| BackgroudOxidative stress is ubiquitous in the biosphere, which due to the imbalances between generation and elimination of ROS (reactive oxygen species) in the body by a variety of noxious stimuli, will lead to human diseases and cell damage. Normal state, the body can produce a small amount of ROS in normal physiological activities, while in vivo the system can eliminate free radicals and inhibit free radical reactions to keep the balance of free radical generation and scavenging.But in some pathological conditions, while the balancing mechanism is damaged, a significant increase in free radicals of the body can cause body damage. ROS generation and accumulation may cause serious cell damage and result in physiological dysfunction and cell death.The basic mechanism of injury is oxygen free radicals damage cells, causing damage to the lipids and membrane, also damage to proteins, enzymes, nucleic acids chromosome, ultimately leading to cell necrosis and apoptosis.Heat shock proteins (heat shock proteins,HSPs) is a group of the high-level expression and the highly conservative proteins, which due to the Hsp genes by the original shock induced under some stress. Hsp can be with a variety of protein formed complexes, and play a role in the cell with the transporter, transmembrane, participate in protein folding and stretching of the poly-complex assembly with protein molecules, it is known as "molecular chaperones"(Molecular Chaprone).Hsps are divided into the macromolecules of Hsp[(110～100)×103], Hsp90, Hsp70, Hsp60, Hsp40, the small molecular HSP [(30～18)×103] and ubiquitin [(7～8)×103] and other family by Christians, etc., according to the the Hsps relative molecular mass. Hsp90is a class of heat shock proteins in the currentl, on the one hand, because of his close relationship with the tumor; the other hand, Hsp90plays an important role in cell signal transduction through a variety of protein kinases and steroid hormone receptor combination. Hsp90in tumor cells than normal cells2to10times, play an important regulatory role in the growth and survival of tumor cells, a variety of important component of cancer gene pathway and anticancer drugs target,a variety of Hsp90inhibitors have entered clinical trials.Hsp90molecular chaperone function, on the one hand, performance for the promotion of the substrate protein folding, On the other hand,degradation of misfolded protein through its co-chaperone and collaborative ubiquitin-proteasome system. CHIP (rcarboxy terminus of Hsc70interacting protein) is an important co-chaperone protein of Hsp90, and also as ubiquitin E3ligase involved in protein quality control.CHIP promote a variety of protein substratescan by the ubiquitin-proteasome system, and play an important role in the pathophysiology process. Ubiquitin-proteasome system (ubiquitin proteasome pathway, UPP) play an important role in many life processes of the cell.Protein quality control (protein quality control, PQC) is essential for maintaining the normal function of the cells under oxidative stress and survival. The protein quality control system consists of a molecular chaperone and proteolytic enzymes.Ubiquitin-proteasome system and molecular chaperones damaged by external,will cause the function was significantly reduced and misfolded proteins can not be completely degradation and aggregation increased,damage the normal function of cells, giving rise to a variety of diseases.The exception of the cell cycle regulation is the main reason for tumor. Inactivation of cell cycle control mechanisms, especially the inactivation of the G1/S and G2/M phase detection point (check point) plays a vital role in the carcinogenesis process.The biological basis of tumor is the persistent proliferation of cell by the abnormal of cycle control mechanisms, hepatocellular carcinoma is also true.The cell cycle will affectd by a variety of tumorigenic factors, leading to cell growth and differentiation out of control. As G2/M cycle regulatory proteins, Cyclin B1activated and form complexes with CDK1, prompting the cells in G2/M phase transition.If cyclin B1accumulation and accompanied phosphorylation block will Hinder cyclin B1into the nuclear, the cells arrest in the G2phase; and if Cyclin B1degradation blocked, the cells will be stagnation in the M phase; Therefore, the degradation disorders of Cyclin B1is a direct result of abnormal of cell mitosis.It is known that cell cycle regulation and protein quality control system closely related to under oxidative stress, Hsp90as a essential chaperone of protein quality control system, it is important for cells to maintenance the normal function and survival under oxidative stress.The molecular chaperone Hsp90and its co-chaperone CHIP play an important role in degradating substrate protein under protein quality control system. State of cell cycle arrest by oxidative stress induced, Cyclin B1degradation regulated by Hsp90and its co-chaperone CHIP, how is the interaction is not clear.The mechanism will provides a new way of thinking for the generation and prevention of the study of the various stress disorders.Objectives1. Established model of G2/M phase arrest under oxidative stress in HepG2cells;. 2. To establish a cell line stably inhibiting the Hsp90a expression by shRNA interference. To research the expression of Hsp90a and Cyclin B1and CHIP at the control group and interference group different time points under oxidative stress in HepG2cells;3. To determine the causal relationship of inhibits of Hsp90a expression and G2/M phase arrest.4. To study the expression by Hsp90a and its co-chaperone CHIP of Cyclin B1under induced G2/M phase arrest by oxidative stress in HepG2cells, and the interactions of them.5. To analysis of the degradation mechanisms regulated of Cyclin B1and G2/M phase arrest by Hsp90α and its co-partner CHIP, and to explore the molecular mechanism of the G2/M phase and the mechanism regulated by Hsp90a involved under oxidative stressMethods1. Established model of oxidative stress in HepG2cells:The cells were divided into control group, group of oxidative stress6h,24h group. Oxidative stress factors:500μmol/L H2O2to stimulate oxidative stress for the appropriate time; Use MTT to observe cell survival. Use Flow cytometry observe the different times of cell cycle arrest under H2O2in HepG2cells. To determine the24h time point for the H2O2induced G2/M phase arrest;2. The experiment was divided into group of not stimulated by H2O2, group of stimulated6h by H2O2, group of stimulated24h by H2O2, group of interference by ShRNA and not stimulate by H2O2, group of interference by ShRNA and stimulated6h by H2O2, group of interference by ShRNA and stimulated24h by H2O2; Use Western blot to observe the protein expression of Hsp90a, Cyclin B1,CHIP at different time points of stimulated by H2O2; Use Flow cytometry to observe the cell cycle arrest at different times of stimulated by H2O2in the HepG2cells;3. Use Quantitative PCR to detect the mRNA of Cyclin B1in HepG2of transfected with empty plasmid and Hsp90a interference by shRNA under oxidative stress;4. Use Western blot to observe the protein expression of Cyclin B1at different time points of oxidative stress and oxidative stress along with the protein synthesis inhibitor CHX in HepG2cells;5. Immunoprecipitation assay were used to detect the interactions of Hsp90a and Cyclin B1, Hsp90α and CHIP, CHIP and Cyclin B1;Results1. The result of MTT colorimetric assay cell viability:Under500μM H2O2concentration,the inhibition of cell proliferation was time dependent(n=3, P<0.05), group of not stimulated by H2O2were:92.25%±4.27%, group of stimulated6h by H2O2were:63.12%±9.69%, group of stimulated24h by H2O2were:53.59%±2.89%; With the time prolongation, the inhibition gradually increased(n=3, P<0.05), group of not stimulated by H2O2were:15.21%±1.18%, group of stimulated6h by H2O2were:14.9%±1.01%, group of stimulated24h by H2O2were:31.89%±2.44%;.2. The protein expression of Hsp90a was increased under Oxidative stress in intracellular, peak of6h, the group of24h decreased gradually to normal(n=3,P<0.05), group of not stimulated by H2O2were:2.00±0.16, group of stimulated6h by H2O2were:3.49±0.14, group of stimulated24h by H2O2were:2.65±0.22,the group of interference by ShRNA and not stimulate by H2O2were:1.09±0.05, group of interference by ShRNA and stimulated6h by H2O2were:1.65±0.04, group of interference by ShRNA and stimulated24h by H2O2were:1.34±0.04; With the time extend of oxidative stress, total protein of Cyclin B1increased of the control group and decreased of interference by ShRNA; Total protein of CHIP reduced of the control group and decreased of interference by ShRNA (n=3,P<0.05), group of not stimulated by H2O2were:1.11±0.01, group of stimulated6h by H2O2were:1.28±0.05, group of stimulated24h by H2O2were:1.39±0.04, group of interference by ShRNA and not stimulate by H2O2were:1.57±0.05, group of stimulated6h by H2O2were:1.40±0.01, group of stimulated24h by H2O2were:1.24±0.04; group of not stimulated by H2O2were:1.91±0.07, group of stimulated6h by H2O2were:1.59±0.05, group of stimulated24h by H2O2were:1.34±0.06, group of interference by ShRNA and not stimulate by H2O2were:1.30±0.02, group of interference by ShRNA and stimulated6h by H2O2were:1.10±0.01, group of interference by ShRNA and stimulated24h by H2O2were:0.89±0.03;3. The G2/M phase arrest in a cell line stably inhibiting the Hsp90a expression by shRNA interference, accompanied by the total protein of Cyclin B1increased (n=3,P<0.05), group of not stimulated by H2O2were:15.54%±1.13%, group of stimulated6h by H2O2were:15.18%±1.73%, group of stimulated24h by H2O2were:32.14%±3.80%, group of interference by ShRNA and not stimulate by H2O2were:40.90%±1.91%, group of interference by ShRNA and stimulated6h by H2O2were:42.77%±1.18%, group of interference by ShRNA and stimulated24h by H2O2were:38.09%±2.34%;4. The mRNA level of Cyclin B1was decreased under Oxidative stress in intracellular(n=3, P<0.05), group of not stimulated by H2O2were:1.00±0.05, group of stimulated6h by H2O2were:0.77±0.10, group of stimulated24h by H2O2were:0.64±0.13, group of interference by ShRNA and not stimulate by H2O2were:0.90±0.20, group of interference by ShRNA and stimulated6h by H2O2were:0.68±0.10, group of interference by ShRNA and stimulated24h by H2O2were:0.41±0.09; The protein expression of cyclin B1in the group of oxidative stress along with the protein synthesis inhibitor CHX less than the group of oxidative stress (n=3,P<0.05), group of not stimulated by H2O2were:1.23±0.03, group of stimulated6h by H2O2were:0.85±0.06, group of stimulated24h by H2O2were:1.38±0.03, group of interference by ShRNA and not stimulate by H2O2were:1.10±0.01, group of interference by ShRNA and stimulated6h by H2O2were:1.53±0.05, group of interference by ShRNA and stimulated24h by H2O2were:1.21±0.02;5. The results of Blue native polyacryamide gel electrophoresis suggest that with the stimulation time extend, the interference of Hsp90a and Cyclin B1in the group of control and the group of Hsp90a change significantly, the interference increased significantly in the group of24h than the group of not stimulated by H2O2(n=3,P<0.05), group of not stimulated by H2O2were:1.18±0.03, group of stimulated6h by H2O2were:1.33±0.03, group of stimulated24h by H2O2were:1.51±0.02, group of interference by ShRNA and not stimulate by H2O2were:1.27±0.02, group of interference by ShRNA and stimulated6h by H2O2were:0.89±0.05, group of interference by ShRNA and stimulated24h by H2O2were:1.16±0.03; Compared to the group of not stimulated by H2O2, the interaction of Hsp90a and CHIP reduce increased (n=3, P<0.05), group of not stimulated by H2O2were:1.31±0.04, group of stimulated6h by H2O2were:1.09±0.03, group of stimulated24h by H2O2were:1.53±0.05; With the time extend of oxidative stress, the interaction of CHIP and cyclin B1gradually reduced (n=3,P<0.05), group of not stimulated by H2O2were:1.39■0.03, group of stimulated6h by H2O2were:1.21±0.07, group of stimulated24h by H2O2were:0.89±0.09;Conclusions1. HepG2cells was stimulated with H2O2in different times, cells produced different degrees of damage, the phase arrest of G2/M appeared at24h2. The expression of Hsp90a, Cyclin B1and CHIP can be affected under severe oxidative stress in cell, the accumulation of Cyclin B1by the inhibition of Hsp90a and CHIP can to result the phase arrest of G2/M;3. There have a clear causal relationship of the expression was inhibited of Hsp90a with the G2/M phase arres;4. Accumulation of Cyclin B1due to the reduced of degradation;5. The combined capacity of Hsp90a, Cyclin B1and CHIP can be affected with Oxidative stress in cell, and leading to the accumulation increased of Cyclin B1, thereby resulting in the phase arrest of G2/M;... |
PDF Full Text Request