Clinical Significance Of NLK Expression And Melanin Content In Malignant Melanoma

Part1 Decreased expression of nemo-like kinase in melanoma is correlated with increased vascularity and metastasisObjective To investigate whether the expression of NLK in melanoma correlates with VEGFR2-related angiogenesis and melanoma metastasis.Methods The expression of NLK and the microvessel density(MVD)of VEGFR2 expression were detected by immunohistochemical staining in one tissue micro-array containing 175 specimens of malignant melanoma using anti-NLK and anti-VEGFR2.Results Using immunohistochemistry analysis,it was found that 129(73.7%)out of 175 patient samples were NLK positive,while 26.3% of the samples showed absent expression of NLK.There was no significant difference in age,gender,TNM stage and lymph node metastasis in malignant melanoma.In metastatic melanoma,expression of NLK was significantly lower than that in primary melanoma(P=0.002).Furthermore,tissues with lower expression of NLK displayed a higher microvessel density as detected by VEGFR2 expression when compared with tissues showing higher NLK expression.Conclusion These data suggest that reduced expression of NLK in melanoma correlates with VEGFR2-related microvessel formation and melanoma metastasis.This study showed that NLK may serve as a novel prognosis marker and revealed new mechanisms in melanoma metastasis.Part2 Melanin content is correlated with melanoma progression and metastasisObjective To investigate the clinical and pathological significance of melanin content and cyclin D1 expression in malignant melanoma.Methods Melanin content and cyclin D1 expression were detected by immunohistochemical staining in one tissue micro-array containing 189 specimens of malignant melanoma between January 2001 and December 2014.Results There were 76 cases(40.2%)showing high melanin content and 80 cases(45.7%)showing high expression of cyclin D1 among 189 malignant melanoma patients.The content of melanin was not correlated with the patients’ age,gender,tumor tissue source,and lymph node metastasis,but instead,it was correlated with tumor invasion depth(P=0.001)and clinical stage(P=0.038).The melanin content was much lower in advanced malignant melanoma(stage Ⅲ and Ⅳ or T3 and T4)than in non-advanced melanoma(stage Ⅰ and Ⅱ or T1 and T2).Regarding cyclin D1 expression,there was no significant difference in age,gender,invasion depth(T stage),clinical stage,lymph node metastasis,and tumor tissue source.Melanin content was negatively correlated with cyclin D1 expression in 58 cases of lymph node metastatic malignant melanoma(r=-0.271,P=0.039).Conclusion Melanin content in melanoma tissues may be involved in the invasion,progression,and metastasis of malignant melanoma.The results will provide new evidence for the prognosis and pathological diagnosis of malignant melanoma.Part3 Effect of sh RNA silencing Nanog gene on transplanted tumor of skin squamous cell carcinoma A431 cell in nude miceObjective To investigate the effect of sh RNA silencing Nanog gene in transplanted tumor of A431 cells in nude mice.Methods Firstly,the plasmid carrying shRNA-Nanog was constructed by using PLKO.1-puro as vector.Then,the virus prepared by 293 T cell line was used to infect A431 cells.Finally screening A431 cell clones with low expression of Nanog determine the interference efficiency from cell model and animal model.Results We obtained the stable transfected cell clones after the selection with puromycin.Compared with the blank control group,the cell morphology of the silenced Nanog group changed.The results of western blot showed that the imprinting of silenced Nanog group was lighter than that of blank control group.The results of CCK-8 showed that the OD value of the Nanog gene silence group was significantly lower than control group on the 3rd and the4 th day,and the difference was statistically significant.Tumor transplantation in nude mice showed that there was no tumor in the Nanog gene silenced group 32 days after transplantation,but in the blank control group,the tumor appeared earlier,the tumor size was larger and the tumor weight was heavier.Conclusion By lentivirus-mediated sh RNA transfection,we obtained A431 cell clones with low expression of Nanog.The silencing of Nanog gene not only inhibited the proliferation of A431 cells,but also inhibited the growth of transplanted tumors in nude mice injected by A431 cells.