Expression Of Tiam1 And Nm23-h1 In Malignant Melanoma

Background and PurposeMalignant melanoma(MM), is a highly malignant tumor that originated from melanoma cells,mainly invades the skin and mucous membranes.Cutaneous Malignant Melanoma(CMM).Compared with other body surface tumor,the most characteristic is the invasion and metastasis, also affect the prognosis of patients.CMM invasion and metastasis mechanism is very complex,it is composed of a variety of pathogenic factors, multiple pathogenetic step and stage the result of joint action.Liotta proposed the metastasis of cancer cells three step hypothesis that adhesion, degradation, mobile,Including cancer cells from primary foci shedding, vascular invasion and lymphatic, migration, adhesion to appropriate sites induced angiogenesis, against the host antitumor immunity, and ultimately the formation of metastases.The research shows that the factors which is particularly important to activate the related oncogenes and tumor suppressor genes decreased or disappeared,lead to the cancer cell proliferation and invasive ability enhancement and cause disease.Therefore, for a variety of malignant melanoma oncogenes and tumor suppressor genes as targets for gene therapy is becoming an effective treatment.T lymphoma invasion and metastasis inducing factor 1(Tiam1),Tiam 1 protein is a guanine nucleotide exchange factor of a series of Roh like GTPase.It can promote the release of GDP and combination of GTP,effects of polymerization and depolymerization of tubulin,In addition to further activate Rac 1,to involve the JNK kinase(c-Jun N-terminal kinase), p38 MAPK kinase(mitogen activated protein kinase) and other downstream signal transduction molecules.realize the regulation of the cytoskeleton structure reorganization,influence the body cell polarization process,with the promotion of cell movement and migration, involved in the regulation of gene expression, cell proliferation and apoptosis, and other biological functions,Play an important role in the occurrence, development, invasion and metastasis of tumors.Non-metastasis 23 homologue 1(Nm23-h1),recently there are lots of the study about Nm23-h1.It plays a negative regulating role in the process of metastasis in breast cancer, gastric cancer, ovarian cancer and other tumors.But reports in neuroblastoma and lymphoma high expression of Nm23-h1 is closely relative to proliferation and tumor metastasis.Nm23-h1 bioactivity primarily through its encoded protein product of the nuclear past diphosphate kinase(NDPK).NDPK directly involved in tubulin polymerization and depolymerization thereby regulating mitosis and cell movement; on the other hand NDPK through a variety of ways to participate in G-protein signal transduction pathways mediated cell proliferation and morphological changes.In addition, it is widely link with Rho family members by regulating the expression of the downstream genes of tumor cell proliferation and infiltration.In this study, understand and Nm23-h1 Tiam1 transcriptional level differences in A375 cells and normal skin tissue cells by RT-PCR method to detect Tiam1 and Nm23-h1 in cutaneous malignant melanoma, skin junctional nevus and normal skin by immunohistochemistry method tissues. Preliminary inquiry with the invasion and metastasis of malignant melanoma relevance, and further understanding of the relevant role Tiam1 between the Nm23-h1. The study reveals that Tiam1 and Nm23-h1 in malignant melanoma, variation of the development process, looking for malignant melanoma gene therapeutic target for the treatment of malignant melanoma clinical targeted to provide experimental evidence.Materials and methods1.Using RT-PCR method to detect and Nm23-h1 Tiam1 transcriptional level differences in A375 cells and normal skin cells and analyze their relationship between the them.2.Collection of cutaneous malignant melanoma wax block 66 cases from 2010 to 2014 during the First Affiliated Hospital of Zhengzhou University, Department of Pathology, archive, junctional nevus 30 cases. Aged 22 to 76 years, with an average age of 58.8±9.2 years; 36 males, 30 females; according to lymph node metastasis into: 31 cases with lymph node metastasis, without lymph node metastasis in 35 cases; by tumor cells Clark grading invasion into: deep and gradeⅠ15 epidermoid and adnexal epithelium, deep and Ⅱ-Ⅲ grade 19 cases of papillary dermis, the deep and Ⅳ grade 21 cases reticular dermis, the deep and invading Ⅴ grade 11 cases of subcutaneous tissue. Junctional nevus 30 cases: 19 males and 11 females, aged 11 to 78 years, with an average age(53.5±10.3) years. 20 cases of excess of normal full-thickness skin tissue from the First Affiliated Hospital of Zhengzhou University, orthopedic surgery as a control group. All of the above subjects with a complete medical history, do not merge any other related diseases, preoperative no lines, refrigeration, chemicals, radiation and other laser and any other relevant treatment. Patients in each group sex, age and lesion such as no significant difference, comparable(P>0.05). Immunohistochemical fluorescent protein streptavidin peroxidase(SP) method to 66 cases CMM, 30 cases of skin junctional nevus and 20 cases of normal skin tissue MTA1, Tiam1 and MMP-9 protein expression levels were detected. Using SPSS17.0 software for data processing; t test to compare the relationship between Tiam1 and Nm23-h1 protein expression rate of both tumor cell invasion and metastasis; Spearman rank correlation analysis to analyze CMM tissue expression of both proteins association..Results1.The relative expression of normal human skin tissue and human malignant melanoma A375 cells Tiam1 m RNA amounts were 1.001 ± 0.074,2.546 ± 0.357, normal skin tissue and A375 cells Nm23-h1 m RNA relative expression levels were 0.999 ± 0.034,0.953 ± 0.022. Tiam1 m RNA expression in human malignant melanoma A375 cells was significantly higher than that in normal human skin tissue; Nm23-h1 expression in human malignant melanoma A375 cells is lower than its expression in normal human skin tissue.2. Tiam1 protein mainly expressed in the cytoplasm, was brown particles. Tiam protein expression in the CMM was 78.79%(52/66), at the junction of the skin nevi positive expression was 46.67%(14/30), the positive expression rate of 10.00% of normal skin(2/20), gradually decreased, the positive expression rate differences in the three groups has statistics significance(P<0.001). Through inter-CMM, junctional nevus and normal skin tissue pairwise comparison shows: CMM group Tiam1 protein expression was significantly higher than its expression in the skin at the junction nevi, which was significantly higher than in normal skin tissue expression in the junctional nevus(P<0.05); expression in the CMM increase with depth of tumor invasion Tiam1 protein gradually increased in CMM patients with lymph node metastasis Tiam1 protein levels higher than those without lymph node metastasis, and the difference were statistically significant(P<0.05).3.Nm23-h1 protein mainly expressed in the cytoplasm, brownish yellow granules. Nm23-h1 protein expression in the CMM was 15.15%(10/66), skin junctional nevus positive expression rate was 44.67%(14/30), the positive rate was 80.00% in normal skin(16/20). Nm23-h1 protein in CMM, junctional nevus skin and normal skin expression gradually increased, the positive expression rate differences in the three groups was statistically significant(P<0.05), through the Inter-CMM, junctional nevus and normal skin tissue pairwise comparison shows: Nm23-h1 protein expression CMM group was significantly lower than its junction expression in the skin nevi, which was significantly lower than in normal skin tissues(P<0.05) junctional nevus of expression; in CMM with the increase in the depth of tumor invasion Nm23-h1 protein expression gradually decreased in CMM patients with lymph node metastasis Nm23-h1 protein levels lower than those without lymph node metastasis, and the differences were statistically significant(P<0.05).4.Analysis of the relationship between the expression of Tiam1 and Nm23-h1 protein: the expression of Tiam1 and Nm23-h1 protein was negatively correlated(r=-0.298, P<0.05).Conclusions1.Tiam1 promotes the occurrence and development of MM.2.Nm23-h1 plays an inhibitory role in MM.3.Tiam1 and Nm23-h1 may act as a target gene for the treatment of malignant melanoma, they also provide a new reference for the diagnosis and prognosis of melanoma.