RESUMO
Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitaminâ E and a photolabile linker at the 5' terminus to inactivate the CRISPR/Cas9 system. The vitaminâ E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitaminâ E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factorâ A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , RNA/genética , DNA/genética , DNA/metabolismo , Células HEK293 , Humanos , RNA/química , RNA/efeitos da radiação , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Raios Ultravioleta , Vitamina E/análogos & derivados , Vitamina E/efeitos da radiaçãoRESUMO
RNA interference (RNAi) technology has been widely used in therapeutics development, especially multiple targeted RNAi strategy, which is a better method for multiple gene suppression. In the study, interfering RNAs (iRNAs) were designed for carrying two or three different siRNA sequences in different secondary structure formats (loop or cloverleaf). By using these types of iRNAs, co-inhibition of survivin and B-cell lymphoma-2 (Bcl-2) was investigated in hepatocellular carcinoma (HCC) cells, and we obtained promising gene silencing effects without showing undesirable interferon response. Furthermore, suppression effects on proliferation, invasion, and induced apoptosis in HCC cells were validated. The results suggest that long iRNAs with secondary structure may be a preferred strategy for multigenic disease therapy, especially for cancer and viral gene therapy and their iRNA drug development.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , RNA Interferente Pequeno/fisiologia , Apoptose/genética , Apoptose/fisiologia , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , SurvivinaRESUMO
The occurrence and development of hepatocellular carcinoma (HCC) is a complicate process involved in genetic mutation and epigenetic regulation. Successful HCC therapy needs multi-targets be involved. The aim of this study was to provide a triple effective RNA (teRNA) which composed of the specific siRNAs targeting NET-1 and VEGF and dsRNA activating TLR3, and explored its anti-HCC roles and mechanism. Real-time quantitative PCR (RT-qPCR), Western blot, immunofluorescence staining, MTT, Annexin V-FITC flow cytometry, Transwell and in-vitro Angiogenesis assay were used to measure the cell biological functions and protein expression analysis. Furthermore in in-vivo mouse model, teRNA inhibited tumor growth were detected by immunohistochemistry and TUNEL assay. Results showed that the proliferation, migration and angiogenesis of HCC cells were inhibited by teRNA effectively, the cell apoptosis also was induced, and further tumor growth was suppressed in-vivo. The gene silencing mechanism of teRNA was in an Ago2-dependent manner with no interferon response. The study suggests that NET-1, VEGF and TLR3 might be better targets for HCC treatment and combined these targets in form of a multi-target small RNA, teRNA could be a stagey for the development of anti-HCC drugs.
RESUMO
Glycogen synthase kinase-3ß (GSK-3ß) is required in the expression of epithelial junction proteins. It was found downregulated in hepatocellular carcinoma (HCC) tissues. The purpose of this study was to investigate the role of GSK-3ß in modulating the metastatic behaviors of human HCC cell lines in vitro. In this study, the expression level of GSK-3ß was measured in 4 human HCC cell lines, and the small interfering RNA (siRNA) vectors against or plasmids encoding GSK-3ß were used to evaluate the responses of target cells to the knockdown or overexpression of this kinase, respectively. Our results showed that GSK-3ß expression was significantly lower in human HCC cell lines with high metastatic potential than that in HCC cell lines without metastatic characteristics or in a normal human liver cell line. The knockdown of GSK-3ß by siRNA led to a decreased expression of the epithelial junction molecules (ZO-1, E-cadherin) and an increase in the expression of a mesenchymal cell marker (α-SMA) and a gene transcription factor (ß-catenin), resulting in enhanced tumor cell dissemination. In contrast, gain-of-function studies revealed that ectopic expression of GSK-3ß reduced invasive and migratory abilities of HCC cells accompanied by decreased HCC cell proliferation and induced apoptosis. More importantly, downregulation of GSK-3ß led to an increase in the expression and accumulation of ß-catenin in the nuclei, promoting gene transcription. In conclusion, GSK-3ß might play a vital role in suppressing HCC dissociation by preventing the disassembly of cancer cell epithelial junctional complex via the GSK-3ß/ß-catenin pathway.
RESUMO
Targeting specificity is an essential issue in the development of CRISPR-Cas technology. Using a luciferase activation assay, off-target cleavage activity of sgRNA was systematically investigated on single nucleotide-mismatched targets. In addition to confirming that PAM-proximal mismatches are less tolerated than PAM-distal mismatches, our study further identified a "core" sequence that is highly sensitive to target-mismatch. This sequence is of 4-nucleotide long, located at +4 to +7 position upstream of PAM, and positioned in a steric restriction region when assembled into Cas9 endonuclease. Our study also found that, single or multiple target mismatches at this region abolished off-target cleavage mediated by active sgRNAs, thus proposing a principle for gene-specific sgRNA design. Characterization of a mismatch sensitive "core" sequence not only enhances our understanding of how this elegant system functions, but also facilitates our efforts to improve targeting specificity of a sgRNA.
Assuntos
Pareamento de Bases , Sequência de Bases , Perfilação da Expressão Gênica , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Sistemas CRISPR-Cas , Genes Reporter , Engenharia Genética , Genoma , Humanos , MutaçãoRESUMO
Multiple targets RNAi strategy is a preferred way to treat multigenic diseases, especially cancers. In the study, multi-target siRNAs were designed to inhibit NET-1, EMS1 and VEGF genes in hepatocellular carcinoma (HCC) cells. And multi-target siRNAs showed better silencing effects on NET-1, EMS1 and VEGF, compared with single target siRNA. Moreover, multi-target siRNA showed greater suppression effects on proliferation, migration, invasion, angiogenesis and induced apoptosis in HCC cells. The results suggested that multi-target siRNA might be a preferred strategy for cancer therapy and NET-1, EMS1 and VEGF could be effective targets for HCC treatments.
RESUMO
RNA interference (RNAi) libraries screens have become widely used for small RNA (sRNA) therapeutic targets development. However, conventional enzymatically libraries, typically prepared using the type 2 restriction enzyme MmeI, produce sRNAs between 18 and 20 bp, much shorter than the usual lengths of 19-23 bp. Here we develop a size unbiased representative enzymatically generated RNAi (SURER) library, which employs type 3 restriction modification enzyme EcoP15I to produce sRNAs ranging from 19 to 23 bp using a group of rationally designed linkers, which can completely mimic the length of sRNAs naturally generated by Dicer enzyme in living cells, and the screening results of SURER libraries showed high recombination rate and knockdown efficiency. SURER library provides a useful tool for RNAi therapeutics screening in a fast and simple way.
Assuntos
Técnicas de Silenciamento de Genes , Interferência de RNA , Sequência de Bases , Biocatálise , DNA Polimerase Dirigida por DNA/genética , Expressão Gênica , Biblioteca Gênica , Terapia Genética , Células Hep G2 , Vírus da Hepatite B/enzimologia , Humanos , Proteínas Inibidoras de Apoptose/biossíntese , Proteínas Inibidoras de Apoptose/genética , Sequências Repetidas Invertidas , RNA Interferente Pequeno/genética , SurvivinaRESUMO
RNA interference (RNAi) has been proven in recent years to be a newly advanced and powerful tool for development of therapeutic agents toward various unmet medical needs such as cancer, in particular, a great attention has been paid to the development of antineoplastic agents. Recent success in clinical trials related to RNAi-based therapeutics on cancer and ocular disease has validated that small interfering RNAs (siRNAs) constitute a new promising class of therapeutics. Currently, a great wealth of multi-target based siRNA structural modifications is available for promoting siRNA-mediated gene silencing with low side effects. Here, the latest developments in RNAi-based therapeutics and novel structural modifications described for siRNAs--in particular multi-target siRNAs--are reviewed.
Assuntos
Antineoplásicos/uso terapêutico , Síndromes do Olho Seco/terapia , Neoplasias/terapia , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Ensaios Clínicos como Assunto , Sistemas de Liberação de Medicamentos/métodos , Humanos , Terapia de Alvo Molecular , Conformação de Ácido NucleicoRESUMO
MicroRNA-200a (miR-200a) has been reported to regulate tumour progression in several tumours; however, little is known about its role in non-small cell lung cancer cells (NSCLCs). Here, we found that miR-200a was up-regulated in A549 and SK-MES-1 cells compared with normal lung cells HELF. By a series of gain-of-function and loss-offunction studies, over-expression of miR-200a was indicated to enhance cells migration, and its knock-down inhibited migration of cells in NSCLC cell lines. Furthermore, miR-200a was identified to induce TSPAN1 expression which was related to migration. TSPAN1 was proved to induce migration, and so up-regulation of TSPAN1 by miR-200a may explain why over-expressing miR-200a promotes NSCLC cells migration.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Movimento Celular/genética , MicroRNAs/genética , Tetraspaninas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Tetraspaninas/metabolismo , Regulação para CimaRESUMO
Simultaneous suppression of multiple oncogenes is an attractive strategy to treat cancers. Herein we present a series of long double-stranded multiplex small interfering RNAs (multi-siRNAs) that is suitable for dual genes silencing through a sequence-specific RNA interference process without inducing significant immune responses. A gap feature structurally designed in either of the nucleotide strands of the multi-siRNAs was proved to be essential toward silencing target genes and avoiding immune responses. Furthermore, the silencing effect of multi-siRNAs against SURVIVIN and BCL-2 genes was shown to be effective and resulted in up-regulation of caspase-3 related apoptosis and, in turn, inhibition of bladder cancer cell proliferation. Our observation suggested that the rationally designed multi-siRNAs would have great potential for therapeutic siRNA design.
Assuntos
Marcação de Genes/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Apoptose/genética , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Interferons/antagonistas & inibidores , Interferons/biossíntese , Interferons/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA de Cadeia Dupla/síntese química , RNA Interferente Pequeno/síntese química , Proteínas de Ligação a RNA , Survivina , Regulação para CimaRESUMO
Over the past two decades, RNA interference (RNAi) has achieved great improvements in medicine, which has benefited the development of innovative cosmeceutical products, particular, to antiaging cosmeceuticals. A variety of ongoing research has tried to employ small RNAs-small interference RNA and microRNA as new cosmeceutical ingredients. Furthermore, several skin care companies have released new small RNA products in cosmetic market. In this review, we will describe the latest and most advanced approaches and strategies of using small RNA as antiaging cosmetics, including investigations on aging-related genes that small RNA target, method of delivering them, and challenges in the development of RNAi-based therapeutics for skin care cosmeceuticals. It is certain that advancement in this direction will evolve a new landscape for innovative antiaging cosmeceuticals.