Antisense RNA molecule represents a unique type of DNA transcript that comprises 19-23 nucleotides and is complementary to mRNA. Antisense RNAs play the crucial role in regulating gene expression at multiple levels, such as at replication, transcription, and translation. In addition, artificial antisense RNAs can effectively regulate the expression of related genes in host cells. With the development of antisense RNA, investigating the functions of antisense RNAs has emerged as a hot research field. This review summarizes our current understanding of antisense RNAs, particularly of the formation of antisense RNAs and their mechanism of regulating the expression of their target genes. In addition, we detail the effects and applications of antisense RNAs in antivirus and anticancer treatments and in regulating the expression of related genes in plants and microorganisms. This review is intended to highlight the key role of antisense RNA in genetic research and guide new investigators to the study of antisense RNAs.
Animals ; Antineoplastic Agents/therapeutic use* ; Antiviral Agents/therapeutic use* ; Gene Expression Regulation ; Genetic Research ; Humans ; MicroRNAs/physiology* ; RNA, Antisense/physiology* ; RNA, Long Noncoding/physiology* ; RNA, Small Interfering/physiology*

RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes or encode proteins for the synthesis desired genes to treat genetic diseases. RNA-based therapeutics are categorized as oligonucleotide drugs (antisense oligonucleotides, small interfering RNA, RNA aptamers), and mRNA drugs. The antisense oligonucleotides and small interfering RNA for treatment of genetic diseases have been approved by the FDA in the United States, while RNA aptamers and mRNA drugs are still in clinical trials. Chemical modifications can be applied to RNA drugs, such as pseudouridine modification of mRNA, to reduce immunogenicity and improve the efficacy. The secure and effective delivery systems such as lipid-based nanoparticles, extracellular vesicles, and virus-like particles are under development to address stability, specificity, and safety issues of RNA drugs. This article provides an overview of the specific molecular mechanisms of eleven RNA drugs currently used for treating genetic diseases, and discusses the research progress of chemical modifications and delivery systems of RNA drugs.
Aptamers, Nucleotide ; RNA, Small Interfering/therapeutic use* ; RNA, Messenger ; Oligonucleotides, Antisense/therapeutic use*

Animals ; Humans ; RNA Interference ; RNA, Antisense ; genetics ; RNA, Small Interfering ; genetics ; SARS Virus ; genetics ; physiology ; Severe Acute Respiratory Syndrome ; virology

OBJECTIVESElevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro.

METHODSAntisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells.

RESULTSThe results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05).

CONCLUSIONRNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.

Animals ; Cells, Cultured ; Dependovirus ; genetics ; Genetic Vectors ; Hepatic Stellate Cells ; metabolism ; RNA, Antisense ; genetics ; RNA, Small Interfering ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism

Animals ; Gene Expression Regulation ; drug effects ; Gene Targeting ; methods ; Humans ; Oligonucleotides, Antisense ; genetics ; pharmacology ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

No abstract available.
Mastocytoma* ; RNA, Antisense*

OBJECTIVETo construct recombinant expression vectors of small interfering RNA (siRNA) targeting survivin and investigate apoptosis of glioma cell line U251 mediated by the survivin-targeting siRNA.

METHODSAccording to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to form hairpin construct as the DNA templates for the target siRNA. The siRNA templates were cloned into siRNA expression vector pGenesil-1, and the resulted vector pGenesil-1/survivin was transfected into U251 cells using Metafectene following the standard protocols. Real-time PCR and Western blotting were performed to evaluate survivin gene silencing induced by siRNA transfection at the RNA and protein levels, respectively. Flow cytometry analysis with Annexin-V/PI double staining was used to determine the cell apoptosis.

RESULTSReal-time RT-PCR and Western blotting revealed significantly lowered survivin expression at both RNA and protein levels in transfected U251 cells, which exhibited a significantly higher apoptosis rate after transfection as shown by flow cytometry analysis.

CONCLUSIONRNA interference mediated by the siRNA expression vector pGenesi-l/survivin can significantly reduce survivin expression and induce remarkable apoptosis in U251 cells.

Apoptosis ; physiology ; Brain Neoplasms ; metabolism ; pathology ; Cell Division ; Cell Line, Tumor ; Genetic Therapy ; Glioma ; metabolism ; pathology ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; RNA, Antisense ; genetics ; RNA, Small Interfering ; Transfection

OBJECTIVETo study the effects of small interfering RNA (siRNA) and antisense oligonucleotides (ASOs) targeting ST6Gal I on adhesion and invasiveness of human colonic carcinoma cell line SW480 over-expressing ST6Gal I.

METHODSsiRNA and ASOs targeting ST6Gal I were constructed and transfected into SW480 cells via lipofectmine 2000. SW480 cells were cultured and divided into 7 groups, namely the blank control group, liposome group, siRNA group (transfected with ST6Gal I siRNA), ASO(1) group (transfected with ST6Gal I ASO whose target site is different from the siRNA), ASO(2) group (transfected with ST6Gal I ASO targeting the same site as siRNA), siRNA+ASO(1) group (transfected with siRNA and ASO(1)), siRNA+ASO(2) group (transfected with siRNA and ASO(2)). RT-PCR was used to examine ST6Gal I mRNA expression following the treatment. Flow cytometry was used to examine the amount of alpha2,6-sialylation on SW480 cell surface. SW480 cell adhesion and invasiveness to the extracellular matrix (ECM) were analyzed using CytoMatrix kit and cell invasion assay kit, respectively.

RESULTSThe expression of ST6Gal I mRNA, the amount of alpha2,6-sialylation on the cell surface and cell adhesion and invasion to ECM decreased remarkably in groups siRNA, ASO(1), ASO(2), siRNA+ASO(1) and siRNA+ASO(2), all significantly lower than those of the blank control and liposome groups (all P<0.05), especially in siRNA+ASO(1) group. Significant difference was noted between siRNA+ASO(1) and siRNA groups (P<0.05), but not between siRNA+ASO(2) and siRNA groups, or between blank control and liposome groups (all P>0.05).

CONCLUSIONChemically synthesized specific siRNA targeting ST6Gal I effectively inhibits SW480 cell ST6Gal I expression and leads to diminished cell adhesion and invasiveness to ECM, suggesting a combined effect of siRNA and ASO with different targeting sites.

Cell Adhesion ; Cell Movement ; Colonic Neoplasms ; genetics ; pathology ; Flow Cytometry ; Gene Silencing ; Humans ; Neoplasm Invasiveness ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sialyltransferases ; genetics ; metabolism ; Transfection

OBJECTIVETo construct a RNA interference vector for human tissue factor (TF) gene.

METHODSHuman TF short hairpin RNA (shRNA) sequence was designed using online design software (Invitrogen) and synthesized into double-strand oligonucleotide (ds oligo), which was cloned into the pENTRTM/U6 plasmid, followed by transformation of the product into competent Top10 E. coli cells. After expansion of the transformed bacteria, the plasmid was extracted and sequenced, which was subsequently transfected into human umbilical vein endothelial cells (HUVECs). The interference effect of the vector on the target gene expression was detected by RT-PCR and immunofluorescence assay.

RESULTSThe sequencing result indicated that the plasmid pENTRTM/U6-RelB-shRNA was constructed correctly, which resulted in effective inhibition of TF expression in HUVECs after transfection.

CONCLUSIONThe RNA interference vector against human TF gene has been constructed successfully, which may provide a stable transfection vector for potential treatment of blood coagulation abnormalities.

Base Sequence ; Cell Line ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Humans ; Molecular Sequence Data ; Oligonucleotides, Antisense ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Thromboplastin ; deficiency ; genetics ; Transfection

With the rapid development of genetic engineering and metabolic regulation, antisense technology displays its fascination to the world as a mild regulation genetic tool. Compared with other loss-of-function research methods (e.g. gene knockout), antisense technologies have advantages such as low cost, short period, and easy operation. It has been increasingly used in bacterial metabolic regulation as a powerful genetic tool. This review briefly summarized the latest progress and problems in antisense technologies that are recently used in metabolic engineering of bacteria, and compares the advantages and disadvantages of these technologies.
Bacteria ; genetics ; metabolism ; Genes, Bacterial ; Genetic Engineering ; Metabolic Networks and Pathways ; genetics ; Oligonucleotides, Antisense ; genetics ; RNA, Antisense ; genetics ; RNA, Catalytic ; genetics