The Role Of Rictor In Vasculogenic Mimicry Of Melanoma

Objectives Vasculogenic mimimcry(VM): independent of endothelium-dependent vessels,which describes the ability of highly invasive melanoma cells to express endothelium-associated genes and form extracellular matrix(ECM)-rich vasculogenic-like channels in three-dimensional culture,blood flow in the pipeline,Outside the channel is a layer of PAS positive basement membrane.Previous studies have confirmed the vasculogenic mimicry(VM)in several malignant tumors with poor survival such as ovarian cancer,breast cancer and other highly invasive tumors.The tumor with VM structure has a high degree of malignancy,and poor prognosis.Because of the high incidence of VM in melanoma and traditional anti-angiogenesis therapy cannot inhibit VM lumen formation by aggressive cutaneous and uveal melanoma cells,lots of researchers have focused on the molecular mechanism underlying this unique process.In this study,we found that Rictor,a core component of mammalian rapamycin complex 2(mTORC2),was highly expressed in human melanoma and was positively correlated with tumor recurrence and metastasis and VM positive rate.However,the role of Rictor in cancer cell VM formation is largely unknown.In our study,we used lentivirus-based small RNA interference technology and a series of molecular biology techniques to explore the influence and possible molecular mechanisms of Rictor underly melanoma Vasculogenic mimicry,looking forward to providing new ideas for its clinical treatment.Methods1)The expression of Rictor in 81 cases of human melanoma tissues and its relationship with angiogenesis mimicry and clinicopathological features were analyzed by immunohistochemistry and CD34 / PAS double staining.2)The effect of four down-regulation sequences of Rictor plasmid on down-regulation of melanoma cells was confirmed by Western Blotting.The expression level of Rictor in human skin melanoma A375 cell line and human uveal melanoma MUM-2B cell line was down-regulated by small RNA interference technique based on lentivirus and the down-regulation of melanoma cells The impact of capacity3)The effect of Rictor expression on the proliferation and cell cycle of A375 and MUM-2B cells was investigated by MTT cell proliferation assay and cell cycle experiment.4)The effect of Rictor expression on the migration ability of melanoma cells A375 and MUM-2B was observed by Wound-healing assay and Transwell migration experiment.The effect of Rictor on the extracellular matrix adhesion of A375 and MUM-2B cells was analyzed by the Adhesion assay.5)The effect of Rictor expression on the activity of downstream AKT and the expression of its dowmstream MMP-2/9 were analyzed by Western Blotting assay.The expression of MMP-2/9 mRNA was analyzed by Real-Time PCR.The effect of Rictor expression down-regulation on MMP-2/9 activity was analyzed by gelatin Zymography.6)To further validate the effect of Rictor on the expression and activity of MMP-2/9 in A375 and MUM-2B cells,we added different concentrations of AKT to A375 and MUM-2B cells through the AKT-MMP-2/9 pathway(MK-2206).The inhibition of AKT was analyzed by gelatin Zymography.Results1)Immunohistochemistry and CD34 / PAS double staining results showed that in 81 cases of human melanoma tissue samples,Rictor high expression were 57 cases,and 36 of them associated with recurrence and metastasis,31 cases were VM positive.In addition,the survival time of the Rictor-positive group was shorter than that of the Rictor-negative group,and the prognosis was worse.2)The results of three-dimensional tube test showed that the down-regulation of Rictor expression inhibited the formation of three-dimensional ducts of A375 and MUM-2B cells compared with the control group.3)Cell proliferation assay showed that Rictor's down-regulation inhibited the proliferation of A375 and MUM-2B cells.Cell cycle experiments further confirmed that the effect of Rictor on the proliferation of A375 and MUM-2B cells was G2 phase cell block,In the G2 phase(late DNA synthesis)and can not enter the M phase(mitosis)inhibited cell proliferation;4)Wound healing assay and Transwell migration invasion and adhesion experiments showed that down-regulation of Rictor could inhibit the invasion and invasion of A375 and MUM-2B cells and the adhesion ability to extracellular matrix.5)Western Blotting results showed that Rictor down-regulation could inhibit the phosphorylation of downstream AKT 473 and 308,and inhibit the expression of MMP-2/9 protein downstream of AKT.Real-Time PCR showed that Rictor The expression of MMP-2/9 in A375 and MUM-2B cells was inhibited by down-regulation of Rictor.The expression of MMP-2/9 in A375 and MUM-2B cells was inhibited by gelatin Zymography.6)Western Blotting results showed that the expression of MMP-2/9 protein was inhibited after AKT was inhibited by different concentrations of AKT inhibitor.The results of gelatin Zymography showed that inhibition of AKT could inhibit the expression of MMP-2/9 activity.Conclusions1)Rictor was highly expressed in human melanoma tissue,and its expression was positively correlated with tumor recurrence and metastasis and VM positive correlation,which was negatively correlated with the survival time of patients,suggesting that Rictor was one of the poor prognostic factors of melanoma;2)In A375 and MUM-2B melanoma cells,Rictor down-regulation could inhibit the formation of VM by inhibiting cell proliferation(G2 arrest),exercise and adhesion to extracellular matrix.3)Further study found that the effect of Rictor on VM was achieved by modulating the activity of downstream AKT and the expression and activity of downstream MMP-2/9 in AKT.