OBJECTIVETo construct the Gcn5 shRNA plasmids and to explore the Gcn5 shRNA role in histone acetylation modification with the differentiation of stem cells.

METHODSSeven shRNA fragments were recombined into pGenesil-1 vector to form 7. Gcn5 shRNA constructions. The mesenchymal stem cells (MSCs) induced for two weeks with 5-aza were transfected by the plasmids with lipofectamine2000. Polyclonal antibodies labeled with TRITC were used to identify the acetylation in MSCs with or without Gcn5 shRNA constructions. The efficiencies of transfection and RNAi were calculated based on the ratio of GFP (green fluorescence)/DAPI (blue fluorescence) and TRITC (red fluorescence)/DAPI, respectively.

RESULTSSeven Gcn5 shRNA plasmids or constructions were identified by restriction endonucleases Pst I/Sal I and DNA sequencing. Acetylation block was observed after Gcn5 shRNA plasmids transfected into cells. Fluorescent intensity of TRITC in nucleuses were decreased remarkably, or even disappeared in MSCs. The efficiencies of transfection and RNAi were 93.7% and 46.6%, respectively.

CONCLUSIONThe Gcn5 shRNA plasmids constructed in the present study can decrease the histone acetylation during cell differentiation. It sets the basis for further exploring the role of acetylation in the regulation of cell differentiation.

Acetylation ; Animals ; Cell Cycle Proteins ; genetics ; Cell Differentiation ; drug effects ; physiology ; Histone Acetyltransferases ; genetics ; Histones ; metabolism ; Plasmids ; genetics ; RNA Interference ; RNA, Small Interfering ; pharmacology ; Rats ; Rats, Wistar ; Stem Cells ; cytology ; drug effects ; Transcription Factors ; genetics ; p300-CBP Transcription Factors

We previously reported that trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, induced DLC-1 mRNA expression and accumulated acetylated histones H3 and H4 associated with the DLC-1 promoter in DLC-1 non-expressing gastric cancer cells. In this study, we demonstrated the molecular mechanisms by which TSA induced the DLC-1 gene expression. Treatment of the gastric cancer cells with TSA activates the DLC-1 promoter activity through Sp1 sites located at -219 and -174 relative to the transcription start site. Electrophoretic mobility-shift assay (EMSA) revealed that Sp1 and Sp3 specifically interact with these Sp1 sites and showed that TSA did not change their binding activities. The ectopic expression of Sp1, but not Sp3, enhances the DLC-1 promoter responsiveness by TSA. Furthermore, the TSA-induced DLC-1 promoter activity was increased by p300 expression and reduced by knockdown of p300. These results demonstrated the requirement of specific Sp1 sites and dependence of Sp1 and p300 for TSA-mediated activation of DLC-1 promoter.
Cell Line, Tumor ; Electrophoretic Mobility Shift Assay ; Histone Deacetylases/*antagonists & inhibitors/metabolism ; Humans ; Hydroxamic Acids/*pharmacology ; Promoter Regions, Genetic ; Sp1 Transcription Factor/genetics/*metabolism ; Sp3 Transcription Factor/genetics/metabolism ; Stomach Neoplasms/*metabolism ; Transcription, Genetic ; Tumor Suppressor Proteins/*biosynthesis/genetics ; p300-CBP Transcription Factors/genetics/metabolism

OBJECTIVETo observe the inhibitory effect of cyclin-dependent kinase inhibitor p21 on regulation of survivin transcription in human liver cancer HepG cells, and explore the related mechanisms.

METHODSDoxorubicin (DOX) was used to treat HepG cells. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cells by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21, p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to determine the cell cycle phases, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 or p300.

RESULTSAfter treatment with DOX, the expression of p53 and p21 was increased, whereas that of survivin was reduced during 24 hours of the treatment. After transfection the p21 level was 2100.1-fold or 980.9-fold enhanced in comparison with that in HepG2 cells or HepG2-pEGFP cells. Survivin level was markedly down-regulated to 0.5% or 0.6% relative to that in the other two groups, nevertheless, significant p53 changes were not observed. Overexpression of p21 resulted in G1/G0 phase arrest (F = 31.59, P < 0.01), meanwhile, E2F-1 mRNA or p300 mRNA were less expressed compared with that in the other controls (F(E2F-1) = 125.28, P < 0.05; Fp300 = 46.01, P < 0.01).

CONCLUSIONp21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

Antibiotics, Antineoplastic ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; physiology ; Doxorubicin ; pharmacology ; E2F1 Transcription Factor ; genetics ; metabolism ; G1 Phase ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Resting Phase, Cell Cycle ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; p300-CBP Transcription Factors ; genetics ; metabolism

OBJECTIVETo investigate the therapeutic effects and mechanisms of using artemisinin (Art) combined with glucocorticoid (GC) to treat lupus nephritis (LN) mice.

METHODSForty hybrid female mice were randomly and equally divided into four groups with the method of random number table: control group, model group, prednisone group administrated with 6.45 mg/(kg·d) prednisone suspension, and Art+prednisone group administrated with 150 mg/(kg·d) Art suspension and 3.225 mg/(kg·d) prednisone suspension. A mice model of LN was established by injection with living lymph cell suspension. The changes of urine protein/24h, the expressions of GC receptor α (GRα) mRNA, GC receptor β (GRβ) mRNA in peripheral blood mononuclear cells (PBMCs), and transcriptional coactivator P300/CBP protein in renal tissue were measured.

RESULTSCompared with the model group, the treatment groups had significant decrease in urine protein/24 h, and renal pathological lesion (P<0.01). In the same groups, the expression of transcriptional coactivator P300/CBP protein in renal tissue and GRα mRNA were significantly increased, and GRβ mRNA expression was significantly decreased (P<0.01). And the Art+prednisone group has a better therapeutic effect than the prednisone group (P<0.01).

CONCLUSIONSArt has therapeutic sensitization effects on GC in the LN mice. The underlying mechanism could be correlated with the effect of Art on the increase of the expressions of GRα mRNA and transcriptional coactivator P300 300/CBP protein in renal tissue and on the decrease of the expression of GRβ mRNA in PBMC.

Animals ; Artemisinins ; administration & dosage ; pharmacology ; Base Sequence ; DNA Primers ; Disease Models, Animal ; Electrophoresis, Agar Gel ; Female ; Lupus Nephritis ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Prednisone ; administration & dosage ; pharmacology ; RNA, Messenger ; genetics ; Receptors, Glucocorticoid ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; p300-CBP Transcription Factors ; metabolism