Shenming Wang

MicroRNA-21 Regulates Vascular Smooth Muscle Cell Function via Targeting Tropomyosin 1 in Arteriosclerosis Obliterans of Lower Extremities

Objective —The goal of this study was to determine the expression signature and the potential role of microRNAs in human arteries with arteriosclerosis obliterans (ASO). Methods and Results —The expression profiles of microRNAs in human arteries with ASO and in normal control arteries were determined by quantitative reverse transcription–polymerase chain reaction array. Among the 617 detected microRNAs, multiple microRNAs were aberrantly expressed in arteries with ASO. Some of these dysregulated microRNAs were further verified by quantitative reverse transcription–polymerase chain reaction. Among them, microRNA-21 (miR-21) was mainly located in arterial smooth muscle cells (ASMCs) and was increased by more than 7-fold in ASO that was related to hypoxia inducible factor 1-&agr;. In cultured human ASMCs, cell proliferation and migration were significantly decreased by inhibition of miR-21. 3′-Untranslated region luciferase assay confirmed that tropomyosin 1 was a target of miR-21 that was involved in miR-21-mediated cellular effects, such as cell shape modulation. Conclusion —The results suggest that miR-21 is able to regulate ASMC function by targeting tropomyosin 1. The hypoxia inducible factor-1 &agr;/miR-21/tropomyosin 1 pathway may play a critical role in the pathogenesis of ASO. These findings might provide a new therapeutic target for human ASO.

MicroRNA Profiling Implies New Markers of Chemoresistance of Triple-Negative Breast Cancer

Objective Triple-negative breast cancer (TNBC) patients with truly chemosensitive disease still represent a minority among all TNBC patients. The aim of the present study is to identify microRNAs (miRNAs) that correlate with TNBC chemoresistance. Methods In this study, we conducted miRNAs profile comparison between triple-negative breast cancer (TNBCs) and normal breast tissues by microRNA array. Quantitative real-time PCR (qRT-PCR) was utilized to confirm the specific deregulated miRNAs change trend. We used starBase 2.1 and GOrilla to predict the potential targets of the specific miRNAs. Cells viability and apoptosis assays were employed to determine the effect of alteration of the specific miRNAs in TNBC cells on the chemosensitivity. Results We identified 11 specific deregulated miRNAs, including 5 up-regulated miRNAs (miR-155-5p, miR-21-3p, miR-181a-5p, miR-181b-5p, and miR-183-5p) and 6 down-regulated miRNAs (miR-10b-5p, miR-451a, miR-125b-5p, miR-31-5p, miR-195-5p and miR-130a-3p). Thereafter, this result was confirmed by qRT-PCR. We predicted the potential targets of the candidate miRNAs and found that they are involved in cancer-associated pathways. For the first time, we found that miR-130a-3p and miR-451a were down-regulated in TNBC. 9 of the 11 specific deregulated miRNAs were found to be associated with chemoresistance. In vitro assays, we found that up-regulation of either miR-130a-3p or miR-451a in MDA-MB-231 cells significantly changed the cells sensitivity to doxorubicin. The results suggest that TNBC chemotherapy might be affected by a cluster of miRNAs. Conclusion The abnormal expression miRNAs in TNBC are mainly chemoresistance related. This might be part of reason that TNBC likely to evade from chemotherapy resulting in early relapse and high risk of death. To alter their expression status might be a potential therapeutic strategy to improve the outcome of chemotherapy for TNBC patients.

MCP-1 Stimulates MMP-9 Expression via ERK 1/2 and p38 MAPK Signaling Pathways in Human Aortic Smooth Muscle Cells

Objective: We investigated the molecular mechanism underlying the role of monocyte chemoattractant protein-1 (MCP-1) in the formation and development of human abdominal aortic aneurysm (AAA). Methods: We examined protein expression profiles using a protein array and found that MCP-1 was the most highly expressed protein in AAA tissues compared with normal aortas. To investigate the potential mechanism of MCP-1 involvement in the pathogenesis of AAA, we treated human aortic smooth muscle cells (HASMCs) with human recombinant MCP-1. Results: MCP-1 was the most highly expressed protein in AAA tissues compared with normal aorta; matrix metalloproteinase-9 (MMP-9) expression was also significantly increased. Treatment with MCP-1 significantly increased the expression and activation of MMP-9 and activated the three major mitogen activated protein kinases (MAPKs) extracellular signal regulated kinase (ERK), c-Jun amino terminal kinase (JNK1/2) and p38 MAPK. Furthermore, MCP-1-induced secretion of MMP-9 was inhibited by U0126 (inhibitor of the ERK 1/2 pathway) and SB203580 (inhibitor of the p38 MAPK pathway), but not SP600125 (inhibitor of the JNK1/2 pathway). Conclusion: These data demonstrate that MCP-1 stimulates secretion of MMP-9 directly through the ERK1/2 and p38 MAPK mediated pathways in HASMCs. Thus, inhibition of this molecular mechanism might be a potential therapeutic target in the non-surgical treatment of AAA.

The inhibition of human bladder cancer growth by calcium carbonate/CaIP6 nanocomposite particles delivering AIB1 siRNA.

Previously, we reported that inorganic amorphous calcium carbonate (ACC) hybrid nanospheres functionalized with Ca(II)-IP6 compound (CaIP6) is a promising gene vector in vitro. Here, nonviral gene carrier, ACC/CaIP6 nanocomposite particles (NPACC/CaIP6), was evaluated for efficient in vitro and in vivo delivery of small interfering RNA (siRNA) targeting human Amplified in breast cancer 1 (AIB1). The nanoparticle is capable of forming ACC/CaIP6 nanoparticle-siRNA complexes and transferring siRNA into targeted cells with high transfection efficiency. Meanwhile the ACC/CaIP6 nanoparticle-siRNA complexes have no obvious cytotoxicity for human bladder cancer T24 cells. Furthermore, NPACC/CaIP6 effectively protected the encapsulated siRNA from degradation, AIB1 knockdown mediated by ACC/CaIP6/siRNA complexes transfection resulted in cells proliferation inhibition, apoptosis induction and cell cycle arrest in vitro. NPACC/CaIP6 exhibited well tissues penetrability in localized siRNA delivering, intratumoral injection of NPACC/CaIP6/siAIB1 could attenuate tumor growth and downregulation of PI3K/Akt signaling pathway in vivo. We conclude that ACC/CaIP6 nanoparticle is a promising system for effective delivery of siRNA for cancer gene therapy.

Expressions of miRNAs in papillary thyroid carcinoma and their associations with the BRAFV600E mutation

OBJECTIVE Alterations in microRNA (miRNA) expression have been described in thyroid tumors, suggesting a role for miRNAs in thyroid carcinogenesis. BRAF(V600E) is the most frequently identified genetic alteration in papillary thyroid carcinoma (PTC). We investigated the link between BRAF(V600E) status and the expression of miRNAs in PTC and analyzed the associations of these factors with clinicopathological characteristics. DESIGN AND METHODS Prospective study of patients who underwent thyroid surgery between October 8, 2008 and November 1, 2010. BRAF(V600E) status was determined by mutant allele-specific amplification PCR and direct sequencing of exon 15 of the BRAF gene in 69 PTC tissues and 69 respective paracancerous normal thyroid tissues. Initially, miRNA expression was analyzed in 12 PTC tissues and three associated paracancerous tissues using a miRNA microarray. miRNAs differentially expressed between BRAF(V600E)-positive and -negative PTC tissues were then validated by real-time quantitative PCR on 69 PTC tissues and 69 paracancerous tissues. We also explored the associations between BRAF(V600E) status or differential miRNA expression and clinicopathological characteristics. RESULTS The mutation rate of BRAF(V600E) in PTC was 47.8%. Twelve miRNAs were upregulated and six were downregulated in PTC tissues, among which miR-15a, 15a*, 34a*, 34b*, 551b, 873, 876-3p, and 1274a were first identified. miR-21* and 203 were significantly dysregulated (P<0.05) in PTC tissues with BRAF(V600E). Additionally, there were significant associations (P<0.05) between BRAF(V600E) and a higher tumor-node-metastasis staging (III/IV), and between miR-21* over-expression and lymph node metastasis. CONCLUSIONS We identified two miRNAs that are differentially expressed in PTC tissues with BRAF(V600E) and revealed their associations with clinicopathological features. These findings may lead to the development of a potential diagnostic biomarker or prognostic indicator of PTC.

The Preparation and Performance of a New Polyurethane Vascular Prosthesis

We investigated the performance of small-caliber polyurethane (PU) small-diameter vascular prosthesis generated using the electrospinning technique. PU was electrospun into small-diameter, small-caliber tubular scaffolds for potential application as vascular grafts. We investigated the effects of electrospinning conditions (solution concentration, mandrel rotation speed) on the microstructure and porosity of the scaffolds for the purpose of preparing scaffolds with optimum microstructures and properties. We evaluated the mechanical properties of the scaffolds by tensile tests and the cytotoxicity of the PU small-diameter, small-caliber PU synthetic vascular graft by the MTT assay. The adhesion of endothelial cells to the PU scaffold was characterized by Hoechst staining and fluorescence microscopy, and we measured endothelial cell proliferation on the PU scaffold by the CCK-8 assay. We analyzed the prosthesis microstructure and endothelial cell morphology using scanning electron microscopy. With increasing PU concentration in the electrospinning solution, the fiber diameter of the vascular graft increased and the porosity decreased. In addition, with increasing electrospinning time, the wall thickness increased and the porosity decreased. We found that regular fiber orientation can be obtained by adjusting the rotation speed of the mandrel. Cell proliferation was not inhibited as the small-caliber PU synthetic vascular grafts showed little cytotoxicity. The endothelial cells had faster adherence to the PU scaffolds than to the PTFE surface during the initial contact. After prolonged cell culture, significantly higher endothelial cell proliferation rate was observed in the PU scaffold groups than the PTFE group. We obtained small-caliber PU vascular grafts with optimal fiber arrangement, excellent mechanical properties, and optimal biocompatibility by optimizing the electrospinning conditions. This study provides in vitro biocompatibility data that is helpful for the clinical application of the PU small-diameter, small-caliber PU vascular grafts.

Promising plasmid DNA vector based on APTES-modified silica nanoparticles

Nanoparticles have an enormous potential for development in biomedical applications, such as gene or drug delivery. We developed and characterized aminopropyltriethoxysilane-functionalized silicon dioxide nanoparticles (APTES-SiNPs) for gene therapy. Lipofectamine® 2000, a commonly used agent, served as a contrast. We showed that APTES-SiNPs had a gene transfection efficiency almost equal to that of Lipofectamine 2000, but with lower cytotoxicity. Thus, these novel APTES-SiNPs can achieve highly efficient transfection of plasmid DNA, and to some extent reduce cytotoxicity, which might overcome the critical drawbacks in vivo of conventional carriers, such as viral vectors, organic polymers, and liposomes, and seem to be a promising nonviral gene therapy vector.

MicroRNA-1298 is regulated by DNA methylation and affects vascular smooth muscle cell function by targeting connexin 43

AIMS Growing evidence links microRNA to the process of peripheral vascular disease. Recently, we have found that microRNA-1298(miR-1298) is one of the most significantly down-regulated microRNAs in human arteries with arteriosclerosis obliterans (ASO) of the lower extremities. However, little is known regarding its role in the process of ASO. The present study aimed to investigate the expression, regulatory mechanisms, and functions of miR-1298 in the process of ASO. METHODS AND RESULTS Using quantitative reverse-transcription PCR and in situ hybridization assays, miR-1298 was observed predominantly expressed in the vascular smooth muscle cells (VSMCs) and was significantly down-regulated in ASO compared with normal arteries. Pyrosequencing analysis revealed that the miR-1298 DNA upstream of CpG sites were hypermethylated in ASO compared with normal arteries. Next, the luciferase reporter assay revealed that miR-1298 down-regulation is related with upstream DNA CpG site hypermethylation. Introducing a miR-1298 mimic into cultured VSMCs significantly attenuated cell proliferation and migration. Connexin 43 (Cx43) was validated to be a functional target of miR-1298 that was involved in the miR-1298-mediated cellular effects. Finally, lentivirus-mediated delivery of miR-1298 and its target Cx43 into a rat carotid balloon injury model indicated that re-overexpression of miR-1298 significantly decreased neointimal formation by targeting connexin 43. CONCLUSION Our data demonstrate a specific role of the upstream DNA methylation/miR-1298/Cx43 pathway in regulating VSMC function and suggest that modulation of miR-1298 levels may offer a novel therapeutic approach for ASO.

Interleukin-8 upregulates integrin β3 expression and promotes estrogen receptor-negative breast cancer cell invasion by activating the PI3K/Akt/NF-κB pathway

Interleukin-8 (IL-8) possesses tumorigenic and proangiogenic properties and is overexpressed in many human cancers. The integrin family regulates a diverse array of cellular functions crucial to the initiation, progression and metastasis of solid tumors. However, the mechanisms of action of IL-8 and integrin in estrogen receptor-negative breast cancer are largely unknown. In this study, IL-8 and integrin β3 expression in human breast cancer cells and tissues was examined by real-time PCR, Western blot and immunochemistry analysis. Integrin β3 expression, invasive ability and the activation of PI3K/Akt and NF-κB pathways in IL-8 knockdown breast cancer cells were evaluated. In addition, reporter assay and ChIP were performed to assess integrin β3 promoter activity in IL-8 knockdown cells. We observed a positive correlation between integrin β3 and IL-8 expression, which was inversely correlated with ER status in breast cancer cell lines and tissues. IL-8 siRNA decreased the invasion and integrin β3 expression in human breast cancer cells. Moreover, IL-8 siRNA attenuated the phosphorylation of PI3K and Akt and inhibited NF-κB activity and binding on integrin β3 promoter. IL-8 siRNA diminished NF-κB nuclear translocation via blocking IκB phosphorylation in the cytoplasm. In conclusion, IL-8 activates the PI3K/Akt pathway, which in turn activates NF-κB, resulting in the upregulation of integrin β3 expression and increased invasion of estrogen receptor-negative breast cancer cells. IL-8/PI3K/Akt/NF-κB/integrin β3 axis may be exploited for therapeutic intervention to breast cancer metastasis.

A novel dendritic nanocarrier of polyamidoamine-polyethylene glycol-cyclic RGD for “smart” small interfering RNA delivery and in vitro antitumor effects by human ether-à-go-go-related gene silencing in anaplastic thyroid carcinoma cells

The application of RNA interference techniques is promising in gene therapeutic approaches, especially for cancers. To improve safety and efficiency of small interfering RNA (siRNA) delivery, a triblock dendritic nanocarrier, polyamidoamine-polyethylene glycol-cyclic RGD (PAMAM-PEG-cRGD), was developed and studied as an siRNA vector targeting the human ether-à-go-go-related gene (hERG) in human anaplastic thyroid carcinoma cells. Structure characterization, particle size, zeta potential, and gel retardation assay confirmed that complete triblock components were successfully synthesized with effective binding capacity of siRNA in this triblock nanocarrier. Cytotoxicity data indicated that conjugation of PEG significantly alleviated cytotoxicity when compared with unmodified PAMAM. PAMAM-PEG-cRGD exerted potent siRNA cellular internalization in which transfection efficiency measured by flow cytometry was up to 68% when the charge ratio (N/P ratio) was 3.5. Ligand-receptor affinity together with electrostatic interaction should be involved in the nano-siRNA endocytosis mechanism and we then proved that attachment of cRGD enhanced cellular uptake via RGD-integrin recognition. Gene silencing was evaluated by reverse transcription polymerase chain reaction and PAMAM-PEG-cRGD-siRNA complex downregulated the expression of hERG to 26.3% of the control value. Furthermore, gene knockdown of hERG elicited growth suppression as well as activated apoptosis by means of abolishing vascular endothelial growth factor secretion and triggering caspase-3 cascade in anaplastic thyroid carcinoma cells. Our study demonstrates that this novel triblock polymer, PAMAM-PEG-cRGD, exhibits negligible cytotoxicity, effective transfection, “smart” cancer targeting, and therefore is a promising siRNA nanocarrier.