Significance Of FOXC2 Expression In Cutaneous Malignant Melanomaand Its Mechanism Of Regulating Lymphangiogenesis Through Exosomes Pathway

Objective:In this study,we investigated the significance of FOXC2 expression in cutaneous malignant melanoma（CMM）and its regulatory mechanism in tumor-associated lymphangiogenesis through the exosomes pathway and presented a new insight into the mechanism of CMM-induced lymphangiogenesis.It provides a new judgment index for lymphatic vessel metastasis and CMM prognosis and a new strategy for the treatment of CMM.Methods:This study was conducted in three main sections.1.Analysis of the differential gene expression in 472 patients between metastatic melanoma and melanoma in situ by TCGA（The Cancer Genome Atlas）database.And the target gene FOXC2 was screened.Analyzing the correlation between FOXC2 expression and lymphatic endothelial cell-related factors expression in melanoma patients through the TCGA database.2.A clinical study was conducted to further analyze the correlation between the expression of FOXC2 in CMM and lymphangiogenesis and clinicopathological characteristics of patients.A total of 36 cases with CMM and 2 normal skin samples in Jiangsu University Hospital were screened.Clinicopathological data（gender,age,site of onset,Breslow thickness,lymph node metastasis,clinical stage,etc.）were collected and all samples were stained by immunohistochemistry（IHC）.Lymphatic vascular density（LVD）was counted by using lymphatic vessel endothelial hyaluronan receptor-1（LYVE-1）to label lymphatic endothelial cells（LECs）in the cancer nests and para-cancer tissues.The correlation between FOXC2expression and LVD,lymph node（LN）metastasis,and the clinicopathological stage was analyzed statistically.3.1 Human cutaneous malignant melanoma cell lines A375 and A875 and normal melanocytes were cultured in vitro,and the FOXC2 expression in melanoma cells and normal melanocytes were detected at the gene and protein levels;Constructed a low FOXC2 expression cell lines(A375^{sh FOXC2},A875^{sh FOXC2})by lentivirus transfection;The cell model construction was examined by real-time fluorescence quantitative PCR（q RT-PCR）and Western Blot technology;The migration ability of A375,A875,A375^{sh FOXC2},and A875^{sh FOXC2}was verified by scratch assay and transwell migration assay;The proliferation ability of melanoma cell lines after the FOXC2knockdown was investigated by using the plate cloning technique and CCK8 cell proliferation technique;The invasion ability of melanoma cell lines after knockdown of FOXC2 was investigated by the transwell invasion assay.3.2 The exosomes(A375-EXO and A375^{sh FOXC2}-EXO)derived from A375 and A375^{sh FOXC2}cells were extracted by the KIT method,respectively.Transmission electron microscopy（TEM）and a Nanosight nanoparticle tracer analyzer were used to observe the structure of exosomes.The expression of exosome-specific markers TSG101,CD9,and CD63 and the expression of FOXC2 were detected by Western blot.The exosomes were labeled by the fluorescent dye PKH26 and then co-cultured with LECs to observe their uptake;Respectively,Phosphate buffer saline（PBS）,A375-EXO,and A375^{sh FOXC2}-EXO were co-cultured with LECs to observe the changes in biological characteristics（proliferation,migration,and tubule formation）.Western blot and immunofluorescence staining were used to observe the effect of A375-EXO and A375^{sh FOXC2}-EXO on the expression of LYVE-1 in LECs.Results:1.A search through the TCGA database revealed that FOXC2 was highly expressed in 368cases of metastatic cutaneous melanoma and lowly expressed in 104 cases of in situ cutaneous melanoma and the p-value was less than 0.05,which was statistically significant.The results showed that m RNA expression level correlations between FOXC2 with lymphatic vascular endothelial cells associated markers（LYVE-1,FLT-4/VEGFR-3,PDPN）were all positively correlated and statistically significant by the TCGA database.2.Clinical study showed that the expression of FOXC2 was negative in 2 cases of normal skin tissue;FOXC2 was mainly expressed in the nucleus and cytoplasm of CMM cells.The high expression rate of FOXC2 in 36 cases of CMM was 58.3%.The high expression rate in the LN metastasis group was 85%,which was significantly higher than that in the non-LN metastasis group（25%,p<0.001）;The high expression rate of FOXC2 in the group with late clinical-stage3C-4 was 73.07%,which was significantly higher than that in the group with clinical stage 0-3B（20%,p<0.05）;The high expression rate of FOXC2 in the infiltration depth≥2 mm group was74.07%,while that in the infiltration depth<2 mm group was 11.11%（p<0.05）;Factors such as gender and age had no statistically significant effect on FOXC2 expression levels in melanoma tissues（p>0.05）;The mean LVD value in the FOXC2 high expression group was 9.86±1.78 and in the low expression group was 4.64±1.34.Pearson correlation analysis showed that the LVD in the FOXC2 high expression group was higher than that in the FOXC2 low expression group,and the results were statistically significant.3.1 The results of q RT-PCR and Western Blot showed that the knockdown FOXC2melanoma cell models(A375^{sh FOXC2},A875^{sh FOXC2})were successfully constructed;Scratch assay and transwell migration assay showed that the migration ability of A375^{sh FOXC2}and A875^{sh FOXC2}cell lines was significantly lower than that of A375 and A875;Plate cloning and CCK8 cell proliferation assay showed that the proliferation ability of A375^{sh FOXC2}and A875^{sh FOXC2}was not significantly different from that of A375 and A875 groups;The results of transwell chamber invasion assay showed that the invasion ability of melanoma cell lines A375^{sh FOXC2}and A875^{sh FOXC2}was weaker than that of A375 and A875.3.2 Western blot results showed that FOXC2 and exosome-specific markers TSG01,CD9,and CD63 were expressed in A375-EXO,and FOXC2 was lower expressed in A375^{sh FOXC2}-EXO.TEM and Nanosight particle tracer analysis observed that exosomes were small Teatro-shaped vesicles with a diameter of about 100 nm.Fluorescence detection revealed that PKH26-labeled exosomes could be taken up by LECs after co-culture with LECs.The results of the CCK8 cell proliferation assay showed that the A375-EXO and A375^{sh FOXC2}-EXO groups significantly promoted the proliferation of LEC compared with the PBS group.However,the ability of A375^{sh FOXC2}-EXO to promote LECs proliferation was not significantly weakened compared with the A375-EXO group.The results of the cell migration assay and tube formation assay showed that A375-EXO and A375^{sh FOXC2}-EXO significantly enhanced the migration and tube formation ability of LECs compared with the PBS group,and the A375^{sh FOXC2}-EXO group had weaker migration and tube formation ability than the A375-EXO group.Western blot and cell fluorescence assay showed that lymphatic endothelial hyaluronic acid receptor-1（LYVE-1）in LECs was significantly up-regulated after co-culture with A375-EXO,and LYVE-1 in LECs was decreased after co-culture with A375^{sh FOXC2}-EXO compared with A375-EXO group.Conclusion:The expression of FOXC2 in cutaneous malignant melanoma tissues is significantly correlated with LVD,LN metastasis,Breslow thickness,and clinicopathological stage;The expression of FOXC2 in melanoma cells is correlated with tumor cells invasion and migration ability;Cutaneous malignant melanoma-derived exosomes may play an important regulatory role in tumor-associated lymphatic vessel metastasis through the FOXC2/LYVE-1 signaling pathway,but the exact regulatory mechanism still needs further study;FOXC2 has potential clinical applications in the diagnosis,treatment,and prognosis assessment of melanoma.