Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long noncoding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMRdepleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.

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.

Long non-coding RNAs (lncRNAs) have been proven to play important roles in diverse cellular processes including the DNA damage response. Nearly 40% of annotated lncRNAs are transcribed in antisense direction to other genes and have often been implicated in their regulation via transcript- or transcription-dependent mechanisms. However, it remains unclear whether inverse correlation of gene expression would generally point toward a regulatory interaction between the genes. Here, we profiled lncRNA and mRNA expression in lung and liver cancer cells after exposure to DNA damage. Our analysis revealed two pairs of mRNA-lncRNA sense-antisense transcripts being inversely expressed upon DNA damage. The lncRNA NOP14-AS1 was strongly upregulated upon DNA damage, while the mRNA for NOP14 was downregulated, both in a p53-dependent manner. For another pair, the lncRNA LIPE-AS1 was downregulated, while its antisense mRNA CEACAM1 was upregulated. To test whether as expected the antisense genes would regulate each other resulting in this highly significant inverse correlation, we employed antisense oligonucleotides and RNAi to study transcript-dependent effects as well as dCas9-based transcriptional modulation by CRISPRi/CRISPRa for transcription-dependent effects. Surprisingly, despite the strong stimulus-dependent inverse correlation, our data indicate that neither transcript- nor transcription-dependent mechanisms explain the inverse regulation of NOP14-AS1:NOP14 or LIPE-AS1:CEACAM1 expression. Hence, sense-antisense pairs whose expression is strongly—positively or negatively—correlated can be nonetheless regulated independently. This highlights the requirement of individual experimental studies for each antisense pair and prohibits drawing conclusions on regulatory mechanisms from expression correlations.

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PurposeThe main objective of this study was to evaluate appropriate doses for elective nodal irradiation (ENI) in head and neck squamous cell carcinoma (HNSCC) patients to optimize the therapeutic ratio.Methods and materialsA matched pair analysis of 2 similar cohorts of HNSCC treated with intensity modulated radiation therapy with different dose prescriptions to the elective nodal regions was conducted. One group received 60 Gy, whereas the other received 50 Gy (ENI60 and ENI50 groups, respectively). Isolated regional recurrences (IRR) and locoregional control were evaluated. Doses received by the parotid and thyroid glands were compared among both groups and were clinically correlated with the trend of salivary function recovery and incidence of hypothyroidism.ResultsOf the 110 patients studied, 97 were eligible for analysis after matching based on propensity scores. The 3-year locoregional control rate was similar in ENI60 and ENI50 (78.7% and 77%, respectively; P = .93). There were no IRR in ENI regions in either group. The mean ipsilateral parotid dose in ENI60 was significantly higher compared with ENI50 (42 vs 35.7 Gy, P = .03). There was no significant difference in the mean contralateral parotid doses (32.5 vs 31.7 Gy, P = .6). The mean thyroid doses were high in ENI60 compared with ENI50 (54.7 vs 43.3 Gy, P < .001). A significant difference in ipsilateral parotid salivary excretory fraction ratio at 1 year (P = .03) was observed with quicker recovery of salivary function. The salivary excretory fractions were poorer in the ENI60 group with higher mean parotid doses (P = .009). At 2 years, 26 patients (54%) in the ENI60 group and 13 patients (26.5%) in the ENI50 group developed biochemical hypothyroidism (P = .007).ConclusionsDoses of 50 Gy equivalent are sufficient to sterilize the uninvolved nodal regions because the rates of IRR are extremely low. Using ENI50 results in clinically meaningful reduction in salivary and thyroid toxicity in HNSCC.

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