Unknown,Transcriptomics,Genomics,Proteomics

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Human primary and secondary glioblastoma vs. non-neoplastic adult brain tissue

ABSTRACT: MicroRNAs (miRNAs) are small (21-25 nucleotide in length) non-coding RNA molecules that negatively regulate protein expression. They are linked to cancer development and maintenance. In this work, studying gene expression profiles of 340 mammalian miRNAs with DNA microarrays, we selected 10 miRNAs gene features able to distinguish primary from secondary glioblastoma type; furthermore we verified that miR-21 and miR-155 up-regulatation seems to characterize the glioblastoma tumour state since it was found up-regulated in all samples analyzed compared to adult brain noneoplastic tissue. Since miR-21 function in glioblastoma cells was addressed previously we concentrated our efforts on miR-155 function. We found that miR-155 levels were markedly elevated both in primary and secondary glioblastomas tumours, in glioblastoma cell cultures and in 4 glioblastoma cell lines (U87, A172, LN229, and LN308) compared with adult brain tissue, CHP212-neuroblastoma cell lines and DAOY-1-medulloblastoma cell line. Since one of the miR-155 target was gamma-aminobutyric acid (GABA) A receptor (GABRA1) we verified if there was a relation between miR-155 up-regulation and GABRA1 expression. We demonstrated that, in cultured glioblastoma cells, knockdown of miR-155, which lower miR-155 expression to normal level, restore the normal expression of the gamma-aminobutyric acid (GABA) A receptor (GABRA1), making glioblastoma cells responsive to GABA cell cycle inhibiting signals. Our data suggest that aberrantly over-expressed miR-155 contribute to the malignant phenotype of the glioblastoma cells, promoting their unlimited growth. Keywords: miRNA expression profile We studied the expression profiles of 340 miRNAs in 97 glioblastoma tissues, of which 66 were primary glioblastomas and 27 were secondary glioblastomas. We have 66 replicates of primary glioblastoma and 27 replicates of secondary glioblastoma, each hybridized with the respective adult non-neoplastic brain tissue as a control.

ORGANISM(S): Homo sapiens

SUBMITTER: Oscar D'Urso

PROVIDER: E-GEOD-13030 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Mantle cell lymphoma (MCL) is an aggressive B-cell non-HodgkinM-bM-^@M-^Ys lymphoma (NHL). In cancers, tumour suppressive microRNAs may be silenced by DNA hypermethylation. By microRNA profiling, miR-155-3p was significantly upregulated upon demethylation treatment of MCL cell lines with 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-azadC). Methylation-specific PCR, verified by pyrosequencing, showed complete methylation of miR-155-3p in one MCL cell line (REC-1). 5-azadC treatment of REC-1 led to demethylation and re-expression of miR-155-3p. Over-expression of miR-155-3p led to increased sub-G1 apoptotic cells and reduced cellular viability, demonstrating its tumour suppressive properties. By luciferase assay, lymphotoxin-beta (LT-M-NM-2) was validated as a miR-155-3p target. In 31 primary MCL, miR-155-3p was found hypermethylated in 6(19%) cases. To test if methylation of miR-155-3p was MCL-specific, miR-155-3p methylation was tested in an additional 191 B-cell, T-cell and NK-cell NHLs, yielding miR-155-3p methylation in 66(34.6%) including 36(27%) non-MCL B-cell, 24(53%) T-cell and 6(46%) of NK-cell lymphoma. Moreover, in 72 primary NHL samples with RNA, miR-155-3p methylation correlated with miR-155-3p downregulation (p=0.030), and LT-M-NM-2 upregulation (p=0.004). Collectively, miR-155-3p is tumour suppressive microRNA hypermethylated in MCL and other NHL subtypes. As miR-155-3p targets LT-M-NM-2, which is an upstream activator of the non-canonical NF-kB signalling, miR-155-3p methylation is potentially important in lymphomagenesis Total RNA isolated from MINO and JEKO-1 before and after 5-azadC treatment were converted into cDNA by MegaplexTM RT Primers and TaqManM-BM-. MicroRNA Reverse Transcription Kit. cDNA was pre-amplified using MegaplexTM PreAmp Primer and loaded onto 384-well format TaqmanM-BM-. human microRNA array A V2.0 & B V3.0. Real-time PCR was performed on 7900HT Real-Time PCR system and raw data were analyzed normalizing to mean of three endogenous controls (U6snRNA, RNU44 and RNU48). Relative microRNA levels were determined by M-NM-^TM-NM-^TCt using endogenous controls and untreated controls using SDS 2.4 and RQ manager 1.2. All experimental procedures and analyses were performed according to manufacturerM-bM-^@M-^Ys instruction, using reagents, system and softwares acquired from Applied Biosystems (Foster City, USA).

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Human primary and secondary glioblastoma vs. non-neoplastic adult brain tissue

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