| BackgroundHepatocellular carcinoma (hepatocellular carcinoma, HCC) is the wold-wide cancer but highlyoccurs in southen Asia including China. In China the incidence of HCC, which is less than lung andesophogus cancers, is the third most cancers that cause people death,There are about110,000people beingdiagnosed and died of liver cancer each year. The infection of Hepatitis B virus, cirrhosis and the foodcontamination of yellow aspergillin are the dominant carcinogens of the primary hepatocellular carcinoma.HCC is characterized with occult incidence and hard to be early diagnosis. Once the patients have thetypical symptoms, they have been developed to the late stage of HCC, and miss the right therapeutictime.Therefore, investigation of the molecular mechanism of HCC will provide more opportunities forHCC ealy diagnosis, and therapy.Heat shock transcription factor1(Hsf1) is one of heat shock transcription factor family members(including Hsf1, Hsf2, HSF3and Hsf4). It can be activated in response of the physiological andpathological stresses and controls the the expression of heat shock proteins, The latter can modulate manyintracellular proceses such as signal transcduction, gene expression, cell cycle and apoptosis et, al bychaperoning the misfolded proteins. The cellular protective response mediated by Hsf1and heat shockproteins is known as heat shock response. Hsf1can be activated by many stressful factors e.g.heat shock,peroxide, pathogen infection and heavy metals. Recently Hsf1is found to be essential in some tissues’scarcinogenesis.. Knock-out mouse Hsf1gene can inhibit the P53-mutantt induced lymphoma, DMBA-TPAinduced skin cancer and DEN induced hepatocellular carcinoma,. Hsf1is assumed to be associated withregulation of Ras-, P53and Insulin pathways. In the other hand, the expression of Hsf1is upregulaed inmost of tumor tissues including HCC, and Hsf1has been potentially targeted for some cancer therapy..However, the signal pathways that activate the Hsf1transcription are still not known during the HCCdevelopment. AimsWe will determine the roles of Hsf1in the Plc/Prf5cells’ proliferation by using shRNA techniqueand identify the Hsf1associating proteins with immunoprecipitation, Gst-pull-down assay,2D gel andMass spectrometry. Our research will provide the fundamental for developing Hsf1inhibitors as HCCtherapy.Methods1. Consturction of pLTH-shRNA recombinant retroviral vector in which the shRNA against Hsf1isexpressed;2. With retroviral system we create the PLC/PRF5/scramble and plc/prf5/shRNA two cell lines that canstably express scramble RNA and anti-Hsf1shRNA;3. Determining the cell proliferation and cell cycles with MTT experiment (MTT assay), flat clonogenicassay (Plate clone formation assay, PCFA) and flow cytometry;4. Determining the protein expression profiles in PLC/PRF5/scramble and PLC/PRF5/shRNA cells.with Gst-pull down, IP and proteomic techniques;Results1. ShRNA-Hsf1can significantly inhibit the expression of Hsf1in PLC/PRF5cells. It can induce PLC/PRF5cells stopping at G1cell cycle and inhibit cell proliferation and colonal formation;2.2D-gel results indicate Hsf1is postmodifed in the PLC/PRF5cells, and Silence Hsf1causes thedifferential protein expression profiles;3. Silencing Hsf1causes deregulation of the expression of tumor suppressor genes Rb and P53in PLC/PRF5cells.Conclusions1. Hsf1participates in the regulation of PLC/PRF5proliferation and colony formation. Silencing Hsf1expression cause PLC/PRF5cell stopping at cell cycle G1phase and induce the protein accumulationof tumor suppressing proteins p53and Rb. The results suggest that Hsf1is involved in regulation ofprotein stability of P53and Rb, which results in tumor cell proliferation;2. Hsf1is post-translational modified, and regulates many proteins expression in PLC/PRF5cells. The protein profiles regulated by hsf1are in elucidation... |
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