1.Syntenin: a novel PDZ domain-containing scaffolding protein associated with human melanoma metastasis.
Jian-Bo YANG ; James B MCCARTHY
Journal of Central South University(Medical Sciences) 2007;32(2):204-212
Syntenin is overexpressed in multiple human cancers and is newly recognized as a novel regulator in melanoma metastasis. It functions as a scaffolding protein, via its two PDZ domains interacting with multiple transmembrane and cytoplasmic partners to regulate many of the major signaling pathways involved in various cellular processes, such as cell surface receptor clustering, protein trafficking, cytoskeleton remodeling, and activation of transcription factor, and results in the increased abilities for tumor cell growth, adhesion, angiogenesis, invasion and metastasis. The present article attempts to review the structure and functions of syntenin by summarizing our current knowledge on the interacting partners and diverse signaling pathways related to syntenin, and highlight the importance of syntenin as a new potential therapeutic target for the aggressive human melanoma.
Animals
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Biomarkers, Tumor
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genetics
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metabolism
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Humans
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Melanoma
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genetics
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metabolism
;
pathology
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Neoplasm Metastasis
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PDZ Domains
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genetics
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Signal Transduction
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Syntenins
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chemistry
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genetics
;
metabolism
2.Functional Annotation of Metastasis-associated MicroRNAs of Melanoma: A Meta-analysis of Expression Profiles.
Jing-Yi LI ; Li-Li ZHENG ; Ting-Ting WANG ; Min HU
Chinese Medical Journal 2016;129(20):2484-2490
BACKGROUNDMelanoma is a type of cancer that develops from the pigment-containing cells. Until now, its pathological mechanisms remain largely unknown. The aim of this study was to identify metastasis-related microRNA (miRNAs) and gain an understanding of the biological functions in the metastasis of melanoma.
METHODSWe searched the PubMed and Gene Expression Omnibus database to collect miRNA expression profiling datasets about melanoma, with key words of "melanoma", "miRNA", "microarray", and "gene expression profiling". Only the original experimental works published before June 2016 for analyzing the metastasis of melanoma were retained, other nonhuman studies, reviews, and meta-analyses were removed. We performed a meta-analysis to explore the differentially expressed miRNA between metastatic and nonmetastatic samples. Moreover, we predicted target genes of the miRNAs to study their biological roles for these miRNAs.
RESULTSWe identified a total of 63 significantly differentially expressed miRNAs by meta-analysis of the melanoma expression profiling data. The regulatory network constructed by using these miRNAs and the predicted targets identified several key genes involved in the metastasis of melanoma. Functional annotation of these genes indicated that they are mainly enriched in some biological pathways such as mitogen-activated protein kinase signaling pathway, cell junction, and focal adhesion.
CONCLUSIONSBy collecting the miRNA expression datasets from different platforms, multiple biological markers were identified for the metastasis of melanoma. This study provided novel insights into the molecular mechanisms underlying this disease, thereby aiding the diagnosis and treatment of the disease.
Computational Biology ; methods ; Gene Expression Profiling ; Humans ; Melanoma ; genetics ; metabolism ; MicroRNAs ; genetics ; Oligonucleotide Array Sequence Analysis ; Signal Transduction ; genetics ; physiology
3.Angiopoietin-like 4 modulates aldolase A expression in human melanoma cell in a PKC dependent manner.
Yang SUN ; Xiancheng WANG ; Borong FANG ; Xiang XIONG ; Jianhong LONG
Journal of Central South University(Medical Sciences) 2015;40(8):851-857
OBJECTIVE:
To investigate the association between angiopoietin-like 4 (ANGPTL4) and aldolase A (ALDOA) in human melanoma cell.
METHODS:
Overexpression or knockdown of ANGPTL4 was performed in WM-115 or WM-266-4 cells, respectively. The expression of ANGPTL4 and ALDOA was measured by RT-PCR and Western blot, respectively. The promoter activity of ALDOA gene was determined by luciferase assay.
RESULTS:
The promoter activity of ALDOA gene and the expression of ALDOA (mRNA and protein) were increased or decreased in the melanoma cells with overexpression or knockdown of ANGPTL4, which was blocked by selective protein kinase C (PKC) inhibitor or restored by PKC agonist, respectively.
CONCLUSION
ANGPTL4 promotes ALDOA expression in human melanoma cell in a PKC dependent manner.
Angiopoietin-like 4 Protein
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Angiopoietins
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genetics
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metabolism
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Blotting, Western
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Cell Line, Tumor
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Fructose-Bisphosphate Aldolase
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metabolism
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Gene Knockdown Techniques
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Humans
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Melanoma
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genetics
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metabolism
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RNA, Messenger
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genetics
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metabolism
4.Influence of the sequence variations of the HLA-DR promoters derived from human melanoma cell lines on nuclear protein binding and promoter activity.
Tae Jin LEE ; Se Jong KIM ; Jeon Han PARK
Yonsei Medical Journal 2000;41(5):593-599
In previous studies we reported that the expression of HLA-DR on melanoma cell lines was differentially modulated by IFN- gamma and that the transcription rate was responsible for this differential modulation. We have also reported the nucleotide sequence variations in the promoter region of HLA-DR genes, and proposed that differences in the promoter activity by the sequence variations of the HLA-DR promoters might contribute to such a differential transcriptional regulation at the promoter level. In this study, in order to assess whether the sequence variations of the HLA-DR promoters affect the factor binding and exert influence on the promoter activity, nuclear factor binding to our previous six HLA-DRA and fourteen HLA-DRB promoter clones was evaluated with the nuclear protein extracted from a B-lymphoblastoid cell line (BLCL), BH, together with the chloramphenicol acetyltransferase (CAT) reporter assay. In the HLA-DRA promoters, clone 35 containing one bp nucleotide sequence variation at the octamer binding site (OCT) (GATTTGC to GATCTGC) showed relatively weak factor binding. In the HLA-DRB promoters, clusters I, III, and IV of our previous HLA-DRB promoter homologues, containing one bp nucleotide sequence variation (GATTCG) in their Y boxes exhibited weak factor binding and CAT activity compared to other clusters (GATTGG) that showed strong factor binding and CAT activity. This data suggests chat the binding patterns of transcription factors influenced by the nucleotide sequence variations of the HLA-DR promoter could affect the promoter activity and the DNA sequence elements in the HLA-DR promoter could mediate transcriptional regulation.
Base Sequence/genetics
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HLA-DR Antigens/genetics*
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Human
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Melanoma/pathology*
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Melanoma/genetics*
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Molecular Sequence Data
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Nuclear Proteins/metabolism*
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Promoter Regions (Genetics)/physiology*
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Promoter Regions (Genetics)/genetics*
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Tumor Cells, Cultured
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Variation (Genetics)*
5.MicroRNA 145 may play an important role in uveal melanoma cell growth by potentially targeting insulin receptor substrate-1.
Yang LI ; Qiming HUANG ; Xuehui SHI ; Xiang JIN ; Li SHEN ; Xiaolin XU ; Wenbin WEI ;
Chinese Medical Journal 2014;127(8):1410-1416
BACKGROUNDMicroRNAs (miRNAs) contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. In this study, we investigated the role of miR-145 in the pathogenesis of uveal melanoma.
METHODSExpression profiles of miRNAs in uveal melanoma were performed using Agilent miRNA array. Quantitative real-time polymerase chain reaction was used to screen the expression levels of miR-145 in normal uveal tissue, uveal melanoma tissue, and uveal melanoma cell lines. Lenti-virus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Cell proliferation, cell cycle, and cell apoptosis of these miR-145 overexpression cell lines were examined by MTT assay and flow cytometry respectively. The target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of insulin-like growth factor-1 receptor (IGF-1R), insulin receptor substrate-1 (IRS-1) proteins was determined by Western blotting analysis. IRS-1 was knocked down in OCM-1 cells. TUNEL, BrdU, and flow cytometry assay were performed in IRS-1 knocked down OCM-1 cell lines to analyze its function.
RESULTSForty-seven miRNAs were up regulated in uveal melanoma and 61 were down regulated. miR-145 expression was significantly lower in uveal melanoma sample and the cell lines were compared with normal uveal sample. Overexpression of miR-145 suppressed cell proliferation by blocking the G1 phase entering S phase in uveal melanoma cells, and promoted uveal melanoma cell apoptosis. IRS-1 was identified as a potential target of miR-145 by dual luciferase reporter assay. Knocking down of IRS-1 had similar effect as overexpression of miR-145.
CONCLUSIONmiR-145 might act as a tumor suppressor in uveal melanoma, and downregulation of the target IRS-1 might be a potential mechanism.
Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; genetics ; physiology ; Humans ; In Vitro Techniques ; Insulin Receptor Substrate Proteins ; genetics ; metabolism ; Melanoma ; genetics ; metabolism ; pathology ; MicroRNAs ; genetics ; metabolism ; Polymerase Chain Reaction ; Uveal Neoplasms ; genetics ; metabolism ; pathology
6.Multi-facet expressions of adenylate cyclase isoforms in B16-F10 melanoma cells differentiated by forskolin treatment.
Du Hyong CHO ; Chang Dae BAE ; Yong Sung JUHNN
Experimental & Molecular Medicine 2000;32(4):235-242
The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.
Adenylate Cyclase/*genetics
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Animal
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Cell Differentiation
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Cyclic AMP/*metabolism
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Forskolin/*pharmacology
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Isoenzymes/genetics
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Melanoma, Experimental/*enzymology/*pathology
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Mice
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Signal Transduction
7.Effect of lentivirus-mediated nestin gene silencing on metastatic potential of human melanoma cell line UACC903 in vitro.
Xuhui YANG ; Tian XIA ; Weihua YU ; Xiaofang LU ; Peng XIANG ; Feng HE
Journal of Southern Medical University 2012;32(1):7-13
OBJECTIVETo study the effect of lentivirus-mediated RNA interference (RNAi) of nestin on the metastatic potential of human melanoma cell line UACC903.
METHODSA lentiviral vector for RNAi targeting the coding region of human nestin mRNA (nestin-RNAi-LV) and another control vector containing a nonsense sequence were constructed. The vectors were transfected into UACC903 cells, and nestin expression in the cells was detected by RT-PCR, immunofluorence assay and Western blotting. The invasive ability and migration of the transfected UACC903 cells was evaluated using Transwell and scrape assay, respectively. Fluorescence assay was used to examine the expressions of E-cadherin, N-cadherin and β-catenin in the cells.
RESULTSThe lentiviral vector nestin-RNAi-LV was constructed successfully. Compared with the control vector, nestin-RNAi-LV resulted in obviously reduced expression of nestin mRNA and protein, lowered migration ability of UACC903 cells, and reduced cell adhesion and invasiveness (P<0.05).
CONCLUSIONLentivirus-mediated nestin RNAi can specifically inhibit nestin expression to cause decreased cell migration and invasiveness of human melanoma cell line UACC903.
Cell Line, Tumor ; Cell Movement ; genetics ; Genetic Vectors ; Humans ; Intermediate Filament Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; Melanoma ; pathology ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; genetics ; metabolism ; Nestin ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics
8.Targeting microRNA-mediated suppression of vascular endothelial growth factor gene expression and proliferation in malignant melanoma cells in vitro.
Yuan JIANG ; Yongzhi HAN ; Jian SUN
Journal of Southern Medical University 2014;34(3):358-363
OBJECTIVETo explore the inhibitory effect of targeting miRNA on the expression of vascular endothelial growth factor (VEGF) and cell proliferation in malignant melanoma (MM) SKmel-28 cells.
METHODSRecombination miRNA plasmid vectors targeting VEGF gene were transfected into SKmel-28 cells via Lipofectamine 2000. The integrity of the inserted fragments was detected using colony PCR and sequence analysis. The expression of VEGF mRNA and protein in SKmel-28 cells was detected by RT-PCR and Western blotting, respectively. MTS assay was used to determine the inhibitory effect of a selected targeting miRNA on SKmel-28 cell proliferation, and the apoptosis of SKmel-28 cells was detected using flow cytometry.
RESULTSTransfection with the targeting miRNAs significantly down-regulated the expressions of VEGF mRNA and protein in SKmel-28 cells (P<0.01), and the miRNA construct X-26-2n-1 showed the highest inhibitory effect. The miRNA X-26-2n-1 significantly suppressed SKmel-28 cell proliferation in a time-dependent manner (P<0.01) and increased the early, late and overall apoptosis rates of the cells (P<0.01).
CONCLUSIONThe targeting miRNA we constructed can effectively suppress the cell proliferation and induce apoptosis of SKmel-28 cells by down-regulating the expressions of VEGF gene.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Humans ; Melanoma ; genetics ; metabolism ; pathology ; MicroRNAs ; genetics ; Skin Neoplasms ; Transfection ; Vascular Endothelial Growth Factor A ; genetics ; metabolism
9.Construction and identification of an RNA interference lentivirus vector targeting the Ras homology C gene of melanoma cells.
Qiying WANG ; Ximei WANG ; Xiaomei ZHAI ; Jianwen ZHANG ; Minjing CHEN ; Linbo LIU
Chinese Medical Journal 2014;127(7):1339-1343
BACKGROUNDMelanoma has the highest mortality among all superficial malignant tumors. The poor prognosis is due to its high metastasis rate and the lack of therapeutic targets. As a molecular switch that controls tumor metastasis, Ras homology C (RhoC) has been correlated with tumor progression, especially tumor invasion and metastasis. However, little research has been done about the effects of RNA interference (RNAi) targeting RhoC on the invasion and metastasis of melanoma. In this study, we constructed an RNAi lentivirus vector targeting the RhoC gene of melanoma cells and identified its silencing effects on the RhoC gene.
METHODSBased on the RhoC gene encoding information, three pGPU6/GFP/Neo-short hairpin (shRNA) plasmids were constructed. After detecting their silencing effects on the RhoC gene of A375 cells, the most effective pGPU6/GFP/Neo-shRNA plasmid was packed with lentivirus to construct the recombinant pLenti6.3-EGFP-453 targeting RhoC. The lentivirus vector was used to infect A375 cells, and then the expression of RhoC mRNA and protein were determined with real-time PCR and Western blotting.
RESULTSThe plasmids pGPU6/GFP/Neo-shRNA 336, pGPU6/GFP/Neo-shRNA 453, and pGPU6/GFP/Neo-shRNA 680 were constructed. After they were transfected into A375 cells, the expressions of RhoC mRNA and protein were 1.47 ± 0.26, 1.13 ± 0.16, 1.39 ± 0.11 and 70.98 ± 9.21, 50.67 ± 6.06, 65.77 ± 4.06, respectively. pGPU6/GFP/Neo-shRNA 453 was the most effective sequence, and was used to successfully construct the pLenti6.3-EGFP-453 lentiviral vector targeting RhoC. pLenti6.3-EGFP-453 was used to infect A375 cells. The expression of RhoC mRNA and protein were 1.05 ± 0.05 and 62.04 ± 15.86 in the lentivirus group, 4.21 ± 0.24 and 220.86 ± 24.07 in the negative lentivirus control group, and 4.63 ± 0.32 and 257.39 ± 12.30 in the normal control group respectively with the difference between the lentivirus group and the control groups being statistically significant (P < 0.05).
CONCLUSIONThe successfully constructed pLenti6.3-EGFP-453 vector targeting the RhoC can effectively infect human melanoma A375 cells in vitro, and significantly inhibit the RhoC mRNA and protein expression.
Cell Line, Tumor ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Melanoma ; genetics ; therapy ; RNA Interference ; physiology ; rho GTP-Binding Proteins ; genetics ; metabolism ; rhoC GTP-Binding Protein
10.A decrease in the expression of CD63 tetraspanin protein elevates invasive potential of human melanoma cells.
Experimental & Molecular Medicine 2003;35(4):317-323
CD63, which belongs to the tetraspanin membrane proteins, has been proposed to play an important role in inhibiting melanoma metastasis. To determine whether reduction of CD63 expression, which frequently occurs in the malignant progression of human melanoma, is responsible for metastasis promotion, we transfected the antisense CD63 cDNA into MelJuso melanoma cells having endogenous CD63 expression. The antisense CD63 transfectant clones showing decreased CD63 expression displayed increased cell motility, matrix-degrading activity, and invasiveness in vitro when compared with the control transfectant cells. The antisense CD63 cDNA-transfected cells also exhibited altered adhesiveness to extracellular matrix. The results suggest that reduced CD63 expression contributes to the invasive and metastatic ability of human melanoma cells.
Antigens, CD/biosynthesis/*genetics
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Gene Expression Regulation, Neoplastic
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Human
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Melanoma/*genetics/metabolism
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Neoplasm Invasiveness/*genetics
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Platelet Membrane Glycoproteins/biosynthesis/*genetics
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Reverse Transcriptase Polymerase Chain Reaction