Notch1 And VEGF Expression And Clinical Significance In Cutaneous Melanoma

Research Background and ObjectiveMelanoma is a highly malignant neoplasm originating from Ectodermal neural crest melanocytes,accounting for about 1.5% of all malignant tumors.Limbs,skin,anus and vulva are the high incidence sites.The most common site of melanoma in China is skin,followed by mucosal sources,and other parts are relatively rare.Cutaneous melanoma(CM)is a melanoma that occurs in the skin,which is characterized by high malignancy,greater likelihood of early local lymph node metastasis,late distant metastasis through bloodstream to distant skin,liver,brain and lung,poor prognosis,and insensitive to radiotherapy and chemotherapy.Because of the poor prognosis,high morbidity and mortality of cutaneous melanoma,it seriously affects human health and quality of life.Clinical experimental studies have confirmed that the occurrence and development of Melanoma is a complex pathological mechanisms which involves not only multiple molecules and signaling pathways,but also environmental factors.Studying the factors and specific mechanisms of the development of Melanoma will provide a theoretical basis for its clinical diagnosis and treatment.The Notch gene was named after the discovery in Drosophila in 1919 that a partial functional deletion of the gene could cause a notch at the edge of the wing.The Notch signaling pathway is composed of four Notch gene receptors(Notchl,2,3,4),five ligands(DLL1,DLL3,DLL4,JAG1,JAG2)and CSL(A Class of DNA binding proteins).When the ligand binds to Notch of adjacent cells,Notch is cleaved by the proteasome,releasing the cytoplasmic region ICN with nuclear localization signal,and enters the nucleus to bind to CLS,regulating gene expression.Recent studies have found that Notch signaling pathway changes are closely related to the occurrence and development of genetic diseases,neurodegenerative diseases,cardiovascular diseases and tumors(including melanoma).Tumor angiogenesis can meet the metabolic needs of tumor growth and provide vascular access for distant metastasis.It is considered to be involved in tumor progression,invasion and metastasis,and generally accepted as a prognostic indicator of tumors.Vascular endothelial growth factor(VEGF)and its receptor,VEGFR,are the most characteristic and typical signal pathways in the process of angiogenesis,They can play an important role in angiogenesis through multiple signaling pathways,such as regulation of vascular basement membrane decomposition and formation of foot ring,promotion of endothelial cell proliferation and migration,participation in vascular network formation and mature remodeling.Microvessel density(MVD)is a quantitative indicator of angiogenesis degree measurement in tumors.As a true predictor of invasive tumor growth and metastasis,it affects the survival of patients.The CD34 monoclonal antibody marker can accurately predict the number of microvessels.A one-way signal or ring-shaped regulatory pathway might exist between Notch1 and VEGF,which coordinates the promotion of angiogenesis in melanoma.The possible mechanism of this pathway is: after activation of VEGFR1 and VEGFR2,the expression of Notch1 and Dll4 is up-regulated through downstream PI3K/Akt signaling cascade pathway,while Dll4/Notch may negatively regulate the expression of vascular endothelial cell signaling pathway by regulating the number of VEGFR2 receptors on endothelial cell membrane through HSER1(HEY1).In addition,it has been reported that hypoxia can up-regulate Notch1 expression.Notch1 gene is clustered with hypoxia inducible factor 1?(IF-1?,a stable transcription factor during hypoxia),which activates several angiogenesis genes such as VEGF.However,the specific mechanism of the two has not yet been elucidated.Notch1 and VEGF have synergistic effects in the occurrence and development of melanoma,suggesting to some extent the biological behavior of CM might be an adverse prognostic factor for melanoma.At present,there have been many studies on Notch1 and Notch signaling pathway and melanoma.However,Researchers hold different views on the role of Notch1 in melanoma,Thus,the exact role of Notch 1 in melanoma still need further exploration and clarification.In addition,the number of reports about the relationship between Notch1 and VEGF expression in melanoma and angiogenesis are relatively few.This study aims to determine the correlation between Notch1 and VEGF in M tissues and their clinicopathological features by detecting the expression intensity and density of Notch1,VEGF and the counting of MVD labeled with CD34(CD34-MVD)in 78 cases of CM,40 cases of intradermal nevus.The clinical significance in the diagnosis and treatment of CM will be explored according to the results.Materials and Methods1.Immunohistochemical SP method was used to detect the expression of Notch1,VEGF in 78 CM tissues,40 intradermal nevus tissues and CD34-labeled MVD(CD34-MVD).2.SPSS19.0 software was used for statistical analysis.It was considered that P < 0.05 had statistical significance.Chi-square test was used to analyze the expression difference of Notch1 and VEGF in CM and intradermal nevus,and independentsamples t-test was used to examine the expression difference of Notch1 and VEGF in CM and intradermal nevus,Correlation analysis of 2×2 paired data analyzes the correlation of Notch1 and VEGF expression in CM,and the correlation between Notch1 expression and CD34-MVD count in CM was performed with Spearman correlation analysis.3.Spearman correlation analysis was used to analyze the relationship between Notch1,VEGF,CD34-MVD counts and the clinicopathological features of CM.Results1.Expression of Notch1,VEGF and CD34-MVD counts and expression differences in cutaneous melanoma and intradermal nevusNotch1 was positive for tan granule-like substances in cell membrane or cytoplasm,which can also be expressed in small amount in the nucleus of melanoma tissue;VEGF was expressed in the cytoplasm of melanoma cells and a few vascular endothelial cells,with uniform brown-yellow granules in cytoplasm or cell membrane as positive cells;CD34 was mostly found in cancer tissue and surrounding infiltrated areas,mainly stained in the cytoplasm of vascular endothelial cells,strongly expressed in the microvessels of tumor tissue,and also in the intradermal nevus tissue.The positive rates of Notch1 expression in CM and intradermal nevus were 84.6%(66/78)and 27.5%(11/40),and the positive rates of VEGF expression in CM and intradermal nevus were 69.2%(54/78)and 40.0%(16/40),The CD34-MVD counts in CM and intradermal nevus were 32.10±8.97?13.05±6.66,differential expression of all three groups had statistical significance(p < 0.05).2.Correlation analysis between Notch1 expression with the expression of VEGF expression and CD34-MVD counts in CM tissuesStatistical analysis showed that the expression of Notch1 and VEGF was correlated(r = 0.283,p = 0.012);Notch1 and CD34-MVD counts were correlated(r = 0.495,p = 0.000).Notch1 expression was positively correlated with both of them.3.The relationship between Notch1 expression,VEGF expression,CD34-MVD counts and clinicopathological factors of CMCompared with CM patients with Clark I to II grade,the expression of Notch1 was significantly increased in patients with Clark III to IV grade(89.1% vs 35.7%,?2=5.417,p<0.05).The difference was demonstrated statistically significant by Spearman analysis.In the lymph node metastasis characteristics,the positive expression rate of Notch1 was higher than in the orthotopic group(92.2% vs 70.4%,?2=6.437,p<0.05),and the difference was statistically significant.Similarly,VEGF had higher expression rate in Clark III~IV,lymph node metastasis group than in Clark I~II grade,and in orthotopic group,as same as CD34-MVD counts.and the differences were also statistically significant(p<0.05).Statistical analysis showed that Notch1,VEGF and CD34-MVD were differentially expressed in terms of gender,age,tumor location and diameter,but they were not statistically significant(p>0.05).Conclusions1.Notch1 may be related to the occurrence,development and metastasis of cutaneous melanoma.2.There may be a one-way or ring-shaped regulatory pathway between Notch1 and vascular endothelial growth factor,which may play an important role in angiogenesis of melanoma.3.Activated Notch1 inhibitors can provide strategies for the treatment of melanoma by blocking tumor-related angiogenesis.