The lncRNA VELUCT strongly regulates viability of lung cancer cells despite its extremely low abundance

Abstract

Little is known about the function of most non-coding RNAs (ncRNAs). The majority of long ncRNAs (lncRNAs) is expressed at very low levels and it is a matter of intense debate whether these can be of functional relevance. Here, we identified lncRNAs regulating the viability of lung cancer cells in a high-throughput RNA interference screen. Based on our previous expression profiling, we designed an siRNA library targeting 638 lncRNAs upregulated in human cancer. In a functional siRNA screen analyzing the viability of lung cancer cells, the most prominent hit was a novel lncRNA which we called Viability Enhancing LUng Cancer Transcript (VELUCT). In silico analyses confirmed the non-coding properties of the transcript. Surprisingly, VELUCT was below the detection limit in total RNA from NCI-H460 cells by RT-qPCR as well as RNA-Seq, but was robustly detected in the chromatin-associated RNA fraction. It is an extremely low abundant lncRNA with an RNA copy number of less than one copy per cell. Blocking transcription with actinomycin D revealed that VELUCT RNA was highly unstable which may partially explain its low steady-state concentration. Despite its extremely low abundance, loss-of-function of VELUCT with three independent experimental approaches in three different lung cancer cell lines led to a significant reduction of cell viability: Next to four individual siRNAs, also two complex siPOOLs as well as two antisense oligonucleotides confirmed the strong and specific phenotype. In summary, the extremely low abundant lncRNA VELUCT is essential for regulation of cell viability in several lung cancer cell lines. Hence, VELUCT is the first example for a lncRNA that is expressed at a very low level, but has a strong loss-of-function phenotype. Thus, our study proves that at least individual low-abundant lncRNAs can play an important functional role.

Figure 1.

Quality assessment of siRNA screens addressing viability of H460 cells. H460 cells were screened for cell viability 72 h after transfection (n = 2). Raw values were normalized to the plate median and Z scores were calculated for each reaction. Replicates were summarized by averaging the Z scores. (A) Boxplots with summarized Z scores of the negative controls (blue), the respective positive controls (red) and siRNAs targeting lncRNAs (gray). (B) Z scores of the two replicates were plotted against each other. R indicates the Pearson correlation coefficient. (C) Image plot of summarized Z scores that were averaged over each well position of the nine screening plates. Red indicates the maximum value, blue the minimum value. Positive and negative controls were excluded for this analysis and were not depicted on the plot.

Figure 2.

Identification of the novel oncogenic lncRNA VELUCT regulating the cell viability. (A) Z scores for siRNAs targeting VELUCT in both screening replicates of H460 cells. (B) Relative cell viability (normalized to plate median) for siRNAs targeting VELUCT in both screening replicates of H460 cells. (C) Genomic location of the annotated and the VELUCT isoforms that were detected by 3΄ RACE using chromatin-associated H460 RNA, PhyloCSF scores for all three frames on the respective strand, conservation by PhyloP and repeating elements by RepeatMasker are shown on the UCSC genome browser version hg19.

Figure 3.

The instable, low abundant VELUCT was only detectable in chromatin-associated lncRNA. (A) RNA deep sequencing was performed with whole cell RNA and chromatin-associated RNA of H460 cells. The read coverage of both runs is shown on a scale from 0 to 120 and from 0 to 5, respectively. The human genome version hg19 was used for alignment of reads. (B) Expression levels in cytoplasmic, nucleoplasmic and chromatin-associated fractions of H460 cells were analyzed by RT-qPCR and normalized to the expression in whole cell RNA. The VELUCT_m amplicon was used for detection of VELUCT. Bars show mean ± SD (n = 3). (C) Determination of VELUCT copy number in chromatin-associated H460 RNA with three qPCR amplicons (location at 5΄ end, middle or 3΄ end) (n = 3). (D) H460 cells were treated with actD or DMSO (-actD) for 1 h and subsequently subcellularly fractionated. Chromatin-associated RNA levels of indicated genes were normalized to RPLP0 and relative to DMSO-treated cells. Bars show mean ± SD (n = 3). Asterisks indicate significant expression difference between treated and untreated cells. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

VELUCT knockdown was not detectable with siRNAs and siPOOLs, but with ASOs. H460 cells were transfected with 30 nM VELUCT-specific siRNAs (A) or siPOOLs (B), 10 nM HDAC4-specific siRNAs or siPOOLs (C) or 30 nM VELUCT-specific ASOs (D) for 24 h. VELUCT RNA levels were analyzed in the chromatin fraction using three independent qPCR amplicons. HDAC4 levels were determined in all three subcellular fractions. All RNA levels were normalized to cyclophilin A and relative to the respective NC. Bars show mean ± SD (n = 3–4). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

Multiple independent VELUCT-specific silencing reagents reduced viability and proliferation of H460 cells. H460 cells were transfected with 30 nM VELUCT-specific siRNAs (A, D), siPOOLs (B, E) or ASOs (C, F). Cell viability (A–C) and proliferation (D–F) was measured 72 h after transfection and normalized to the respective NC. Bars show mean ± SD (n = 3–4). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Multiple independent VELUCT-specific silencing reagents reduced viability of other lung cancer cell lines. H1437 and H1944 cells were transfected with 30 nM siRNAs (A), siPOOLs (B) or ASOs (C) targeting VELUCT. Cell viability was measured 72 h after transfection and normalized to the respective NC. Bars show mean ± SD (n = 4–5). *P < 0.05, **P < 0.01, ***P < 0.001.