The purpose of this study was to clone human HSP90beta cDNA and construct its eukaryote expression vector . The total RNA was isolated by TRIzol Reagent (Invitrogen) from human NPC and its cDNA was gained by RT-PCR. The purified RT-PCR products and PGEM-T Easy Vector were ligated and transformed into XL1-blue E. coli bacteria. The white clones were selected and the plasmid was purified, which was further identified by double enzyme digestion and sequenced. PGEM-hHSP90beta and pcDNA3.1(+) DNA were digested by AflII and Xbal respectively. After purification, the two fragments obtained were ligased by using T4 DNA ligase (Fermentas). This recombinant DNA was then transformed into E. coli Competent Cells XL1-blue and positive clones were selected on the LB agarose plate containing Ampr (100 microg/ml). Single clones were identified by double digestion with AflII and Xbal, and two fragments with the size 5.4 kb and 2.1 kb were produced as expected. The hHSP90beta gene was successfully inserted into the eukaryote expression vector pcDNA3.1(+) by the recombination technique in vitro.
Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; HSP90 Heat-Shock Proteins ; biosynthesis ; genetics ; Humans ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

To explore the anti-apoptotic role of electroacupuncture (EA) and its molecular mechanisms after cerebral ischemia/reperfusion (IR) of rats, by using animal model of middle cerebral artery occlusion (MCAO), the changes of the cleavage of PARP were observed by Western blot and the mRNA of heat shock protein (Hsp) 70 and Hsp90 beta detected by competitive RT-PCR after cerebral IR and EA treatment. The results were as follows: (1) The cleavage of PARP was increased in ischemic hippocampus, and EA treatment could attenuate the level of the cleavage remarkably; (2) The mRNA expression of Hsp70 was increased in the ischemic cortex and hippocampus and was further increased after EA treatment; (3) The mRNA expression of Hsp90 beta was decreased in ischemic cortex and hippocampus and the decrease was relatively slight after EA treatment. The above results demonstrated EA treatment could protect neurons from apoptosis after cerebral IR. One of the molecular mechanisms was the promotion of the inducible expression of Hsp70 and the improvement of the inhibition of the expression of Hsp90.
Animals ; Apoptosis ; Brain ; metabolism ; Brain Ischemia ; genetics ; metabolism ; Electroacupuncture ; Female ; HSP70 Heat-Shock Proteins ; biosynthesis ; genetics ; HSP90 Heat-Shock Proteins ; biosynthesis ; genetics ; Male ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; genetics ; metabolism

This study was purposed to investigate the mechanism of C-reactive protein (CRP) on proliferation of U266 cells. The human multiple myeloma cell line U266 was incubated with human CRP (0, 5, 10, 20 mg/L) for 24 hours, then the proliferation level of U266 cells was detected by using blood analyser. The mRNA expressions of survivin and HSP90alpha were examined by RT-PCR. The results showed that the proliferation ratio was increased, as compared with the control group (p<0.05); furthermore, the mRNA levels of survivin and HSP90alpha were up-regulated in proportion to the increased CRP concentrations. There was significant correlation between expression of survivin and HSP90alpha (r=0.737, p<0.0001) in incubated cells. It is concluded that CRP can stimulate the proliferation of MM cells directly by up-regulating the expression of survivin and HSP90alpha in MM cells. CRP can be regarded as a potential target for MM treatment.
Apoptosis ; C-Reactive Protein ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; HSP90 Heat-Shock Proteins ; metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; RNA, Messenger ; genetics

AIMTo construct underexpression HSP90alpha and overexpression HSP90beta human hepatoma cell line HepG2.

METHODSThe combined plasimid pSilencerHSP90alpha and pSmycHSP90beta were introduced into HepG2 by electroporation, respectively. The result of transfection was identified by Western-blotting and the curve of cell growth was drew by MTT. Observe the cell vitality and expression of HSP90.

RESULTSExpression of HSP90 in transfected cell line was shown by Western-blotting: Compared with control, expression of HSP90 in the cells transfected with pSilencerHSP90alpha decreased, whereas that in the cells transfected with pSmycHSP90beta increased.The growth curves of the two groups of transfected cells was as the same as that of the control group.

CONCLUSIONThe stable overexpression HSP90beta and underexpression HSP90alpha HepG2 cell lines were established.

Base Sequence ; Electroporation ; HSP90 Heat-Shock Proteins ; genetics ; metabolism ; Hep G2 Cells ; metabolism ; Humans ; Molecular Sequence Data ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo probe the mechanism of acupuncture for anti-aging.

METHODSIn the senescence accelerated mouse the SAMP10 and the SAMR1, by using RT-PCR and DIG-labeled Northern blot technique, the expression differences of HSP84 and HSP86 genes in whole brain, cortex and hippocampus in the 4 groups,8-month SAMR1 control group, 8-month SAMP10 control group, 8-month SAMP10 acupuncture group and 8-month SAMP10 non-point acupuncture group were investigated.

RESULTSIn the SAMP10 control group, the expression of HSP84 and HSP86 were down-regulated in the whole brain, the cortex and the hippocampus, and they were up-regulated after acupuncture, tending to the normal group.

CONCLUSIONBrain aging of the SAMP10 mouse is related with abnormal expression of HSP84 and HSP86 genes, and acupuncture can strengthen the protection of cells, inhibit apoptosis and anti-oxidative stress through regulating expression of HSP84 and HSP86, hence anti-aging.

Acupuncture Therapy ; Aging ; metabolism ; Animals ; Blotting, Northern ; Brain ; metabolism ; Cerebral Cortex ; metabolism ; Female ; HSP90 Heat-Shock Proteins ; genetics ; Hippocampus ; metabolism ; Male ; Mice ; RNA, Messenger ; analysis

OBJECTIVETo investigate the mechanism by which heat shock protein 90 (HSP90) regulates 26S proteasome in hyperthermia.

METHODSHyperthermic HepG2 cell models established by exposure of the cells to 42 degrees celsius; for 3, 6, 12, and 24 h were examined for production of reactive oxygen species (ROS) and cell proliferation, and the changes in Hsp90α and 26S proteasome were analyzed.

RESULTSROS production in the cells increased significantly after hyperthermia (F=28.958, P<0.001), and the cell proliferation was suppressed progressively as the heat exposure time extended (F=621.704, P<0.001). Hyperthermia up-regulated Hsp90α but decreased the expression level (F=164.174, P<0.001) and activity (F=133.043, P<0.001) of 26S proteasome. The cells transfected with a small interfering RNA targeting Hsp90α also showed significantly decreased expression of 26S proteasome (F=180.231, P<0.001).

CONCLUSIONThe intracellular ROS production increases as the hyperthermia time extends. Heat stress and ROS together cause protein denature, leading to increased HSP90 consumption and further to HSP90 deficiency for maintaining 26S proteasome assembly and stability. The accumulation of denatured protein causes unfolded protein reaction in the cells to eventually result in cell death.

HSP90 Heat-Shock Proteins ; metabolism ; Hep G2 Cells ; Hot Temperature ; Humans ; Proteasome Endopeptidase Complex ; metabolism ; RNA, Small Interfering ; genetics ; Reactive Oxygen Species ; metabolism ; Up-Regulation

Biomarkers, Tumor ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Transformation, Neoplastic ; metabolism ; HSP90 Heat-Shock Proteins ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; metabolism ; pathology

OBJECTIVETo establish a RT-PCR system for detecting mRNA expression level of 4 hsp genes in human cells.

METHODSRT-PCR system was established with gene cloning, gene recombination, in vitro transcription and RT-PCR techniques; The detection of expression level of 4 hsp genes in SW13 cell was carried out with this system.

RESULTSIn SW13 cell, hsp70 and hsp90 alpha were typical heat shock induced genes, while hsp60 and hsp90 beta were efficiently expressed and further induced by heat-shock to various extent.

CONCLUSIONSIn our hands novel RT-PCR system can be used to detect mRNA expression level of 4 human hsp genes.

Chaperonin 60 ; biosynthesis ; genetics ; HSP40 Heat-Shock Proteins ; HSP70 Heat-Shock Proteins ; biosynthesis ; genetics ; HSP90 Heat-Shock Proteins ; biosynthesis ; genetics ; Heat-Shock Proteins ; biosynthesis ; genetics ; Humans ; Leukemia, T-Cell ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of p53 binding site (+31/+60) of hsp90 beta gene on its transcriptional regulation.

METHODSThe binding site was first inserted into pBS-SK. After the plasmid annealing and elongation with mutagenic and selective primers, nuclease digestion and bacteria transformation was performed twice to select the positive mutated plasmid. Electrophoretic mobility shift assays (EMSA) was employed to detect the binding of hsp90 beta gene fragment containing mutated p53 binding site and Jurkat cell nuclear extract transfected by p53 expression vector.

RESULTSThe sequence analysis profile confirmed a successful mutation of two bases on the core sequence of the second half binding site. EMSA results showed the specific DNA-protein complex band disappeared after the mutation.

CONCLUSIONSThe core sequence of p53 binding site plays a key role in the trans binding of p53 to hsp90 beta gene.

Binding Sites ; HSP90 Heat-Shock Proteins ; genetics ; Humans ; Leukemia, T-Cell ; pathology ; Mutagenesis, Site-Directed ; Mutation ; Transcription, Genetic ; Transcriptional Activation ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo evaluate the effect of heat shock protein 90 (HSP90) on hepatitis B virus (HBV) replication in hepatocytes and to investigate the related molecular mechanism.

METHODSA eukaryotic plasmid expressing human HSP90 was constructed (designated as HA-HSP90). HepG2 cells were co-transfected with HA-HSP90 and the HBV replicative plasmid HBV1.3. Expression of the exogenous HSP90 was assessed by Western blotting. Expression of the HBV surface antigen (HBsAg) was determined by enzyme-linked immunosorbent assay, and HBV replicative intermediates were detected by Southern blotting. Small interfering (si)RNAs were designed against HSP90 and TBK1 and transfected into the HepG2 cells to further assess the effects of HSP90 and its underlying mechanism. HSP90-mediated effects on the expression of interleukins IL-1b and IL-6 and the interferon response gene IFIT1 were assessed by quantitating mRNA levels with real time RT-PCR.

RESULTSThe HA-HSP90 plasmid successfully expressed exogenous HSP90 protein in HepG2 cells. The exogenous HSP90 was able to inhibit HBV replication and HBsAg expression. IFIT1 expression was up-regulated after HA-HSP90 transfection, but neither IL-1b nor IL-6 were affected. The siRNA-mediated TBK1 down-regulation had no effect on the HSP90-inhibited HBV replication.

CONCLUSIONHSP90 can inhibit HBV replication and TBK1 is not involved in this process.

HSP90 Heat-Shock Proteins ; genetics ; Hep G2 Cells ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; Protein-Serine-Threonine Kinases ; genetics ; Transfection ; Virus Replication