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Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy ( TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies ( mAbs) in either dendritic cells ( DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1 µm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3 T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (∼50 nm) pathogen, dengue virus, leading to infection of host cells. [ABSTRACT FROM AUTHOR]

MicroRNA 520c and 373 (miR-520c and miR-373) have been characterized as oncogenes and play critical roles in cancer cell metastasis. However, the relationship between these two microRNAs and matrix metalloproteinases (MMPs), which are important in cancer cell metastasis, remains unknown. Here, we report new evidence in which miR-520c and miR-373 effects in human fibrosarcoma HT1080 cells are associated with MMP9 activity, and this upregulation of MMP9 is not only at the activity and protein levels, but also at that of its mRNA. Our experimental data demonstrate that these effects occur not by direct binding to the MMP9 promoter, but by miR-520c and miR-373 directly targeting the 3′-untranslational region (UTR) of mRNAs of mTOR and SIRT1 (negative regulators of expression of MMP9 via inactivating the Ras/Raf/MEK/Erk signaling pathway and transcription factor NF-κB activity); and thus suppressing translation levels of SIRT1 and mTOR. Moreover, inhibition of key kinases of the Ras/Raf/MEK/Erk signaling pathway and Western blots for selected proteins further identified miR-520c and miR-373 as activating this signaling pathway and NF-κB. In conclusion, miR-520c and miR-373 increased the expression of MMP9 by directly targeting the 3′-UTRs of mRNAs of mTOR and SIRT1 and suppressing their translation; resulting in activation of the Ras/Raf/MEK/Erk signaling pathway and NF-κB; and, finally, increasing the mRNA, protein, and activity of MMP9 and enhancing cell migration and cell growth in 3D type I collagen gels. J. Cell. Physiol. 227: 867-876, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Objective: Temozolomide is extensively applied in chemotherapy for glioblastoma with unclear exact action mechanisms. This article seeks to address the potential molecular mechanisms in temozolomide therapy for glioblastoma involving LINC00470. Methods: Bioinformatics analysis was conducted to predict the potential mechanism of LINC00470 in glioblastoma, which was validated by dual‐luciferase reporter, RIP, ChIP, and RNA pull‐down assays. LINC00470 expression and the predicted downstream transcription factor early growth response 2 (EGR2) were detected in the collected brain tissues from glioblastoma patients. Following temozolomide treatment and/or gain‐ and loss‐of‐function approaches in glioblastoma cells, cell viability, invasion, migration, cycle distribution, angiogenesis, autophagy, and apoptosis were measured. In addition, the expression of mesenchymal surface marker proteins was assessed by western blot. Tumor xenograft in nude mice was conducted for in vivo validation. Results: Mechanistic analysis and bioinformatics analysis revealed that LINC00470 transcriptionally activated SRY‐related high‐mobility‐group box 4 (SOX4) through the transcription factor EGR2. LINC00470 and EGR2 were highly expressed in brain tissues of glioblastoma patients. LINC00470 and EGR2 mRNA expression gradually decreased with increasing concentrations of temozolomide in glioblastoma cells, and SOX4 expression was reduced in cells by temozolomide and LINC00470 knockdown. Temozolomide treatment induced cell cycle arrest, diminished cell viability, migration, invasion, and angiogenesis, and increased apoptosis and autophagy in glioblastoma, which was counteracted by overexpressing LINC00470 or SOX4 but was further promoted by LINC00470 knockdown. Temozolomide restrained glioblastoma growth and angiogenesis in vivo, while LINC00470 or SOX4 overexpression nullified but LINC00470 knockdown further facilitated these trends. Conclusion: Conclusively, temozolomide repressed glioblastoma progression by repressing the LINC00470/EGR2/SOX4 axis. [ABSTRACT FROM AUTHOR]

Cutaneous squamous‐cell carcinoma (cSCC) is the second most common skin cancer, with an increasing incidence in recent years. To define the molecular basis that drive cSCC development and progression, this study aimed at identifying potential novel molecular targets for the diagnosis and therapy of patients with cSCC. Two data sets with the accession number GSE45164 and GSE66359 were downloaded from Gene Expression Omnibus (GEO) database. After the identification of differentially expressed genes (DEGs) from these two data sets, respectively, between cSCC samples and controls, a combination of DEGs from these two data sets were subjected to the following analyses, including functional annotation, protein–protein interaction (PPI) network and module construction, transcription factor (TF)‐target regulation prediction, and drug–gene interaction predictive analysis. A total of 204 upregulated genes and 213 downregulated genes were found in two data sets which were used for the follow‐up analysis. Upregulated and downregulated genes were mainly involved in the functions such as cell division, mitotic nuclear division, cell cycle, and p53 signaling pathway. Interferon induced protein family members and proteasome subunit members were involved in the TF‐target regulatory network, such as PSMB8, CXCL10, and IFIT3. Eight upregulated genes (TOP2A, CXCL8, RRM2, PSMB8, PSMB9, PBK, CXCL10, and ISG15) that were hub genes in the PPI network and significant modules were identified in the predicted drug–gene interaction. In conclusion, TOP2A, CXCL8, RRM2, PSMB8, PSMB9, PBK, CXCL10, and ISG15 may be potential targets for the diagnosis and therapy of patients with cSCC. [ABSTRACT FROM AUTHOR]

Background: The five‐year survival rate of non‐small cell lung cancer (NSCLC) patients is very low. MiR‐873 is involved in the growth, metastasis, and differentiation of tumors. Herein, we determined the target gene and influence of miR‐873 in NSCLC. Methods: MiRanda and Targetscan websites were used to predict the target gene of miR‐873 in NSCLC. Luciferase activity was examined using a dual luciferase reporter gene assay kit. The viability, tube formation, and proliferation of cells were analyzed by cell counting kit‐8, angiogenic analysis, and flow cytometry, respectively. The levels of miR‐873 and GLI1 were evaluated using quantitative real‐time PCR and Western blot assays. Results: Low levels of GLI1 and high levels of miR‐873 were observed in an NSCLC cell line (PC9) highly sensitive to EGFR‐tyrosine kinase inhibitors. There was a negative correlation between miR‐873 and GLI1 expression in PC9 and PC9/GR cells. The inhibition of miR‐873 enhanced GLI1 levels. MiR‐873 expression was inhibited by gefitinib. Gefitinib markedly reduced the viability, tube formation, and cell number in PC9 cells. However, suppression of miR‐873 enhanced the resistance and knockdown of GLI1 enhanced the sensitivity of PC9 cells to gefitinib. Conclusions: GLI1 is a target gene of miR‐873 in NSCLC. The inhibition of miR‐873 increased gefitinib resistance of NSCLC cells via the upregulation of GLI1. These results indicate that miR‐873‐GLI1 signaling is involved in gefitinib resistance in NSCLC. [ABSTRACT FROM AUTHOR]

Loss of function mutations in the human ether-a-go-go-related gene ( hERG) cause long QT syndrome type 2 ( LQT2). Most LQT2 patients are heterozygous mutation carriers in which the mutant hERG exerts potent dominant-negative effects. 1, 3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea ( NS1643) is known to enhance IKr in WT- hERG. We investigated its actions following lipofectamine-induced expression of both mutant G604S- and WT- hERG in the heterologous HEK293 expression system. Cells transfected with pc DNA3-G604S- hERG did not lead to any expression of detectable currents whether before or following NS1643 challenge. Cells transfected with both pc DNA3- WT- hERG and pc DNA3-G604S- hERG showed reduced hERG currents compared to those transfected with pc DNA3-G604S- hERG consistent with the reduced trafficking and formation of modified heteromeric WT-G604S channels reported on earlier occasions. Nevertheless, NS1643 then continued to produce concentration- and voltage-dependent increases in hERG current amplitude. It did not affect the voltage dependence of activation, recovery from inactivation and deactivation. However, NS1643 (30 μmol/L) slowed steady state inactivation and shifted the steady state half maximal activation voltage ( V1/2) of the inactivation curve by +10 mV, and significantly increased the time constants of inactivation. Our present experimental results suggest that NS1643 significantly increases ion current and attenuates its inactivation in cells co-expressing G604S- hERG and WT- hERG. These findings raise the possibility that hERG channel activators offer potential treatment strategies for inherited LQT2. [ABSTRACT FROM AUTHOR]

Chloride intracellular channel 1 (CLIC1) is expressed in many human tissues and has been reported to be involved in the regulation of cell cycle, cell proliferation, and differentiation. Its roles in human hepatic tumor, however, remain unclear. The aim of this study was to investigate the clinicopathological significance and expression pattern of CLIC1 in human primary hepatic tumors. We examined the expression pattern of CLIC1 mRNA and protein in hepatic tumors using real-time quantitative RT-PCR and Western blot, respectively. CLIC1 protein and mRNA levels were significantly higher in cancerous tissues compared with corresponding normal tissue. In 85 hepatic tumor tissues, CLIC1 was significantly higher in 69 cases (81.2%), as determined by immunohistochemical staining. Increased CLIC1 expression was correlated with tumor size (p = 0.021), distant metastasis (p = 0.025), pathological TNM (pTNM) stage (p = 0.023), and poor survival (25.11 ± 2.27 vs 45.29 ± 4.28 months, p = 0.001). Our data show that increased CLIC1 protein expression is associated with clinicopathological factors and a poor prognosis of hepatic tumors, and suggest that CLIC1 might represent a valuable prognostic marker for human hepatic tumors. [ABSTRACT FROM AUTHOR]

Objective To investigate the relationship between ectonucleotide pyrophosphatase phosphodiesterase-1( ENPP-1) expression and transforming growth factor beta 1 ( TGF-β1) of end-plate chondrocytes after stimulation with intermittent cyclic mechanical tension ( ICMT) by using an FX-4000T Flexercell Tension Plus unit. Methods Rat end-plate chondrocytes were cultured and ICMT (strain at 0.5 Hz sinusoidal curve at 10% elongation) applied for 7 days for 4 h/day and cultured for a further 2 days. End-plate chondrocytes were also exposed to 10 ng/mL of TGF-β1. Then, using small interfering RNA technology, small interfering TGF-β1 (si TGF-β1) was transfected. Expression of ENPP-1 and TGF-β1 was measured by real-time reverse-transcriptase polymerase chain reaction ( RT-PCR) and western blotting. Results Expression of both ENPP-1 and TGF-β1 was up-regulated after ICMT. Both RT-PCR and western blot showed that ENPP-1 expression decreases with si RNA TGF-β1 after 3% elongation 40 min, and cultured for an additional 2 days. Conclusion It was found that down-regulation of ENPP-1 gene expression induced by ICMT is likely dependent on TGF-β1 in end-plate chondrocytes. [ABSTRACT FROM AUTHOR]

Both the deregulation of microRNAs and epidermal growth factor receptor ( EGFR) are emerging as important factors in non-small-cell lung cancer ( NSCLC). Here, miR-133b was found to be associated with tumor stage, the extent of regional lymph node involvement, stage, visceral pleura or vessel invasion and EGFR mRNA expression in Chinese patients with NSCLC. Bioinformatic analysis and luciferase reporter assay revealed that miR-133b can interact specifically with the 3′- UTR of EGFR mRNA. Functionally, miR-133b transfection showed regulatory activity in translationally repressing EGFR mRNA. Moreover, miR-133b transfection may modulate apoptosis, invasion and sensitivity to EGFR-TKI through the EGFR signaling pathways, especially in EGFR-addicted NSCLC cells. Taken together, our findings show that miR-133b can inhibit cell growth of NSCLC through targeting EGFR and regulating its downstream signaling pathway. This finding has important implications for the development of targeted therapeutics for a number of EGFR-addicted cancers. [ABSTRACT FROM AUTHOR]