signal pathway for the key rate-limiting enzyme of mitochondrial fatty acid β-oxidation, carnitine palmitoyltransferase (CPT) 1, in regulating PASMC proliferation in PAH. We found that CPT1 was highly expressed in rat lungs and pulmonary arteries in monocrotaline-induced PAH, accompanied by decreased adenosine triphosphate (ATP) production and downregulation of the AMPK-p53-p21 pathway. Platelet-derived growth factor (PDGF)-BB upregulated the expression of CPT1 in a dose- and time-dependent manner. PASMC proliferation and ATP production induced by PDGF-BB were partly reversed by the CPT1 inhibitor etomoxir (ETO). The overexpression of CPT1 in PASMCs also promoted proliferation and ATP production and subsequently inhibited the phosphorylation of AMPK, p53, as well as p21 in PASMCs. Furthermore, AMPK was activated by ETO, which increased the expression of p53 and p21, and the proportion of cells in the cell cycle G₂/M phase in response to PDGF-BB stimulation in PASMCs. Our work reveals a novel mechanism of CPT1 regulating PASMC proliferation in PAH, and regulation of CPT1 may be a potential target for therapeutic intervention in PAH.

     

Bioinformatic analysis and validation of microRNA-508-3p as a protective predictor by targeting NR4A3/MEK axis in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) featured a debilitating progressive disorder. Here, we intend to determine diagnosis-valuable biomarkers for PAH and decode the fundamental mechanisms of the biological function of these markers. Two mRNA microarray profiles (GSE70456 and GSE117261) and two microRNA microarray profiles (GSE55427 and GSE67597) were mined from the Gene Expression Omnibus platform. Then, we identified the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs), respectively. Besides, we investigated online miRNA prediction tools to screen the target gene of DEMs. In this study, 185 DEGs and three common DEMs were screened as well as 1266 target genes of the three DEMs were identified. Next, 16 overlapping dysregulated genes from 185 DEGs and 1266 target gene were obtained. Meanwhile, we constructed the miRNA gene regulatory network and determined miRNA-508-3p-NR4A3 pair for deeper exploring. Experiment methods verified the functional expression of miR-508-3p in PAH and its signalling cascade. We observed that ectopic miR-508-3p expression promotes proliferation and migration of pulmonary artery smooth muscle cell (PASMC). Bioinformatic, dual-luciferase assay showed NR4A3 represents directly targeted gene of miR-508-3p. Mechanistically, we demonstrated that down-regulation of miR-508-3p advances PASMC proliferation and migration via inducing NR4A3 to activate MAPK/ERK kinase signalling pathway. Altogether, our research provides a promising diagnosis of predictor and therapeutic avenues for patients in PAH.     

MiR-20a regulates the PRKG1 gene by targeting its coding region in pulmonary arterial smooth muscle cells

Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with de-differentiation of pulmonary artery smooth muscle cells (PASMC). Here, we show that miR-20a expression is up-regulated in response to hypoxia in both mouse and human PASMC. We also observed that miR-20a represses the protein kinase, cGMP-dependent, type I (PRKG1) gene and we identified two crucial miR-20a binding sites within the coding region of PRKG1. Functional studies showed that miR-20a promotes the proliferation and migration of human PASMC, whereas it inhibits their differentiation. In summary, we provided a possible mechanism by which hypoxia results in decreased PRKG1 expression and in the phenotypic switching of PASMC.     

Regulation and Methylation of Tumor Suppressor MiR-124 by Androgen Receptor in Prostate Cancer Cells

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.     

15-oxo-Eicosatetraenoic acid prevents serum deprivation-induced apoptosis of pulmonary arterial smooth muscle cells by activating pro-survival pathway

Pulmonary arterial hypertension (PAH) is a progressive condition in which remodeling of the pulmonary vasculature plays an important role. The vascular remodeling involves pulmonary arterial smooth muscle cell (PASMC) proliferation and apoptosis, which is affected by several arachidonic acid metabolites. 15-oxo-Eicosatetraenoic acid (15-oxo-ETE) is one of the metabolites. However, the biological role of 15-oxo-ETE in PASMCs remains unknown. Here we show evidence for the modulation of PASMC apoptosis by 15-oxo-ETE. We found that 15-oxo-ETE increased rat and human PASMC viability. Consistently, 15-oxo-ETE attenuated nuclear fragmentation and DNA strand breaks, decreased caspase-3 activity, reduced mitochondrial depolarization, and increased Bcl-2 expression. Interestingly, the anti-apoptotic effect of 15-oxo-ETE was lost when the Akt intracellular signaling pathway was blocked. Taken together, we have established that 15-oxo-ETE protects PASMCs against apoptosis through the Akt pathway. These results suggest that 15-oxo-ETE seems to be a potential agent for PAH controls by preventing unwanted PASMC death.