Front Matter

This file contains cover for Volume II, Issue II, Editorial Board, Acknowledgements

VEGF siRNA Delivery System Using Arginine-Grafted Bioreducible Poly(disulfide amine)

Small interfering RNAs (siRNAs) are able to silence their target genes when they are successfully delivered intact into the cytoplasm. Delivery systems that enhance siRNA localization to the cytoplasm can facilitate gene silencing by siRNA therapeutics. We describe an arginine-conjugated poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH-R)), a bioreducible cationic polymer, as an siRNA carrier for therapeutic gene silencing for cancer. After intracellular uptake of the siRNA/poly(CBA-DAH-R) polyplexes, the reductive environment of the cytoplasm cleaves the disulfide linkages in the polymeric backbone, resulting in decomplexing of the siRNA/poly(CBA-DAH-R) polyplexes and release of siRNA molecules throughout the cytoplasm. The siRNA/poly(CBA-DAH-R) polyplexes, which demonstrate increased membrane permeability with arginine modification, have a similar level of cellular uptake as siRNA/bPEI polyplexes. The VEGF siRNA/poly(CBA-DAH-R) polyplexes, however, inhibit VEGF expression to a greater degree than VEGF siRNA/bPEI in various human cancer cell lines. The improved RNAi activity demonstrated by the VEGF siRNA/poly(CBA-DAH-R) polyplexes is due to enhanced intracellular delivery and effective localization to the cytoplasm of the VEGF siRNAs. These results demonstrate that the VEGF siRNA/poly(CBA-DAH-R) polyplex delivery system may useful for siRNA-based approaches for cancer therapy

Inflammatory gene expression patterns in response to LPS stimulation and after LPS withdrawal in BV-2 microglial cells.

<p>(A, B) Quantitative real-time reverse transcriptase-PCR analysis of inflammatory gene expression in BV-2 microglial cells stimulated with LPS (10 ng/ml) and after LPS was washed away (LPS withdrawal). The expression of inflammatory genes was significantly up-regulated in cells treated with LPS and significantly decreased after the removal of LPS compared with untreated cells (<i>*P<0</i>.<i>05 and **P<0</i>.<i>001</i>) at the indicated times. Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are shown as the means ± SD of triplicate wells.</p

Confirmation of differentially expressed genes by quantitative reverse transcription-polymerase chain reaction in BV-2 microglial cells.

<p>ccl12, ccl7, irak3, ptgs2, il1a, irg1, irf9, irf1, relb, p65, cxcl10, and ccl2 genes were significantly up-regulated in LPS-treated BV-2 microglia cells. Gene expression was normalized to the GAPDH transcript levels. <i>*P<0</i>.<i>05</i>, <i>**P<0</i>.<i>001</i> and ns stands for no significant difference compared with control. The data represent three independent experiments.</p

List of primers used in q-RT-PCR studies.

<p>List of primers used in q-RT-PCR studies.</p

Effect of Aβ₄₂ on the expressions of inflammatory mediators in microglial cells.

<p>Quantitative real-time reverse transcriptase-PCR analysis of inflammatory gene expression in BV-2 and primary microglial cells stimulated with Aβ₄₂. The expression of inflammatory genes were significantly up-regulated in primary microglial cells treated with Aβ₄₂ compared with untreated cells (<i>*P<0</i>.<i>05</i>, <i>**P<0</i>.<i>001</i> and ns stands for no significant difference compared with control) at the indicated times. Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are shown as the means ± SD of triplicate wells.</p

RNA-Seq analysis reveals that LPS-stimulated pro-inflammatory gene expression in BV-2 microglial cells.

<p>(A) Heat map representing RNA-Seq gene expression of up-regulated (≥ 1.5 log₂-fold) inflammatory genes at 2 and 4 h after LPS stimulation in BV-2 microglia cells compared with controls. (B) Venn diagram displaying the number of inducible or repressible (≥ 1.5 log₂-fold) genes after LPS stimulation in BV-2 microglia cells. (C, D) UCSC Browser images representing normalized RNA-Seq read densities at 2 and 4 h after LPS stimulation in BV-2 microglia cells compared with controls. (E, F) Gene Ontology analysis of functional annotations associated with up-regulated genes at 2 and 4 h after LPS stimulation in BV-2 microglia.</p

Dual RNA Sequencing Reveals the Expression of Unique Transcriptomic Signatures in Lipopolysaccharide-Induced BV-2 Microglial Cells

<div><p>Microglial cells become rapidly activated through interactions with pathogens, and the persistent activation of these cells is associated with various neurodegenerative diseases. Previous studies have investigated the transcriptomic signatures in microglia or macrophages using microarray technologies. However, this method has numerous restrictions, such as spatial biases, uneven probe properties, low sensitivity, and dependency on the probes spotted. To overcome this limitation and identify novel transcribed genes in response to LPS, we used RNA Sequencing (RNA-Seq) to determine the novel transcriptomic signatures in BV-2 microglial cells. Sequencing assessment and quality evaluation showed that approximately 263 and 319 genes (≥ 1.5 log₂-fold), such as cytokines and chemokines, were strongly induced after 2 and 4 h, respectively, and the induction of several genes with unknown immunological functions was also observed. Importantly, we observed that previously unidentified transcription factors (TFs) (irf1, irf7, and irf9), histone demethylases (kdm4a) and DNA methyltransferases (dnmt3l) were significantly and selectively expressed in BV-2 microglial cells. The gene expression levels, transcription start sites (TSS), isoforms, and differential promoter usage revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with LPS. In addition, gene ontology, molecular networks and pathway analyses identified the top significantly regulated functional classification, canonical pathways and network functions at each activation status. Moreover, we further analyzed differentially expressed genes to identify transcription factor (TF) motifs (−950 to +50 bp of the 5’ upstream promoters) and epigenetic mechanisms. Furthermore, we confirmed that the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in LPS treated primary microglial cells. This transcriptomic analysis is the first to show a comparison of the family-wide differential expression of most known immune genes and also reveal transcription evidence of multiple gene families in BV-2 microglial cells. Collectively, these findings reveal unique transcriptomic signatures in BV-2 microglial cells required for homeostasis and effective immune responses.</p></div

Confirmation of differentially expressed genes and release of pro-inflammatory mediators in primary microglial cells.

<p>(A, B, and C) irg1, il1a, il1b, ccl7, ccl12, ccl2, cxcl10, irf1, and irf7 genes were significantly up-regulated in LPS (10 ng/mL) treated primary microglia cells. Gene expression was normalized to the GAPDH transcript levels. (D) Primary microglial cell culture supernatants of LPS (10 ng/mL) treated cells were subjected to ELISA to detect the levels of pro-inflammatory cytokines/chemokines. Therefore, primary microglial cells were treated with LPS for 2 h and 4 h, followed by quantification of ccl2, ccl7, and cxcl10 levels. Values are given in pg/ml. Means and standard deviations of the mean of three independent experiments are shown (<i>*P value <0</i>.<i>05</i>, <i>**P value <0</i>.<i>001</i>, ns stands for no significant difference compared with control).</p

Distribution of all TF-encoding genes that are altered by ethanol.

A) Venn diagram representing the overall distribution of all differentially expressed TF-encoding genes. A total of 42, 15 and 35 differentially expressed genes were identified from NCCIT vs. EB, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, respectively. A total of 2,16 and 4 genes were common between NCCIT vs. EB and NCCIT vs. EB+EtOH, NCCIT vs. EB+EtOH and EB vs. EB+EtOH, and NCCIT vs. EB and EB vs. EB+EtOH, respectively. B), C) and D) represent heat maps of differential TF-encoding gene expressions between NCCIT vs. EB, EB vs. EB+EtOH and NCCIT vs. EB+EtOH, respectively. Gene expression level of each gene in the heat map is scaled and represented as relative expression value. E) Represents heat maps for TF-encoding genes found common between NCCIT vs. EB and EB vs. EB+EtOH. Genes with specific expression patterns were clustered into 4 groups–cluster 1, 2, 3 and 4 represent up-up, up-down, down-down and down-up, respectively. Only up-down and down-up relationships were found enriched. F) Line graph representing relative expression pattern of those TF-encoding gene clusters defined from the transcriptomic profiling. Genes with specific expression patterns were clustered according to their relative expression values. G), H) and I) represent transcription motif analysis of selected 42, 15 and 35 TF-encoded genes, respectively. Significantly enriched top 5 motifs are presented here (p-value < 0.05). The sequence logos are illustrated in the third column of each table.</p