Data_Sheet_1_Comprehensive Analysis of a Competing Endogenous RNA Network Identifies Seven-lncRNA Signature as a Prognostic Biomarker for Melanoma.docx

Long non-coding RNAs (LncRNAs) can act as competing endogenous RNA (ceRNA) involving in tumor initiation and progression. Nevertheless, the prognostic roles of lncRNAs in lncRNA-related ceRNA network of melanoma remain elusive. In this study, RNA sequence profiles were downloaded from The Cancer Genome Atlas (TCGA) database, and there were 2020 differentially expressed messenger RNAs (DEmRNAs), 438 differentially expressed lncRNAs (DElncRNAs) and 65 differentially expressed microRNAs (DEmiRNAs) between primary and metastasis melanoma patients. A ceRNA regulatory network was constructed based on the DElncRNAs-DEmiRNAs and DEmiRNAs-DEmRNAs interactions, which contained 39 lncRNAs, 10 miRNAs, and 16 mRNAs. Furthermore, univariate and multivariate Cox regression analysis were carried out to establish a 7-lncRNA prognostic signature. Subsequently, the area under the curve (AUC) value of the receiver operating characteristic (ROC) curve and the Kaplan-Meier risk survival analysis revealed the significant performance of this signature. Finally, pathway enrichment analyses implied that lncRNA MIR205HG and MIAT were associated with multiple cancer-related pathways, especially epidermis development and immune response. The current study provides novel insights into the lncRNA-related ceRNA network and the potential of lncRNAs to be candidate prognostic biomarkers and therapeutic targets in melanoma.</p

AQP3 is a transcriptional target of Notch signaling.

<p>(A) HKCs reverse transfected with siRNAs against CSL or scrambled control were analyzed 1 week later (in differentiating condition) by qRT-PCR and immunoblot of the indicated genes and proteins. mRNA levels were normalized to reference ribosomal gene RPLP0, and presented as mean fold-change over control ± S.E.M. n=3. ** p< 0.01, *** p < 0.001, **** p<0.0001, Hes1, Keratin 10 and Involucrin were used as indicators of endogenous Notch activity, γ-tubulin served as loading control. (B) Differentiating HKCs were treated with DAPT (10μM) or DMSO control for 72 hours followed by qRT-PCR and immunoblot analysis of the indicated genes and proteins. The analysis was done as in (A). **** p<0.0001, n=3. (C) Proliferating HKCs were infected with retroviruses expressing activated Notch1 or GFP followed, 72 hours later, by qRT-PCR and immunoblot analysis of AQP3. Hey1 was used as an indicator of endogenous Notch activity. The analysis was done as in (A). * p<0.05, *** p<0.001, n=3. (D) Chromatin immuno-precipitation (ChIP) analysis of CSL binding to the regulatory region of the <i>AQP3</i> gene. Left panel: schematic representation of the <i>AQP3</i> gene locus based on ChIP-seq information on different chromatin states as provided by the ENCODE project. The location of CSL binding site in the <i>AQP3</i> gene was analyzed using Mat-inspector software. Right panel: HKCs were processed for ChIP analysis using CSL antibody or non immune IgG. Results are expressed as mean fold of enrichment ± S.E.M. n=2, for each indicated binding site, after immunoprecipitation with CSL antibody or non immune IgG, relative to enrichment for the negative control region. </p

AQP3 expression is down-regulated during late differentiation concurrent with Notch signaling activation.

<p>(A) Immunofluorescence analysis of AQP3 expression in normal human skin. Frozen sections (8 μm) of normal human skin were stained with an antibody against RORα (green) and Hoechst (blue) for DNA. Images are representatives of several independent fields. Bar=100 μm. (B) qRT-PCR analysis of expression of the indicated genes in HKCs under proliferating condition (70% confluence, cf) and at various time (days, D) of differentiation induced by high cell density. Values are normalized to reference ribosomal gene RPLP0, and presented as fold-changes relative to cells under proliferating condition ± S.E.M. n=3. ** p< 0.01, *** p < 0.001, **** p<0.0001. (C) Immunoblot analysis of indicated proteins in the same time course experiment as in (B). </p

Attenuated AQP3 expression promotes differentiation through activating Notch1.

<p>(A) HKCs reversely transfected with siRNAs against AQP3 or scrambled control were analyzed 72 week later (in proliferating condition) by qRT-PCR and immunoblot of AQP3. The analysis was done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080179#pone-0080179-g002" target="_blank">Figure 2A</a>. **** p<0.0001 n=3. (B) Alamar blue cell proliferation assay of HKCs with AQP3 silencing. HKCs reverse transfected with siRNAs against AQP3 or scrambled control as in (A). 48 hours post-transfection, cells were trypsinized and plated in 96-well plate at a density of 1000 cells/well in triplicate. Fluorescence intensity was measure for four consecutive days. Data are presented as mean fold change of fluorescence intensity ± S.E.M. over day 1. * p<0.0001, n=3. (C) Same samples as in (A) were analyzed by qRT-PCR of the indicated genes. (D) Same samples as in (A) were analyzed by qRT-PCR and immunoblot of the indicated genes and proteins. * p<0.0001, n=3. (E) HKCs were reverse transfected with siRNAs against AQP3 or scrambled control, followed by 48 hours later, incubation with DMSO or DAPT (10μM) for additional 3 days. The expression of indicated genes were analyzed by qRT-PCR. Results are presented as mean fold-change over control ± S.E.M. n=3. One-way ANOVA was used for statistical analysis. ** p= 0.0023, *** p=0.0001. </p

Increased AQP3 expression suppresses the expression of differentiation markers and induces the expression of pro-inflammatory cytokines.

<p>(A) HKCs were infected with retroviruses expressing AQP3 or GFP followed, 72 hours later (differentiating condition), by qRT-PCR and immunoblot analysis of the indicated genes and proteins. The analysis was done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080179#pone-0080179-g001" target="_blank">Figure 1</a>. * p< 0.0001, n=3. (B) HKCs were either reverse transfected with siRNAs against AQP3 or scrambled control and analyzed 96h later (differentiating condition) or infected with retroviruses expressing AQP3 or GFP and analyzed 72 hours later (differentiating condition) by qRT-PCR analysis of the indicated genes. In parallel, differentiating HKCs were treated with DAPT (10μM) or DMSO control for 72 hours followed by qRT-PCR analysis of the indicated genes. The analysis was done as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080179#pone-0080179-g001" target="_blank">Figure 1</a>. ** p< 0.01, *** p < 0.001, **** p<0.0001, n=3. </p

Weyl Semimetal TaAs: Crystal Growth, Morphology, and Thermodynamics

Tantalum arsenide is experimentally verified as a Weyl semimetal recently. However, it is difficult to grow large TaAs single crystals due to the decomposition prior to reaching its melting point. Here we report an improved chemical vapor transport method using iodine as an agent to get large-size, high-quality TaAs single crystals up to 1 cm. X-ray diffraction confirmed that they are tetragonal TaAs. Specific heat of TaAs was measured from 2 K to room temperature, and hence the entropy and enthalpy were obtained, which are helpful in designing the optimal growth conditions. The as-grown crystals exhibit polyhedral morphology consisting of {101}, {001}, {103}, and {112} facets. The key points in crystal growth include using tantalum in the form of foils instead of powder as the starting material, tilting ampule to enhance convections and controlling the concentration of agent iodine. These measures should be applicable to the growth of other transition metal arsenides and phosphides

Inhibition of Cell Proliferation in an NRAS Mutant Melanoma Cell Line by Combining Sorafenib and α-Mangostin

<div><p>α-Mangostin is a natural product commonly used in Asia for cosmetic and medicinal applications including topical treatment of acne and skin cancer. Towards finding new pharmacological strategies that overcome NRAS mutant melanoma, we performed a cell proliferation-based combination screen using a collection of well-characterized small molecule kinase inhibitors and α-Mangostin. We found that α-Mangostin significantly enhances Sorafenib pharmacological efficacy against an NRAS mutant melanoma cell line. The synergistic effects of α-Mangostin and Sorafenib were associated with enhanced inhibition of activated AKT and ERK, induced ER stress, and reduced autophagy, eventually leading to apoptosis. The structure of α-Mangostin resembles several inhibitors of the Retinoid X receptor (RXR). MITF expression, which is regulated by RXR, was modulated by α-Mangostin. Molecular docking revealed that α-Mangostin can be accommodated by the ligand binding pocket of RXR and may thereby compete with RXR-mediated control of MITF expression. In summary, these data demonstrate an unanticipated synergy between α-Mangostin and sorafenib, with mechanistic actions that convert a known safe natural product to a candidate combinatorial therapeutic agent.</p></div

α-Mangostin activates Sorafenib induced apoptosis.

A, Clonogenic survival assay was performed to examine the synergistic effect of the combination of Sorafenib and α-Mangostin on the survival of SK-MEL-2 cells. Cell colonies ware stained in blue. B, Colony numbers were counted. The combination of Sorafenib and α-Mangostin significantly suppressed colony formation (p = 0.001). C, TUNEL staining detects cell apoptosis. Slices were cultured in medium containing Sorafenib (5 μM), α-Mangostin (2 μM) or their combination for 3 hours and stained for TUNEL (red) or 4′, 6-diamidino-2-phenylindole (DAPI; blue). Representative images of three independent experiments are shown. D. Apoptotic cells were counted. Student’s T-test showed that a significant increase of apoptotic cells appeared in the drug combinatorial treatment (p = 0.0003).</p