Molecular Mechanisms Of CUTL1 In The Tumorigenesis And Progression Of Malignant Melanoma

Malignant melanoma is one of the most common fetal malignancies of nonepithelial origin, whose incidence has strongly increased during the last few years. Although many genes and molecular pathways have been reported to be involved in the carcinogenesis of malignant melanoma, such as CDKI2A (cyclin-dependent kinase inhibitor2A) and CDK4 (cyclin-dependent kinase 4), the molecular mechanisms of malignant melanoma has not been clarified. So it is important to identify new oncogenes and therapeutic targets to improve the survival rate of patients with malignant melanoma.Transcription factors plays important roles in physiological and pathological processes by regulating one or more genes. And one gene might be regulated by many transcription factors. So it is obvious that transcription factors are the key factors in gene regulations. NF-κB(Nuclear transcription factor-κB, NF-κB)6-8 and(Activactor Protein, AP-1) have been reported which might participate in the genesis and progress of malignant melanoma. So it is important to investigate the possible mechanisms of transcription factors in genesis and progress of malignant melanoma.CUTL1(also known as CDP or CCAAT displacement protein) belongs to the homeodomain transcrption factor family. CUTL1 proteins are an evolutionarily conserved family of proteins containing several DNA binding domains:one Cut homeodomain and one, two or three Cut repeats. The CUTL1 gene in human was mapped to 7q22, a chromosomal region that is frequently rearranged in various cancers. CUTL1 is involved with development, cell differentiation, cell cycle progression, tumorigenesis, tumor invasion and migration, and so no. As we all know Wnt5A is an key molecule in the genesis of malignant melanoma, and it is also one of the targets of CUTL1. So we are interested in if CUTL1 participates in the process of malignant melanoma, and which pathway was the most important one during the process. Howere the answers for these questions are still unknown.In this study, we attempted to detect the expression of CUTL1 in the tissues of malignant melanoma and intradermal nevus, and detect the expression of CUTL1 in malignant melanoma cell lines and menalocyte. In addition, we would investigate the regulation between the expression of CUTL1 and the biological activities of malignant melanoma cells by transfection and infection. And we would primarily explore the possible mechanisms of CUTL1 in malignant melanoma. [Aims]1. To evaluate the expression of CUTL1 in malignant melanoma tissues and intradermal nevus tissues.2. To investigate the effect of CUTLl on the biological activities of malignant melanoma cells.3. To explore the possible mechanisms by which CUTLl would promote the proliferation of melanoma cell[Methods]1. Immunohistochemistry was performed to examine the expression of CUTL1 in melanoma and intradermal nevus tissues.2. RT PCR and western blot analysis were performed to examine the mRNA and protein expression of CUTL1 in malignant melanoma cell lines.3. Liposome 2000 was used to transfect CUTL1 siRNA vector and CUTL1 sense retrovirus vector was used to infect the melanocyte.4. MTT assay, clone formation assay and FCM were used to analyze the effect of CUTL1 on the proliferation of malignant melonoma cells in vitro.5. Subcutaneous tumor formative assay was used to investigate the effect of CUTL1 on proliferative ability in vivo.6. Western blot analysis was carried out to detect cell cycle related molecules in transfected cells and control cells, such as p21, p27.[Results]1. The expression of CUTL1 in pancreatic cancer tissues and pancreatic cancer cells.CUTL1 staining was found positive in 82.4%(112/136) cases of malignant melanoma tissues, compared to 16.7%(4/24) cases of intradermal nevus tissues (P<0.05). The expression of CUTL1 was higher in low degree TNM stage and migration groups (P< 0.05). The mRNA and protein expression of CUTL1 was higher in malignant melanoma cell lines than that in melanocyte.2. The effect of CUTL1 on the biological activities of malignant melanoma cells in vitro and in vivo.After the CUTL1 sense retrovirus vector was successfully infected into melanocytes, CUTL1 enhanced melanocytes growth and tumorigenesis ability in vitro, and promoted more melanocytes cells entering into S phase from G1 phase. When CUTL1-siRNA vector was transfected into Libr cells, Libr cell growth, the ability of colony formation, G1/S cell cycle progression and tumorigenesis ability in vivo were decreased.3. CUTL1 regulated various cell cycle related proteins which control G1 to S transition.Suppression of CUTL1 could up-regulate p27 and p21, Overexpression of CUTL1 led to opposite results.(Conlusions]1. The expression of CUTL1 was significantly higher in malignant melanoma tissues, compared with intradermal nevus tissues. The overexpression of CUTL1 was associated with the malignant phenotype of malignant melanoma.2. CUTL1 could enhance the biological activities of malignant melanoma cells in vitro and in vivo.3. CUTL1 enhance malignant melanoma cell growth and proliferation partly by regulating cell cycle specific molecules p21 and p27.