The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-Regulated by N-Myc

<div><p>The worst subtype of neuroblastoma is caused by <i>MYCN</i> oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc-induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin immunoprecipitation and luciferase assays showed that N-Myc suppressed <i>linc00467</i> gene expression through direct binding to the <i>linc00467</i> gene promoter and reducing <i>linc00467</i> promoter activity. While N-Myc suppressed the expression of <i>RD3</i>, the protein-coding gene immediately down-stream of <i>linc00467</i> gene, through direct binding to the <i>RD3</i> gene promoter and reducing <i>RD3</i> promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings therefore demonstrate that N-Myc-mediated suppression of <i>linc00467</i> gene transcription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression.</p></div

Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell survival.

<p>(<b>A</b>) BE(2)-C and Kelly cells were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (<b>B</b>) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (<b>C</b>) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 alone, DKK1 siRNA alone, or combination of linc00467 siRNA-1 and DKK1 siRNA was expressed as a percentage change, compared with control siRNA-transfected samples. (<b>D</b>) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by staining with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. *, ** and *** indicated p<0.05, 0.01 and 0.001 respectively.</p

Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as identified by lncRNA microarray.

<p>Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as identified by lncRNA microarray.</p

linc00467 enhances neuroblastoma cell survival.

<p>(<b>A</b>) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 was expressed as a percentage change in the number of viable cells after transfection with linc00467 siRNA-1, compared with control siRNA-transfected samples. (<b>B</b>) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA-1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of time and siRNAs were expressed as percentages of the number of viable cells after transfection with control siRNA for 0 hour. (<b>C</b>) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with propodium iodide, and subjected to flow cytometry analyses of the cell cycle. The percentage of cells at sub-G1 phase was calculated. (<b>D</b>) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p<0.05, and ** p<0.01.</p

N-Myc represses <i>linc00467</i> gene expression by direct binding to the linc00467 gene promoter.

<p>(<b>A–B</b>). BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours, followed by RNA and protein extraction, real-time RT-PCR and immunoblot analyses of N-Myc mRNA, protein expression (<b>A</b>) or linc00467 RNA expression (<b>B</b>). (<b>C</b>) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of N-Myc and linc00467 RNA expression. (<b>D</b>) Schematic representation of the <i>linc00467</i> gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (<b>E</b>) ChIP-Seq data from Dr. Michael Snyder's group at Yale University for the ENCODE/SYDH project generated from K562 cells. (<b>F</b>) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the <i>linc00467</i> gene core promoter region enriched in Sp1-binding sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. (<b>G</b>) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control construct plus empty vector or <i>linc00467</i> gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. *, ** and *** indicated <i>P</i><0.05, 0.01 and 0.001 respectively.</p

Amino acid sequence comparison of TRIM16 and MID1 and modeling of TRIM16 B-boxes.

<p>The conserved residues in the zinc binding regions are in bold underlined type and are; cysteine; C, histidine; H, alanine; A aspartic acid; D. (<b>A</b>) TRIM16 and MID1 share the zinc binding consensus sequence for B-box1. (B) TRIM16 and MID1 share the Zinc binding consensus sequence for B-box2. (C/D) Modeling of B-boxes reveals zinc binding capability. Superimposition of the alpha-carbon backbone of the B-boxes from the MDM1 NMR structure (purple) and the homology model of TRIM16 (blue-grey). These two structures overlay with an average root-mean-square deviation of 0.4 Å.</p

B-boxes are required for TRIM16’s E3 ligase activity.

<p>(A) TRIM16 <i>in vivo</i> polyubiquitination assay. In HEK293 cells, HA-Ub was co-transfected with various TRIM16-GFP domain deletion expression plasmids. The protein lysate was subjected to immunoprecipitation by GFP antibody, and subjected to Western blot and probed with anti-HA antibody for Ub (right panel) and anti-GFP antibody for TRIM16 (left and middle panel). Two exposure times are shown. GFP antibodies detect both un-ubiquitinated and polyubiquitinated forms of TRIM16. Polyubiquitinated smear is present in the sample transfected with wild-type TRIM16 and shown by anti-GFP and anti-HA antibodies. (B) <i>In vitro</i> ubiquitination assay with myc-His tagged TRIM16 together with a panel of E2 enzymes, showing activity with the UbcH5 family. (C) <i>In vitro</i> ubiquitination assay with full-length TRIM16, TRIM16 domain deletion mutants or empty vector showing extensive polyubiquitination with full-length TRIM16 as detected by Western blot with anti-myc antibodies. Numbers indicate protein size in kDa. (D) Recombinant TRIM16 (Abnova) was evaluated for E3 activity in the presence of recombinant E1, UbcH5b, and HA-Ub as indicated. The capacity to catalyse auto-ubiquitination <i>in vitro</i> was observed only in the presence of ZnCl₂ and ATP. Western Blot (lower panel) with TRIM16 antibody showed amount of TRIM16 protein in each lane.</p

TRIM16 can heterodimerize with MID1, TRIM24 and PML.

<p>(A) Schematic structures of TRIM proteins used in heterodimerization studies. (B) TRIM16 binds MID1. Co-transfection of MID1-GFP and TRIM16-myc-His in HEK293 cells and subsequent immunoprecipitation by anti-myc antibody and Western blot with anti-GFP antibody. (C) MID1 was pulled down via its GFP antibody and a Western blot was performed to detect TRIM16-myc-His in the immunoprecipitated protein complex. (D) Whole cell lysates (WCL) of HEK293 cells transfected with empty vector (EV) or TRIM16-GFP were immunopreciptated with anti-GFP antibody. An anti-PML antibody was used to detect PML as a binding partner of TRIM16. (E) Lysates containing both TRIM16-GFP and TRIM24-His proteins were immunopreciptated with anti-GFP antibody. Anti-His antibody was used to detect the presence of TRIM24 in the TRIM16-associated complex.</p

N-Myc represses <i>RD3</i> gene transcription by direct binding to the <i>RD3</i> gene promoter.

<p>(<b>A</b>) BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT-PCR analyses of RD3 mRNA expression. (<b>B</b>) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of RD3 RNA expression. (<b>C</b>) Schematic representation of the <i>RD3</i> gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (<b>D</b>) ChIP-Seq data from Dr. Michael Snyder's group at Yale University for the ENCODE/SYDH project generated from K562 cells. (<b>E</b>) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the <i>RD3</i> gene core promoter region enriched in Sp1-binding sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. (<b>F</b>) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control construct plus empty vector or <i>RD3</i> gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. * indicated <i>P</i><0.05.</p

linc00467 reduces mRNA expression of its neighbouring protein-coding RD3.

<p>BE(2)-C and Kelly cells were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and and real-time RT-PCR analysis of the expression of linc00467 (<b>A</b>) or RD3 (<b>B</b>). Error bars represented standard error. * indicated <i>P</i><0. 05, and *** indicated <i>P</i><0.001.</p