The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells

Abstract

In recent years, long non-coding RNA (lncRNA) research has identified essential roles of these transcripts in virtually all physiological cellular processes including tumorigenesis, but their functions and molecular mechanisms are poorly understood. In this study, we performed a high-throughput siRNA screen targeting 638 lncRNAs deregulated in cancer entities to analyse their impact on cell division by using time-lapse microscopy. We identified 26 lncRNAs affecting cell morphology and cell cycle including LINC00152. This transcript was ubiquitously expressed in many human cell lines and its RNA levels were significantly upregulated in lung, liver and breast cancer tissues. A comprehensive sequence analysis of LINC00152 revealed a highly similar paralog annotated as MIR4435-2HG and several splice variants of both transcripts. The shortest and most abundant isoform preferentially localized to the cytoplasm. Cells depleted of LINC00152 arrested in prometaphase of mitosis and showed reduced cell viability. In RNA affinity purification (RAP) studies, LINC00152 interacted with a network of proteins that were associated with M phase of the cell cycle. In summary, we provide new insights into the properties and biological function of LINC00152 suggesting that this transcript is crucial for cell cycle progression through mitosis and thus, could act as a non-coding oncogene.

Figure 1

High-throughput RNA interference screen for cell cycle regulators. (a) Schematic overview of the siRNA screening strategy. (b) Bioinformatical analysis was performed by calculating the z-scores for MitosisCount. Black lines represent the median of z-scores. Dashed lines indicate the cut-off for potential phenotypes. (c) Manual analysis was accomplished by assigning a manual score representing the strength of any kind of aberrant phenotype. 1 = normal; 5 = strong phenotype. (d) Venn diagram of hits from the primary screen and secondary validation. (e) Image sections of exemplary phenotypes discovered by time-lapse microscopy (red: histone H2B, green: tubulin). The siRNA IDs are stated above the images, phenotypes are described below the images. Images were acquired at 10x magnification. Scale bar represents 100 μm.

Figure 2

LINC00152 transcripts and their subcellular localization. (a) The exonic regions of LINC00152 and MIR4435-2HG differed only by 13 base pairs (upper panel, mismatch numbers were counted for each exon separately). Exons were numbered according to their genomic order and transcripts were named according to their comprised exons. Both paralogs were transcribed into several isoforms (lower panel) and had splice variants ex15 and ex145 in common (right panel). (b) Relative expression of all identified LINC00152 splice variants determined by RT-qPCR normalized to Cyclophilin A expression. Note that the graph is depicted in Log10 scale. (c) Subcellular localization of LINC00152 was determined using cell fractionation. After separation of cytoplasm, nucleoplasm and chromatin, RNA was extracted from all fractions and LINC00152 expression was measured by RT-qPCR. tRNA-Lys, RNU1 and NEAT1 were used as cytoplasmic, nucleoplasmic and chromatin marker, respectively. N = 3. Error bars indicate SEM.

Figure 3

LINC00152 expression in different human tumour tissues. (a) Microarray profiling of the LINC00152 expression in 150 patient samples (7 normal liver vs. 32 liver cancer, 27 normal lung vs. 27 matched lung cancer, 13 normal breast vs. 44 breast cancer). (b) Relative LINC00152 expression profiles in liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) obtained from The Atlas of non-coding RNA in Cancer (TANRIC). Boxes show the median, 25^th and 75^th percentiles, and whiskers range from minimum to maximum. FC = fold change. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Live-cell microscopy revealed a cell division phenotype with high temporal resolution. (a) Relative expression of LINC00152 detected by RT-qPCR and normalized to Cyclophilin A expression in untreated HeLa cells and cells transfected with 30 nM siPOOLs. (b) Endpoint images (upper panel) and magnified time-lapse microscopy pictures (lower panel) of HeLa cells treated with 30 nM siPOOLs. Images were acquired at 20x magnification and scale bar represents 200 μm. Live-cell imaging revealed a mitotic arrest in prometaphase and reduced cell viability after LINC00152 depletion. Time depicts hours after transfection. (c) Quantification of mitotic (significance *) and apoptotic (significance ^#) cells at three different time points after RNAi knockdown. (d) Validation of mitotic arrest by quantification of P-Lamin A/C (Ser22) levels detected by Western blot and normalized to β-Actin levels in HeLa cells. (e) Relative cell viability detected by CellTiter-Glo Luminescent Cell Viability Assay in different cell lines treated with siPOOLs. UT = untreated; tfx = transfection reagent only. N = 3. Error bars indicate SEM. *^/#p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant.

Figure 5

Analysis of cell fates after LINC00152 depletion. (a) Images from movies of HeLa cells after LINC00152 knockdown by 30 nM siPOOL showed prometaphase arrest followed by mitotic death, mitotic exit or cytokinesis failure. Some cells also underwent apoptosis at an early stage without prior mitotic arrest. Time depicts hours after transfection. Images were acquired at 20x magnification and scale bar represents 20 μm. (b) Left circle portrays the effect on cells 72 hours after transfection with siLINC00152 (compare with Fig. 4b). Right circle represents a quantification of the cell fates depicted in (A) occurring in phenotypic cells. N = 3.

Figure 6

Proteins pulled down with LINC00152 were significantly associated with M phase. (a) Enrichment analysis identified gene ontology (GO) terms based on biological processes that were significantly over-represented within proteins detected in LINC00152 pulldowns. Mitosis and cell cycle-related GO terms are highlighted in orange. (b) String database revealed interaction networks between the proteins associated with M phase by GO analysis and their functional enrichment for microtubule cytoskeleton organization (green nodes) and ubiquitin-protein ligase activity (blue nodes) with cyclin B (CCNB1) being involved in both (cyan). Line thickness indicates the strength of protein interaction by data support.