| Hepatocellular carcinoma(HCC) is one of the highest incidence and mortality cancers worldwide. The incidence of HCC ranks fifth in all of the malignant tumors. Due to the high metastasis and relapse rate, the overall prognosis of patients with HCC is poor. In China, the mortality of HCC ranks second in all of the malignant tumors. Currently, due to insidious onset and early metastasis, most of patients with HCC have missed the best time of resection. HCC is insensitive to chemotherapy and radiotherapy treatments. Even if given liver cancer cure resection, 60-70% of patients with HCC will be recurrent in five years, making HCC treatment still great challenging. Therefore, in order to improve the overall survival, it’s very important and necessary to explore new and effective adjuvant treatment for HCC.With the development in molecular biology and genetics technology, we have gradually deepened the understanding of the carcinogenesis mechanisms. Many studies have iindicated that tumor cells around the immune microenvironment, including immune cells and immune factors are involved in the process of tumor development and closely related to tumor progression and prognosis of cancer. Cancer cells just like a seed, which can not leave without the right soil to germinate. Therefore, tumorigenesis and development depends not only on the characteristics of the tumor cells themselves, but also on the soil of tumor cell survival, that is, the tumor microenvironment(Tumor microenvironment).Galectin(galectin) namely β-galactoside binding protein, is an animal lectin family, presents in the nematode, sponges to mammals and other animals, is widely distributed in various tissues and participates in many physiological processes, including growth, hematopoietic lineage differentiation of neural stem cells and muscles, and is involved in cell adhesion, proliferation, differentiation, various pathophysiological processes such as apoptosis and inflammation, and tumor metastasis, invasion and growth. Thus, it is likely to become a new target for the treatment of cancer and inflammatory.Galectin-1 is the first to be reported in a mammal galectin. galectin-1 is encoded by the LSGALS1 gene located on chromosome 22q12 in approximately 14 k D(1 D = 1 u) proteins. Galectin-1 is widely expressed in the central and peripheral immune tissue cells by inducing apoptosis of thymocytes and activated T cells. Thus, the T cells were transferred galectin-1 in the regulation of T cell proliferation and apoptosis and played an important role in maintaining T cells stability of the environment.Galectin-1 not only can inhibit T cell to be effective, as well as antiinflammatory effect in the body. Galectin-1 plays an important role in various biological behaviors of cancer, including tumor transformation, cell cycle regulation, apoptosis, cell adhesion, migration, inflammation and tumors evading the immune response. Studies have confirmed that galectin-1 expression in a variety of tumors is increased, and that its expression is closely associated with tumor invasion and metastasis,tumor occurrence, development, immune escape, thus it may determine the prognosis of cancer. Clinical experiments also have shown that galectin-1 expression is related to the development of tumor, compared with the corresponding normal tissues. The expression of galectin-1in many tumor tissues was significantly increased, including pancreatic cancer, breast cancer, ovarian cancer, prostate cancer and so on. In most cases, its expression was closely associated with tumor invasion and metastasis phenotype.Galectin-1 is involved in tumor progression of the disease, mainly from the following aspects:1) Studies have shown that galectin-1 can promote the growth of cancer cells. Galectin-1 plays a key role in the growth of Lewis lung carcinoma cells. Using transient transfection, stable transfection or galectin-1-specific antibodies can cause a down-regulated expression of galectin-1, resulting in a growth inhibitory effect in lung, breast, head and neck cancer, ovarian cancer, cervical cancer and Kaposi’s sarcoma and other in vivo models. In addition, researchers found that anti-vascular endothelial growth factor treatment of cancer through the promotion of tumor cells to secrete inflammatory cytokines and hypoxia may promote the occurrence of this process.2) Studies have shown that galectin-1 in vitro can enhance glioblastoma migration and invasion, and the expression of galectin-1 in vivo was higher, and the prognosis is likely worse. Meanwhile, some reports indicated that galectin-1 could regulated chemotactic protein synthesis and secretion, induced tumor-associated fibroblasts, thereby enhancing the invasion and migration of malignant tumor cells.3) Galectin-1 regulates tumor angiogenesis through enhancing angiogenesis(VEGF signal) or promoting vascular endothelial cell activation and proliferation. Some studies have showedthat galectin-1 may also interact with VEGF and combined NRP1, thereby enhancing the proliferation and migration of endothelial cells. Tumor cells secrete galectin-1 against oxidative stress response in the tumor microenvironment, thereby exerting a protective effect on vascular endothelial cells.4) Galectin-1 is closely associated with tumor immune escape. The correlation between galectin-1 immunoregulatory function and tumor cells suggested that tumor cells could secret galectin-1 to achieve the destruction of T cell effector, resulting in the formation of tumor location immunosuppressive environment. Closed galectin-1 in melanoma tissue can reduce the tumor size, and can stimulate in vivo tumor-specific T cell response. Therefore, galectin-1 significantly contribute to tumor immune escape.Numerous studies show that galectin-1 plays an important role in the regulation of malignant process. Galectin-1 can promote tumor cell growth, invasion, metastasis and angiogenesis, which may be an early diagnosis of malignant tumors,and an important target for the treatment of cancers. The targeting-galectin-1 therapy can block the signal transduction, inhibit tumor development, and enhance the sensitivity of tumor cells to chemotherapeutic drugs. Therefore, studying the mechanism of galectin-1 in malignant tumors is important, and, the galectin-1 as a target for cancer therapy also has broad application prospects.TRAIL(TNF-related apoptosis-inducing ligand), also known as Apo2L(apoptosis-2 ligand), is one of the members of the superfamily of TNF(tumor necrosis factor), equal to 1995 by Wiley from human cardiac c DNA library obtained [7]. TRAIL selectively induces apoptosis in cancer cells, but does not present a killing effect in normal cells, which makes TRAIL have broad application prospects in the cancer treatment. Thus, TRAIL, become a research focus in recent years.Some tumor cells presented a low sensitivity to TRAIL, but some common drugs can significantly improve the anti-tumor activity of TRAIL. With the development of molecular imaging techniques such as bioluminescence imaging technology, screening chemical libraries from enhanced TRAIL apoptosis-inducing activity of molecules has become possible. Any change of apoptosis-related factors are likely to have an impact on TRAIL-induced apoptosis, thereby influencing the understand of the regulation of TRAIL and its receptors, TRAIL apoptosis pathway and active regulation. Once TRAIL anti-tumor clinical trials were adopted, TRAIL formulation will become the drug of choice of human grams cancer. It effectively prevents other anticancer drugs due to strong side effects caused by the tragic death of cancer patients. Therefore, the successful clinical application of TRAIL is bound to make a significant contribution to the cause of human health [10]. The selective apoptosis for HCC treatment induced by TRAIL has brought new ideas. Recent studies have demonstrated that six kinds of HCC cell lines could express TRAILR1, TRAILR2 and TRAILR4, but TRAILR3 was expressed in only two kinds of HCC cell lines. Most of HCC cells strains were resistant to the presence of a single TRAIL, but chemotherapy can enhance the cancer-killing effect of TRAIL.Based on the above background, the present study will detect the expressions of galectin-1 in HCC cells and tissues, explore the correlations between galectin-1 expression levels and clinical outcomes in HCC patients, and study the possibility of galectin-1 gene as hepatocellular carcinoma molecular diagnostic and prognostic indicatorfor HCC. Meanwhile, this study will also research the influence of the down-regulation of galectin-1 on HCC biological behaviors, and the influence of HCC regulation on the sensitivity to TRAIL. Thus, this study will demonstrate the possibility of galectin-1 protein as a potiential therapy target for HCC treatment, thereby providing a theoretical basis for the further experimental research in vivo and clinical applications. hepatoma cell lines, demonstrated the possibility of a potential target of galectin-1 protein as a molecular Le to treat liver cancer, in order to further the body and provide a.Part 1 Galectin-1 expression associates with the poor prognosis inHCC patients Aim:To investigate the expression of galectin-1 in the normal hepatocyte line, HCC cell lines and HCC tissues, and its correlation with clinical outcomes in HCC patients. Methods:1.The Western Blot method was performed to detect the expression of galectin-1 in the normal liver cell lines, HCC cell lines and HCC tissues.2.Immunohistochemistry was used to detect galectin-1 expression in HCC tissues.3. The relationship between galectin-1 expression and clinical characterristics of the HCC patients was analyzed. Results:1.The expressions of m RNA and protein for galectin-1 were higher in the HCC tissues than in normal tissues.2.Galectin-1 was highly expressed in the HCC patients with metastasis and recurrence.3. The high expression of galectin-1 in HCC patients indicated a shorter overall survival, suggesting that galectin-1 may play an important role in the progression of HCC.Part 2 The effects on HCC cells biological behavior aftergalectin-1 knockdown in vitro Aim:To investigate the effects of galectin-1 gene silencing by RNA interference technology on HCC cell growth, apoptosis, migration and invasion. Methods:1. Targeting si RNA galectin-1 was transfected into hepatoma cell lines, including SK-HEP-1 and HUH-7.2. Galectin-1 m RNA and protein expression levels were detected by RT-PCR and Western blot techniques.3.MTT assay was performed to investigate the influence of galectin-1 gene knocking out on HCC cells proliferation.4.Transwell assay was used to analyze the influence of galectin-1 gene knocking out on HCC cells invasion.5.The wound healing assay was performed to detect the influence of galectin-1 gene knocking out on HCC cells migration.6.Colony formation assay was used to investigate the influence of galectin-1 gene knocking out on HCC cells. Results:1.After transfection of si RNA, galectin-1 m RNA and protein expressions were reduced, suggesting a successful knockdown test..2.Galectin-1 gene knockdown did not have a significant influence on the proliferation of HCC cell lines, but the, migration and invasion ability as well as tumorigenic capacity were significantly decreased.3. The apoptosis of HCC cell lines was significantly increased after galectin-1 gene knockdown.Part 3 Galectin-1 modulates the sensitivity of HCC cells to tumornecrosis factor-related apoptosis-inducing ligand(TRAIL) Aim:Based on building bcl-2 and survivin over-expression plasmid vector, this study aims to research galectin-1 sensitivity to TRAIL and explore the preliminary mechanism of HCC cells regulating Bcl-2 and surviving. Methods:1. The overexpression plasmids of bcl-2 and survivin were constructed and identified.2. The overexpression plasmids of bcl-2 and survivin were transfected into hepatoma cell line, and the bcl-2 and survivin m RNA and protein levels were detected.3. Influence of TRAIL receptor down galectin-1 DR-4, DR-5’s was analyzed.4. MTT assay was performed to detect the sensitivity to TRAIL after galectin-1 gene down.5. The apoptosis rate induced by TRAIL therapy was detected by flow cytometry after galectin-1 genedown-regulation..6. The influences of galectin-1 gene down-regulation and over-expression of bcl-2 and survivin on HCC cells apoptosis were detected. Results:1. The plasmids of Bcl-2 and survivin overexpression were successfully constructed by the identification of RT-PCR and Western-blot assays.2. The down-regulation of galectin-1 has no significant impact on the TRAIL receptor DR-4 and DR-5.3. The over-expression of Bcl-2 and survivin can reverse cells apoptosis induced by the downregulation of galectin-1.4. The Silence of galectin-1 can affect the sensitivity of HCC cells to TRAIL.5. Galectin-1 regulates the sensitivity of HCC cells to TRAIL by Bcl-2 and Survivin In conclusion: 1. Galectin-1 expression was significantly increased in HCC, and closely associatedwith clinical characteristics including HCC metastasis and recurrence. 2. After down-regulation of galectin-1, the proliferation ability of HCC presented nosignificant alterations, the apoptosis rate was significantly increased, but theability of migration and invasion as well as tumor-forming were remarkablydecreased. 3. The down-regulation of galectin-1 could increase the sensitivity of HCC cells toTRAIL. 4. Galectin-1 regulates the sensitivity of HCC cells to TRAIL by regulating Bcl-2and Survivin expression. |
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