Identification of differentially expressed genes in uveal melanoma using suppressive subtractive hybridization.
ABSTRACT: Uveal melanoma (UM) is the most common primary cancer of the eye, resulting not only in vision loss, but also in metastatic death. This study attempts to identify changes in the patterns of gene expression that lead to malignant transformation and proliferation of normal uveal melanocytes (UVM) using the Suppressive Subtractive Hybridization (SSH) technique.The SSH technique was used to isolate genes that are differentially expressed in the TP31 cell line derived from a primary UM compared to UVM. The expression level of selected genes was further validated by microarray, semi-quantitative RT-PCR and western blot analyses.Analysis of the subtracted libraries revealed that 37 and 36 genes were, respectively, up- and downregulated in TP31 cells compared to UVM. Differential expression of the majority of these genes was confirmed by comparing UM cells with UVM by microarray. The expression pattern of selected genes was analyzed by semi-quantitative RT-PCR and western blot, and was found to be consistent with the SSH findings.We demonstrated that the SSH technique is efficient to detect differentially expressed genes in UM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor.
Project description:Uveal melanoma (UM) has been the subject of intense interest due to its distinctive metastatic pattern, which involves hematogenous dissemination of cancerous cells toward the liver in 50% of patients. To search for new UM prognostic markers, the Suppressive Subtractive Hybridization (SSH) technique was used to isolate genes that are differentially expressed between UM primary tumors and normal uveal melanocytes (UVM).A subtracted cDNA library was prepared using cDNA from uncultured UM primary tumors and UVM. The expression level of selected genes was further validated by cDNA microarray, semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), and immunofluorescence analyses.One hundred-fifteen genes were identified using the SSH technique. Microarray analyses comparing the gene expression profiles of UM primary tumors to UVM validated a significant differential expression for 48% of these genes. The expression pattern of selected genes was then analyzed by semi-quantitative RT-PCR and was found to be consistent with the SSH and cDNA microarray findings. A down-regulation of genes associated with melanocyte differentiation was confirmed in UM primary tumors. Presence of undifferentiated cells in the UM was demonstrated by the expression of stem cell markers ATP-binding cassette sub-family G member 2 (ABCG2) and octamer-binding protein 4 (OCT4).We demonstrated that the SSH technique is efficient to detect differentially expressed genes between UM and UVM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor. In addition, deregulation of the melanocyte differentiation pathway revealed the presence of UM cells exhibiting a stem cell-like phenotype.
Project description:BAP1 is a tumor suppressor gene important to the development and prognosis of many cancers, especially uveal melanoma (UM). Its role in more common cancers such as breast and colon cancer is largely unknown. We collected the transcriptome profiling data sets from the TCGA uveal melanoma (TCGA-UVM), breast cancer (TCGA-BRCA), and colon cancer (TCGA-COAD) projects to analyze the expression of BAP1. We found that patients with UM and breast cancer, but not colon cancer, who died had a lower level of BAP1 gene expression compared to surviving patients. Importantly, in breast cancer patients, the lowest BAP1 expression levels corresponded to the dead young patients (age at diagnosis < 46). Since the number of cases in TCGA-BRCA was much higher than TCGA-UVM, we obtained highly correlated genes with BAP1 in invasive breast carcinomas. Then, we tested if these genes are also highly correlated with BAP1 in UM and colon cancer. We found that BAP1 is highly positively correlated with RBM15B and USP19 expression in invasive breast carcinoma, UM, and colon adenocarcinoma. All three genes are located in close proximity on the 3p21 tumor suppressor region that is commonly altered in many cancers.
Project description:Purpose: Uveal melanoma (UM) is the most frequent metastatic ocular tumor in adults. Therapeutic intervention remains ineffective since none of the novel procedures used to treat this disease increased survival rates. To deal with this limitation, additional studies are required to clarify its pathogenesis. The current study focused on describing how epigenetic modulation by miR-142-3p affects changes in some cellular functions underlying UM pathogenesis. Methods and results: Microarray analysis identified 374 miRNAs which were differentially expressed between UM cells and uveal melanocytes. miR-142-3p was one of the 10 most downregulated miRNAs. Quantitative RT-PCR analysis confirmed that miR-142-3p expression levels were significantly decreased in both UM cell lines and clinical specimens. The results of the MTS, clone formation, scratch wound, transwell assays, and in vivo biofluorescence imaging showed that miR-142-3p overexpression significantly inhibited cell proliferation, migration, and invasiveness. Nevertheless, miR-142-3p did not affect cell apoptotic activity or sensitivity to doxorubicin. Cell cycle and EdU analysis showed that miR-142-3p overexpression induced G1/G2 cell cycle arrest and reduced DNA synthesis in UM cells. Microarray analysis showed that miR-142-3p mainly regulates the TGF? signaling pathway, and those in which MAPK and PI3K-Akt are constituents. Functional interactions between miR-142-3p and CDC25C, TGF?R1, GNAQ, WASL, and RAC1 target genes were confirmed based on the results of the luciferase reporter assay and Western blot analysis. CDC25C or RAC1 downregulation is in agreement with cell cycle arrest and DNA synthesis disorder induction, while downregulation of TGF?R1, GNAQ, WASL, or RAC1 accounts for declines in cell migration. Conclusion: miR-143-3p is a potential therapeutic target to treat UM since overriding its declines in expression that occur in this disease reversed the pathogenesis of this disease. Such insight reveals novel biomarker for decreasing UM vitality and for improved tracking of tumor progression.
Project description:The present study aimed to assess the expression of legumain in uveal melanoma (UM) cell lines and primary UM specimens, and to determine the possible association between legumain expression and clinical as well as pathological characteristics to reveal its impact on the prognosis of patients with UM. Records of primary UM cases treated at Beijing Tongren Hospital and Tianjin Eye Hospital between 1996 and 2005 were retrieved for analysis and a total of 82 patients with uveal melanoma were included in the study. The expression of legumain in the formalin‑fixed and paraffin‑embedded surgical specimens of these 82 patients was determined using immunohistochemical analysis. In addition, the expression of legumain was examined in two uveal melanoma cell lines using polymerase chain reaction and western blot analyses. The association of legumain expression with clinical/pathological characteristics was analyzed using the χ2 and Fisher's exact test. In addition, the impact of legumain on the prognosis of patients with uveal melanoma was examined. Upregulation of legumain was more predominant in the highly invasive uveal melanoma cell line MUM‑2B compared with that in the MUM‑2C with low invasiveness. Of 82 primary uveal melanoma tissues, 35 exhibited high expression of legumain, while the other 47 specimens exhibited low or negative expression of legumain. High legumain expression was primarily associated with local invasion of UM. Overall survival analysis revealed that the patients with high legumain expression exhibited poorer survival than patients with low/negative legumain expression. These findings suggested that upregulation of legumain is associated with malignant behavior of UM and that legumain may be used as an negative prognostic factor as well as a therapeutic target.
Project description:Background: Uveal melanoma (UM) is the most common primary intraocular cancer in adults. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of UM that may lead to novel therapeutic strategies. Methods: Dataset TCGA-UVM, download from TCGA portal, were taken as the training cohort, and dataset GSE22138, obtained from GEO database, was set as the validation cohort. In training cohort, Kaplan-Meier analysis and univariate Cox regression model were applied to preliminary screen prognostic genes. Besides, the Cox regression model with LASSO was implemented to build a multi-gene signature, which was then validated in the validation cohorts through Kaplan-Meier, Cox, and ROC analyses. In addition, the correlation between copy number aberrations and risk score was evaluated by Spearman test. GSEA and immune infiltrating analyses were conducted for understanding function annotation and the role of the signature in the tumor microenvironment. Results: A ten-gene signature was built, and it was examined by Kaplan-Meier analysis revealing that significantly overall survival, progression-free survival, and metastasis-free survival difference was seen. The ten-gene signature was further proven to be an independent risk factor compared to other clinic-pathological parameters via the Cox regression analysis. Moreover, the receiver operating characteristic curve (ROC) analysis results demonstrated a better predictive power of the UM prognosis that our signature owned. The ten-gene signature was significantly correlated with copy numbers of chromosome 3, 8q, 6q, and 6p. Furthermore, GSEA and immune infiltrating analyses showed that the signature had close interactions with immune-related pathways and the tumor environment. Conclusions: Identifying the ten-gene signature (SIRT3, HMCES, SLC44A3, TCTN1, STPG1, POMGNT2, RNF208, ANXA2P2, ULBP1, and CA12) could accurately identify patients' prognosis and had close interactions with the immunodominant tumor environment, which may provide UM patients with personalized prognosis prediction and new treatment insights.
Project description:Uveal melanoma (UM) represents the most frequent primary tumor of the eye. Despite the development of new drugs and screening programs, the prognosis of patients with UM remains poor and no effective prognostic biomarkers are yet able to identify high‑risk patients. Therefore, in the present study, microRNA (miRNA or miR) expression data, contained in the TCGA UM (UVM) database, were analyzed in order to identify a set of miRNAs with prognostic significance to be used as biomarkers in clinical practice. Patients were stratified into 2 groups, including tumor stage (high‑grade vs. low‑grade) and status (deceased vs. alive); differential analyses of miRNA expression among these groups were performed. A total of 20 deregulated miRNAs for each group were identified. In total 7 miRNAs were common between the groups. The majority of common miRNAs belonged to the miR‑506‑514 cluster, known to be involved in UM development. The prognostic value of the 20 selected miRNAs related to tumor stage was assessed. The deregulation of 12 miRNAs (6 upregulated and 6 downregulated) was associated with a worse prognosis of patients with UM. Subsequently, miRCancerdb and microRNA Data Integration Portal bioinformatics tools were used to identify a set of genes associated with the 20 miRNAs and to establish their interaction levels. By this approach, 53 different negatively and positively associated genes were identified. Finally, DIANA‑mirPath prediction pathway and Gene Ontology enrichment analyses were performed on the lists of genes previously generated to establish their functional involvement in biological processes and molecular pathways. All the miRNAs and genes were involved in molecular pathways usually altered in cancer, including the mitogen‑activated protein kinase (MAPK) pathway. Overall, the findings of the presents study demonstrated that the miRNAs of the miR‑506‑514 cluster, hsa‑miR‑592 and hsa‑miR‑199a‑5p were the most deregulated miRNAs in patients with high‑grade disease compared to those with low‑grade disease and were strictly related to the overall survival (OS) of the patients. However, further in vitro and translational approaches are required to validate these preliminary findings.
Project description:Uveal melanoma (UVM) is the most common primary intraocular malignancy in adults. With over 50% of patients developing metastatic disease, there is an unmet need for improved diagnostic and therapeutic options. Efforts to understand the molecular biology of the disease have revealed several markers that correlate with patient prognosis, including the copy number of chromosome 3, genetic alterations in the BAP1, EIF1AX and SF3B1 genes, and other transcriptional features. Here, we expand upon previous reports by comprehensively characterizing the short RNA-ome in 80 primary UVM tumor samples. In particular, we describe a previously unseen complex network involving numerous regulatory molecules that comprise microRNA (miRNAs), novel UVM-specific miRNA loci, miRNA isoforms (isomiRs), and tRNA-derived fragments (tRFs). Importantly, we show that the abundance profiles of isomiRs and tRFs associate with various molecular phenotypes, metastatic disease, and patient survival. Our findings suggest deep involvement of isomiRs and tRFs in the disease etiology of UVM. We posit that further study and characterization of these novel molecules will improve understanding of the mechanisms underlying UVM, and lead to the development of new diagnostic and therapeutic approaches.
Project description:Currently, there is no effective treatment for metastatic uveal melanoma (UVM). Here, we aimed to identify the mechanism involving intrinsic chemoresistance of metastatic UVM and the relevant therapeutic targets for UVM. We analyzed cohorts of 80 and 67 patients with primary UVM and skin cutaneous melanoma (SKCM), respectively, using The Cancer Genome Atlas dataset. Mutational burdens identified by whole exome sequencing were significantly lower in UVM than in SKCM patients. COSMIC mutational signature analysis identified that most of the mutations in UVM patients (>90%) were associated with spontaneous deamination of 5-methylcytosine or defective mismatch repair. Transcriptome analysis revealed that the MYC signature was more enriched in UVM patients, as compared to SKCM patients. Fifty-nine (73.8%) of 80 UVM patients showed gains in MYC copy number, and a high MYC copy number was associated with aggressive clinicopathological features of tumors and poor survival. Kinome-wide siRNA library screening identified several therapeutic targets, reported as synthetic lethal targets for MYC-addicted cancers. Notably, UVM cell lines showed high susceptibility to a WEE1 inhibitor (MK-1775; adavosertib) at a clinically tolerable dose. Overall, our study identified high MYC activity in UVM, and suggested G2/M checkpoint inhibitors as effective therapeutic targets for UVM.
Project description:BACKGROUND:This study was to investigate some new pathological genes in colorectal adenocarcinoma of human. METHODS:Human colorectal adenocarcinoma tissues and normal colorectal tissues were taken and suppression subtractive hybridization (SSH) and cDNA microarray techniques were employed. From differentially expressed 86 expressed sequence tags (EST), 10 EST of the SSH were selected as seed sequence for bioinformatics analyses, semi-quantitative RT-PCR and PCR-sequencing. Each lane of semi-quantitative RT-PCR was analyzed by Q1 software. RESULTS:Among these 10 EST, it has been found that ES274070, ES274071, ES274076 and ES274081 may play role in the onset of colorectal adenocarcinoma in human. CONCLUSIONS:The ES274070, ES274071, ES274076 and ES274081 are related to the onset of human colorectal adenocarcinoma.
Project description:BACKGROUND: Uveal melanoma (UM) is a rare eye tumor. There are two classes of UM, which can be discriminated by the chromosome 3 status or global mRNA expression profile. Metastatic progression is predominantly originated from class II tumors or from tumors showing loss of an entire chromosome 3 (monosomy 3). We performed detailed EFS (embryonal Fyn-associated substrate) methylation analyses in UM, cultured uveal melanocytes and normal tissues, to explore the role of the differentially methylated EFS promoter region CpG island in tumor classification and metastatic progression. METHODS: EFS methylation was determined by direct sequencing of PCR products from bisulfite-treated DNA or by sequence analysis of individual cloned PCR products. The results were associated with clinical features of tumors and tumor-related death of patients. RESULTS: Analysis of 16 UM showed full methylation of the EFS CpG island in 8 (50%), no methylation in 5 (31%) and partial methylation in 3 (19%) tumors. Kaplan-Meier analysis revealed a higher risk of metastatic progression for tumors with EFS methylation (p = 0.02). This correlation was confirmed in an independent set of 24 randomly chosen tumors. Notably, only UM with EFS methylation gave rise to metastases. Real-time quantitative RT-PCR expression analysis revealed a significant inverse correlation of EFS mRNA expression with EFS methylation in UM. We further found that EFS methylation is tissue-specific with full methylation in peripheral blood cells, and no methylation in sperm, cultured primary fibroblasts and fetal muscle, kidney and brain. Adult brain samples, cultured melanocytes from the uveal tract, fetal liver and 3 of 4 buccal swab samples showed partial methylation. EFS methylation always affects both alleles in normal and tumor samples. CONCLUSIONS: Biallelic EFS methylation is likely to be the result of a site-directed methylation mechanism. Based on partial methylation as observed in cultured melanocytes we hypothesize that there might be methylated and unmethylated precursor cells located in the uveal tract. The EFS methylation of a UM may depend on which type of precursor cell the tumor originated from.