A cautionary tale of sense-antisense gene pairs: independent regulation despite inverse correlation of expression.

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.

The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer.

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding 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, lincNMR-depleted 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.

RNA motifs and combinatorial prediction of interactions, stability and localization of noncoding RNAs.

Although the number of documented noncoding RNAs (ncRNAs) is rapidly increasing, knowledge of their molecular function is lagging behind. The identification of specific RNA motifs that mediate transcript stability, interactions and localization may aid in the prediction of these features in new transcripts and may have potential implications for ncRNA function. Here, we review RNA motifs, focusing on four recent studies identifying nuclear-retention motifs, and discuss the limited specificity of short-RNA motifs and the resulting challenge for effective functional prediction. Future approaches may succeed by integrating combinatorial and cooperative effects of additional partially sequence-based properties.

Submandibular function recovery after IMRT in head and neck cancer: A prospective dose modelling study.

PURPOSE: To estimate the dose response relationship for submandibular gland (SMG) recovery using salivary scintigraphy in patients diagnosed with head and neck cancer treated with curative image guided chemoradiation. MATERIAL AND METHODS: Ninety newly diagnosed head and neck cancer patients (T1-3, N0-2c, M0) treated with intensity modulated radiotherapy on a prospective clinical trial were assessed for salivary toxicity at predefined intervals using dynamic salivary scintigraphy. The SMG function was measured using salivary excretion fraction (SEF) ratios at baseline and 6 monthly. Tolerance dose (TD) 50 for submandibular gland was estimated from dose response curves. RESULTS: The mean SEF ratio of 180 SMGs decreased at 6 months with a nadir at 12 months after treatment (SEF ratio 15%) and progressively recovered over time reaching 38% over 24 months. There was significant inverse correlation between SEF ratio and mean SMG dose at 6 months (r = -0.18, p = 0.04); 12-months (r = -0.36, p < 0.001); 18-months (r = -0.48, p < 0.001); 24-months (r = -0.42, p < 0.001); and more than 24-months (r = -0.56, p < 0.001). The estimated TD 50 values at 1 year and 2 year post treatment were 36 Gy and 44 Gy respectively with SEF ratio of ≤45% used to define severe xerostomia. For every 1 Gy reduction in mean dose below 54 Gy, there is 2-2.5% reduction in the probability of severe xerostomia. CONCLUSION: The submandibular gland function declines after radiotherapy with a nadir at 12 months and there is incomplete recovery over time with continued improvement over 24 months. The TD 50 at 1 year and 2 year was 36 Gy and 44 Gy with a 2-2.5% reduction in the probability of severe xerostomia for every 1 Gy reduction in mean dose.

Elective nodal dose of 60 Gy or 50 Gy in head and neck cancers: A matched pair analysis of outcomes and toxicity.

PURPOSE: The 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 MATERIALS: A 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. RESULTS: Of 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). CONCLUSIONS: Doses 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.

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

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.