| Background:Hepatocellular carcinoma (HCC) is one of the most common digestive system malignant tumors in China, the new cases and deaths of which are the highest in the world. HCC threatens human health seriously, also brings heavy burden to the family and the society, because of its difficulty to diagnose at the onset, quick progression, high grade and poor prognosis. In recent years, with the cognition of new treatment concept and promotion of standardization of diagnosis and treatment in HCC, comprehensive treatment strategy greatly reduce the postoperative recurrence rate and improve the patients survival rate, however five-year survival rate is also not satisfying. How to perform more effectively prevention and treatment of HCC has become a bottleneck problem to further improve patients’ survival rate. Protein is the executor of the life functions. Proteomics, which mainly focus on expression level of proteins, modification of proteins after translation and protein-protein interaction, has gradually become the hotspot of life science. It is playing an increasingly important role in the molecular mechanism research of tumor development, screening of diagnostic or prognostic molecular marker, targets and molecular mechanism research of antitumor drugs and cell signal transduction research.Objectives:The aim of this study is screening differentially expressed proteins between HCC tissues and matched adjacent tissues with proteomic technology. Next, verify the differentially expressed protein aminoacylase1(ACY1) on more tissues and serum samples, analyze the relationship between ACY1expression and clinical pathological parameters of HCC patients. We also want to further define the biology function of ACY1on HCC cell lines and possible underlying molecular mechanism.Methods:1. Screening of differentially expressed proteins between3pairs of HCC tissues and matched adjacent tissues were performed with the proteomic technology of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). And interested proteins were identified by MALDI-TOF mass spectrometry.2.128pairs of HCC tissues and matched adjacent tissues were collected to further verify the mRNA and protein expression of ACY1with the methods of real-time fluorescent quantitative PCR (qRT-PCR), western blotting and immunohistochemical staining. The relationship between ACY1protein expression level and clinical pathological parameters was analyzed.3. Serum sample from21cases of healthy people and40cases of HCC patients were collected. And serum ACY1expression level was detected using enzyme-linked immune sorbent assay (ELISA).4. HCC cell line SMMC7721was transfected with small interfering RNA (siRNA) to interfere ACY1expression, while HCC cell line BEL7402was transfected with adenovirus to overexpress ACY1in vitro. And then, the influence of ACY1on cell proliferation, cell apoptosis, cell cycle, cell invasive ability and tumorigenicity were detected using cell counting kit-8(CCK8) assay, apoptosis and cell cycle assay, tumor invasion assay and nude mouse tumorigenicity assay.5. Gene expression profiles of HCC cell line SMMC7721transfected with ACY1-siRNA or negative control were analyzed using gene chips (Affymetrix U133Plus2.0) to further explore the possible molecular mechanism of ACYl on HCC.Results:1.2D-DIGE analysis showed30differentially expressed proteins (fold change>1.5,p<0.05) between HCC tissues and matched adjacent tissues in this experiment. And11proteins (fold change>2.5, p<0.05) containing ACY1and keratin8were identified by MALDI-TOF mass spectrometry.2. qRT-PCR showed that ACY1mRNA expressed lower in HCC tissues than in matched adjacent tissues (p<0.05). The result of western-blot confirmed that HCC tissues expressed lower ACY1than matched adjacent tissues (p<0.05). Immunohistochemical staining also showed the same expression trend of ACY1. The ACY1expression was correlated with the serum alpha fetoprotein (AFP) level (p=0.033) and vascular invasion (p=0.027). However, there is no significant difference between ACY1expression and age, gender, HBV infection, tumor size, or histological differentiation.3. Serum ACY1was significantly higher in HCC patients than in healthy volunteers (p<0.001).4. Cellular experiments in vitro revealed that ACY1silencing promoted the viability of the SMMC7721cells, while restoring ACY1expression decreased the viability of BEL7402cells. Knockdown of ACY1induced shift from G1to S phase in SMMC7721cells. The invasive potential of SMMC7721cells was markedly increased after down-regulation of ACY1. In contrast, up-regulation of ACY1markedly decreased the invasive potential of BEL7402cells. Animal experiment revealed that overexpression of ACY1could suppress tumorigenicity of BEL7402cells in vivo.5. Microarray analysis showed1726differentially expressed genes in ACY1-slienced cell line SMMC7721compared with negative control group. Several critical genes, such as TGFβ1, phosphatase and tensin homolog (pten), fibroblast growth factor21(FGF21) and wnt5A, were differentially expressed. The silence of ACY1in SMMC7721cells also increased the protein expression of TGFβ1and ERK1.Conclusion:1.ACY1was expressed lower in HCC tissues compared with adjacent tissues, ACY1may play an important role in the development of HCC.2. Serum ACY1level of HCC patients is higher than that of healthy volunteers, prompting it as a potential diagnostic molecular marker for HCC.3. Silence of ACY1enhanced viability and invasive potential of SMMC7721cells, while the overexpression of ACY1inhibited the viability and invasive potential of BEL7402cells. 4. TGFβ1and ERK1were increased after silence of ACY1in SMMC7721, indicating an important role of ACY1in HCC. |
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