Strand antagonism in RNAi: an explanation of differences in potency between intracellularly expressed siRNA and shRNA

Strategies to regulate gene function frequently use small interfering RNAs (siRNAs) that can be made from their shRNA precursors via Dicer. However, when the duplex components of these siRNA effectors are expressed from their respective coding genes, the RNA interference (RNAi) activity is much reduced. Here, we explored the mechanisms of action of shRNA and siRNA and found the expressed siRNA, in contrast to short hairpin RNA (shRNA), exhibits strong strand antagonism, with the sense RNA negatively and unexpectedly regulating RNAi. Therefore, we altered the relative levels of strands of siRNA duplexes during their expression, increasing the level of the antisense component, reducing the level of the sense component, or both and, in this way we were able to enhance the potency of the siRNA. Such vector-delivered siRNA attacked its target effectively. These findings provide new insight into RNAi and, in particular, they demonstrate that strand antagonism is responsible for making siRNA far less potent than shRNA

Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

Abstract Background Increasing evidence reveals a significant correlation between gamma-synuclein (SNCG) level and tumor invasion and metastasis in various human cancers. Our previous investigation showed that SNCG could secrete into extracellular environment and promoted tumor cell motility, but the mechanism is unknown. Methods The membrane binding ability of SNCG was characterized by immunohistochemical staining, immunofluorescence staining and fractionation of colorectal cancer (CRC) cell membrane. Association between SNCG and β1 integrin was validated by coimmunoprecipitation and far Western blot. After inhibition of β1 integrin and focal adhesion kinase (FAK), effect of SNCG on cell motility was measured by transwell chamber assays and changes of protein levels were detected by Western blot. Association between SNCG and activated β1 integrin levels in human CRC tissues was determined by Spearman’s rank correlation analysis. Secreted proteins in conditioned medium (CM) were screened by antibody array. Results Extracellular SNCG bound β1 integrin on CRC cell membrane and increased levels of activated β1 integrin and FAK. Correspondingly, SNCG-enhanced cell motility was counteracted by knockdown or inhibition of β1 integrin or FAK. Further study revealed that high SNCG level indicated poor outcome and SNCG levels positively correlated with those of activated β1 integrin and phospho-FAK (Tyr397) in human CRC tissues. Additionally, extracellular SNCG promoted secretion of fibronectin (FN), vitronectin (VN), matrix metalloproteinase (MMP)-2, and MMP-24 from HCT116 cells. Protease activity of MMP-2 in the CM of HCT116 cells was increased by treatment with SNCG, which was abolished by inhibiting β1 integrin. Conclusion Our results highlight the potential role of SNCG in remodeling extracellular microenvironment and inducing β1 integrin-FAK signal pathway of CRC cells

Additional file 1: of Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

Table S1. Detailed information of antibodies used in the study. Table S2. Clinicopathologic characteristics of patients (n=250). Table S3. Small interfering RNA sequences for β1 and FAK. Figure S1. Effect of small interfering RNA sequence on β1 protein expression. Figure S2. β1 integrin and FAK mediate SNCG-promoted tumor cell migration. Figure S3. Up-regulation of phospho-FAK induced by SNCG is blocked by β1 integrin knockdown, but has no effect on Src or Erk phosphorylation. Figure S4. Exogenously added SNCG promotes MMP-24 and MMP-2 secretion from colorectal cancer cells. (DOC 1280 kb

Integrated analysis of genome-wide association studies and 3D epigenomic characteristics reveal the BMP2 gene regulating loin muscle depth in Yorkshire pigs.

BackgroundThe lack of integrated analysis of genome-wide association studies (GWAS) and 3D epigenomics restricts a deep understanding of the genetic mechanisms of meat-related traits. With the application of techniques as ChIP-seq and Hi-C, the annotations of cis-regulatory elements in the pig genome have been established, which offers a new opportunity to elucidate the genetic mechanisms and identify major genetic variants and candidate genes that are significantly associated with important economic traits. Among these traits, loin muscle depth (LMD) is an important one as it impacts the lean meat content. In this study, we integrated cis-regulatory elements and genome-wide association studies (GWAS) to identify candidate genes and genetic variants regulating LMD.ResultsFive single nucleotide polymorphisms (SNPs) located on porcine chromosome 17 were significantly associated with LMD in Yorkshire pigs. A 10 kb quantitative trait locus (QTL) was identified as a candidate functional genomic region through the integration of linkage disequilibrium and linkage analysis (LDLA) and high-throughput chromosome conformation capture (Hi-C) analysis. The BMP2 gene was identified as a candidate gene for LMD based on the integrated results of GWAS, Hi-C meta-analysis, and cis-regulatory element data. The identified QTL region was further verified through target region sequencing. Furthermore, through using dual-luciferase assays and electrophoretic mobility shift assays (EMSA), two SNPs, including SNP rs321846600, located in the enhancer region, and SNP rs1111440035, located in the promoter region, were identified as candidate SNPs that may be functionally related to the LMD.ConclusionsBased on the results of GWAS, Hi-C, and cis-regulatory elements, the BMP2 gene was identified as an important candidate gene regulating variation in LMD. The SNPs rs321846600 and rs1111440035 were identified as candidate SNPs that are functionally related to the LMD of Yorkshire pigs. Our results shed light on the advantages of integrating GWAS with 3D epigenomics in identifying candidate genes for quantitative traits. This study is a pioneering work for the identification of candidate genes and related genetic variants regulating one key production trait (LMD) in pigs by integrating genome-wide association studies and 3D epigenomics

¹²⁵I–F56 Peptide as Radioanalysis Agent Targeting VEGFR1 in Mice Xenografted with Human Gastric Tumor

¹²⁵I-Radiolabeled F56 peptide was designed as a radioactive analogue of F56 (peptide WHSDMEWWYLLG) to bind with VEGFR1 receptor. It was synthesized in high radiochemical yield and specific activity. The <i>in vitro</i> stability of ¹²⁵I–F56 was tested, and the bioactivity of ¹²⁵I–F56 was confirmed by both cell uptake and binding affinity measurement in VEGFR1 positive BGC-823 cells. The time–radioactivity relationship and biodistribution of ¹²⁵I–F56 tracer were conducted using nude mice bearing human gastric carcinoma BGC-823, by noninvasive micro-SPECT/CT imaging. The tracer’s tumor uptake was further confirmed by autoradiography and HE stain of ¹²⁵I–F56 in tumor tissues ex vivo. Those results demonstrated that ¹²⁵I–F56 holds great potential as a diagnostic agent in both molecular imaging and radioanalysis probe for gastric cancer

SW inhibits cell scratch wound healing.

<p><i>In vitro</i> scratch assay was used to evaluate the effect of SW on the migration of HepG2 (A), MDA-MB-231 (B) and T24 cells (C). Representative images were displayed in left panel; quantification of the data in left panel was shown in right panel, shown were composite results of three independently experiments with triplicate parallel samples. The migration index represents migration speed in relative to control group. Columns, mean; bars, SD.</p

SW promotes cell adhesion.

<p>Left panels: HepG2 (A), MDA-MB-231 (B) and T24 cells (C) were pretreated with indicated doses of SW for 15 or 30 min before seeding into the matrigel-coated wells, and 2 h later, adhered cells were counted after removing the floating cells by PBS. Representative images were displayed. Scale bar, 200 μm. Right panels: quantification of the data in the left panel, shown were composite results of three independently experiments with triplicate. Columns, mean; bars, SD.</p

SW increases size and fluorescence intensity of focal adhesion.

<p>(A) HepG2 and T24 cells were treated with SW (3.5 μg/μl) for indicated times and then immuno-stained with F-actin (FITC-phalloidin) and vinculin (red). Nuclei were counterstained by DAPI (blue). Representative images were displayed. Scale bar, 100 μm. (B) The area of adhesion sites per cell (where F-actin and vinculin merged). Shown were composite results of two independently experiments (n = 50 cells). Columns, mean; bars, SD. (C) The fluorescence intensity of vinculin in the adhesion patches per cell. Shown were composite results of two independently experiments (n = 50 cells). Columns, mean; bars, SD.</p

Sarsaparilla (<i>Smilax Glabra</i> Rhizome) Extract Inhibits Migration and Invasion of Cancer Cells by Suppressing TGF-β1 Pathway

<div><p>Sarsaparilla, also known as <i>Smilax Glabra</i> Rhizome (SGR), was shown to modulate immunity, protect against liver injury, lower blood glucose and suppress cancer. However, its effects on cancer cell adhesion, migration and invasion were unclear. In the present study, we found that the supernatant of water-soluble extract from SGR (SW) could promote adhesion, inhibit migration and invasion of HepG2, MDA-MB-231 and T24 cells <i>in vitro</i>, as well as suppress metastasis of MDA-MB-231 cells <i>in vivo</i>. Results of F-actin and vinculin dual staining showed the enhanced focal adhesion in SW-treated cells. Microarray analysis indicated a repression of TGF-β1 signaling by SW treatment, which was verified by real-time RT-PCR of TGF-β1-related genes and immunoblotting of TGFBR1 protein. SW was also shown to antagonize TGF-β1-promoted cell migration. Collectively, our study revealed a new antitumor function of Sarsaparilla in counteracting invasiveness of a subset of cancer cells by inhibiting TGF-β1 signaling.</p></div