ABSTRACT
Objective To explore the miRNA regulating the potential cancer-promoting gene CCL18 in cutaneous malignant melanoma.Methods Bioinformatics analysis was conducted by using online software miRanda and TargetScan,so as to predict the miRNA targeting CCL18 gene.Three kinds of C CL18 3'UTR dual-luciferase reporter vectors,including mutant 3'UTR vector (mutant 3'UTR group),wildtype 3'UTR vector (wild-type 3'UTR group) and empty vector (blank control group),as well as miRNA vectors carring selected miRNAs were constructed according to human gene sequence analysis,and then were used to co-transfect 293T cells.After 48-hour treatment,the cells were lysed for detection of luciferase activity.Real-time fluorescence-based quantitative PCR was performed to measure the expression of CCL 18 and selected miRNA in 14 fresh malignant melanoma tissue specimens and 14 paracancerous normal skin tissue specimens (control tissues),and their correlations were analyzed.Results Online software analysis showed that some miRNAs were identified to target the 3'UTR of CCL18 gene,including miR-183,miR-128 and miR-33a.Luciferase reporter vectors and miRNA vectors were constructed successfully.As luciferase activity assay showed,when miR-183 and miR-128 were bound to the CCL18 3'UTR,the luciferase activities were significantly higher in their mutant 3'UTR groups (11.63 ± 0.42;8.80 ± 0.49) than in their wild-type 3'UTR groups (4.86 ± 0.39;5.01 ± 0.54;both P ＜ 0.05) and blank control groups (2.41 ± 0.13;2.39 ± 0.05;both P ＜ 0.01),while there were no significant differences between miR-33a-hinding mutant 3'UTR group (6.41 ± 0.47) and miR-33a-binding wild-type 3'UTR group (6.16 ± 0.22,P ＞ 0.05).Real-time fluorescence-based quantitative PCR revealed higher mRNA expression of the CCL18 gene (3.52 ± 1.68),but lower expression of miR-183 (0.49 ± 0.32),miR-128 (0.30 ± 0.20) and miR-33a (0.46 ± 0.40) in the malignant melanoma tissues compared with the control tissues.The mRNA expression of the CCL18 gene was negatively correlated with the expression of miR-128 (rs =-0548,P ＜ 0.05),but showed no significant correlations with the expression of miR-183 and miR-33a (both P ＞ 0.05).Conclusion miR-128 may play a role in regulating the potential malignant melanoma-promoting gene CCL18.
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Objective To measure the expression of CC chemokine ligand 18(CCL18)in cutaneous malignant melanoma (CMM) tissues, and to explore its clinical significance, as well as relationship with vascular endothelial growth factor (VEGF) and Ki67 antigen expressions. Methods Immunohistochemistry was performed to measure CCL18, VEGF and Ki67 expressions in 58 paraffin?embedded CMM tissue specimens, as well as CCL18 expression in 20 paraffin?embedded pigmented nevus specimens, and immunofluorescence assay to confirm the expression of CCL18 in fresh CMM tissue specimens. Correlations of CCL18 expression with CMM clinicopathologic features, VEGF and Ki67 expressions were analyzed. Results CCL18 was detected in 49 (84.48%) of 58 paraffin?embedded CMM specimens, but in none of the 20 paraffin?embedded pigmented nevus specimens, with a significant difference in the positive rate of CCL18 between the CMM group and pigmented nevus group(χ2=45.46, P0.05). In addition, the expression of CCL18 in CMM tissues was positively correlated with that of VEGF(rs = 0.727, P 0.05). Immunofluorescence assay showed CCL18 expression in the cytoplasm of tumor cells in CMM tissues. Conclusion CCL18 is highly expressed in CMM tissues, and may be involved in tumor invasion and metastasis.
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Objective To explore the role of chemokine CCL18 in the occurrence and development of children allergic diseases. Methods The serum levels of CCL18 in 87 children cases of allergic asthma,64 cases of allergic rhinitis,46 cases of allergic con-junctivitis and contemporaneous 50 health students with physical examination as the control group were measured by ELISA and re-measured after 6-month treatment.The detection results were statistically analyzed with combining the clinical related detection data by adopting SPSS18.0 statistical software and t-test.Results The serum levels of CCL18 in the children allergic asthma,allergic rhinitis and allergic conjunctivitis groups were remarkably higher than those in the control group with statistical differences(P <0.05);the levels of CCL18 in the severe groups were remarkably higher than those in the mild and moderate groups with statistical difference(P <0.05);the levels of CCL18 after 6-month treatment in 3 groups were significantly decreased compared with before treatment with statistical difference(P <0.05).Conclusion The serum level of CCL18 is significantly associated with the severity of illness and significantly decreased after treatment,which indicating that the CCL18 detection has the important value in the diag-nosis,severity evaluation and treatment effect monitoring of children allergic diseases.
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Objective To investigate the effects of chemokine CCL18 on the proliferation and invasion of a human melanoma cell line A375. Methods Human peripheral blood monocytes were isolated from healthy volunteers, cultured in vitro and divided into two groups to be induced by IL-4 for 48 hours or remain untreated. A375 cells were classified into 3 groups to be cultured with IL-4-induced monocytes, untreated monocytes or CCL18 of 200 g/L for various durations. A375 cells receiving no treatment served as the control.MTT assay was performed to detect the proliferation of A375 cells, chemotaxis test and Matrigel-transwell assay to evaluate the chemotaxis and invasion ability of A375 cells, and chicken chorioalllantoic memebrane (CAM)was used to detect the effect of CCL18 on tumor angiogenesis. Results The proliferation of A375 cells was statistically accelerated by IL-4-induced monocytes and untreated monocytes, but unaffected by CCL18.Matrigel-transwell assay revealed that IL-4-induced monocytes and CCL18 promoted the chemotaxis and invasion ability of A375 cells (all P＜0.05). Tumor angiogenesis was also increased by IL-4-induced monocytes and CCL18 (both P ＜ 0.05). Conclusion The chemokine CCL18 can promote the invasion ability of A375 cells and tumor angiogenesis.