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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and CANT1 Overexpression Retinoblastoma Transcriptomes

ABSTRACT: PURPOSE: Retinoblastoma (RB) is the most common malignant intraocular tumor of childhood. Recent studies have shown that long non-coding RNAs (lncRNAs), which are longer than 200 bp and without protein-coding ability, are key regulators of tumorigenesis. However, the role of lncRNAs in retinoblastoma remains to be elucidated. METHODS: LncRNA CASC15-New-Transcript 1 (CANT1) expression was determined by real-time PCR in RB cells. Tumor characteristics in CANT1-overexpressing RB cells were determined through CCK8 cell viability assay, soft agar assay and mouse xenograft experiment. RNA transcriptome-sequencing was performed to find downstream target genes of CANT1. CANT1's interactions with phosphoinositide 3-kinase gamma (PI3Kγ) were studied through chromatin oligonucleotide precipitation (ChOP) and chromatin immunoprecipitation (ChIP) assays. RESULTS: In this study, we found that the expression of lncRNA CANT1 was significantly downregulated in RB. Notably, CANT1 overexpression significantly inhibited RB growth both in vitro and in vivo. Furthermore, lncRNA CANT1, which was mainly located in the nucleus, occupied the promoter of PI3Kγ and blocked histone methyltransferase hSET1 from binding to the PI3Kγ promoter, thus abolishing hSET1-mediated histone H3K4 trimethylation of the PI3Kγ promoter and inhibiting PI3Kγ expression. Furthermore, we found that silencing PI3Kγ either by CANT1 overexpression or by PI3Kγ siRNA, reduced the activity of PI3K/Akt signaling and suppressed RB tumorigenesis. CONCLUSIONS: LncRNA CANT1 acts as a suppressor of RB progression by blocking gene-specific histone methyltransferase recruitment. These findings outline a new CANT1 modulation mechanism and provide an alternative option for RB treatment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141327 | GEO | 2020/02/05

REPOSITORIES: GEO

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Project description:Once thought to be transcriptional noise, large non-coding RNAs (lncRNAs) have recently been demonstrated to be functional molecules. Cell type-specific expression patterns of lncRNAs suggest that their transcription may be regulated epigenetically. Using a custom-designed microarray, here we examine the expression profile of lncRNAs in embryonic stem (ES) cells, lineage-restricted neuronal progenitor cells (NPC), and terminally differentiated fibroblasts. In addition, we also analyze the relationship between their expression and their promoter H3K4 and H3K27 methylation patterns. We find that numerous lncRNAs in these cell types undergo changes in the levels of expression and promoter H3K4me3 and H3K27me3. Interestingly, lncRNAs that are expressed at lower levels in ES cells exhibit higher levels of H3K27me3 at their promoters. Consistent with this result, knockdown of the H3K27me3 methyltransferase Ezh2 results in derepression of these lncRNAs in ES cells. Thus, our results establish a role for Ezh2-mediated H3K27 methylation in lncRNA silencing in ES cells and reveal that lncRNAs are subject to epigenetic regulation in a similar manner to that of protein-coding genes. lncRNA expression analysis was performed in 5 different cell types: (1) ES cells, (2) neuroprogenitors, (3) tail tip fibroblasts, (4) Ezh2 knockdown #1 ES cells, and (5) Ezh2 knockdown #2 ES cells. Total RNA was extracted and RNA quality was assessed via Agilent Bioanlayzer. mRNA was amplified using the Low RNA Linear Amplification Kit (Agilent Technologies) by standard manufacturer's protocol. Hybridization of experimental RNA labeled with Cy5 and Cy3-labeled Universal Reference RNA (Ambion) was carried out by standard protocol on a custom-designed array manufactured by Agilent Technologies. Briefly, three unique 60nt probes were designed for each entry in the Fantom3 database and were synthesized along with lineage marker control probes on a 2x105K format array. Upon array scanning, data was quality filtered, normalized by the Lowess method, and all probes corresponding to a given lncRNA were averaged using the UNC Microarray Database (UNCMD). Intraclass correlation analysis was carried out in the UNCMD.

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Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and CANT1 Overexpression Retinoblastoma Transcriptomes

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