The Study Of IL-23 In Vasculogenic Mimicry Formation Of Malignant Melanoma

ObjectiveBi-directional differential malignant tumors show both the epithelial-like tissue differentiation and mesenchymal-like tissue differentiation which refers to the different tumor cell morphology and organization arrangement.And malignant melanoma is a kind of common bi-directional differential malignant tumors.Vasculogenic mimicry(VM)is a functional microcirculation formed by tumor cells to describe the unique ability of highly aggressive tumor cells to form capillary-like and extracellular matrix-rich tubular networks without the participation of endothelial cells.VM networks are functional vascular-like structures that carry plasma and red blood cells from host blood vessels.Thus,VM was associated with proliferation,invasion and metastasis.As an indispensable part of the tumour microenvironment,immune cells and inflammatory factors play an important role in proliferation,invasion and metastasis.As an immunomodulatory factor,the heterodimeric cytokine IL-23 may participate in that process by regulating the expression and modification of STAT3 and NF-?? P65,with different effects in different cell types.As early as 1863,Virchow speculated tumor originated from chronic inflammation.The early hypothesis raised following a series of research on relationship between tumor and inflammation,but it not covered the role of IL-23 in bi-directional differential and Vasculogenic mimicry process.This study is to examine the expression of IL-23 in melanoma cells and its role in bi-directional differential and Vasculogenic mimicry process,and then study its effect on melanoma cell migration and invasion ability.Methods1.Based on IL-23 expression level in A375,A875,MUM-2B and MUM-2C cells,A375,MUM-2B and MUM-2C cells were selected as in vitro models.The expression levels of IL-23 were assessed by Western blot analysis,and the results showed that MUM-2C cells had low-level IL-23 expression,while A375 and MUM-2B cells presented high levels.To establish stable IL-23 knockdown or IL-23-overexpressing cells,A375 and MUM-2B cells were stably transfected with sh-IL-23 and sh Control vectors,and MUM-2C cells were stably transfected with IL-23 c DNA and control vectors.These effects were confirmed by Western blotting and immunofluorescence.2.The changes of A375,MUM-2B and MUM-2C cells morphology were detected after IL-23 transfection by using a microscope.3.The proliferation of A375,MUM-2B and MUM-2C cells were determined after IL-23 transfection by MTT.4.The migration of A375,MUM-2B and MUM-2C cells were determined after IL-23 transfection by the wound healing assay.5.The invasion of A375,MUM-2B and MUM-2C cells were detected after IL-23 transfection by Transwell experiment.6.The ability of tube structure formation in A375,MUM-2B and MUM-2C cells were detected after IL-23 transfection by three-dimensional culture.7.The changes of the expression of related protein(NF-???N-cadherin?Vimentin?Snail?MMP-2 and VE-cadherin)in A375,MUM-2B and MUM-2C cells were detected after IL-23 transfection by Western blot and immunofluorescence.8.The expression activities of MMP-2 and MMP-9 in A375 and MUM-2B cells were analyzed after IL-23 transfection by Gelatin Zymography.9.To validate the function of IL-23 in vivo,A375-Control,A375-sh IL-23,MUM-2C-Control and MUM-2C-IL-23 cells were subcutaneously injected into BALB/c-nu/nu mice,and recorded the tumor formation rate and volume.10.The changes of VM and the expression of related protein(NF-???Vimentin?Snail and VE-cadherin)in xenografts were detected by Endomucin/periodic acid–Schiff(PAS)double staining assays and immunohistochemical(IHC)staining.Results1.Establish stable IL-23 knockdown A375 and MUM-2B cells and IL-23-overexpressing MUM-2C cells.2.After IL-23 transfection,the morphology of A375,MUM-2B and MUM-2C cells showed significant changes.A375 and MUM-2B cells become big with decreased pseudopods.MUM-2C cells become small with increased pseudopods.3.The MTT results showed that inhibition of IL-23 expression suppressed the growth of A375 and MUM-2B cells.In contrast,promotion of IL-23 expression accelerated the growth of MUM-2C cells.4.The wound healing assay results showed that IL-23 knockdown significantly decreased the abilities of A375 and MUM-2B cells to migrate,whereas IL-23 overexpression had an inverse effect on MUM-2C cells.5.The Transwell experiment results demonstrated that IL-23 knockdown significantly decreased the abilities of A375 and MUM-2B cells to invade,whereas IL-23 overexpression had an inverse effect on MUM-2C cells.6.The three-dimensional culture results showed that Knockdown of IL-23 expression in A375 and MUM-2B cells inhibited tube structure formation,whereas IL-23 overexpression in MUM-2C cells facilitated the trend of tube-like structures.7.The Western blot and immunofluorescence results showed that MUM-2C cells overexpressing IL-23 demonstrated increased levels of the transcription factors NF-?? P65 and Snail,up-regulated the mesenchymal markers N-cadherin and vimentin,and increased VM–related proteins VE-cadherin and MMP-2 protein expression.In contrast,knockdown of IL-23 expression resulted in contrasting results of these genes in A375 and MUM-2B cells.8.The Gelatin Zymography results showed that knockdown of IL-23 expression in A375 and MUM-2B cells demonstrated decreased the expression activities of MMP-2 and MMP-9.9.Compared with the Control group,the tumors grew at a significantly slower rate and the volume of the tumors was significantly reduced in IL-23 transfection group.10.The Endomucin/periodic acid–Schiff(PAS)double staining assays results showed that IL-23 knockdown in A375 cells strongly inhibited the formation of VM channels.The immunohistochemical(IHC)staining results showed that increased Snail expression,vimentin expression and VE-cadherin expression was observed in high IL-23-expressing tumors,while the converse relationship was observed in low IL-23-expressing tumors.Conclusions1.IL-23 facilitates the proliferation,migration and invasion of melanoma cells,contributing to translate mesenchymal-like phenotype and promoting vasculogenic mimicry formation.2.IL-23 may contributes to translate mesenchymal-like phenotype via the NF-??P65/Snail/ vimentin signaling pathway in malignant melanoma.3.IL-23 promotes metastasis via increased levels of the mesenchymal markers N-cadherin and vimentin,increased the expression activities of MMP-2 and MMP-9,and up-regulated the expression of VE-cadherin and MMP-2 in malignant melanoma,thereby promoting vasculogenic mimicry formation.