ABSTRACT
MicroRNA-21 (miR-21) expression in glioblastoma inhibits the expression of pro-apoptotic genes, thereby promoting tumor growth. A previous study showed that the amphiphilic R3V6 peptide is an efficient carrier of the anti-miR-21 antisense oligodeoxynucleotide (antisense-ODN) into cells in vitro. In the current study, in vivo delivery of antisense-ODN using the R3V6 peptide was evaluated in a glioblastoma animal model. In vitro transfection showed that the R3V6 peptide delivered antisense-ODN more efficiently than polyethylenimine (25 kDa, PEI25k) in C6 glioblastoma cells. For in vivo evaluation, antisense-ODN/R3V6 complex was injected intratumorally into a C6 glioblastoma xenograft animal model. Tumor growth was suppressed by the injection of the antisense-ODN/R3V6 complex, compared with the antisense-ODN/PEI25k and scrambled-antisense-ODN (scr-antisense-ODN)/R3V6 complexes. Real-time RT-PCR showed that miR-21 levels were reduced most efficiently by the antisense-ODNR3V6 complex in tumors. Due to inhibition of miR-21, expression of the programed cell death 4 (PDCD4) gene was promoted in tumors, resulting in the induction of apoptosis of tumor cells. These results suggest that delivery of antisense-ODN using R3V6 peptides may be useful for the development of antisense-ODN therapy for glioblastoma.
Subject(s)
Genetic Therapy/methods , Glioblastoma/drug therapy , MicroRNAs/antagonists & inhibitors , Neoplasms, Experimental/drug therapy , Oligonucleotides, Antisense/therapeutic use , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Gene Transfer Techniques , Male , Mice , Mice, Nude , MicroRNAs/genetics , MicroRNAs/metabolism , Peptides , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Inhibition of microRNA-21 (miR-21) has been shown to promote apoptosis of cancer cells and to reduce tumor size in glioblastoma. However, efficient carriers for antisense-oligodeoxynucleotide (antisense-ODN) against miR-21 have not yet been developed. In this study, the R3V6 peptide (R3V6) was evaluated as a carrier of antisense-ODN. In a gel retardation assay, R3V6 formed a complex with an antisense-ODN. The serum stability assay showed that R3V6 protected it from nucleases more efficiently than polyethylenimine (PEI; 25 kDa, PEI25k). A Renilla luciferase gene with a 3'-untranslated region (3'-UTR) recognizable by miR-21 (psiCHECK2-miR-21-UTR) was constructed for the antisense-ODN assay. psiCHECK2-miR-21-UTR expressed less Renilla luciferase in the cells with a higher level of miR-21 due to the effect of miR-21. In an in vitro transfection assay, the R3V6 peptide delivered anti-miR-21 antisense-ODN into cells more efficiently than PEI (25 kDa, PEI25k) and lipofectamine. As a result, antisense-ODN/R3V6 complex inhibited miR-21 and increased Renilla luciferase expression more efficiently than antisense-ODN/PEI25k or antisense-ODN/Lipofectamine complexes in both C6 and A172 glioblastoma cells. Furthermore, the antisense-ODN/R3V6 complexes reduced the level of miR-21 and induced apoptosis of glioblastoma cells. These results suggest that the R3V6 peptide may be a useful carrier of antisense-ODN for glioblastoma gene therapy.
Subject(s)
Genetic Therapy/methods , Glioblastoma/therapy , MicroRNAs/genetics , Oligonucleotides, Antisense/administration & dosage , Animals , Apoptosis/genetics , Cell Line, Tumor , Glioblastoma/genetics , Humans , Luciferases/genetics , Peptides/chemistry , Polyethyleneimine/chemistry , Rats , Renilla/genetics , Transfection/methodsABSTRACT
High mobility group box-1 (HMGB-1) is a DNA binding nuclear protein and pro-inflammatory cytokine. The box A domain of HMGB-1 (rHMGB-1A) exerts an anti-inflammatory effect, inhibiting wild-type HMGB-1 (wtHMGB-1). In this study, HMGB-1A was evaluated as an siRNA carrier with anti-inflammatory effects. HMGB-1A was expressed and purified by consecutive nickel chelate chromatography, cationic exchange chromatography, and polymixin B chromatography. Purified rHMGB-1A demonstrated an anti-inflammatory effect, reducing tumor necrosis factor-α (TNF-α) in wtHMGB-1 or lipopolysaccharide (LPS) activated macrophages. In gel retardation assay, rHMGB-1A formed a stable complex with siRNA at or above a 1:2 weight ratio (siRNA:rHMGB-1A). A heparin competition assay showed that an siRNA/rHMGB-1A complex released siRNA more easily than an siRNA/polyethylenimine (PEI, 25 kDa) complex. Luciferase siRNA/rHMGB-1A reduced firefly luciferase expression at a similar level as luciferase siRNA/PEI complex. Furthermore, TNF-α siRNA/rHMGB-1A synergistically reduced TNF-α expression in LPS activated macrophages. Therefore, rHMGB-1A may be useful as an siRNA carrier with anti-inflammatory effects in siRNA therapy for various inflammatory diseases.
Subject(s)
HMGB Proteins/genetics , HMGB Proteins/metabolism , Inflammation Mediators/metabolism , RNA, Small Interfering/metabolism , Animals , Cell Line , Cytokines , DNA-Binding Proteins , Gene Expression Regulation , Genetic Therapy/methods , HEK293 Cells , Humans , Inflammation/immunology , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/immunology , Mice , NF-kappa B/metabolism , RNA Interference , Tumor Necrosis Factor-alpha/metabolismABSTRACT
A non-toxic and efficient gene carrier is one requirement for clinical gene therapy. In this study, amphiphilic peptides composed of arginines and valines were synthesized and characterized as plasmid DNA (pDNA) carriers. The peptides have a cationic region containing 1-4 arginines and a hydrophobic region containing 6 valines. The arginine-valine peptides (RV peptides) formed micelles in aqueous solution with a critical micelle concentration (CMC) of 1.35 mg/ml. In gel retardation assay, the RV peptides retarded all pDNA at weight ratios (pDNA:RV peptide) of 1:3 for R1V6, 1:2 for R2V6 and R3V6, and 1:1 for R4V6. A heparin competition assay showed that the R3V6 peptide formed tighter complexes with pDNA than poly-L-lysine (PLL). In vitro transfection assay into HEK293 cells showed that the R1V6 and R2V6 peptides had the highest transfection efficiencies at 1:30 weight ratios (pDNA:RV peptide), while the R3V6 and R4V6 peptides had the highest efficiencies at 1:20 weight ratios. Under optimal conditions, the R3V6 peptide had the highest transfection efficiency of all the RV peptides and PLL. MTT assay showed that the RV peptides did not have any detectable toxicity to cells. Therefore, the RV peptide may be useful for the development of non-toxic gene carriers.
Subject(s)
Arginine/chemistry , Genetic Vectors/chemistry , Peptides/chemistry , Surface-Active Agents/chemistry , Transfection/methods , Valine/chemistry , Animals , DNA/administration & dosage , Genetic Therapy , HEK293 Cells , Humans , Peptides/chemical synthesis , Plasmids/administration & dosage , Rats , Surface-Active Agents/chemical synthesisABSTRACT
Liver fibrosis results from chronic damage to the liver by chronic hepatitis, alcohol, and toxic agents. A characteristic of liver fibrosis is an accumulation of extracellular matrix (ECM) protein, which distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of regenerating nodules defines cirrhosis. Transforming growth factor (TGF)-beta1, one of the most powerful profibrogenic mediators, plays a major role in the development of liver cirrhosis and regulates ECM gene expression and matrix degradation. This study elucidates the changes of TGF-beta1-mediated signals during liver fibrogenesis by using RNA interference. In this experiment, the TGF-beta1 siRNAs reduced the expression of TGF-beta1 in the livers of CCl(4) injection compared with those of control group, and the expression of type I collagen and alpha-smooth muscle actin was decreased. In conclusion, this study demonstrates that TGF-beta1 siRNAs inhibit TGF-beta1 expression in the murine model of liver cirrhosis and might be a good therapeutic strategy to prevent liver cirrhosis in human.
