Small interfering RNA (siRNA) has been used to treat various skin diseases. However, siRNA is limited in application due to its electronegativity, strong polarity, easy degradation by nuclease and difficulty in breaking through the skin barrier. Therefore, safe and efficient siRNA delivery vector is the premise of effective treatment of skin diseases by siRNA. In recent years, with the deepening of research on siRNA, great progress has been made in the development of delivery systems based on lipids, polymers, peptides and nanoparticles, some new transdermal delivery vectors of siRNA have emerged, such as liposomes, dendrimers, cell penetrating peptides, and spherical nucleic acid nanoparticles. This review will focus on the recent advance in siRNA transdermal delivery vectors.
Administration, Cutaneous ; Genetic Vectors ; administration & dosage ; Humans ; RNA, Small Interfering ; administration & dosage ; Skin Diseases ; therapy

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) downregulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.
Amines/chemistry* ; Antineoplastic Agents/adverse effects* ; Breast Neoplasms/pathology* ; Chitosan/chemistry* ; Doxorubicin/adverse effects* ; Drug Delivery Systems ; Epithelial-Mesenchymal Transition/drug effects* ; Female ; Humans ; MCF-7 Cells ; Neoplasm Metastasis/prevention & control* ; Oxidation-Reduction ; RNA, Small Interfering/administration & dosage* ; Reactive Oxygen Species/metabolism* ; rac1 GTP-Binding Protein/physiology*

Tumor necrosis factor-alpha (TNF-α) has been found to be centrally involved in the development of ischemia-reperfusion injury (IRI)-induced inflammation and apoptosis. Knockdown of TNF-α gene using small interfering RNA (siRNA) may protect renal IRI. Renal IRI was induced in mice by clamping the left renal pedicle for 25 or 35 min. TNF-α siRNA was administered intravenously to silence the expression of TNF-α. The therapeutic effects of siRNA were evaluated in terms of renal function, histological examination, and overall survival following lethal IRI. A single systemic injection of TNF-α siRNA resulted in significant knockdown of TNF-α expression in ischemia-reperfusion injured kidney. In comparison with control mice, levels of BUN and serum creatinine were significantly reduced in mice treated with siRNA. Pathological examination demonstrated that tissue damage caused by IRI was markedly reduced as a result of TNF-α siRNA treatment. Furthermore, survival experiments showed that nearly 90% of control mice died from lethal IRI, whereas more than 50% of siRNApretreated mice survived until the end of the eight-day observation period. We have demonstrated for the first time that silencing TNF-α by specific siRNA can significantly reduce renal IRI and protect mice against lethal kidney ischemia, highlighting the potential for siRNA-based clinical therapy.
Animals ; Apoptosis ; Disease Models, Animal ; Genetic Therapy ; Humans ; Inflammation ; genetics ; pathology ; therapy ; Kidney ; drug effects ; injuries ; pathology ; Mice ; RNA, Small Interfering ; administration & dosage ; genetics ; Reperfusion Injury ; genetics ; pathology ; therapy ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors ; genetics

To investigate the effect of RAD18-siRNA on cell proliferation and chemotherapy sensitivity of esophageal squamous cell carcinoma (ESCC) ECA-109 cells.RAD18-siRNA was transfected into human ECA-109 cells by Lipofectamine 3000. Quantitative PCR and Western blot were performed to detect RAD18 and CyclinD1 expression; CCK-8 assay was used to determine cell proliferation and chemotherapy drug sensitivity; flow cytometry was used to determine cell cycle. Correlation between RAD18 and CyclinD1 mRNA expression was analyzed by Pearson's correlation.Compared with non-transfected cells, the expression of RAD18 in RAD18-siRNA group was significantly decreased (<0.05). The cell proliferation was inhibited (<0.05) and the cell number of G1 phase was increased, G2/M phase cells decreased (<0.05) in RAD18-siRNA group. After treatment with different concentrations of cisplatin or 5-FU, the survival rate of the two cell groups was reduced (all<0.05), and the IC50 of RAD18-siRNA group was significantly lower than that of non-transfected group (<0.05). The mRNA expression of RAD18 was positively correlated with CyclinD1 expression in ESCC tissues(=0.478,<0.01).Down-regulated expression of RAD18 can decrease the cell proliferation and increase chemo-sensitivity of ESCC cells, and CyclinD1 may participate in the process.
Adjuvants, Pharmaceutic ; pharmacology ; Carcinoma, Squamous Cell ; drug therapy ; physiopathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cisplatin ; pharmacology ; Cyclin D1 ; drug effects ; genetics ; DNA-Binding Proteins ; administration & dosage ; pharmacology ; Down-Regulation ; drug effects ; genetics ; Drug Resistance, Neoplasm ; drug effects ; Drug Screening Assays, Antitumor ; methods ; Drug Synergism ; Esophageal Neoplasms ; drug therapy ; physiopathology ; Fluorouracil ; pharmacology ; G1 Phase ; drug effects ; G2 Phase ; drug effects ; Humans ; Metaphase ; drug effects ; RNA, Small Interfering ; administration & dosage ; pharmacology ; Transfection ; Ubiquitin-Protein Ligases ; administration & dosage ; pharmacology

OBJECTIVETo investigate the action mechanisms of the FZD5 gene in prostate cancer bone metastasis and search for some new treatments for this disease.

METHODSWe determined the expression level of the FZD5 gene in prostate cancer PC3 cells and, after transfection of siRNA into the PC3 cells and silence of the FZD5 gene, observed the changes in the migration and proliferation of the cells. We established the model of prostate cancer bone metastasis by tibial injection of prostate cancer cells in the nude mice. Then we injected control siRNA and FZD5-silenced siRNA into the tibia of the mice followed by evaluation of tumor-induced bone destruction by X-ray imaging at 0, 1, and 3 weeks and by HE staining at 3 weeks after injection.

RESULTSAfter transfection of FZD5-silenced siRNA into the prostate cancer PC3 cells, the expression of the FZD5 gene was decreased about 70%. The rate of cell proliferation was significantly lower in the gene silencing group than in the control (P < 0.05), and that of cell migration dropped by 30% in the former as compared with the latter group at 48 hours after FZD5 silencing (P < 0.05). At 3 weeks after injection of control siRNA or FZD5-silenced siRNA into the tibia of the mice, osteolytic damage was observed in both groups, though less in the FZD5 silencing group, with only a few remaining bone trabeculae visible.

CONCLUSIONSilencing the FZD5 gene can reduce the migration and proliferation of prostate cancer cells, help to suppress bone metastasis and destruction, and thereby improve the survival rate and quality of life of the patients.

Animals ; Bone Neoplasms ; genetics ; prevention & control ; secondary ; Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Frizzled Receptors ; genetics ; physiology ; Gene Expression ; Gene Silencing ; Male ; Mice ; Mice, Nude ; Osteolysis ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Quality of Life ; RNA, Small Interfering ; administration & dosage ; genetics ; Transfection

Evidence suggested that glycogen synthase kinase-3β (GSK-3β) is involved in Nogo-66 inhibiting axonal regeneration in vitro, but its effect in vivo was poorly understood. We showed that stereotactic injection of shRNA GSK-3β-adeno associated virus (GSK-3β-AAV) diminished syringomyelia and promoted axonal regeneration after spinal cord injury (SCI), using stereotactic injection of shRNA GSK-3β-AAV (tested with Western blotting and RT-PCR) into the sensorimotor cortex of rats with SCI and by the detection of biotin dextran amine (BDA)-labeled axonal regeneration. We also determined the right position to inject into the sensorimotor cortex. Our findings consolidate the hypothesis that downregulation of GSK-3β promotes axonal regeneration after SCI.
Animals ; Axons ; drug effects ; metabolism ; Dependovirus ; genetics ; Glycogen Synthase Kinase 3 beta ; genetics ; metabolism ; Humans ; Nerve Regeneration ; genetics ; RNA, Small Interfering ; administration & dosage ; genetics ; Rats ; Sensorimotor Cortex ; drug effects ; pathology ; Spinal Cord Injuries ; genetics ; pathology ; therapy ; Syringomyelia ; genetics ; pathology ; therapy

Hepatocellular carcinoma (HCC) is a major cause of cancer-related mortality in part due to its high resistance to chemotherapeutic drugs. The anti-apoptotic Mcl-1 expression has been reported as a resistance factor in various types of tumors. Here, we investigated the expression of Mcl-1 in hepatoma cells and HCC tissues and its relationship with p53, and analyzed the possibility of the gene as a molecular target for HCC therapy. HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression. Mcl-1 expression in hepatoma cell lines was measured by RT-PCR and Western blotting. The suppression of Mcl-1 by RNA interference or specific phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, was evaluated as monotherapy, and it was combined with mitomycin C (MMC) in treating hepatoma cell line HepG2. Cell viability and apoptosis were assessed by MTT and FACS analysis. Finally, changes of Mcl-1 or p53 expression in various hepatoma cell lines were examined after transfection with Mcl-1 siRNA, the Mcl-1 expression plasmid, or the wide-type p53 expression plasmid, respectively. Mcl-1 protein was remarkably enhanced in HCC tissues as compared with adjacent non-tumor liver tissues. In addition, Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly in SMMC7721 cells, and not in L02 cells. P53 protein was also overexpressed in HCC tissues and there was a significant correlation between the expression of p53 and Mcl-1. Silencing Mcl-1 by RNAi or LY294002 downregulated Mcl-1 expression and led to decreased cell viability and increased apoptosis. Combination of MMC and Mcl-1 RNAi or LY294002 exhibited a significant chemosensitizing effect. The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfection with the Mcl-1 expression plasmid in L02 cells. Furthermore, the expression of Mcl-1 in Hep3B cells was also not significantly changed after transfection with the wild-type p53 expression plasmid. It is concluded that Mcl-1 is overexpressed in HCC tissues. The mechanisms by which silencing Mcl-1 sensitizes hepatoma cells towards chemotherapy may be not attributed to the upregulated expression of p53 but the dysfunction of p53 through Mcl-1/p53 interaction. Mcl-1 may be a potential target of gene therapy for HCC.
Adenoma, Liver Cell ; drug therapy ; genetics ; pathology ; Apoptosis ; drug effects ; Biomarkers, Tumor ; biosynthesis ; genetics ; Chromones ; administration & dosage ; Gene Expression Regulation, Neoplastic ; drug effects ; Hep G2 Cells ; Humans ; Liver Neoplasms ; drug therapy ; genetics ; pathology ; Morpholines ; administration & dosage ; Myeloid Cell Leukemia Sequence 1 Protein ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVETo study the molecular mechanism of cisplatin to enhance the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in reversing multidrug resistance in vincristine-resistant human gastric cancer SGC7901/VCR cells.

METHODSMTT assay was used to measure the 50% inhibiting concentration (IC₅₀) and cell survival in SGC7901 and SGC7901/VCR cells after different treatments. SGC7901/VCR cells were treated with different concentrations of DDP, different concentrations of TRAIL alone or in combination, and then the mRNA and protein levels of several genes were determined by RT-PCR, RT-qPCR and Western-blot analysis. After targeted silencing with specific siRNA and transfection of recombinant plasmid c-myc into the SGC7901/VCR cells, the mRNA and protein levels of DR4, DR5 and c-myc were determined by RT-PCR and Western-blot analysis.

RESULTSAfter combined treatment with TRAIL and DDP of the SGC7901/VCR cells, the IC₅₀ of VCR, DDP, ADM, and 5-Fu treatment was significantly decreased compared with the control group or TRAIL-treated group (P < 0.05). After treatment with 0, 10, 50 ng/ml TRAIL in combination with 0.4 µg/ml DDP, the SGC7901/VCR cells showed significantly higher activation of caspase 3, down-regulation of DNA-PKcs/Akt/GSK-3β signaling pathway, and higher inhibition of MDR1(P-gp) and MRP1 than those treated with TRAIL alone (P < 0.01 for all). The mRNA and protein levels of DR4, DR5, c-myc were significantly decreased after silencing c-myc with specific siRNA in the SGC7901/VCR cells (P < 0.01 for all), and were significantly increased after transfection of recombinant plasmid c-myc into the SGC7901/VCR cells (P < 0.01 foe all). After the treatment with 10 ng/ml TRAIL, 0.25 µg/ml DDP + 10 ng/ml TRAIL and 0.5 µg/ml DDP + 10 ng/ml TRAIL, the relative expression level of c-myc protein in the SGC7901/VCR cells was 0.314 ± 0.012, 0.735 ± 0.026, and 0.876 ± 0.028, respectively, and the relative expression of cytochrome C was 0.339 ± 0.036, 0.593 ± 0.020 and 0.735 ± 0.031, respectively, and the relative expression levels of DR4, DR5, active-caspase 3 and active-caspase 9 in the SGC7901/VCR cells were also increased along with increasing DDP concentrations.

CONCLUSIONSThe activation of DNA-PKcs/Akt/GSK-3β signaling pathway and high expression of MDR1 and MRP1 play an important role in the multi-drug resistance properties of SGC7901/VCR cells. After combining with TRAIL, DDP can enhance the expression of DR4 and DR5 through up-regulating c-myc and enhancing the activation of caspase 3 and caspase 9 by facilitating mitochondrial release of cytochrome C. It may be an important molecular mechanism of DDP-induced sensitization of TRAIL to reverse the multidrug resistancein SGC7901/VCR cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; administration & dosage ; pharmacology ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Line, Tumor ; Cisplatin ; administration & dosage ; pharmacology ; Down-Regulation ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; administration & dosage ; pharmacology ; Formazans ; Genes, myc ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Inhibitory Concentration 50 ; Multidrug Resistance-Associated Proteins ; metabolism ; Neoplasm Proteins ; metabolism ; Plasmids ; Proto-Oncogene Proteins c-myc ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Stomach Neoplasms ; drug therapy ; pathology ; TNF-Related Apoptosis-Inducing Ligand ; administration & dosage ; pharmacology ; Tetrazolium Salts ; Transfection ; methods

Bmi1 is a member of the polycomb group family of proteins, and it drives the carcinogenesis of various cancers and governs the self-renewal of multiple types of stem cells. However, its role in the initiation and progression of bladder cancer is not clearly known. The present study aimed to investigate the function of Bmi1 in the development of bladder cancer. Bmi1 expression was detected in human bladder cancer tissues and their adjacent normal tissues (n=10) by immunohistochemistry, qRT-PCR and Western blotting, respectively. Bmi1 small interference RNA (siRNA) was synthesized and transfected into human bladder carcinoma cells (EJ) by lipofectamine 2000. The Bmil expression at mRNA and protein levels was measured in EJ cells transfected with Bmil siRNA (0, 80, 160 nmol/L) by qRT-PCR and Western blotting, respectively. Cell viability and Ki67 expression (a marker of cell proliferation) were determined in Bmi1 siRNA-transfected cells by CCK-8 assay and qRT-PCR, respectively. Cell cycle of transfected cells was flow-cytometrically determined. Immunofluorescence and Western blotting were used to detect the expression levels of cell cycle-associated proteins cyclin D1 and cyclin E in the cells. Pro-apoptotic proteins Bax and caspase 3 and anti-apoptotic protein Bcl-2 were detected by Western blotting as well. Additionally, xenograft tumor models were established by inoculation of EJ cells (infected with Bmil shRNA/pLKO.1 lentivirus or not) into nude mice. The tumor volumes were measured every other day for 14 days. The results showed that the Bmil expression was significantly increased in bladder tumor tissues when compared with that in normal tissues (P<0.05). Perturbation of Bmi1 expression by using siRNA could significantly inhibit the proliferation of EJ cells (P<0.05). Bmi1 siRNA-transfected EJ cells were accumulated in G1 phase and the expression levels of cyclin D1 and cyclin E were down-regulated. Bax and caspase-3 expression levels were significantly increased and Bcl-2 levels decreased after Bmi1 knockdown. Tumor volume was conspicuously reduced in mice injected with EJ cells with Bmi1 knockdown. Our findings indicate that Bmi1 is a potential driver oncogene of bladder cancer and it may become a potential treatment target for human bladder cancer.
Animals ; Apoptosis ; genetics ; Carcinogenesis ; genetics ; metabolism ; pathology ; Carcinoma ; genetics ; metabolism ; pathology ; therapy ; Caspase 3 ; genetics ; metabolism ; Cell Line, Tumor ; Cyclin D1 ; antagonists & inhibitors ; genetics ; metabolism ; Cyclin E ; antagonists & inhibitors ; genetics ; metabolism ; G1 Phase Cell Cycle Checkpoints ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Injections, Intralesional ; Ki-67 Antigen ; genetics ; metabolism ; Mice ; Mice, Nude ; Polycomb Repressive Complex 1 ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; genetics ; metabolism ; RNA, Small Interfering ; administration & dosage ; genetics ; metabolism ; Signal Transduction ; Tumor Burden ; Urinary Bladder ; metabolism ; pathology ; Urinary Bladder Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; agonists ; genetics ; metabolism

OBJECTIVETo construct, express and purify human fusion proteins composed of a single-chain antibody fragment scFv that recognizes the prostate specific membrane antigen (PSMA) protein, Fdt, HA2 and tp, and to analyze the binding activity of the expressed fusion proteins.

METHODSThe fusion protein genes scFv, scFv-tp, and scFv-Fdt-HA2-tp were amplified by PCR, and the genes obtained were then cloned into the expression vector pET28 and expressed in E. coli BL21. The expressed products were identified by SDS-PAGE and Western blot and purified with Ni(2+)-NTA chelating agarose. The antigen-binding activity of the fusion proteins was determined by ELISA.

RESULTSThe human anti-PSMA fusion gene was successfully constructed and expressed in M15 as the inclusion body after induced with IPTG. All the target proteins expressed could bind the PSMA antigen.

CONCLUSIONFusion proteins can specifically bind the PSMA antigen. This finding contributes to the study of the targeted delivery of siRNA.

Antigens, Surface ; immunology ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; immunology ; Glutamate Carboxypeptidase II ; immunology ; Humans ; Male ; Polymerase Chain Reaction ; RNA, Small Interfering ; administration & dosage ; immunology ; Recombinant Fusion Proteins ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; immunology