OBJECTIVETo explore the anti-cancer effects of siRNAs targeting hTERT in SMMC-7721 cells.

METHODSTwo siRNAs targeting hTERT mRNA were designed and synthesized by T7 transcription system in vitro. MMT, RT-PCR and Western blot were applied to evaluate effects on inhibiting cell growth, hTERT mRNA and protein expression in SMMC-7721 cells.

RESULTSsiRNAs decreased cell proliferation in a dose-dependent manner. At a concentration of 100 nmol/L, siRNAs exhibited obvious effects on inhibiting hTERT mRNA and protein expression in SMMC-7721 cells.

CONCLUSIONsiRNAs targeting hTERT have significant inhibitory effects on hTERT gene expression in SMMC-7721 cells. siRNA has the possibility to become a new anti-cancer agent

DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; Gene Targeting ; Genetic Therapy ; Humans ; Liver Neoplasms ; pathology ; therapy ; RNA, Small Interfering ; genetics ; Telomerase ; antagonists & inhibitors ; genetics

OBJECTIVETo assess the telomerase activity inhibitory effect of ham merhead ribozyme targeting the 5'-end of human telomerase reverse transcriptase mRNA (hTERT-5'RZ), to compare it with the effect of another ribozyme teloRZ, and the combine the applications of the two ribozymes.

METHODShTERT-5'RZ gene was synthesized and cloned into pcDNA3.1(+); the ribozyme was produced by in vitro transcription. The teloRZ ribozyme was produced in the same way by in vitro transcription of p(SPT19-teloRZ) which had been constructed by the present authors. The ribozymes were transiently transfected into HeLa cells by liposome every 24 hours. After 72 hours, the cells were collected and their telomerase activities were assayed.

RESULTSThe ribozyme targeting the 5'-end of hTERT mRNA exhibited a very strong telomerase-inhibitory activity, the combined use of hTERT-5'RZ and teloRZ also showed clear inhibitory activity, but the inhibitory effect of teloRZ used alone was not so strong.

CONCLUSIONThese observations suggest that the use of hTERT-5'RZ and the combined use of hTERT-5'RZ and teloRZ are more effective in telomerase inhibition as compare with the use of teloRZ alone. They may find applications in cancer therapy.

5' Untranslated Regions ; Cloning, Molecular ; DNA-Binding Proteins ; HeLa Cells ; Humans ; RNA, Catalytic ; metabolism ; RNA, Messenger ; Telomerase ; antagonists & inhibitors ; genetics ; Transcription, Genetic

Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA-Directed RNA Polymerases ; antagonists & inhibitors ; Drug Discovery ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; metabolism ; Humans ; Influenza A virus ; drug effects ; genetics ; metabolism ; Influenza, Human ; drug therapy ; Molecular Targeted Therapy ; Neuraminidase ; antagonists & inhibitors ; RNA-Binding Proteins ; antagonists & inhibitors ; Signal Transduction ; drug effects ; Viral Core Proteins ; antagonists & inhibitors ; Viral Matrix Proteins ; antagonists & inhibitors

The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.
Animals ; Catalytic Domain ; Cell Differentiation ; drug effects ; physiology ; Cell Line, Tumor ; DNA-Binding Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; GTPase-Activating Proteins ; genetics ; metabolism ; Immunohistochemistry ; Mice ; Microscopy, Fluorescence ; Neuroblastoma ; metabolism ; pathology ; Protein Isoforms ; antagonists & inhibitors ; genetics ; metabolism ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; metabolism ; Valproic Acid ; pharmacology

OBJECTIVETo clarify growth inhibition in pancreatic cancer cells by interference with the hTR component of the telomerase reverse transcriptase enzymatic complex.

METHODSA 593 bp full length hTR cDNA was subcloned into a mammalian expression vector pcDNA3.1(-) in the antisense orientation to construct an antisense hTR expression plasmid. These were introduced into panc1 cells, a human pancreatic carcinoma cell line, by lipofectin and G418-resistant stable transformants were expanded. Resulting stable clones were screened for the presence of the hTR insert by PCR with T7 and BGH reverse primers located on the flanks of the multiclonal site of the pcDNA3.1 vector. Cell growth rate, hTR expression, telomerase activity and anchorage-independent growth properties were analyzed.

RESULTSSignificant downregulation of endogenous hTR was evident in the antisense-hTR transformed cells and telomerase activity was markedly decreased compared to control cells in standard TRAP assays. Furthermore, cell proliferation and the anchorage-independent growth ability in antisense-hTR expressing cells were significantly decreased compared with control parental cells. However, no crisis or senescence phenomena were observed.

CONCLUSIONSThese data indicate that hTR may be a critical component of human telomerase activity and suggest that downregulation of the RNA component of human telomerase is a possible target for anticancer strategies.

DNA-Binding Proteins ; Humans ; Pancreatic Neoplasms ; pathology ; therapy ; RNA, Antisense ; therapeutic use ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; antagonists & inhibitors ; genetics ; Tumor Cells, Cultured

OBJECTIVEMajor histocompatibility complex class I C-related molecules A and B (MICA and MICB) are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. Our previous study showed that 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell), and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells.

METHODSCells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR.

RESULTSMICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment (P less than 0.05), and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase (P less than 0.05).

CONCLUSIONOur data suggest that 5-aza-dC induces the expression of MICA by a DNA damage-dependent mechanism.

Ataxia Telangiectasia Mutated Proteins ; Azacitidine ; analogs & derivatives ; pharmacology ; Caffeine ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; Cell Cycle Proteins ; antagonists & inhibitors ; metabolism ; Cell Line ; Cell Membrane ; metabolism ; DNA Damage ; DNA-Binding Proteins ; antagonists & inhibitors ; metabolism ; Flow Cytometry ; Hep G2 Cells ; Hepatocytes ; metabolism ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; Protein-Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Proteins ; antagonists & inhibitors ; metabolism ; Up-Regulation

The zinc-finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses, including murine leukemia virus, Sindbis virus and Ebola virus, by targeting the viral mRNAs for degradation. ZAP directly binds to the target viral mRNA and recruits the cellular RNA degradation machinery to degrade the RNA. No significant sequence similarity or obvious common motifs have been found in the so far identified target viral mRNAs. The minimum length of the target sequence is about 500 nt long. Short workable ZAP-binding RNAs should facilitate further studies on the ZAP-RNA interaction and characterization of such RNAs may provide some insights into the underlying mechanism. In this study, we used the SELEX method to isolate ZAP-binding RNA aptamers. After 21 rounds of selection, ZAP-binding aptamers were isolated. Sequence analysis revealed that they are G-rich RNAs with predicted stem-loop structures containing conserved "GGGUGG" and "GAGGG" motifs in the loop region. Insertion of the aptamer sequence into a luciferase reporter failed to render the reporter sensitive to ZAP. However, overexpression of the aptamers modestly but significantly reduced ZAP's antiviral activity. Substitution of the conserved motifs of the aptamers significantly impaired their ZAP-binding ability and ZAP-antagonizing activity, suggesting that the RNA sequence is important for specific interaction between ZAP and the target RNA. The aptamers identified in this report should provide useful tools to further investigate the details of the interaction between ZAP and the target RNAs.
Aptamers, Nucleotide ; chemistry ; genetics ; metabolism ; Base Sequence ; Carrier Proteins ; antagonists & inhibitors ; metabolism ; Genes, Reporter ; HEK293 Cells ; Humans ; Leukemia Virus, Murine ; genetics ; Molecular Sequence Data ; Nucleic Acid Conformation ; Protein Binding ; RNA ; chemistry ; metabolism ; RNA, Viral ; genetics ; Response Elements ; SELEX Aptamer Technique

Chloroquine ; pharmacology ; Down-Regulation ; Gene Expression ; drug effects ; HEK293 Cells ; Humans ; Leupeptins ; pharmacology ; Membrane Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Microscopy, Fluorescence ; Protein Binding ; Protein Subunits ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Shab Potassium Channels ; genetics ; metabolism

ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; antagonists & inhibitors ; genetics ; Adenoviridae ; genetics ; Breast Neoplasms ; therapy ; Cell Line, Tumor ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Gene Silencing ; Humans ; Mitoxantrone ; pharmacokinetics ; Neoplasm Proteins ; antagonists & inhibitors ; genetics ; RNA Interference ; RNA, Small Interfering ; pharmacology

Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; Apoptosis ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Neoplasms ; pathology ; Phosphorylation ; Protein Isoforms ; RNA-Binding Proteins ; chemistry ; metabolism ; Transcription Factors ; antagonists & inhibitors