The Mechanism Of MiR-128 Targets CCL18 To Regulate Invasion And Metastasis Of Melanoma Cells

Cutaneous malignant melanoma(CMM)is a highly malignant cutaneous tumor which is characterised by aggressive progression,early metastasis,and high lethality.Tumor-associated microenvironment plays an important role in tumor progression and metastasis in a bidirectional manner with tumor cells through molecular signals such as cytokines.Due to their significant roles in tumor's diagnosis and target treatment,key molecules in tumor-associated microenvironment become one of the important research areas of cancer.Previous research of our group discovered that chemokine CCL18 was up-regulated in CMM tissues,and CCL18 promoted invasion and metastasis of melanoma cells in vitro.CCL18 was one of potential cancer promoting genes in melanoma.In the current study,we conducted research to investigate the regulation mechanism of CCL18 expression from the aspect of RNAi.We discovered the expressions of miR-128 and CCL18 in CMM,and analyzed their relationship.Then the effects of miR-128 targeting CCL18 on invasion and metastasis of melanoma cells were clarifed.This study aimed to explore the miRNA which could effectively silence CCL18 expression,and find a new therapeutic potential for melanoma.Part 1 The expression of CCL18 in cutaneous malignant melanoma tissues and serumsOur group previously discovered that there was significantly higher expression of gene CCL18 in acral lentiginous melanoma(ALM)tissues of Chinese patients by an whole-genome expression microarray.By immunohistochemical staining,they discovered that the expression of CCL18 in CMM was obviously higher than nevus,and CMM of higher Clark level had obviously higher CCL18 levels than CMM of Clark I.To explore the relationship of CCL18 expression and the development of CMM,real-time PCR and ELISA analysis were used to detect the expression of CCL18 mRNA and protein,respectively.The results showed that both mRNA and protein of CCL18 was overexpressed in CMM compared with paracancerous normal skin and healthy human serum.Furthermore,immunohistochemical staining was performed on 58 CMM tissue specimens and 50 benign nevi tissue specimens,followed with clinicopathological significance analysis.We founded that CCL18 was apparently up-regulated in CMM,and the expression level of CCL18 was positively correlated with Clark level,Breslow thickness,and expression of VEGF in primary melanoma.Moreover,patients with ulceration or lymphatic metastasis presented high CCL18 levels.These results confirmed the abnormal high expression of CCL18 in melanoma,and suggested a relationship of CCL18 expression and tumor's malignant behaviours.Furthermore,immunofluorescence assay showed CCL18 expression in the cytoplasm of tumor cells in CMM tissues.Part 2 Bioinformatics prediction and analysis of miRNAs targeting gene CCL18Bioinformatics analysis was applied to predict the miRNAs targeting gene CCL18.Luciferase reporter gene assay was used to verify the possible interaction of miRNA and CCL18 3' UTR.Real-time PCR was used to detect the expression patterns of CCL18 and selected miRNAs in 14 CMM tissues,followed with their correlation analysis.We confirmed miR-128 was significantly down-regulated in CMM.There was an prominently inverse correlation between miR-128 and CCL18 expression.These results suggested miR-128 was the possible miRNA negatively regulating the expression of gene CCL18 in CMM.Part 3 Regulating effects analysis of miR-128 targeting gene CCL18We chosed miR-128 as target miRNA as it presented possiblity of negtively regulating the expression of gene CCL18.We evaluated the regulation of miR-128 on CCL18,the effects of inhibition of CCL18 on biological behaviours of A375 cells,and the possible involved mechanisms.Human melanoma cell lines as A375,M14,MV3,SK-mel-28 had no expression of CCL 18,while human peripheral blood monocytes induced by IL-4(20ng/ml)presented high expression level.Therefore we created a tumor-associated microenvironment by co-cultivation of monocytes and A375 cells.Monocytes were transfected with adenoviral vector containing miR-128 mimics,while a mock adenoviral vector as a negtive control.Biological behaviours of A375 cells in each group were evaluated and compared.Cell proliferation ability was detected by CCK8 assay;Cell apoptosis were detected by Flow cytometer;Colony formation ability was tested by Clonogenic assay;Cell migration ability was tested by Transwell study.Western blot was used to detect expression of E-cadherin,N-cadherin and ?-catenin.These results manifested that transfection of miR-128 mimics in monocytes obviously down-regulated the expression of CCL 18,which had promotive effects on apoptosis of A3 75 cells and significantly suppressive effects on migration and colony formation,but affected cell proliferation weakly.MiR-128 tageting CCL 18 down-regulated N-cadherin expression,suggesting the possible role of inhibiting epithelial-mesenchymal transition(EMT).ConclusionChemokine CCL18 was significantly overexpressed in CMM tissues and serums,and its expression was positively correlated with tumor's aggressive progression.MiR-128 could target gene CCL18 by interaction with its 3' UTR.The in vitro results manifested that miR-128 targeting CCL18 had promotive effects on the apoptosis of A375,significantly suppressive effects on migration and colony formation,and regulation of EMT,suggesting a suppressive role of miR-128 on the oncogene effects of CCL18 in melanoma.These results indicated chemokine CCL18 may play an important impact in development and progression of CMM.MiR-128 might be the miRNA effectively targeted down-regulating gene CCL18.CCL18 may be a predictable new target for the diagnosis and treatment of melanoma.