Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling of human HEK cells transfected with the calcium regulated gene HcARG reveals HCaRG increases renal cell migration by a TGF-alpha autocrine loop mechanism

ABSTRACT: HEK293 cells were transfected with control plasmid (pcDNA1/Neo; Invitrogen) or with the plasmid encoding HCaRG by a standard calcium phosphate co-precipitation method. The clones expressing the highest levels of HCaRG, HCaRG clone 8 and 9 were used in this experiment, while clone transfected with vector alone, Neo clone, served as controls. Stable transfectants were synchronized and grown in the presence of 10% FBS for 48 h. Total RNAs were purified with the mini RNeasy kit (Qiagen).

ORGANISM(S): Homo sapiens

SUBMITTER: Johanne Tremblay

PROVIDER: E-GEOD-4737 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

HCaRG increases renal cell migration by a TGF-alpha autocrine loop mechanism.

El Hader Carlos C Tremblay Sandra S Solban Nicolas N Gingras Denis D Béliveau Richard R Orlov Sergei N SN Hamet Pavel P Tremblay Johanne J

American journal of physiology. Renal physiology 20050720 6

We have shown previously that the hypertension-related, calcium-regulated gene (HCaRG) is involved in the control of renal cell proliferation and differentiation (Devlin AM, Solban N, Tremblay S, Gutkowska J, Schurch W, Orlov SN, Lewanczuk R, Hamet P, and Tremblay J. Am J Physiol Renal Physiol 284: F753-F762, 2003). To determine whether HCaRG plays a role in kidney repair after injury, we extended our studies on the cellular function of HCaRG by comparing cell migration of two kidney cell lines ...[more]

PMID: 16033922

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Project description:Many classes of noncoding RNAs (ncRNAs) are known to be involved in gene expression regulation; however, long intronic ncRNAs have received little attention. In previous works, our group has provided evidence that intronic regions, transcribed from 80% of all RefSeq gene loci, are key sources of potentially regulatory ncRNAs. Intronic transcripts were shown to be correlated to the degree of prostate cancer differentiation, regulated by physiological stimuli, and highly expressed in genomic loci related to transcriptional regulation. In the present work we aimed at functional characterization of KLHL29 intronic transcript, one of the most highly expressed in human tissues. Although the protein-coding KLHL29 transcripts are still poorly studied, genes from this family are involved in a wide variety of biological processes, including cell cycle regulation mechanisms. We have observed that the long intronic ncRNA is predominantly transcribed from the opposite strand at the KLHL29 locus, and that the ncRNA overexpression does not affect the levels of protein-coding mRNAs from the same locus. Intriguingly, we found 2,002 genes with significant (q< 5.3%) differential expression between HEK293 cells overexpressing KLHL29 sense (S) or antisense (AS) intronic transcript. Within these genes, GO categories such as ‘Regulation of cell cycle’ and ‘Transcription’ were significantly enriched. Furthermore, human tumorigenic cell lines overexpressing KLHL29 AS intronic transcript showed increased proliferation rates in vitro and increased tumorigenic capacity in nude mice. In conclusion, we suggest that KLHL29 long intronic ncRNA controls the cell cycle mechanisms, probably through a fine-tuning regulation of gene expression. Genes were defined as expressed when their intensity signals were above the average negative control value plus three standard deviations; a set of 153 negative controls (Agilent Technologies) was present on the array. Only genes that were detected as expressed in all four replicates of the same HEK293 transfection (or not expressed in the four replicates) were used in further analysis. The intensity values between experiments were normalized by the 40% trimmed mean intensity. For each transfection, one entire replicate experiment was excluded from the analyses, based on evaluating the coefficient of variance among the samples (for S KLHL29 intronic transcript, exclusion of the outlier replicate decreased the coefficient of variance from 0.21-0.24 (obtained by excluding any other replicate) to 0.19; for AS transcript, the coefficient of variance decreased from 0.17-0.18 to 0.12). Differentially expressed genes were identified using three transfection replicates for each construction, employing the Significance Analysis of Microarrays (SAM) tool (Tusher et al., 2001) with the following parameters: two-class response, 100 permutations and K-Nearest Neighbors Imputer. We applied 5.4 or 10.3% FDR (False Discovery Rate) cutoffs, as described in the results. A fold-change filter (1.4-fold cutoff) was applied after identification of significant differentially expressed genes.

Dataset Information

Transcription profiling of human HEK cells transfected with the calcium regulated gene HcARG reveals HCaRG increases renal cell migration by a TGF-alpha autocrine loop mechanism

Publications

HCaRG increases renal cell migration by a TGF-alpha autocrine loop mechanism.

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