RNA interference (RNAi) is a post-transcriptional gene silencing process by targeting mRNA for degradation in a sequence-specific manner. This powerful platform has enormous potential in functional genomics and medical research. As a tool to knock out expression of specific genes in a variety of organisms, RNAi was used to investigate gene function in a high throughput fashion. Highly conserved in evolution RNAi appears to have evolved as a cellular defense mechanism in plants and animals to suppress viral infection, transposon jumping and endogenous aberrant genes. Exploiting the natural mechanism, the researchers can shut down disease-causing genes and develop novel therapeutics against infection, tumor and other disease.
Gene Expression Regulation ; Gene Silencing ; Genomics ; RNA Interference ; RNA, Small Interfering ; RNA-Induced Silencing Complex

The role of suppression of small RNA molecules in the management of malignity and viral infection on human was studies and discussed. SiRNA (small interference RNA) suppressing gene expression was described. In the year 2001, Ribopharma AG researchers had first demontrated the function of RNAi in mammal cells. SIRPLEX is appropriate with target gene, for using in the treatment of suppression of pathological gene in various genera, including human.
RNA ; Genes ; Gene Silencing

OBJECTIVETo find some effective short interfering RNA's sites targeting HBV surface gene sequence using shRNA expression vectors.

METHODSFour shRNA expression vectors targeting HBV surface gene sequence were constructed based on pAVU6 + 27 vector, and cotransfected into AD293 cells with HBs-EGFP fusion gene plasmid. The changes of HBs-EGFP image were detected by FACS and microscopy. The HBs-EGFP mRNA expression was evaluated by RT-PCR.

RESULTSFour shRNA expression vectors and HBs-EGFP fusion gene plasmid were successfully constructed. pAVU6 + 4sh579 vector inhibited the HBs-EGFP expression by 69.8% in AD293 and suppressed the HBs-EGFP mRNA expression by 74.6%.

CONCLUSIONSThe results showed that the 579 site of HBV surface gene sequence was an effective target and pAVU6 + 4sh579 vector could suppress the HBs-EGFP expression in AD293 cells

Gene Expression Regulation, Viral ; Gene Silencing ; Gene Targeting ; methods ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; RNA-Induced Silencing Complex ; genetics

Animals ; Gene Silencing ; physiology ; Gene Targeting ; Genetic Therapy ; methods ; Genomics ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA-Induced Silencing Complex

OBJECTIVEThe aim of this review was to assess RNA interference (RNAi) and its possibility as a potential and powerful tool to develop highly specific double-stranded RNA (dsRNA) or small interfering RNA (siRNA) based gene-silencing therapeutics.

DATA SOURCESThe data used in this review were obtained from the current RNAi-related research reports.

STUDY SELECTIONdsRNA-mediated RNAi has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms. The discovery that synthetic duplexes of 21 nucleotides siRNAs trigger gene-specific silencing in mammalian cells has further expanded the utility of RNAi in to the mammalian system.

DATA EXTRACTIONThe currently published papers reporting the discovery and mechanism of RNAi phenomena and application of RNAi on gene function in mammalian cells were included.

DATA SYNTHESISSince the recent development of RNAi technology in the mammalian system, investigators have used RNAi to elucidate gene function, and to develop gene-based therapeutics by delivery exogenous siRNA or siRNA expressing vector. The general and sequence-specific inhibitory effects of RNAi that will be selective, long-term, and systemic to modulate gene targets mentioned in similar reports have caused much concern about its effectiveness in mammals and its eventual use as a therapeutic mordality.

CONCLUSIONSIt is certain that the ability of RNAi in mammals to silence specific genes, either when transfected directly as siRNAs or when generated from DNA vectors, will undoubtedly accelerate the study of gene function and might also be used as a potentially useful method to develop highly gene-specific therapeutic methods. It is also expected that RNAi might one day be used to treat human diseases.

Animals ; Antigens, Neoplasm ; Gene Silencing ; Genes, abl ; Genetic Therapy ; Humans ; Neoplasm Proteins ; genetics ; RNA Interference

Acute myeloid leukemia is a well characterized blood cancer in which the unnatural growth of immature white blood cell takes place, where several genes transcription is regulated by the micro RNAs (miRNAs). Argonaute (AGO) protein is a protein family that binds to the miRNAs and mRNA complex where a strong binding affinity is crucial for its RNA silencing function. By understanding pattern recognition between the miRNAs-mRNA complex and its binding affinity with AGO protein, one can decipher the regulation of a particular gene and develop suitable siRNA for the same in disease condition. In the current work, HOXA9 gene has been selected from literature, whose deregulation is well-established in acute myeloid leukemia. Four miRNAs (mir-145, mir-126, let-7a, and mir-196b) have been selected to target mRNA of HOXA9 (NCBI accession No. NM_152739.3). The binding interaction between mRNAs and mRNA of HOXA9 gene was studied computationally. From result, it was observed mir-145 has highest affinity for HOXA9 gene. Furthermore, the interaction between miRNAs-mRNA duplex of all chosen miRNAs are docked with AGO protein (PDB ID: 3F73, chain A) to study their interaction at molecular level through an in silico approach. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding of AGO-assisted miRNA based gene silencing mechanism in HOXA9 gene associated in acute myeloid leukemia computationally.
Computer Simulation ; Gene Silencing ; Humans ; Hydrogen Bonding ; Leukemia, Myeloid, Acute* ; Leukocytes ; MicroRNAs* ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering

Gene Silencing ; Genetic Therapy ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; therapy ; virology ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate the effects of lentivirus-mediated RNA interference targeting HOXA10 gene on the proliferation, apoptosis and morphology of leukemic cell line U937.

METHODSFour different shRNA plasmids were designed and built to interfere with HOXA10 gene. The four interference plasmids were transfected into 293T cells with the HOXA10 over expression plasmid and then the RNAi efficiency of the four interference plasmids was determined by Western blot. The best one was chosen to transfect 293T cells with lentiviral helping plasmids to produce packaged lentivirus (lenti-shHOXA10). U937 cells were divided into interference group (lenti-shHOXA10), negative control group and untreated group. After infection with the packaged lentivirus, infection efficiency of lentivirus for U937 was detected by flow cytometry, and the expression of HOXA10 gene mRNA and protein was detected by real-time PCR and Western blot. Cell survival was determined by MTT assay. Apoptosis rate was detected by flow cytometry.

RESULTSLentiviral-shRNA vector of HOXA10 gene was successfully constructed. Compared with the negative control and untreated groups, mRNA level of HOXA10 decreased by (92.3±1.3)%, protein levels decreased by 91.1%, and the inhibition rate of U937 cells [(43.9±0.7)%] increased in the interference group (P<0.05). Wright's staining showed that the ratio of karyon to cytoplasm was reduced and mitotic phase was rare in the interference group. Apoptosis rate in the interference group [(27.1±1.4)%] was significantly higher than in the negative [(19.4±1.9)%] and untreated groups [(5.5±1.3)%] (P<0.05).

CONCLUSIONSLentivirus mediated RNAi can reduce the expression level of HOXA10, effectively inhibit proliferation and promote apoptosis of U937 cells. HOXA10 gene is expected to become a new target for the treatment of leukemia at gene level.

Apoptosis ; Cell Proliferation ; Gene Silencing ; Homeodomain Proteins ; antagonists & inhibitors ; genetics ; Humans ; Lentivirus ; genetics ; RNA Interference ; Sequence Analysis, DNA ; U937 Cells

Chemotherapy remains at the first line for the treatment of leukemia. However, the multi-drug resistance of the tumor cells caused by chemotherapeutic drugs has seriously affected the effect of chemotherapy. And this is the main reason for the failure of the leukemia treatment. Therefore, to explore an effective way of reversing drug resistance has become the key of leukemia treatment. RNA interference, a system within living cells, helps to determine which genes are active and how active they are. It is a process in which translation of some cell messenger RNA (mRNA) sequences is prevented, because of the presence of (and consequent destruction of) matching double-stranded RNA sequences. RNA interference is also called post-transcriptional gene silencing (PTGS), since its effect on gene expression occurs after the production of mRNA during transcription. It is believed that RNA interference can protect the cell against viruses and other threats. The greatest advantage of RNAi is the specificity and high efficiency which can induce suppression of specific genes of interest but the unrelated genes are not affected. The selective and robust effect of RNAi on gene expression makes it a valuable research tool both in cell culture and living organisms because synthetic dsRNA introduced into cells can induce the suppression of specific genes of interest. Nowadays, the technology has been widely used in biomedical fields, especially in the diagnosis and treatment of blood system disease. However, besides the stability, targeting and biological safety in genetics, the immune response induced by exogenous RNA is also one of the key factors to limit the clinical practice of this emerging technology. In this review, the breakthrough of the technology in multi-drug resistance reversal in leukemia is summarized with the RNA interference technology as a starting point.
Cell Line, Tumor ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Gene Silencing ; Humans ; Leukemia ; genetics ; therapy ; RNA Interference

BACKGROUND: Small interfering RNAs (siRNAs) have been used to knockdown specific gene expression in various cells. Astrocytes and microglial cells play a key role in fundamental central nervous system functions and in chronic neuroinflammation. The aims of this study were to determine the optimal concentration of siRNA demonstrating efficient transfection and inhibition of gene expression via RNA interference (RNAi) and lower cytotoxicity, in primary cultured astrocytes and microglial cells of rats. METHODS: Astrocytes and microglial cells were isolated from the cerebral cortices of 2-day-old rats. Both the cells were transfected using transfection reagent (Lipofectaminetrade mark 2000), and fluorescein-labeled double-stranded RNA (dsRNA) or siRNA targeting green fluorescent protein. Transfection efficiency and cytotoxicity of dsRNA, and the degrees of RNAi induced by siRNA in these cells, were evaluated at various concentrations of RNA. RESULTS: Transfection efficiencies of dsRNA in both astrocytes and microglial cells were significantly higher (P < 0.05) at the concentrations of 20, 40, and 80 nM than at the concentrations of 0, 5, and 10 nM. There were no significant cytotoxicities within the applied concentrations of dsRNA (0-80 nM). The degrees of RNAi induced by siRNA were significantly higher (P < 0.05) at the concentrations of 5, 10, 20, 40, 80 nM, and 20, 40, 80 nM in astrocytes and microglial cells, respectively, compared with the control (0 nM). CONCLUSIONS: The siRNA concentration of 20 nM may be appropriate to induce RNAi in both astrocytes and microglial cells, while demonstrating low cytotoxicity, high transfection efficiency, and effective RNAi.
Animals ; Astrocytes ; Central Nervous System ; Cerebral Cortex ; Gene Expression ; Gene Silencing ; Rats ; RNA Interference ; RNA, Double-Stranded ; RNA, Small Interfering ; Transfection