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Objective To construct a recombinant lentiviral vector carrying the casitas B-lineage lymphoma (Cbl)-b short hairpin RNA (shRNA),and to evaluate its effect on the biological behavior of A375 melanoma cells in vitro.Methods Three specific shRNAs targeting Cbl-b gene and a negative control shRNA were designed and synthesized,and recombinant lentiviral vectors were constructed.A375 cells were divided into 5 groups to be transfected with 3 kinds of lentiviral vector expressing Cbl-b genespecific shRNAs (CBLB-shRNA-1 group,CBLB-shRNA-2 group and CBLB-shRNA-3 group),a lentiviral vector containing negative control shRNA (negative control group),and an empty vector (blank control group).Real-time fluorescence-based quantitative PCR and Western blot analysis were performed to determine the silencing efficiency at 72 hours after transfection.Cell counting kit-8 (CCK-8)assay was conducted to evaluate cellular proliferative activity at 24,48,72 and 96 hours after transfection,flow cytometry to detect cell apoptosis and cell cycle at 72 hours after transfection,and Transwell invasion assay to assess cellular invasive activity at 72 hours after transfection.Results Three recombinant lentiviral vectors containing Cbl-b shRNA were constructed successfully.As Western blot analysis revealed,the CBLB-shRNA-3 showed the highest silencing efficiency.CCK-8 assay indicated that the proliferative activity of A375 cells was significantly lower in the CBLB-shRNA-3 group than in the negative control group and blank control group at 72 and 96 hours after transfection(all P ＜ 0.01).Flow cytometry showed that the apoptosis rate of A375 cells was significantly higher in the CBLB-shRNA-3 group (22.73％ ± 6.58％) than in the negative control group (6.08％ ± 1.35％,P ＜ 0.01) and blank control group (6.34％ ± 1.07％,P ＜ 0.01).The CBLB-shRNA-3 group showed a significantly higher proportion of A375 cells at G1 phase,but a significantly lower proportion of A375 cells at S phase compared with the negative control group and blank control group(all P ＜ 0.01).Transwell assay showed that there were significant differences in the number of A375 cells crossing the artificial basement membrane (matrigel) at 72 hours after transfection among the negative control group,blank control group and CBLB-shRNA-3 group (76.60 ± 1.82,73.20 ± 3.83,19.60 ± 1.14,respectively;F =794.50,P ＜ 0.01).Conclusions A recombinant CBLB-shRNA-3-expressing lentiviral vector which can efficiently silence Cbl-b gene has been successfully constructed.It can inhibit the proliferation,cell cycle progression and invasive activity of A375 cells,but promote the apoptosis of A375 cells.
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Objective To analyze differentially expressed proteins in A375 melanoma cells before and after short hairpin RNA (shRNA)-mediated Cbl-b gene silencing.Methods The label-free quantitative proteomics approach was performed to identify differentially expressed proteins between A375 cells transfected with lentiviral vectors containing Cbl-b shRNA (Cbl-b shRNA group) and those with control lentiviral vectors (control group).Then,the properties of differentially expressed proteins were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analysis.Western blot analysis was conducted to determine the expression of differential proteins (EphA2 and GSK3β) and phosphorylated protein kinase (p-AKT) after shRNA-mediated Cbl-b gene silencing.Statistical analysis was carried out by t test of two independent-samples for comparison of protein expression abundance between the two groups with SPSS 23.0 software.Additionally,the results of GO and KEGG enrichment analysis were analyzed by Fisher's exact test.Results A total of 3 449 proteins were identified and quantified,and 74 of them were differentially expressed between the Cbl-b shRNA group and control group.Compared with the control group,52 proteins were up-regulated and 22 were down-regulated in the Cbl-b shRNA group.GO enrichment analysis of differential proteins revealed that the top five significantly enriched biological processes were integrin-mediated cell adhesion,single-organism metabolic process,regulation of integrin-mediated cell adhesion,regulation of protein-targeting mitochondria and nucleic acid metabolic process.The top five significantly enriched molecular functions included DNA binding,2-iron,2-sulfur cluster binding,signaling receptor activity,cadherin binding and cell adhesion molecule binding.The top five significantly enriched cell components included nucleosome,DNA packaging complex,photoreceptor connecting cilium,DNA-protein complex and extracellular region part.KEGG enrichment analysis demonstrated that the top five significantly enriched melanoma-related signaling pathways were folate biosynthesis,axon guidance,extracellular matrix-receptor interaction,adherens junction and Wnt signaling pathways.As Western blot analysis revealed,the Cbl-b shRNA group showed lower protein expression of EphA2 (0.369),but higher protein expression of GSK3β (3.524) compared with the control group (1),which were consistent with the results of proteomics analysis.Additionally,the protein expression of p-AKT was down-regulated in Cbl-b shRNA group (0.453) compared with the control group (1).Conclusion Cbl-b may be involved in the occurrence of melanoma through a variety of biological pathways,and the EphA2/PI3K/AKT signaling pathway may be one important pathway.
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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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An 88?year?old man presented with multiple nodules and ulcers on both lower limbs for 6 months. Dermatological examination showed multiple dark purple hard nodules with central crusted ulceration. Histopathological examination revealed a diffuse infiltration of atypical lymphoid cells with large hyperchromatic nuclei in the dermis and subcutis, as well as small necrotic foci in some regions. Conspicuous mitoses were seen. The large lymphoid cells were positive for CD20, CD79a and Bcl?2. Epstein?Barr virus?encoded RNA was detected by in situ hybridization. These findings were consistent with Epstein?Barr virus?positive diffuse large B?cell lymphoma. The patient gave up systemic chemotherapy and died six months later.
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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 investigate the diagnosis and differential diagnosis of dermatofibrosarcoma protuberans (DFSP). Methods Totally, 50 patients with DFSP visiting the Institute of Dermatology, Chinese Academy of Medical Sciences from 1998 to 2014 were enrolled. The clinical manifestations, histopathological and immunohistochemical features, treatment and prognosis of DFSP were retrospectively reviewed. Results The average age at onset of DFSP was (29.5 ± 15.9)years in the 50 patients, with a mean disease duration of 9.57 years. Skin lesions most frequently occurred on the trunk(n = 33, 66.0%), followed by the extremities, head and neck. DFSP was characterized by atrophic patches or plaques in 13 cases (26.0%), multiple nodules varying in size and arising on atrophic plaques or patches in 30 cases (60.0%), single or multiple nodules arising on normal skin in 7 cases (14.0%). Histologically, the tumor consisted of uniform infiltrative spindle cells arranged in a storiform or cartwheel pattern. In addition, the tumor cells expressed CD34 and vimentin. Twenty patients experienced recurrence at the primary site after resection of skin lesions with a recurrence rate of 43.5%. No distant metastasis or death occurred in these patients. Conclusions DFSP usually has various skin manifestations, is easily misdiagnosed, and can be confirmed based on histopathological and immunohistochemical findings. Local recurrence of DFSP is common, and may occur for many times after surgical excision, but lymphatic and distant metastases are rare.