| BackgroundHCC(Hepatocellular carcinoma, HCC) is one of the most common malignant tumor in the world. Studies have reported the main etiology of HBV and HCV infection for HCC, in Europe and the United States and other developed countries or regions, alcoholic liver cirrhosis associated with obesity and nonalcoholic fatty liver disease is also the main cause of the risk of HCC. The initiation and progression of malignant tumor is usually jointly by many factors in vivo and in vitro effect of multi-stage is involved in complex process. The occurrence of HCC process is made up of many factors in vivo and in vitro as well as the outcome of combined action of multiple signaling pathways, although at present has been widely used in clinical routine radiotherapy and chemotherapy technology to effectively control the occurrence and development of HCC, but the majority of patients after receiving the therapy prognosis is still not optimistic. Targeted therapy is a new discovery, the new way of effective treatment for HCC, based tumor in the medical community has become a research hotspot and difficulty of the content. The broad masses of medical workers are trying to find new targets to be clinically effective treatment for HCC, in order to effectively control the prognosis of patients with HCC and improve lay the foundation.ARK5 abnormal expression and a variety of clinical closely linked the occurrence and development of malignant tumor, is associated with the invasion and metastasis of malignant tumor. At present, the research has confirmed that ARK5 gene in human various high expression in malignant tumor tissues, especially in some high invasion and metastatic malignant tumor, but its expression in HCC research is relatively small. For Akt in HCC occurrence and development process of how to regulate ARK5 with HCC proliferation, apoptosis and tumor related mechanism in the process of research is still less, TGF- beta 1 abnormal signal channel is a malignant tumor occurrence, development, invasion and metastasis process, the important factors that TGF- beta 1 abnormal signal channel is a malignant tumor occurrence, development, invasion and metastasis process of the important factors.The present study was designed to investigate the expression of ARK5 in tumor tissues of HCC(Hepatocellular Carcinoma, HCC) patients. We analyze the changes of ARK5 expression and its influence to HCC tumor cell in proliferation, apoptosis, invasion, and explore the regulation of TGF-β1 and Akt activation on HCC tumor cells ARK5 expression and its impact on HCC biological behavior of tumor cells, designed to understand the occurrence and development mechanisms of HCC, and it provide a reliable basis for clinical diagnosis and targeted therapy. ObjectiveThe present study was designed to investigate the expression of ARK5 in tumor tissues of HCC(Hepatocellular Carcinoma, HCC) patients. We analyze the changes of ARK5 expression and its influence to HCC tumor cell in proliferation, apoptosis, invasion, and explore the regulation of TGF-β1 and Akt activation on HCC tumor cells ARK5 expression and its impact on HCC biological behavior of tumor cells, designed to understand the occurrence and development mechanisms of HCC, and it provide a reliable basis for clinical diagnosis and targeted therapy. Methods(1) Quantitative PCR has used to detect the expression of ARK5 mRNA in 20 cases of HCC tissues and corresponding adjacent normal tissue samples; we have used western blot assay to detect the expression of ARK5 protein in HCC tissue and corresponding adjacent normal tissues; we have used immunohistochemical to detect the expression of ARK5 protein, and analysised the expression of ARK5 protein in HCC patients with clinical and pathological parameters and patient survival correlation.(2) We used siRNA-ARK5 transfected human hepatoma cell AMMC-7721, thereby reducing ARK5 expression in cells; expression using quantitative PCR and western blot assay ARK5 mRNA and the corresponding protein in AMMC-7721 cells after transfection; after use invasiveness Transwe II chamber was detected in transfected cells; MTT assay using cell proliferative activity after transfection; Annexin V-FITC / PI double staining its impact on apoptosis after transfection; cell scratch assay turn its impact on cell migration after transfection; we detect Caspase-3 activity in cells after transfection.(3) The TGF-β1 inhibitor Ly364947 has used to effect on human hepatoma cell AMMC-7721, thereby reducing cell expression of TGF-β1, BrdU cell proliferation assay cell proliferation; small room with Transwe II assay Cell invasion ability; Cell western blot assay cells TGF-β1, Akt, ARK5 protein expression; scratch migration assay cells. Results(1) The expression of ARK5 mRNA and its corresponding protein in HCC tissue was significantly higher than that in normal liver tissue adjacent the respective cancer; and the expression of ARK5 was significantly associated with HCC tumor size, histological grade, tumor stage(P <0.05); but had onthing on age, cirrhosis, HBsAg, AFP(P> 0.05); ARK5 protein expression can be used as an independent prognostic factor for HCC.(2) After transfection of siRNA-ARK5 ARK5 mRNA and expression of the corresponding protein was significantly decreased in AMMC-7721 cells(P <0.05); the proliferation of AMMC-7721 cells, siRNA-ARK5 group was significantly lower than siRNA-NC group and the control group, and siRNA-ARK5 cell growth inhibition rate highest in 48 h, significant differences compared with the siRNA-NC group(P <0.01); 48 h after transfection, apoptosis rate of siRNA-ARK5 group was significantly higher, and compared to the control group were significantly different(P <0.01); the invasion ability of cells to siRNA-ARK5 group was significantly lower compared with the control group were significantly different(P <0.05); compared with the control group, cell group of siRNA-ARK5 migration was significantly lower(P <0.01); siRNA-ARK5 group of cells, caspase-3 activity was significantly higher than the control group(P <0.05).(3) Ly364947 role of TGF-β1 inhibitors in post-AMMC-7721 cell proliferation activity of the cells TGF-β1 inhibitor group were significantly lower than the control group(P <0.01); the invasion ability of cells to TGF-β1 inhibitor group were significantly lower than the control group(P <0.05); the migration of cells TGF-β1 inhibitor group were significantly lower than the control group(P <0.01); after the expression of TGF-β1 was inhibited, the expression of Akt and ARK5 protein were significantly decreased compared with the control group(P <0.05). Conclusions(1)ARK5 expression was connected with tumor size, histological grade, tumor staging in HCC.(2) ARK5 expression may be due to the TGF-β1 stimulation of Akt / ARK5 signal transduction pathway, thereby promoting the proliferation, invasion and metastasis in AMMC-7721 cells. |
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