Gastric Hyperplasia in MN/ Carbonic Anhydrase IX Deficient Mice

Title Contents 1\. Introduction 1 1.1. Carbonic Anhydrases 1 1.2. Carbonic Anhydrase IX (MN) 5 1.3. Development of the Mouse Stomach and Architecture of the Gastric Mucosa 13 2\. Aim of the Work 22 3\. Materials 23 4\. Methods 31 4.1. Preparation and Analysis of DNA 31 4.2. Enzymatic Manipulations of Nucleic Acids 38 4.3. Preparation and Analysis of RNA 43 4.4. Analysis of Proteins 49 4.5. Cell Culture Methods, ES Cells Handling and Use 52 4.6. Analysis if Phenotypic Changes in the Mice 60 5\. Results 62 5.1. Isolation and Characterization of Murine MN/CA9 cDNA 62 5.2. Isolation and Characterization of MN/CA9 Gene 70 5.3. Construction of MN/CA9 Deficient Mouse 72 5.4. Analysis of MN/CA9 Deficient Mice 81 5.5. Conclusions 89 6\. Discussion 90 7\. Summary 100 7.1. Summary 100 7.2. Zusammenfassung 102 8\. References 104 9\. Annexe 116Human MN/CA IX protein localized on the surface of epithelial cells is a member of the carbonic anhydrase family isozymes. Its multidomain composition implies rather broad functionality. Under normal conditions MN/CA IX is expressed in several tissues of the alimentary tract, notably in the gastric mucosa. Furthermore, its aberrant expression has been connected to carcinogenesis. Recently MN/CA IX protein was linked to the regulation of cell proliferation and identified as a molecule involved in the cell adhesion. Despite the increasing knowledge about MN/CA IX protein, its physiological relevance in the gastrointestinal tract and its role in oncogenesis has not been understood. In order to assess the function of the MN/CA IX protein in vivo, mice lacking the MN/CA IX protein were generated by gene targeting. In order to fulfil this approach, cDNA and genomic DNA, coding for the murine MN/CA IX protein, had to be identified, isolated and characterized. The murine counterpart of human MN/CA IX was identified on the basis of its sequence homology and expression pattern. The full length of the murine MN/CA9 cDNA is 1982bp. The open reading frame of 1311bp has a coding capacity for a 437 amino acid protein with a theoretical molecular mass of 47,3kDa. The alignment of the predicted amino acid sequence of the murine and human MN/CA9 cDNAs showed only 69,5% sequence identity. However, all the extracellular and intracellular domains were conserved in both. Expression of the murine MN/CA9 mRNA is highest in mouse stomach, then in the proximal intestine and distal colon. The MN/CA9 cDNA was also detected in several tumor cell lines (TS/A; GR/3; and MM5) and one tumor sample. This observation hints at a possible implication of the MN/CA IX protein in oncogenesis also in mouse. For further investigation the murine MN/CA IX protein was isolated, purified, and the identity validated by MALDI-MS. Since the used purification method is applicable only for enzymatically active carbonic anhydrases, this result at the same time sustains the conservation of at least some carbonic anhydrase activity of the murine MN/CA IX protein. The apparent molecular mass of the murine MN/CA IX was estimated at 54kDa. This result was confirmed by Western blot analysis with prepared polyclonal rabbit antiserum which was raised against an oligopeptide from the proteoglycan-like domain. The murine MN/CA9 gene was isolated from a mouse genomic library. The gene is divided into 11 exons and 10 introns, spanning 6,7kb of the mouse genomic DNA. A comparison of exon/intron boundaries of the mouse and human genes showed identical gene structure suggesting their high evolutionary conservation. A replacement type vector with a targeted interruption of the first exon of the murine MN/CA9 gene was introduced into the mouse embryonic stem cells by homologous recombination. The mice, homozygous in the MN/CA9 gene mutation, were normal in terms of size, activity, behavior, ability to reproduce and life span. The most remarkable changes were found in the gastric mucosa of MN/CA9-/- mice. Severe hyperplasia in adult mutant mice concerned all major glandular and superficial epithelial cells. In addition to hyperplastic changes, large pathological cysts were observed. The first mild changes in the thickness of the gastric epithelium were detected already in newborn MN/CA9-/- mice. Immunohistochemical staining for PCNA indicated a massive enlargement and disorganization of the proliferative zone of the stomach epithelium. A similarly disorganized pattern was observed by E-cadherin immunostaining of the gastric mucosa of mutant mice. The ratio of the number of proliferative cells stained by PCNA and total number of cells in the proliferative area was approximately the same as in the MN/CA9+/+ mice. Since a defect in the cellular death was excluded, the alterations in the gastric epithelium are most likely caused by deregulated cell proliferation. Whether E-cadherin mediated cell adhesion is affected by the absence of MN/CA IX remains still to be determined. Although the precise physiological role of the MN/CA IX protein in the gastrointestinal tract is still not clear, significant insight has been gained. We propose that the null mutation of MN/CA IX protein perturbs the process of proper mediation and/or processing of the extracellular signaling that normally defines the level of the proliferation and maintenance of the architecture of gastric epithelial cells. Among the carbonic anhydrase isozymes MN/CA9-/- mice are the first animal model for carbonic anhydrase deficiency constructed by gene targeting. Moreover, MN/CA IX is the first targeted adhesion-related molecule expressed in the gastrointestinal epithelium that revealed involvement in morphogenesis of the mucosa. The MN/CA9-/- deficient mouse provide an excellent system to elucidate the role of this molecule in the morphogenesis of the gastric epithelium and its involvement in carcinogenesis.Das humane MN/CA IX Protein, welches sich auf der Oberfläche von Epithelzellen befindet, ist ein Mitglied der Familie der Carboanhydraseisozymen. Seine multidomäne Zusammensetzung deutet auf eine vielseitige Funktion. Unter normalen Bedingungen wird MN/CA IX in verschiedenen Geweben exprimiert, hauptsächlich in den Schleimhäuten des Gasterointestaltrakts. Eine gestörte Expression ist mit Krebsentwicklung in Zusammenhang gebracht worden. MN/CA IX Protein beeinflusst auch die Regulation der Zellproliferation und der Zelladhäsion. Obwohl die Erkenntnis über MN/CA9 zunimmt, bleibt seine physiologische Relevanz im Gastrointestaltrakt und seine Rolle bei der Krebsentwicklung unverstanden. Um Verständnis für die Funktion des MN/CA IX Proteins in vivo zu erlangen, wurden Mäuse mit einer Nullmutation im MN/CA9 Gen durch gezielte Mutagenese hergestellt. Vorbereitend wurden die cDNA und die genomische DNA Sequenzen der Maushomologen isoliert und charaktersisiert. Das maushomologe MN/CA9 Gen wurde basierend auf DNA Sequenzhomologie und anhand von Expressionsmustern identifiziert. Die murine cDNA von MN/CA9 ist 1982 Basenpaare lang. Das offene Leseraster besteht aus 1311 Basenpaaren kodiert ein Protein mit 437 Aminosäuren und einer theoretischen Masse von 47,3kDa. Die Sequenzhomologie der humanen und murinen cDNA Sequenz von MN/CA9 ist nur 69,5%. Alle intra- und extrazellulären Domänen sind jedoch konserviert. Die Expression der murinen MN/CA9 mRNA ist im Magen am höchsten, gefolgt vom proximalen und distalen Darm. MN/CA9 cDNA konnte auch in einer Reihe von Tumorzellinien (TS/A, GR/3 und MM5) und in einer Tumorprobe nachgewiesen werden. Dies deutet auf eine mögliche Rolle von MN/CA IX Protein in der Krebsentstehung hin. Zur weiteren Untersuchung wurde das murine MN/CA IX Protein isoliert, aufgereinigt und seine Identität mit MALDI-MS bestätigt. Die verwendete Aufreinigungsmethode ermöglicht lediglich die Isolation von enzymatisch aktiven Carboanhydrasen. Dies bezeugt eine mindestens teilweise Carboanhydrase-Aktivität der murinen MN/CA IX. Die Masse des isolierten Proteins wurde mit 54kDa abgeschätzt. Dieses Resultat wurde mit einer Westernblot Analyse mit polyklonalem Kaninchenantiserum, welches gegen Oligopeptide aus der proteoglykan-ähnlichen Domäne des murinen MN/CA IX Protein hergestellt wurde, bestätigt. Das murine MN/CA9 Gen wurde aus einer murinen genomischen Bibliothek isoliert. Es reicht über 6,7kBasen und ist aufgeteilt in 11 Exons und 10 Introns. Der Vergleich der Exon/Intron Abgrenzung des humanen und murinen Gens zeigt die identische Genstruktur, was auf eine hohe evolutionäre Konservierung hindeutet. Mittels homologer Rekombination wurde ein Ersatzvektor mit einer Unterbrechung im ersten Exon des murinen MN/CA9 in embryonale Stammzellen eingeführt. MN/CA9-/- Mäuse waren normal in Bezug auf Körpergrösse, Aktivität, Verhalten, Fortpflanzungsfähigkeit und Lebensspanne. Die bemerkenswertesten Unterschiede wurden in der Magenschleimhaut gefunden. Erwachsene MN/CA9-/- Tiere zeigten eine ausgeprägte Hyperplasie in Drüsen- und oberflächlichen Epithelzellen. Grosse pathologische Zysten wurden auch beobachtet. Die ersten Anzeichen der Verdickung des gastrischen Epithels wurden bereits in Neugeborenen MN/CA9-/- Mäusen gefunden. Eine immunohistochemische Anfärbung für PNCA zeigte eine massive Expansion und Desorganisation in der proliferierenden Zone des Magenepithels. Ein ähnlich unorganisiertes Muster wurde mit E-Cadherin- Immunoanfärbung des gastrischen Epithels in den mutierten Mäusen gefunden. Das Verhältnis der Anzahl proliferierender und der Gesamtzahl der Zellen in der proliferierenden Zone blieb vergleichbar zu MN/CA9+/+ Tieren. Da eine Störung der Apoptose ausgeschlossen werden konnte, sind die Unterschiede wahrscheinlich auf eine Störung in der Zellproliferation zurückzuführen. Ob eine durch E-Cadherin vermittelte Zelladhäsion durch die Abwesenheit von MN/CA IX beeinflusst wird, muss noch ermittelt werden. Obwohl die genaue physiologische Rolle von MN/CA IX Protein im Gastrointestaltrakt immer noch nicht klar ist, sind wesentliche Fortschritte erzielt worden. Wir vermuten, dass die Nullmutation von MN/CA IX Protein den Prozess der Übermittlung und Verarbeitung der extrazellulären Signale, der normalerweise den Grad der Zellproliferation und der Integrität des gastrischen Epithelzellen regelt, stört. Diese MN/CA9-/- Mäuse sind das erste Tiermodel für ein Carboanhydrase, das mit gezielter Mutagenese erstellt wurde. MN/CA IX ist das erste adhäsionsvermittelnde Molekül, welches im Gastrointestaltrakt exprimiert wird und eine Rolle in der Morphogenese der Magenschleimhaut zeigt. MN/CA9-/- Mäuse sind ein ausgezeichnetes Tiermodel um die Rolle von MN/CA IX in der Morphogenese des gastrischen Epithels und seinen Zusammenhang mit der Krebsentwicklung zu untersuchen

Innovative Escapement-Based Mechanism for Micro-Antenna Boom Deployment

This paper presents the prototype of a tubular boom antenna developed for the Polish BRITE-PL satellite by the Space Research Center of the Polish Academy of Sciences (CBK PAN). What is unique about our work is that we developed an original type of the tubular boom antenna deployment mechanism that can be used widely as a basic solution for compact electrical antennas, booms deploying sensitive instruments, ultra-light planetary manipulators etc. The invented electromagnetic driving unit provides a dual complementary action - it adds extra energy to the driving spring, making the system more reliable, and at the same time it moderates the deployment speed acting as a kind of damper. That distinguishing feature predetermines the mechanism to be applied wherever the dynamic nature of a spring drive introducing dangerous vibrations and inducing severe local stress in the structure needs to be mitigated. Moreover, the paper reveals a product unique in Europe - a miniature beryllium bronze tubular boom free of geometry and strain defects, which is essential for stiffness and fatigue resistance. Both the deployment mechanism and the technology of tubular boom manufacturing are protected by patent rights

Novel cinnamic acid/4-aminoquinoline conjugates bearing non-proteinogenic amino acids: Towards the development of potential dual action antimalarials

A series of cinnamic acid/4-aminoquinoline conjugates conceived to link, through a proper retro-enantio dipeptide, a heterocyclic core known to prevent hemozoin formation, to a trans-cinnamic acid motif capable of inhibiting enzyme catalytic Cys residues, were synthesized as potential dual-action antimalarials. The effect of amino acid configuration and the absence of the dipeptide spacer were also assessed. The replacement of the D-amino acids by their natural L counterparts led to a decrease in both anti-plasmodial and falcipain inhibitory activity, suggesting that the former are preferable. Molecules with such spacer were active against blood-stage Plasmodium falciparum, in vitro, and hemozoin formation, implying that the dipeptide has a key role in mediating these two activities. In turn, compounds without spacer were better falcipain-2 inhibitors, likely because these compounds are smaller and have their vinyl bonds in closer vicinity to the catalytic Cys, as suggested by molecular modeling calculations. These novel conjugates constitute promising leads for the development of new antiplasmodials targeted at blood-stage malaria parasites

Information recovery from low coverage whole-genome bisulfite sequencing.

The cost of whole-genome bisulfite sequencing (WGBS) remains a bottleneck for many studies and it is therefore imperative to extract as much information as possible from a given dataset. This is particularly important because even at the recommend 30X coverage for reference methylomes, up to 50% of high-resolution features such as differentially methylated positions (DMPs) cannot be called with current methods as determined by saturation analysis. To address this limitation, we have developed a tool that dynamically segments WGBS methylomes into blocks of comethylation (COMETs) from which lost information can be recovered in the form of differentially methylated COMETs (DMCs). Using this tool, we demonstrate recovery of ∼30% of the lost DMP information content as DMCs even at very low (5X) coverage. This constitutes twice the amount that can be recovered using an existing method based on differentially methylated regions (DMRs). In addition, we explored the relationship between COMETs and haplotypes in lymphoblastoid cell lines of African and European origin. Using best fit analysis, we show COMETs to be correlated in a population-specific manner, suggesting that this type of dynamic segmentation may be useful for integrated (epi)genome-wide association studies in the future

Genetic Diversity and Population Structure of Rice Varieties Cultivated in Temperate Regions

Background After its domestication, rice cultivation expanded from tropical regions towards northern latitudes with temperate climate in a progressive process to overcome limiting photoperiod and temperature conditions. This process has originated a wide range of diversity that can be regarded as a valuable resource for crop improvement. In general, current rice breeding programs have to deal with a lack of both germplasm accessions specifically adapted to local agro-environmental conditions and adapted donors carrying desired agronomical traits. Comprehensive maps of genome variability and population structure would facilitate genome-wide association studies of complex traits, functional gene investigations and the selection of appropriate donors for breeding purposes. Results A collection of 217 rice varieties mainly cultivated in temperate regions was generated. The collection encompasses modern elite and old cultivars, as well as traditional landraces covering a wide genetic diversity available for rice breeders. Whole Genome Sequencing was performed on 14 cultivars representative of the collection and the genomic profiles of all cultivars were constructed using a panel of 2697 SNPs with wide coverage throughout the rice genome, obtained from the sequencing data. The population structure and genetic relationship analyses showed a strong substructure in the temperate rice population, predominantly based on grain type and the origin of the cultivars. Dendrogram also agrees population structure results. Conclusions Based on SNP markers, we have elucidated the genetic relationship and the degree of genetic diversity among a collection of 217 temperate rice varieties possessing an enormous variety of agromorphological and physiological characters. Taken together, the data indicated the occurrence of relatively high gene flow and elevated rates of admixture between cultivars grown in remote regions, probably favoured by local breeding activities. The results of this study significantly expand the current genetic resources available for temperate varieties of rice, providing a valuable tool for future association mapping studies

Wartość diagnostyczna wybranych markerów biochemicznych w diagnostyce wznowy raka rdzeniastego tarczycy — porównanie kalcytoniny, prokalcytoniny, chromograniny A i antygenu karcynoembrionalnego

Introduction: Medullary thyroid cancer (MTC) is a malignancy of the thyroid gland, which derives from parafollicular C cells. Periodic measurement of biochemical markers of MTC remains a crucial part of patient follow-up and disease monitoring. The aim of the study was to compare the diagnostic value of four selected markers — calcitonin (Ct), procalcitonin (PCT), chromogranin A (CgA), and carcinoembryonic antigen (CEA). Material and methods: Patients with histopathologically confirmed MTC hospitalised in a single department between January 2015 and December 2015 were included in the study. Patients were subdivided into two groups: a remission group and an active disease group, based upon serum markers of MTC and imaging. Levels of Ct, PCT, CgA, and CEA were compared between the groups. Results: Forty-four patients were included; 20 patients presented active disease and 24 were in remission. All patients with active disease had Ct exceeding the upper limit of normal range (10 pg/mL) — for that threshold the sensitivity was 100.0% and the specificity was 73.9%; for the best-fit threshold of 121.0 pg/mL the specificity was 95.8% with sensitivity 100.0%. There was significant correlation between Ct and PCT — p < 0.000001, r = 0.93. All patients with active disease exceeded the upper limit of the normal range (0.5 ng/mL) — for that threshold the sensitivity was 100.0% and the specificity was 83.3%; for the best-fit threshold of 0.95 ng/mL the specificity was 95.8% with sensitivity 100.0%. In case of CEA for the best-fit threshold of 12.66 ng/mL the specificity was 100.0% with sensitivity 57.9%; for CgA the best-fit threshold was 75.66 ng/mL with specificity 83.3% and sensitivity 75.0%. Conclusions: Our study confirms that PCT can be considered as an equivalent alternative for measurement of calcitonin. On the other hand, it is also worth noting that MTC can be a rare cause of very high levels of PTC not resulting from infectious diseases. The diagnostic value of CEA and chromogranin A is much lower and can be within the normal range even in patients with advanced, metastatic MTC. They should be used only as accessory markers.Wstęp: Rak rdzeniasty tarczycy (RRT) to nowotwór złośliwy tarczycy wywodzący się z przypęcherzykowych komórek C. Okresowa kontrola markerów biochemicznych RRT stanowi kluczowy element prowadzenia pacjenta i monitorowania choroby. Celem obecnej pracy było porównanie wartości diagnostycznej czterech wybranych markerów — kalcytoniny (Ct), prokalcytoniny (PCT), chromograniny A (CgA) i antygenu karcynoembrionalnego (CEA). Metody: Do badania włączono pacjentów z histopatologicznie potwierdzonym RRT hospitalizowanych w jednym oddziale szpitalnym w 2015 roku. Pacjentów podzielono na dwie podgrupy — grupę pacjentów w remisji oraz z aktywną chorobą zależnie od wartości markerów osoczowych oraz wykonanej diagnostyki obrazowej. Wyniki: Włączono czterdziestu czterech pacjentów; 20 pacjentów prezentowało cechy aktywnej choroby, 24 było w remisji. Wszyscy pacjenci z aktywną chorobą wykazywali stężenia Ct przekraczające górną granicę normy (10 pg/ml); dla optymalnego punktu odcięcia 121,0 pg/ml swoistość wyniosła 95,8% przy czułości 100,0%. Wykazano istotną korelację pomiędzy Ct i PCT- p < 0.000001, r = 0.93. Dla optymalnego punktu odcięcia wynoszącego 0.95 ng/ml swoistość wyniosła 95.8% przy czułości 100.0%. W przypadku CEA dla najlepiej dopasowanego progu 12,66 ng/ml swoistość wyniosła 100,0% przy czułości 57,9%; dla CgA optymalny punkt odcięcia wyniósł 75,66 ng/ml przy swoistości 83,3% i czułości 75,0%. Wnioski: Nasze badanie potwierdza, że PCT może być uważana za równoważną alternatywę dla oznaczenia kalcytoniny. Wartość diagnostyczna CEA i chromograniny A jest znacznie mniejsza i parametry te mogą pozostawać w granicach normy nawet u pacjentów z zaawansowanym RRT z przerzutami odległymi. Powinny być one traktowane jedynie jako pomocnicze markery

Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eightythree genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes

Transcriptome characterization by RNA sequencing identifies a major molecular and clinical subdivision in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein-coding genes, noncoding RNAs, and pseudogenes. Transposable elements are globally derepressed in CLL cells. In addition, two thousand genes-most of which are not differentially expressed-exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome, and ribosome were among the most down-regulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences

Benchmarking of Whole Exome Sequencing and Ad Hoc Designed Panels for Genetic Testing of Hereditary Cancer

Acknowledgements: We thank all patients who contributed to this study. The work was supported by grants from the Instituto de Salud Carlos III (ISCIII, MINECO) (operating grants: PI13/00285 and RD12/0036/0008 awarded to C.L. and PIE13/00022 and RD12/0036/0031 awarded to G.C.) and confunded by FEDER funds/European Regional Development Fund (ERDF) - a way to Build Europe-"// FONDOS FEDER "una manera de hacer Europa", the Generalitat de Catalunya (Government of Catalonia) (operating grant 2014SGR338, awarded to G.C.) and the Asociación Española Contra el Cáncer (operating grants, 2010 Grupos Estables, awarded to G.C.). J.B. received a Spanish Society of Medical Oncology grant. This activity is sponsored by the ISCIII Ministerio de Economía y Competitividad (PT13/0001/0044).Next generation sequencing panels have been developed for hereditary cancer, although there is some debate about their cost-effectiveness compared to exome sequencing. The performance of two panels is compared to exome sequencing. Twenty-four patients were selected: ten with identified mutations (control set) and fourteen suspicious of hereditary cancer but with no mutation (discovery set). TruSight Cancer (94 genes) and a custom panel (122 genes) were assessed alongside exome sequencing. Eightythree genes were targeted by the two panels and exome sequencing. More than 99% of bases had a read depth of over 30x in the panels, whereas exome sequencing covered 94%. Variant calling with standard settings identified the 10 mutations in the control set, with the exception of MSH6 c.255dupC using TruSight Cancer. In the discovery set, 240 unique non-silent coding and canonic splice-site variants were identified in the panel genes, 7 of them putatively pathogenic (in ATM, BARD1, CHEK2, ERCC3, FANCL, FANCM, MSH2). The three approaches identified a similar number of variants in the shared genes. Exomes were more expensive than panels but provided additional data. In terms of cost and depth, panels are a suitable option for genetic diagnostics, although exomes also identify variants in non-targeted genes

Differential expression of long non-coding RNAs are related to proliferation and histological diversity in follicular lymphomas

Long non‐coding RNAs (lncRNAs) comprise a family of non‐coding transcripts that are emerging as relevant gene expression regulators of different processes, including tumour development. To determine the possible contribution of lncRNA to the pathogenesis of follicular lymphoma (FL) we performed RNA‐sequencing at high depth sequencing in primary FL samples ranging from grade 1‐3A to aggressive grade 3B variants using unpurified (n = 16) and purified (n = 12) tumour cell suspensions from nodal samples. FL grade 3B had a significantly higher number of differentially expressed lncRNAs (dif‐lncRNAs) with potential target coding genes related to cell cycle regulation. Nine out of the 18 selected dif‐lncRNAs were validated by quantitative real time polymerase chain reaction in an independent series (n = 43) of FL. RP4‐694A7.2 was identified as the top deregulated lncRNA potentially involved in cell proliferation. RP4‐694A7.2 silencing in the WSU‐FSCCL FL cell line reduced cell proliferation due to a block in the G1/S phase. The relationship between RP4‐694A7.2 and proliferation was confirmed in primary samples as its expression levels positively related to the Ki‐67 proliferation index. In summary, lncRNAs are differentially expressed across the clinico‐biological spectrum of FL and a subset of them, related to cell cycle, may participate in cell proliferation regulation in these tumours