The Effects Of CXCL12/CXCR4 And CXCL12/CXCR7 Axes On The Progression Of Melanoma

Melanoma is a kind of highly invasive tumor with a high capacity for metastasis. Recent studies have found the close relationship among the chemokines, their receptors, and the progression of melanoma. Among these chemokines and their receptors, CXCL12/CXCR4 (C-X-C motif chemokine ligand 12/C-X-C chemokine receptor type 4) and CXCL12/CXCR7(C-X-C motif chemokine ligand 12/C-X-C chemokine receptor type 7) axes play an important role in the progression of melanoma. However, the potential function of these two axes remains to be fully understood. In this study, we conducted a further research to investigate the expression of CXCR4 and CXCR7 in cutaneous malignant melanoma tissues and the effects of CXCL12/CXCR4 and CXCL12/CXCR7 axes on the biological behavior of human melanoma M14 cell line to enrich the theory on pathogenesis of melanoma.Part 1 The expressions of CXCR4 and CXCR7 in cutaneous malignant melanoma and melanocytic nevus tissuesObjective To detect the expressions of CXCR4 and CXCR7 in cutaneous malignant melanoma(CMM) and melanocytic nevus tissues. Methods Immunohistochemistry was used to determine the expression of CXCR4 and CXCR7. Results Immunohistochemistry revealed that the expression rate of CXCR4 and CXCR7 significantly differed between melanoma tissue and nevus tissue [CXCR4:(-):20% vs. 50%, (+):12.5% vs. 42.5%, (++):32.5% vs. 7.5%, (+++):35%vs.0%, all P< 0.05; CXCR7: (-):15% vs.35%, (+):10% vs.62.5%, (++):22.5% vs.2.5%, (+++):52.5% vs.0%, all P< 0.05]. The expression of CXCR4 and CXCR7 showed poor correlation in melanoma tissue and normal correlation in nevus tissue by Kappa test(P< 0.001). As far as CMM was concerned, the CXCR4 expression was uncorrelated with age, sex, Breslow depth, presence of ulcer, and infiltration of lymphocytes(all P> 0.05), but positively correlated with Clark level, location, and lymph node metastasis(all P< 0.05). The CXCR7 expression was uncorrelated with age, sex, presence of ulcer, infiltration of lymphocytes, and lymph node metastasis(all P> 0.05), but positively correlated with location, Breslow depth, and Clark level(all P< 0.05). Conclusions The expression of CXCR4 and CXCR7 in melanocytic nevus tissues is negative or low, the expression of CXCR4 and CXCR7 in CMM tissues is medium or high, which may be closely associated with the progression and prognosis of CMM, but there is no obvious correlation between CXCR4 and CXCR7.Part 2 The effects of CXCL12 on the biological behavior of human melanoma M14 cell lineObjective To investigate the effects of CXCL12 on the biological behavior of human melanoma M14 cell line. Methods Real-time PCR and Western blot were used to detect the expression of CXCR4 and CXCR7. The human melanoma M14 cell line were incubated or induced by different concentrations of CXCL12(0,10,50,100,200ng/ml).The Cell Counting Kit-8(CCK8), transwell migration and invasion assays were used to evaluate the proliferation,migration, and invasion of M14 cells. The Western blot was used to detect the protein expression of ERK, p-ERK, AKT, and p-AKT of M14 cells treated with 100ng/ml CXCL12. Results The concentrations of CXCL12(10,50,100,200ng/ml) promoted the proliferation and migration of M14 cells, and the concentrations of CXCL12(50,100,200ng/ml) promoted the invasion of M14 cells, to a certain extent, a dose-dependent increased the proliferation, migration, and invasion of M14 cells. But there was no obvious difference in the proliferation and migration numbers of M14 cells between 100ng/ml and 200ng/ml concentration. The concentration of 100ng/ml activated the ERK and AKT signaling pathways. Conclusions CXCL12 is capable of promoting the proliferation, migration, and invasion of M14 cells, furthermore, the activation of the ERK and AKT signaling pathways has been observed. CXCL12 may be associated with the progression of melanoma.Part 3 The effects of CXCR4 siRNA and CXCR7 siRNA on biological behavior of human M14 melanoma cell lineObjective To study the effect and their mechanism of CXCL12/CXCR4 and CXCL12/CXCR7 axes modulating proliferation, migration, and invasion in human M14 melanoma cells. Methods The small interfering RNA(siRNA) was applied to silence CXCR4 and CXCR7 in human M14 melanoma cells. Q-PCR and Western blot were used to observe the inhibitory effect. The Cell Counting Kit-8(CCK8), transwell migration and invasion assays were used to evaluate the proliferation,migration, and invasion of M14 cells. The Western blot was used to detect the protein expression of ERK, p-ERK, AKT, and p-AKT. Results Downregulation of the expression levels of CXCR4 and CXCR7 using siRNA was confirmed at mRNA and protein level. CXCR4 and CXCR7 siRNA inhibited the proliferation, migration, and invasion of human M14 melanoma cells.In addition, the activation of the ERK and AKT signaling pathways was also suppressed by CXCR4 and CXCR7 siRNA. Conclusions Theses observations suggest that CXCL12/CXCR4 and CXCL12/CXCR7 axes may differently participate in the processes of proliferation, migration, and invasion of melanoma, and may be associated with the activation of ERK and AKT signaling pathways.