To determine whether levels of HSF1 expression and phospho-S326/HSF1 were upregulated in HCC tissues, nine primary human HCC tissues and their corresponding adjacent normal tissues were assessed by immunoblotting. of a hepatitis B virus infection and the expression of alpha-fetoprotein and carcinoembryonic antigen. Knockdown of with shRNA induced the protein expression of tumor suppressor retinoblastoma protein, resulting in attenuated plc/prf5 cell growth and colony formation (7) reported that Col4a2 hyperphosphorylation of HSF1/S326, which is upregulated in breast cancer compared with the normal counterparts, was used as a biomarker to indicate HSF1 activation in breast cancer. The constitutive activation 1-Methylpyrrolidine of HSF1 in breast cancer contributes to the expression of a group of malignant program genes in addition to the heat shock proteins and this HSF1-regulated malignant program was also active in colon and lung cancer (7). Hepatocellular carcinoma is the fifth most common, with the third highest mortality rate of all cancer types worldwide. It dominantly occurs in Asian countries, including China, Japan and Southeast Asian countries (24). HCC is closely correlated with the infection of hepatitis B virus (HBV), HCV, aspergillus flavus infections, as well as cirrhosis and obesity (25). 1-Methylpyrrolidine A number of proteins have been identified as biomarkers for HCC diagnosis and prognosis, including alpha-fetoprotein (AFP), AFPLens culinaris agglutinin-reactive AFP, des-gamma-carboxy prothrombin, glypican-3, osteopontin, and others, including squamous cell carcinoma antigen-immunoglobulin M complexes, alpha-1-fucosidase, chromogranin A, human hepatocyte growth factor and insulin-like growth factor (26). However, none of these biomarkers are efficacious for the early diagnosis of HCC, and therefore, further studies are required to identify novel specific biomarkers of HCC to improve the prognosis. The accumulative evidence indicates that HSF1 and its downstream HSPs are upregulated in HCC tissues. Knockdown HSP70 and HSF1 triggered apoptosis of an HCC cell line (27) and the inhibition of DEN-induced HCC (16). HSP27, which is upregulated in HCC tissues, is also elevated in HCC patient serum and is correlated with HCC prognosis (28). It was reported that HSF1 is upregulated in prostate cancer and HCC (27). However, the possible role of HSF1 as a prognostic marker of HCC has not been well studied. The present study investigated HSF1 protein expression and its phosphorylaton at S326 in HCC tumor tissues and HCC cell lines. Knockdown of HSF1 in the HCC cell line plc/pfr5 was achieved with small hairpin (sh)RNA, and its effects on protein expression, cell growth and colony formation were assessed. It was explored whether of HSF1 and phospho-S326 may be used as biomarkers of HCC progression and as potential candidate targets for HCC therapeutics. Materials and methods Cell culture and plasmids The cell lines SMMC7042 (Chinese Academy of Sciences, Shanghai, China), HepG2 (ATCC, Manassas, VA, USA), plc/prf5 (ATCC), SM7721 (Chinese Academy of Sciences) and Chang liver cells (China Military Medical Science Academy, Beijing, China) were routinely grown in Dulbeccos modified Eagles medium (DMEM) containing 10% FBS and 100 g/ml ampicillin-streptomycin mixture in a 37C incubator with 5% CO2. The cells were passaged every two days. The pLTHR-shRNA-HSF1 plasmid was used as a retroviral vector expressing the shRNA targeting the human HSF1 sequence CAG GAG CAG CTC CTT GAG A (29). The pLTHR-shRNA-enhanced green fluorescence protein (EGFP) was used as the scrambled shRNA. Recombinant retrovirus expresses shRNA-HSF1 The pLTHR-shRNA-HSF1 and pLTHR-scramble constructs were transiently transfected into 293 amph cells for retrovirus packaging. The cell supernatants, which were collected and mixed with 2 g/ml polybrane, were used to infect the plc/prf5 cells for 12 h. Following selection with 2 g/ml of puromycin for three days, the live cells were pooled and used for the experiments (e.g. immunoblotting, cell growth and colony formation assay and cell cycle analysis). Immunohistochemical staining 1-Methylpyrrolidine Primary HCC tissues were kindly provided by Dr. Song Zhenshun (Department of Surgery, Shanghai Tenth Hospital Affiliated to Tongji University, Shanghai, China) were imbedded in paraffin and selected for immunohistochemical staining using the standard method. Briefly, following deparaffinization, rehydration and antigen retrieval, the slides were blotted in 3% bovine serum albumin/phosphate-buffered saline buffer for 1 h and incubated with primary rabbit polyclonal antibody against Hsf1 for 1C2 h. Following washing out unbound primary antibody, the slides were then incubated with secondary antibody conjugated with alkaline phosphatase (AP). The slides were developed in DAB buffer and counterstained with hematoxylin. The slides were photographed with the ZEISS 540 microscope under 40x-index (Carl Zeiss, Jena, Germany). The study was.