Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Lack of microRNA-101 causes E-cadherin expression and function deregulation through EZH2 up-regulation in gastric cancer

ABSTRACT: Background & Aims: E-cadherin expression disruption is commonly observed in epithelial cancers and metastasis. Such event is also recognized as a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression and function of pivotal cancer-related genes, we characterized and dissected a pathway initiated by loss of microRNA-101 that causes E-cadherin dysfunction through upregulation of EZH2 expression in GC. Methods: Microarrays were used to profile the expression of microRNAs in human GC. Array-CGH revealed DNA copy number changes that were validated by genomic quantitative PCR and Snapshot. Expression levels of microRNAs, mRNA and protein were determined by quantitative-real time PCR and western-blot/co-immunofluorescence. CDH1 inactivating mechanisms were analyzed. Gain and loss of function experiments were done in KatoIII cells. E-cadherin functionality was assessed by immunofluorescence and flow cytometry. Results: MiR-101 expression was significantly decreased in tumors in comparison with normal gastric mucosas (P<.0001). In 65% of the analyzed GC cases, miR-101 downregulation was caused by deletions and/or microdeletions at miR-101-2 locus. EZH2 overexpression and consequent loss/aberrant E-cadherin expression was found in concomitance with miR-101 downregulation in 41% of the analyzed GC cases. This occurred preferentially in cases retaining allele(s) untargeted by classical CDH1 inactivating mechanisms. MiR-101 gain of function experiments or direct inhibition of EZH2, led to a strong depletion of endogenous EZH2 protein and consequent rescue of functional E-cadherin at the cell membrane, mimicking results obtained with clinical GC samples. Conclusions: Deletions and/or microdeletions at miR-101-2 locus underlying mature miR-101 downregulation and consequent EZH2 overexpression represented a novel cascade of genetic events leading to E-cadherin disruption, preferentially affecting the intestinal-type of GC. In the present study, 37 primary GCs and a pool of 10 normal gastric mucosas were used to acquire the expression of 703 known human mature miRNAs deposited in the Sanger miRBase Sequence Database, Release 10.0 (http://microrna.sanger.ac.uk), as well as 393 novel human miRNAs discovered through deep sequencing and validated by qRT-PCR and array profiling (NCodeM-bM-^DM-" Human miRNA microarray probe set V3 from Invitrogen). The 10 normal gastric mucosa RNA samples were pooled to obtain the normal gastric reference sample, which was labelled and hybridised four times to check arrays' reproducibility.

ORGANISM(S): Homo sapiens

SUBMITTER: PatrM-CM--cia Pereira

PROVIDER: E-GEOD-33743 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background & Aims: E-cadherin expression disruption is commonly observed in epithelial cancers and metastasis. Such event is also recognized as a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression and function of pivotal cancer-related genes, we characterized and dissected a pathway initiated by loss of microRNA-101 that causes E-cadherin dysfunction through upregulation of EZH2 expression in GC. Methods: Microarrays were used to profile the expression of microRNAs in human GC. Array-CGH revealed DNA copy number changes that were validated by genomic quantitative PCR and Snapshot. Expression levels of microRNAs, mRNA and protein were determined by quantitative-real time PCR and western-blot/co-immunofluorescence. CDH1 inactivating mechanisms were analyzed. Gain and loss of function experiments were done in KatoIII cells. E-cadherin functionality was assessed by immunofluorescence and flow cytometry. Results: MiR-101 expression was significantly decreased in tumors in comparison with normal gastric mucosas (P<.0001). In 65% of the analyzed GC cases, miR-101 downregulation was caused by deletions and/or microdeletions at miR-101-2 locus. EZH2 overexpression and consequent loss/aberrant E-cadherin expression was found in concomitance with miR-101 downregulation in 41% of the analyzed GC cases. This occurred preferentially in cases retaining allele(s) untargeted by classical CDH1 inactivating mechanisms. MiR-101 gain of function experiments or direct inhibition of EZH2, led to a strong depletion of endogenous EZH2 protein and consequent rescue of functional E-cadherin at the cell membrane, mimicking results obtained with clinical GC samples. Conclusions: Deletions and/or microdeletions at miR-101-2 locus underlying mature miR-101 downregulation and consequent EZH2 overexpression represented a novel cascade of genetic events leading to E-cadherin disruption, preferentially affecting the intestinal-type of GC.

Project description:Cullin 4B (CUL4B), a scaffold protein that assembles CRL4B ubiquitin ligase complexes, has been reported to be overexpressed in several types of solid tumors and contributes to epigenetic silencing of tumor suppressor. However, its clinical significance and the molecular mechanisms underlying its regulation in gastric cancer (GC) remain largely unknown. Here, we showed that CUL4B was elevated in GC tissues and its overexpression was positively correlated with poor prognosis and lymph node metastasis. CUL4B knockdown in GC cells decreased proliferation, mesenchymal transition and invasion in vitro, as well as tumor growth and metastasis in nude mice, and CUL4B overexpression induced the opposite results. Further studies showed that miR-101 could inhibit CUL4B expression by directly targeting its 3’-UTR and they were inversely correlated in clinical GC specimens. Notably, a positive relationship between CUL4B and HER2 was found in GC clinical specimens, GC cells and GC xenograft tumors. Through bioinformatics analysis of miRNA-seq data and target prediction, we nominated miR-125a as a direct target of CUL4B. ChIP assays demonstrated that CUL4B directly repressed miR-125a expression by physically binding to its promoter. In addition, we confirmed miR-125a is the target of HER2. Consequently, we demonstrated that CUL4B can up-regulate HER2 expression through repressing miR-125a. Most importantly, silencing of HER2 by Herceptin or siRNA partially reversed CUL4B-induced epithelial-to-mesenchymal transition (EMT), cell invasion and metastasis in vitro and in vivo. These findings define a CUL4B-miR-125a-HER2 regulatory mechanism shed light on CUL4B oncogenic mechanisms and reveals promising therapeutic targets for progressive GC. CUL4B proteins are frequently upregulated in human cancer, yet little is known about the underlying molecular mechanisms of CUL4B induced gastric cancer(GC) carcigenesis.Here, we uncover the critical role of CUL4B in gastric cancer growth and metastasis through the regulation of HER2 expression.CUL4B contributes to GC invasion and metastasis by transcriptional repression of HER2 targeting miR-125a, which provide a new insight into how CUL4B regulates GC progression and metastasis.

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Lack of microRNA-101 causes E-cadherin expression and function deregulation through EZH2 up-regulation in gastric cancer

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