Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Effects of the insulin degrading enzyme silencing on the transcriptome of HepG2 cells

ABSTRACT: Insulin degrading enzyme (IDE) is a major enzyme responsible for insulin degradation in the liver. The modulation of insulin degrading enzyme activity is hypothesized to be a link between T2DM and liver cancer. Results provide insight into role of IDE in proliferation and other cell functions. HepG2 cells were transfected with 96nM siRNA for IDE or AllStars Negative Control siRNA (Qiagen) using Lipofectamine 2000 (Invitrogen). 16 h after transfection, cells were treated with 10 nM insulin (Sigma Aldrich) or vehicle for 24 h in serum starvation condition. Total RNA was extracted. For each of the 4 conditions, 3 biological replicates were included.

ORGANISM(S): Homo sapiens

SUBMITTER: Olga Pivovarova

PROVIDER: E-GEOD-61142 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Malignant glioma is the most common type of primary brain tumor diagnosed annually in 16,000 individuals in the United States. We performed a systematic large-scale transcriptomics data mining study of 9,783 Affymetrix samples from the GeneSapiens database in order to identify those genes that are most glioma-specific as compared to other cancers and normal tissues. We searched for genes that are highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared to 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (Hairy and Enhancer of Split 6) emerged as one of the most glioma-specific genes. In immunostaining of a tissue microarray series, HES6 was expressed in 335 (98.8%) out of the 339 clinical glioma samples. Recurrent grade 2 astrocytomas and grade 2-3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. Functional studies implied a critical role for HES6 in supporting survival of glioma cells, as evidenced by 60% reduced cancer cell viability and induction of Caspase 3/7 activity after HES6 silencing by RNA interference in A172 and LN405 cells. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which indicated a key role for HES6 in e.g. p53, c-myc, and NF-?B transcriptional networks. We conclude that HES6 has a critical role in sustaining glioma cell growth, survival, migration and possibly angiogenesis. HES6 is a potential therapeutic target and biomarker for glioma. A172 and LN405 cells (240 000 per well on 6-well plates) were transfected for 12-24 h with siHES6 pool, or three individual siRNAs (HES6_1, HES6_2, HES6_3 from Qiagen) or AllStars negative control siRNA (Qiagen) at 30 nM using SiLentFect (Bio-Rad Laboratories, Hercules, CA). One to two biological replicate transfections were performed. LN405 cells were also used for creating stable cell lines by transfecting either pEYFP-C1-mock or pEYFP-HES6 with Fugene6 (Roche) and selecting the positive cells with 600ug/ml of G418 (Sigma). The cells were kept in culture in the presence of 400ug/ml of G418. Total RNA was isolated with RNeasy (Qiagen) or MiRVana? Total RNA Isolation kit (Ambion). RNA quality was evaluated by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).

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Effects of the insulin degrading enzyme silencing on the transcriptome of HepG2 cells

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