Hep G2 Cells ; Hepatitis B virus ; physiology ; Humans ; RNA Interference ; RNA, Small Interfering ; Virus Replication

RNA interference provides a new approach for elucidation of gene function. It holds the advantages of quickness, convenience, high effect and high specificity. In spite of these, the application of RNA interference technique in studying the mammalian cells and human disease is still in the beginning. In this paper, a review of the development of RNA interference in mammalian cells and human disease is presented.
Animals ; Genes ; physiology ; Genetic Vectors ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics

OBJECTIVETo investigate the role of antiapoptotic Bcl-x(L) protein in megakaryocyte differentiation and maturation.

METHODSRNA interference was used to block the expression of Bcl-x(L) when K562 cells were induced to differentiate into megakaryocyte (CD61 + cells) by PDBu, and the expression of Bcl-x(L) was evaluated with flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). The CD34 + cell fraction was positively isolated by using the MiniMACS system from normal bone marrow. Immunochemical staining and flow cytometry were used to detect the expression of Bcl-x(L) in the differentiation (CD41 + cells) of CD34 + cells induced by trombopoietin (TPO).

RESULTSAmong K562 cells induced by PDBu, the percentage of CD6L + cells rapidly increased in 24 hours and maintained at a high positive level in 72 hours. When exposured to si-Bcl-x(L), the percentage of CD6 1 + cells only slightly increased in 72 hours. The expression of Bcl-x(L) mRNA was significantly decreased after transfection compared with that of control group, and Bcl-x(L) protein expression decreased correspondingly. After the CD34 + bone marrow cells having been treated with TPO for 5 days to 20 days, the Bcl-x(L)-megakaryocytes increased as the culture time prolonged, and there was a strong expression of Bcl-x(L) in immature megakaryocyte and an obviously decreased expression in degenerating megakaryocytes maturation.

CONCLUSIONSIncreased expression of antiapoptotic Bcl-x(L) may be essential to mature megakaryocyte. The down-regulation of antiapoptotic Bcl-x(L) in mature megakaryocyte may be crucial to platelets formation.

Cell Differentiation ; Humans ; K562 Cells ; Megakaryocytes ; physiology ; RNA Interference ; bcl-X Protein ; biosynthesis ; genetics ; physiology

Pseudogenes, which have long been described as "fossils", play a very important role in eukaryotic genomes. Recently, studies on the so called "junk gene" have attracted more attention. Far from being silent, pseudogenes participate in various biological activities, including being a part in the transcription process, or participating in the formation of small interfering RNA (siRNA) which regulated gene expression by means of the RNA-interference pathway. Recent studies have also shown that pseudogenes regulate tumor suppression through competing for the microRNA (miRNA) with their parent genes. However, a deeper understanding of function analysis of pseudogenes depends on the comprehensive and accurate identification. With the sequencing completion of many genomes and the innovation of bioinformatics tools, efficient and precise identification of pseudogenes have become available in a genome-wide scale. Our review focused particularly on the method of pseudogene identification, the mechanism of its regulatory roles and its potential to be applied in directed evolution. Besides, the promising research direction of pseudogenes was proposed.
Gene Expression Regulation ; Genes, Tumor Suppressor ; physiology ; MicroRNAs ; genetics ; Pseudogenes ; genetics ; physiology ; RNA Interference ; RNA, Small Interfering ; genetics

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

To investigate the inhibitory effect of RNA interference (RNAi) on dengue virus I (DENV-1) replication. Small interfering RNA (siRNA) against the PreM gene of dengue virus was synthesized and transfected into C6/36 cells with liposome, which was then attacked by DENV-1 virus. The antiviral effect of siRNA was evaluated by cytopathic effect (CPE), the cell survival rate measured by MTT, and virus RNA quantified by real-time RT-PCR. The results showed that after 7 days post infection of dengue virus, the transfected C6/36 cells showed less CPE. The cell survival rate of the transfected C6/36 cells increased by 2.26 fold, and the amount of virus RNA in the transfected cells was reduced by about 97.54% as well. These findings indicated that the siRNA could effectively inhibit dengue virus RNA replication, and protect C6/36 cells from viral attack, indicating its potential role in prevention and treatment of dengue fever.
Animals ; Cell Line ; Dengue Virus ; genetics ; physiology ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Virus Replication ; genetics ; physiology

Prostate cancer is one of the common cancers in old men. Androgen ablation is a major option for the treatment of the metastatic diseases. However, most of the cancers progress to a more aggressive stage, so-called androgen-independent (or hormone refractory) relapse beyond any cure. The androgen receptor (AR) is an important factor in regulating the differentiation and proliferation of prostate epithelial cells, and also plays a critical role in cellular survival. Studies have demonstrated that aberrant activation of the AR is a major determinant in prostate cancer progression. We have provide a brief summary of AR-mediated cellular survival and an introduction to the advances of RNA interference techniques in silencing AR expression as a novel therapy for prostate cancer.
Apoptosis ; physiology ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Prostatic Neoplasms ; genetics ; pathology ; physiopathology ; RNA Interference ; Receptors, Androgen ; genetics ; physiology

This study was aimed to examine the effect of TREK-1 silencing on the function of astrocytes. Three 21-nucleotide small interfering RNA (siRNA) duplexes (siT1, siT2, siT3) targeting TREK-1 were constructed. Cy3-labeled dsRNA oligmers were used to determine the transfection efficiency in cultured astrocytes. TREK-1-specific siRNA duplexes (siT1, siT2, siT3) at the optimal concentration were transfected into cultured astrocytes, and the most efficient siRNA was identified by the method of immunocytochemical staining and Western blotting. The proliferation of astrocytes tranfected with TREK-1-targeting siRNA under hypoxia condition was measured by fluorescence-activated cell sorting (FACS). The results showed that TREK-1 was expressed in cultured astrocytes. The dsRNA oligmers targeting TREK-1 could be transfected efficiently in cultured astrocytes and down-regulate the expression of TREK-1 in astrocytes. Moreover, the down-regulation of TREK-1 in astrocytes contributed to the proliferation of astrocytes under hypoxia condition as determined by cell cycle analysis. It was concluded that siRNA is a powerful technique that can be used to knockdown the expression of TREK-1 in astrocytes, which helps further investigate the function of TREK-1 channel in astrocytes under physicological and pathological condition.
Animals ; Astrocytes ; physiology ; Cells, Cultured ; Gene Silencing ; physiology ; Potassium Channels ; Potassium Channels, Tandem Pore Domain ; genetics ; RNA Interference ; physiology ; RNA, Small Interfering ; genetics ; Rats

SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.
Cells, Cultured ; Humans ; Plasmids ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; RNA, Viral ; metabolism ; Ribonuclease III ; physiology ; SARS Virus ; genetics

BACKGROUNDCalreticulin (CRT) is major Ca 2+ -binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes.

METHODSApoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules.

RESULTSCompared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis.

CONCLUSIONSOur results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.

Animals ; Apoptosis ; genetics ; physiology ; Calreticulin ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Myocytes, Cardiac ; cytology ; metabolism ; RNA Interference ; Rats