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Preliminary investigation of the effects of silencing the non-coding RNA, NEAT1, on the Burkitt's lymphoma cell line BJAB

Preliminary investigation of the effects of silencing the non-coding RNA, NEAT1, on the Burkitt's... BioscienceHorizons Volume 6 2013 10.1093/biohorizons/hzt006 Research article Preliminary investigation of the effects of silencing the non-coding RNA, NEAT1, on the Burkitt’s lymphoma cell line BJAB Christopher Halford School of Life Sciences, Huxley Building, Keele University, Keele, Staffordshire ST5 5BG, United Kingdom *Corresponding author: 17 Pelican Close, Crewe, Cheshire CW1 5YA, UK. Email: chrishalford27@gmail.com Project supervisor: Dr Mirna Mourtada-Maarabouni Nuclear enriched abundant transcript 1 (NEAT1) is a long non-coding RNA with two isoforms. Both are expressed constitu- tively and have been shown to play a crucial structural role in the formation of paraspeckles. Paraspeckles are subnuclear bodies which retain certain mRNAs, preventing their translation in the cytoplasm, thereby silencing the corresponding genes. This study aimed to assess the effects of knocking down NEAT1 by RNA interference on the Burkitt’s lymphoma cell line, BJAB. The results have shown that a targeted siRNA depletion of NEAT1 resulted in an increased number of cells displaying aberrant morphology, including the appearance of ‘giant cells’, multinucleated cells and cells with cytoplasmic vacuoles. Furthermore, NEAT1 down-regulation was accompanied with an increased level of apoptosis and decreased cell viability, assessed by acri- dine orange morphology staining, vital dye exclusion and (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assay. Taken together, the study strongly suggests that modulation of NEAT1 expression has a significant effect on the BJAB cell line. Such remarkable effects of changes of NEAT1 expression on the control of cell morphol - ogy reported here indicate the potential significance of this gene in the development and progression of cancer and highlight the importance of further investigation into the role of NEAT1 dysregulation in autoimmune disease and oncogenesis. Key words: lncRNA, NEAT1, MEN, BJAB, lymphoma, cancer Received 12 November 2012; revised 27 March 2013; accepted 30 April 2013 Introduction generating a short, poly-A rich tract instead of the classical poly-A tail. This also generates a smaller, independent tRNA- Nuclear enriched abundant transcript 1 (NEAT1) or multiple like molecule called menRNA (Sunwoo et al., 2009; Novikova endocrine neoplasia ε/β, a long non-coding RNA (lncRNA), is Hennelly, and Sanbonmatsu, 2012). NEAT1 is widely expressed polyadenylated (poly-A) and encoded on chromosome 11q13.1. in many tissue types, especially in the ovary, prostrate, colon It is produced from a single, intergenic exon and has two small and pancreas (Hutchinson et al., 2007) and Sasaki et al. (2009) regions that are highly conserved within the mammalian lin- report that both isoforms are expressed at similar levels. eage (Hutchinson et al., 2007). Two isoforms exist: a smaller NEAT1 lncRNAs are up-regulated upon differentiation of 3.7 kb isoform (MEN ε) and a larger 23 kb isoform (MEN β) human embryonic stem cells (Chen and Carmichael, 2009), (Hutchinson et al., 2007; Clark and Mattick, 2011). Both share muscle differentiation (Sunwoo et al., 2009) and in vitro neuro- a single transcriptional start site, and MEN β is a continuation nal differentiation (Mercer et al., 2010). of the shorter MEN ε isoform (Hutchinson et al., 2007). MEN β contains a conserved 3′ cloverleaf secondary structure, which Numerous studies show that the NEAT1 lncRNAs are is cleaved by RNaseP to form the 3′ end of the mature transcript, essential for the formation and structural integrity of © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 6 2013 subnuclear bodies called ‘paraspeckles’ (Bond and Fox, 2009; implicated in amyotrophic lateral sclerosis and frontotemporal Clemson et al., 2009; Sasaki et al., 2009; Sunwoo et al., lobar degeneration (Tollervey et al., 2011). The significance of 2009; Fox and Lamond, 2010). Paraspeckles are ‘dynamic’, these results is unclear; the NEAT1 knockout mice are viable, because the nucleating bodies, the NEAT1 lncRNAs (Bond despite the absence of paraspeckles (Nakagawa et al., 2011). and Fox, 2009; Clemson et al., 2009), are unstable (Clark Despite this, due to the roles that NEAT1 has been implicated et al., 2012) and maintenance of the paraspeckle requires in, it may still have value as a diagnostic or therapeutic target that they are continuously transcribed (Nakagawa and (Lipovich, Johnson and Lin, 2010). Hirose, 2012). Paraspeckles are ribonucleoprotein complexes Since the significance of changes in the level of NEAT1 characterized by the NEAT1 lncRNAs and three RNA- expression have not yet been investigated, we were prompted binding proteins, P54, PSF and PSP1, members of the to analyse the functional effects of silencing NEAT1 on the Drosophila melanogaster behaviour, human splicing (DBHS) B-cell lymphoma cell line, BJAB using RNA interference. These family (Bond and Fox, 2009; Novikova, Hennelly and analyses clearly suggest a potential role for NEAT1 in the con- Sanbonmatsu, 2012; Nakagawa and Hirose, 2012). The trol of cell morphology and, consequently, cell function. function of paraspeckles is not fully understood. Prasanth et al. (2005) suggest that paraspeckles retain and store ade- nosine to inosine hyper-edited mRNAs, silencing the corre- Materials and methods sponding gene. Nakagawa and Hirose (2012) note that P54 Materials preferentially recognizes A-to-I edited RNAs. Others suggest that the function of paraspeckles is to localize/sequestrate its NEAT1 siRNA1 (ID: S238175), NEAT1 siRNA2 (ID: protein components, many of which have functions outside S238174) and NEAT1 siRNA3 (ID: S238176) from Life of the nucleus (Nakagawa and Hirose, 2012). Technologies (Ambion), catalogue no.: 4399665 Custom Synthesis (named to differentiate them from each other). Paraspeckle assembly starts with the targeting of newly syn- siRNA1 targets nucleotide 1352, near the centre of the thesized NEAT1 by DBHS dimers (Bond and Fox, 2009). The NEAT1 transcript; siRNA2 targets nucleotide 3373, towards NEAT1–DBHS complexes build up the paraspeckle; the fin - the 5′ end of the transcript and siRNA3 targets nucleotide ished structure consisting of multiple RNA–DBHS complexes, 120 near to the 3′ end of the transcript. Control siRNA from facilitated by DBHS oligomerization. Although both isoforms Life Technologies (Ambion), catalogue no.: AM4635. This is have structural roles, their overlap is unclear. The smaller an RNA molecule designed to have no significant sequence MEN ε transcript may have the ability to form large structural similarity to human sequences used to ensure that observed complexes by itself, as NEAT1 complexes form quickly at the effects from RNAi were not by-products of the transfection transcription site, before PSP1 or P54 are detectable within process (Ambion, Inc., 2007). Cy3-siRNA Labelling Reagent them (Clemson et al., 2009). The MEN ε transcript is self- also from Life Technologies (Ambion), catalogue no.: complementary in many places, which may allow the forma- AM1632. RPMI-1640 medium from Sigma-Aldrich, cata- tion of intramolecular and intermolecular structures (Clemson logue no.: R0883; Phenol Red, 200 mM l-glutamine, 10% et al., 2009). However, MEN β preferentially associates with foetal calf serum, 100 mg streptomycin and 100 units of pen- DBHS proteins in vivo, indicating that MEN β forms the core icillin/ml were pre-added to indicate pH, provide growth- of the paraspeckle and then recruits MEN ε (Sasaki et al., encouraging factors and prevent bacterial contamination, 2009). Souquere et al. (2010) show that MEN ε and the 3′ end respectively. OptiMEM and GlutaMAX solution from Life of MEN β are localized to the periphery of the paraspeckle, Technologies (Invitrogen), catalogue no.: 51985-026 (Lot: and the central sequence of MEN β runs through the centre of 905272). Iscove’s Modified Dulbecco’s Medium from Sigma- the paraspeckle. They also show that while the MEN ε overex- Aldrich, catalogue no.: I3390. Solution V from Lonza, as pression increases the number of paraspeckles, and transient part of Amaxa Nucleofector transfection kit. CellTiter 96 overexpression does not rescue a total NEAT1 knockdown, solution for MTS assay from Promega, catalogue no.: G3580 the MEN β knockdown causes only residual paraspeckles to (Lot: 326240). Trypan blue powder from Sigma-Aldrich, remain. At present, it is currently unclear how the structural catalogue no.: T6146. Acridine orange powder from Sigma- roles of each isoform overlap. Aldrich, catalogue no.: A6014. Various non-coding RNA molecules, along with subnuclear disorganization, have been implicated in a number of diseases Methods (Mehler and Mattick, 2007; O’Rourke and Swanson, 2009; Cell culture Qureshi, Mattick and Mehler, 2010; Gibb et al., 2011; BJAB cell lines were maintained in the RPMI-1640 medium Wapinski and Chang, 2011; Johnson, 2012). This suggests (sigma) in a 1:9 ratio. Cells were incubated at 37°C and 5% that aberrant NEAT1 function may be associated with disease. CO . NEAT1 is down-regulated in lung, liver, oesophageal and reti- 2 nal cancers (Gibb et al., 2011) and is a novel target of P53 Preparation of Cy3 labelling reagent (Lipovich, Johnson and Lin, 2010). The MEN ε isoform is up- Hundred microlitres of reconstitution solution was added to regulated in the Huntington’s caudate nucleus and the nucleus dry Cy3 labelling reagent (Ambion). Solution was vortexed accumbens of heroin users (Johnson, 2012). NEAT1 is also 2 Bioscience Horizons • Volume 6 2013 Research article and after 5 min was re-vortexed to ensure full suspension. carded and the pellet washed with 1 ml of phosphate-buff- Labelling reagent was then stored in the dark at −20°C. ered saline (PBS). The solution was centrifuged (settings above) and washed with 1 ml PBS. The whole sample was Labelling siRNA pipetted onto a microscope slide, a cover slip applied and To eppendorf tubes, 18.3 µ l nuclease-free water was added, viewed under a microscope, with normal light and fluores - followed by 5 µ l 10× labelling buffer, 19.2 µ l siRNA (20 µ M) cence to estimate the transfection efficiency. and finally 7.5 µ l labelling reagent. Finally, the solution was MTS assay incubated at 37°C in the dark for 1 h. The reagent was then stored at −20°C. CellTiter 96 solution was removed from freezer and allowed to thaw. BJAB suspensions in a 6-well plate were gently agitated to Cell count mix. In a 96-well plate, 100 µ l of transfected BJAB suspension Cell suspension was agitated gently to ensure mixing. Twenty was added to wells from each siRNA used. This was repeated microlitres of suspension was transferred to a 96-well plate. three times. Twenty microlitres of the CellTiter 96 solution was Twenty microlitres of trypan blue was added and mixed gen- added to each 96-well plate suspension. The 96-well plate was tly. Cells were counted under microscopy. incubated at 37°C for 2 h. Absorbance readings were taken at 490 nm wavelength on a Victor Wallac 1420 machine. Electroporation Acridine orange (% apoptosis) Cells were counted, and the volume of suspension required for electroporation was calculated (10–20 × 10 cells/ml minimum). One millilitre samples of transfected BJAB suspensions, one Suspension was centrifuged at 1200 rpm (220 g) and room tem- from each siRNA used, were centrifuged at 2000 rpm (600 g) perature (23–25°C) for 8 min. Supernatant was discarded and the for 5 min. Most of the supernatant was discarded and the pellet pellet suspended in 5 ml Optimem (Invitrogen). The solution was resuspended in the remaining solution. Twenty microlitres of centrifuged again (settings above). Supernatant was then dis- the solution was added to a slide to which 5 µ l of acridine carded and the pellet resuspended in 1.2 ml Optimem (400 µ l for orange (50 µ g/ml) was added. Acridine orange is fluorescent, each electroporation). Four hundred microlitres of the solution nucleic-acid selective dye that interacts with DNA by intercala- was added to one electroporation cuvette. siRNA of 1.56 µ l to be tion, emitting green fluorescence. A cover slip was placed on the used was added to the cuvette. The electroporation cuvettes were slide and was viewed under a microscope with fluorescence. allowed to rest for 10 min, and 5 ml of Iscove’s Modified Preparation of cells for transmission electron microscopy Dulbecco’s Medium (Sigma-Aldrich) was added to wells in a (TEM) 6-well plate. After 10 min, the cuvettes were placed in the BioRad Gene Pulser II machine and electroporated at 1050 µ f capacitance The cells were counted and centrifuged (settings as above). The and 292 V. Ten minutes after electroporation, the solutions from supernatant was discarded and the pellet resuspended in 2.5% each cuvette were pipetted into separate wells in the 6-well plate. glutaraldehyde tannic acid (GTA) and incubated at 37°C for The plate was then incubated at 37°C and 5% CO for 48 h. 1 h. Glutaraldehyde is a popular fixing agent for electron microscopy but alone does not provide adequate preservation Nucleofection of cellular structures. In combination with tannic acid and Cells were counted and the volume required for nucleofection heavy metal treatment, the cellular structures are protected was calculated (2 × 10 cells/ml minimum). This solution was from shrinkage by dehydration and drying (Svitkina and centrifuged at 1500 rpm (350 g) and room temperature for Borisy, 1998). The cells were span down, supernatant dis- 8 min and 1.5 ml of Iscove’s Modified Dulbecco’s Medium was carded and the pellet resuspended in 0.5–1% GTA to store as added to wells in a 6-well plate, and the plate was incubated at needed. For use: the 0.5–1% GTA supernatant was discarded 37°C. After 8 min, the supernatant was discarded and the pel- and 1 ml of sodium cacodylate buffer (pH 7.4) with 2 mM let suspended in 0.3 ml Solution V (Lonza) (100 µ l for each calcium chloride was added. The solutions were vortexed to nucleofection). Hundred microlitres of solution was trans- disperse the cells and were centrifuged. Osmium tetroxide, ferred to an eppendorf tube, where 0.39 µ l of the siRNA to be which acts as a fixing agent and stain ( Bozzola and Russell, transfected was added and mixed. The mixture was pipetted 1999), was added to the solutions, and then vortexed, to dis- into nucleofection cuvettes. These cuvettes were placed into perse the cells. The solutions were left to stand for 1 h. After the Lonza Nucleofector Device II machine, and nucleofected 1 h, the solutions were centrifuged to remove the excess using program T-001. A small amount of the incubated Iscove’s osmium. One millilitre of sodium cacodylate buffer was added Modified Dulbecco’s Medium was pipetted into each cuvette. and the solutions were centrifuged. The supernatant was dis- Using a fine pipette, the entire solution in the cuvettes was carded and liquid agar was added to the eppendorf tubes. transferred to separate wells in the 6-well plate. The plate was Finally, they were dehydrated using 70% ethanol. then incubated at 37°C and 5% CO for 48 h. Statistical analysis Fluorescent microscopy (transfection efficiency) All statistical analyses were carried out using a two-sample 1 ml of Cy3-labelled negative control siRNA was centrifuged t-test at a 95% confidence level (unless stated otherwise). at 2000 rpm (600 g) for 8 min. The supernatant was dis- All results are ±standard error of the mean. 3 Research article Bioscience Horizons • Volume 6 2013 conductive pore produced by the field exceeds a certain, crit - Results ical size (dependent on local factors such as mechanical stress and lipid bilayer edge energy) it can rapture the bilayer, lead- To determine the most efficient method of siRNA transfec - ing to cell death (Joshi and Schoenbach, 2000). The effect of tion, two techniques were investigated. Electroporation and each transfection method on cell viability was, therefore, also nucleofection both move charged molecules into the cell, via investigated. water-filled membranous pores induced by an applied electri - cal field ( Neumann et al., 1982; Melikov et al., 2001). Figure 1 shows that the average transfection efficiency for Electroporation is a general technique that can be used to electroporation was lower than that for nucleofection. transfect cells with any serum-free cell medium. Nucleofection, Transfection by nucleofection had a higher efficiency and was on the other hand, uses cell-type specific reagents and electri - more consistent than transfection by electroporation. Cell cal parameters and transfers the molecules directly into the viability was determined by vital dye exclusion, 72 and 96 h nucleus. These are ‘harsh’ techniques and have some detri- post-transfection, as shown in Fig. 2. At 72 h cell viability mental effects regarding cell survival, more specifically, if the Figure 1. The average transfection efficiency (%) for the methods of electroporation (blue) and nucleofection (red). * P < 0.05. Figure 2. The average cell viability for the methods of electroporation (yellow) and nucleofection (green), at 72 and 96 h post-transfection, allowing 48 h for RNA knockdown to occur. 4 Bioscience Horizons • Volume 6 2013 Research article was higher for the nucleofected cells. At 96 h, cell viability was also higher for the nucleofected cells. Nucleofection was chosen as the method for siRNA transfection for its higher efficiency and higher cell viability rates. Transfection with the NEAT1 siRNAs caused down-regu- lation of NEAT1 by 65–75% as was confirmed by qRT-PCR, performed in a subsequent study using the same methodol- ogy (Dr Mourtada-Maarabouni, personal communication). It is unclear how the silencing affected the different isoforms of the NEAT1 transcript. Cell viability was measured to determine the number of living cells present in suspension. This indicates whether transfection of NEAT1 siRNAs has any direct consequences for cell survival. Figure 3 shows cell viability measured by vital dye exclusion using trypan blue. Non-viable cells cannot remove the dye, appearing blue under microscopy. Overall, the samples transfected with NEAT1 siRNAs showed a decreased cell viability. At 72 h, the samples trans- fected with the NEAT1 siRNAs had a lower viability than the respective controls. At 96 h, NEAT siRNAs 2 and 3 had a lower viability than their controls, and NEAT1 siRNA1 had an equal viability with the negative control siRNA. These results indicate that NEAT1 knockdown may adversely affect cell viability. Cell viability was also measured by an MTS assay. The MTS dye (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth- oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is reduced by mitochondrial enzymes to a chromogenic formazan dye. As mitochondrial components are involved, the MTS assay is also an indicator of metabolic activity. A higher the rate of activity produces more formazan dye; therefore the activity of the cell can be measured colourimetrically. The results of the MTS assays (Fig. 4) were similar to the vital dye exclusion. At 72 h, NEAT1 siRNAs 1 and 3 had lower absorbance readings than the respective controls, indi- cating less viability. NEAT1 siRNA2 had a higher absorbance reading compared with its control sample; however, the error bar for the control exceeds the result and error bar of the siRNA sample, indicating the possibility that the control sample had a higher viability. At 96 h, all NEAT1 siRNAs had lower absorbance values than the respective controls. Overall, these results similarly indicate that cells transfected with NEAT1 siRNA had decreased viability. BJAB cells were stained with acridine orange and their mor- phology was viewed under microscopy. The morphology of a cell is a good indicator of its health, and any abnormal features can indicate a pathological process. The number of cells under- Figure 3. T he viable cell number for BJAB suspensions transfected going apoptosis and the number of giant/multinucleated cells with NEAT1 siRNAs, 72 and 96 h post-transfection, as measured by vital were counted (Figs 5 and 6) and interesting morphological dye exclusion. The respective negative controls (blue) are shown with features (such as the appearance of cytoplasmic vacuoles, giant (A) NEAT1 siRNA1 (red), (B) NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). *P < 0.05. cells and multinucleated cells) were observed (Fig. 7). At 72 and 96 h post-transfection, all samples transfected undergoing apoptosis, compared with controls. These results with NEAT1 siRNA contained a higher number of cells clearly demonstrate that samples transfected with NEAT1 5 Research article Bioscience Horizons • Volume 6 2013 Figure 4. The cell viability for BJAB suspensions transfected with NEAT1 siRNAs, 72 and 96 h post-transfection, as measured by the MTS assay. Higher absorbance readings correspond with increased viability. The respective negative controls (blue) are shown with (A) NEAT1 siRNA1 (red), (B) NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). Figure 5. T he percentage of apoptosis present in BJAB suspensions transfected with NEAT1 siRNAs, 72 and 96 h post- siRNAs, contain a higher percentage of apoptotic cells. transfection, as determined by acrid orange morphology staining. The At 72 h, samples transfected with NEAT siRNAs 1 and 2 respective negative controls (blue) are shown with (A) NEAT1 siRNA1 clearly contain a higher number of giant cells with aberrant (red), (B) NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). morphology, compared with the respective control samples. *P < 0.05. 6 Bioscience Horizons • Volume 6 2013 Research article For cells transfected with NEAT1 siRNA3, at 72 h there were fewer cells with aberrant morphology than the control. At 96 h, the NEAT1 siRNA1 sample still clearly has a higher number of giant cells than its control. At 96 h, the samples transfected with siRNAs 2 and 3 contained fewer giant cells compared with the controls. Overall, samples transfected with the NEAT1 siRNAs all showed a marked increase in the percentage of apoptotic cells compared with controls. Although not as clear, NEAT1 knockdown also appears to influence the appearance of giant cells and aberrant morphology. Figures 7 and 8 clearly show atypical cells with irregular morphology. Figure 8B and D indicate the formation of ‘spaces’ inside the cytoplasm, bordered by a membrane-like structure on at least one side. In Figure 8B, these ‘spaces’ almost appear like swollen mitochondria, the entities inside could be the cristae of the inner-membrane. Some of these ‘spaces’, however, are bordered completely and have no inner ‘artefacts’ and appear like vacuoles (Fig. 8D). Another fea- ture of Fig. 8 is the aberrant nuclear morphology. Figure 8D illustrates this well, with a large ‘U-shaped’ nucleus with a smaller nuclear body that may have ‘budded off’ from the main nucleus. Although the cell in Fig. 8C also shows irregu- lar nuclei, the shape and position suggest that the cell is sim- ply undergoing mitosis. Another prominent example of irregular nuclear morphology is Fig. 7D, a giant cell with a huge number of nuclear entities. Figure 9 shows confocal microscopy images of BJAB cells transfected with (A) the negative control siRNA and (B) NEAT1 siRNA2. In the control sample, the cell nuclei are regular: large, mostly round, consistent densities and no obvious cytoplasmic aberrations. In the NEAT1 siRNA2 sample, cells display nuclei with irregular shape and inconsis- tent densities, cytoplasmic vacuole-like structures and a num- ber of the cells are multinucleate. These results suggest that aberrant morphology occurs more often in cells transfected with the NEAT1 siRNAs, compared with the negative control siRNA. This suggests that NEAT1 knockdown could influence the processes that cause this morphology to appear and, consequently, cell func- tion. Discussion We were prompted to analyse the functional effects of silenc- ing NEAT1 on the B-cell lymphoma cell line BJAB. The results show that cells in which NEAT1 was silenced, had lower viability, increased levels of apoptosis and more cells displayed atypical morphology. While the atypical morphol- Figure 6. The percentage of giant, multinucleate or otherwise ogy of the cells can be explained as a facet of cancer physiol- morphologically aberrant cells, present in BJAB suspensions ogy (see below), the increased number of cells cannot. This transfected with NEAT1 siRNAs, 72 and 96 h post-transfection, as may be explained by two general mechanisms: first, NEAT1 determined by acrid orange morphology staining. The respective knockdown may directly affect the malignancy of the cells. negative controls (blue) are shown with (A) NEAT1 siRNA1 (red), (B) This implies that paraspeckles loss may release mRNA that NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). 7 Research article Bioscience Horizons • Volume 6 2013 Figure 7. A selection of nucleofected BJAB cells stained with acridine orange and viewed under fluorescent microscopy. Magnification ×400. Transfected with (A and B) NEAT1 siRNA1, (C) NEAT1 siRNA2 and (D) NEAT1 siRNA3 and (E and F) negative control siRNA. (A) and (D) both show a huge cell with numerous subnuclear entities and (B) and (C) clearly show numerous vacuoles in the cytoplasm. The majority of cells in (E) and (F) display regular morphology. Figure 8. (A) Selection of nucleofected BJAB cells under electron microscopy (scale bar included). Transfected with the (A) negative control siRNA, (B) and (C) NEAT1 siRNA3 and (D) NEAT1 siRNA1. Aberrant morphology is apparent in (B) and (D), with ‘spaces’ forming in the cytoplasm and in (D), the nucleus appears disorganized. The cytoplasmic ‘spaces’ may represent swollen or burst mitochondria. 8 Bioscience Horizons • Volume 6 2013 Research article Figure 9. Confocal microscopy images of cells transfected with the (A) negative control siRNA and (B) NEAT1 siRNA2. In (A), there are very few cytoplasmic vacuole-like structures whereas in (B), many of these structures appear in a number of cells. In (B), it can also be seen that the nuclear morphology is irregular for many cells. codes for a mutated/oncogenic protein. Secondly, can revert back to normal parental cells. Apoptosis, mitotic NEAT1 knockdown could activate normal cellular pathways catastrophe and necrosis have been shown to follow the for- (e.g. cell-cycle progression), where the aberrant morphology mation of giant cells; however, some studies have shown that arises simply because the BJAB cell line is cancerous. This giant cells can escape cell death and give rise to new cancer implies that paraspeckle loss would release mRNA that is cells (Rengaswami et al., 2005, 2006). The physical cell produced by cells as part of their normal function. This latter enlargement can be induced by anti-mitotic/cancer agents mechanism is supported by the fact that NEAT1 has been that interfere with microtubule organization [(such as vin- found to be down-regulated in various cancers but not up- blastine (Horbay and Stoika, 2011) and docetaxel (Morse regulated (Gibb et al., 2011). et al., 2005)] and occurs during cell-cycle arrest. This is often preceded by mitotic catastrophe due to spindle disorganiza- It is significant to note that a number of cell-cycle compo - tion. Horbay and Stoika, 2011, speculate that the cell cycle of nents (cdk10, cyclin F, E3 ubiquitin-protein ligase) have alu cancer cells is halted in this giant state to survive unfavour- sequences in their mRNA transcript (Levy, Sela and Ast, able conditions. 2007), which could represent a target for A-I editing and sub- sequent nuclear retention (Chen, DeCerbo and Carmichael, Frequently, cancer cells are poly/aneuploid, associated 2008). In particular, cyclin F has an alu sequence in its 3′UTR with their genomic instability (Geddis, 2008). This is caused and is known to regulate the timing of the G /M transition by endomitosis, where mitosis occurs, but the cell fails to (Bai, Richman and Elledge, 1994; Tetzlaff et al., 2004). While undergo teleophase or cytokinesis, resulting in DNA repli- purely speculative, it is not entirely unreasonable to suggest cation but no cell division (Lee, Davidson and Duronio, that the mRNA of cell-cycle components/regulators is 2009). This explains the altered nuclear morphology in the retained in a paraspeckle and that NEAT1 knockdown dis- above figures. While endomitosis can give rise to the poly - rupts the cell cycle by allowing inappropriate mRNA export ploidization of giant cells, Vakifahmetoglu, Olsson and from the nucleus. This may accelerate cells through their cell Zhivotovsky (2008) state that it is endocycling (the uncou- cycle, giving rise to an increased number of cells displaying pling of DNA synthesis from cell division) which is the atypical morphology, as observed. cause of polyploidy. Another hypothesis for giant cell for- mation, is simply formation via cell fusion, which could Perhaps the most interesting phenomenon observed dur- occur spontaneously (Woodgett and Jin, 2005). There is ing this study was the presence of ‘giant cells’. These gigantic evidence for and against both mechanisms of giant cell for- cells had irregular nuclear morphology and size and were mation; however, endocycling seems to be more likely for often multinucleated (see Fig. 7). Shown by the figures above, two main reasons: First, endocycling has been observed giant cells occur more readily in samples transfected with the widely in tumours/cancer cells, particularly in those NEAT1 siRNAs, suggesting that NEAT1 knockdown may with dysfunctional P53 (Vakifahmetoglu, Olsson and influence their appearance. Without further analysis, it is Zhivotovsky, 2008) and secondly, reports of spontaneous hard to define the etiology of these cells; however, several cell fusion in the BJAB cell line do not exist in the literature. hypotheses (below) have been put forward in the literature In light of its relation to mitotic catastrophe (see below) and which may be relevant. the consequences of aberrant cell-cycle progression, endo- Giant cells appear in most cancers (Horbay and Stoika, cycling seems a more likely explanation for multinucleate 2011). It has been suggested that they are terminal cells, but giant cell formation. 9 Research article Bioscience Horizons • Volume 6 2013 Implicated in the formation and fate of giant cells is a pro- Translocation of c-myc to immunoglobulin loci and its subse- cess known as mitotic catastrophe. While there is no clear defi - quent massive increase in expression drive excessive prolifera- nition of mitotic catastrophe, it is a form of cell death that tion through the E2F1 transcription factor. E2F1 expression shares some hallmarks of apoptosis (Castedo et al., 2004). It is stimulates concomitant expression of tumour suppressor ARF unclear whether mitotic catastrophe is a special case of apopto- P14 , which promotes P53 activation by binding MDM2 sis or pre-process that leads to cell death by apoptosis or necro- (Bates et al., 1998). Because oncogene expression seemingly sis. Some studies have even shown mitotic catastrophe to be a drives activation of P53, Burkitt’s lymphoma cell lines must survival mechanism for tumour cells and a way of switching have a mutation in P53 (Hollstein et al., 1991) or at least the ARF from an abnormal cell cycle to mitosis (Castedo et al., 2004). P14 -MDM2-P53 pathway (Smardova et al., 2008). Cells cannot undergo mitotic catastrophe without prematurely Although P53 is considered the ‘guardian of the genome’, entering mitosis, making the abrogation of the G or G check- there are other ways to halt the cell cycle and initiate apopto- 1 2 points in the cell-cycle essential (Vakifahmetoglu, Olsson and sis (Vakifahmetoglu, Olsson and Zhivotovsky, 2008), includ- Zhivotovsky, 2008). In tumour cells, where cell-cycle check- ing activation of the proapoptotic proteins APAF-1, P73 [both points are often faulty or absent, cells can die by apoptosis after are driven by E2F1 expression (Castedo et al., 2004)] and repeated mitotic divisions and so this does not imply that abro- PML, and activation of the CDC25A and CDC25C phospha- gated checkpoints will automatically lead to mitotic catastro- tases to halt the cell cycle (Castedo et al., 2004). It seems more phe (Vakifahmetoglu, Olsson and Zhivotovsky, 2008). likely that apoptosis has occurred through one of these mech- anisms than through the P53 pathway. The ultimate fate for the majority of giant cells is death. Mitotic catastrophe and apoptosis share some features and As the above discussion indicates, aberrant morphology differ in others (Vakifahmetoglu, Olsson and Zhivotovsky, and giant cells seem to be the normal product of cancer phys- 2008). Previous studies show that caspases are not required iology. The reduced viability and increased apoptosis levels for initiation of mitotic catastrophe, but are required for its could be considered an extension of this. The results suggest termination, suggesting it is a ‘pre-stage’ of apoptosis that NEAT1 knockdown increases the appearance of this (Vakifahmetoglu, Olsson and Zhivotovsky, 2008). Castedo aberrant morphology and the number of giant cells, possibly et al. (2004) indicate that although cell death by apoptosis by causing the premature release of paraspeckle-retained can follow mitotic catastrophe, death by necrosis can also components that, for example, may progress the cell through occur. Similar morphology has been observed and could be a its cell cycle. product of aneuploid instability (Vakifahmetoglu, Olsson In conclusion, the present study provides evidence that and Zhivotovsky, 2008; Horbay and Stoika, 2011). NEAT1 knockdown causes an increase in the number of cells In the present study, a prominent feature of these giant displaying atypical morphology in the BJAB cell line, which cells is the presence of cytoplasmic bodies that appear to be ultimately may affect cell fate and survival. These observa- vacuoles (Figs 7B and C, 8B and D and 9B). While vacuoles tions should stimulate further investigation into the potential classically appear as part of autophagy (Mansilla, Bataller involvement of NEAT1 dysregulation in leukaemias and lym- and Portugal, 2006), other studies have shown that they also phomas, and other cancers. occur in certain Burkitt’s lymphomas (Liu et al., 2007) and more readily in Epstein-Bar Virus-negative Burkitt’s lym- Acknowledgements phoma cells (Ishii et al., 1997). Without further analysis, it is The author would like to acknowledge the following people hard to define the etiology of these vacuoles, it appears that for their advice and guidance through the course of the proj- NEAT1 knockdown may have influenced their appearance. ect: Dr Mirna Mourtada-Maarabouni (project supervisor Cell viability results show that NEAT1 siRNA transfected and for performing the confocal microscopy), Dr Mark cells were consistently less viable than the control sample. Prichard (lab procedure guidance), Prof Gwyn Williams, This indicates that NEAT1 knockdown may adversely effect Karen Walker (for performing the electron microscopy and cell survival. This contrasts with studies by Nakagawa et al. imaging) and fellow students Dave Lambley, Jay Coombes (2011), who show that NEAT1 − /− mice are still viable and and Elizabeth Clipson. fertile under laboratory growth conditions concluding that paraspeckles are non-essential nuclear bodies. In light of the Funding above discussion concerning giant cells and their fate, it may be the case that paraspeckle loss in combination with other This project was supported by the School of Life Sciences, factors is the cause of the reduced viability. Keele University, Keele, Staffs, UK. The results also indicate that there is a consistently higher amount of apoptosis in the NEAT1 siRNA transfected cells. Author biography Considering the cell viability results and the above discussion regarding giant cells, this may not come as a surprise; C.H. studied BSc (Hons) Biomedical Science at Keele however, it is significant to note that P53 is often dysfunc - University, achieving a first class degree and graduating top tional in Burkitt-type lymphomas (Smardova et al., 2008). of the class. The project was completed as a contribution to 10 Bioscience Horizons • Volume 6 2013 Research article Hutchinson, J. N., Ensminger, A. W., Clemson, C. M. et al. (2007) A screen the degree and was chosen due to an interest in blood malig- for nuclear transcripts identifies two linked noncoding RNAs associ - nancies and the techniques of RNA interference. Other fields ated with SC35 splicing domains, BMC Genomics, 8 (39). of particular interest include endocrinology, immunology and the potential applications of stem cell engineering and Ishii, H., Gobe, G., Joshita, T. et al. 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(2009) MEN ε/β noncoding RNAs are essential for structural integrity of nuclear paraspeckles, Proceedings Wapinski, O. and Chang, H. Y. (2011) Long noncoding RNAs and human from the National Academy of Science of the United States of disease, Trends in Cell Biology, 21 (6), 354–361. America, 106, 2525–2530. Woodgett, J. R. and Jin, J. (2005) Chronic activation of protein kinase Smardova, J., Grochova, D., Fabian, P., et al. (2008) An unusual p53 muta- Bb/Akt2 leads to multinucleation and cell fusion in human epithe- tion detected in Burkitt’s lymphoma: 30 bp duplication, Oncology lial kidney cells: events associated with tumorigenesis, Oncogene, Reports, 20, 773–778. 24, 5459–5470. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Preliminary investigation of the effects of silencing the non-coding RNA, NEAT1, on the Burkitt's lymphoma cell line BJAB

Bioscience Horizons , Volume 6 – Jun 7, 2013

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Abstract

BioscienceHorizons Volume 6 2013 10.1093/biohorizons/hzt006 Research article Preliminary investigation of the effects of silencing the non-coding RNA, NEAT1, on the Burkitt’s lymphoma cell line BJAB Christopher Halford School of Life Sciences, Huxley Building, Keele University, Keele, Staffordshire ST5 5BG, United Kingdom *Corresponding author: 17 Pelican Close, Crewe, Cheshire CW1 5YA, UK. Email: chrishalford27@gmail.com Project supervisor: Dr Mirna Mourtada-Maarabouni Nuclear enriched abundant transcript 1 (NEAT1) is a long non-coding RNA with two isoforms. Both are expressed constitu- tively and have been shown to play a crucial structural role in the formation of paraspeckles. Paraspeckles are subnuclear bodies which retain certain mRNAs, preventing their translation in the cytoplasm, thereby silencing the corresponding genes. This study aimed to assess the effects of knocking down NEAT1 by RNA interference on the Burkitt’s lymphoma cell line, BJAB. The results have shown that a targeted siRNA depletion of NEAT1 resulted in an increased number of cells displaying aberrant morphology, including the appearance of ‘giant cells’, multinucleated cells and cells with cytoplasmic vacuoles. Furthermore, NEAT1 down-regulation was accompanied with an increased level of apoptosis and decreased cell viability, assessed by acri- dine orange morphology staining, vital dye exclusion and (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) assay. Taken together, the study strongly suggests that modulation of NEAT1 expression has a significant effect on the BJAB cell line. Such remarkable effects of changes of NEAT1 expression on the control of cell morphol - ogy reported here indicate the potential significance of this gene in the development and progression of cancer and highlight the importance of further investigation into the role of NEAT1 dysregulation in autoimmune disease and oncogenesis. Key words: lncRNA, NEAT1, MEN, BJAB, lymphoma, cancer Received 12 November 2012; revised 27 March 2013; accepted 30 April 2013 Introduction generating a short, poly-A rich tract instead of the classical poly-A tail. This also generates a smaller, independent tRNA- Nuclear enriched abundant transcript 1 (NEAT1) or multiple like molecule called menRNA (Sunwoo et al., 2009; Novikova endocrine neoplasia ε/β, a long non-coding RNA (lncRNA), is Hennelly, and Sanbonmatsu, 2012). NEAT1 is widely expressed polyadenylated (poly-A) and encoded on chromosome 11q13.1. in many tissue types, especially in the ovary, prostrate, colon It is produced from a single, intergenic exon and has two small and pancreas (Hutchinson et al., 2007) and Sasaki et al. (2009) regions that are highly conserved within the mammalian lin- report that both isoforms are expressed at similar levels. eage (Hutchinson et al., 2007). Two isoforms exist: a smaller NEAT1 lncRNAs are up-regulated upon differentiation of 3.7 kb isoform (MEN ε) and a larger 23 kb isoform (MEN β) human embryonic stem cells (Chen and Carmichael, 2009), (Hutchinson et al., 2007; Clark and Mattick, 2011). Both share muscle differentiation (Sunwoo et al., 2009) and in vitro neuro- a single transcriptional start site, and MEN β is a continuation nal differentiation (Mercer et al., 2010). of the shorter MEN ε isoform (Hutchinson et al., 2007). MEN β contains a conserved 3′ cloverleaf secondary structure, which Numerous studies show that the NEAT1 lncRNAs are is cleaved by RNaseP to form the 3′ end of the mature transcript, essential for the formation and structural integrity of © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Research article Bioscience Horizons • Volume 6 2013 subnuclear bodies called ‘paraspeckles’ (Bond and Fox, 2009; implicated in amyotrophic lateral sclerosis and frontotemporal Clemson et al., 2009; Sasaki et al., 2009; Sunwoo et al., lobar degeneration (Tollervey et al., 2011). The significance of 2009; Fox and Lamond, 2010). Paraspeckles are ‘dynamic’, these results is unclear; the NEAT1 knockout mice are viable, because the nucleating bodies, the NEAT1 lncRNAs (Bond despite the absence of paraspeckles (Nakagawa et al., 2011). and Fox, 2009; Clemson et al., 2009), are unstable (Clark Despite this, due to the roles that NEAT1 has been implicated et al., 2012) and maintenance of the paraspeckle requires in, it may still have value as a diagnostic or therapeutic target that they are continuously transcribed (Nakagawa and (Lipovich, Johnson and Lin, 2010). Hirose, 2012). Paraspeckles are ribonucleoprotein complexes Since the significance of changes in the level of NEAT1 characterized by the NEAT1 lncRNAs and three RNA- expression have not yet been investigated, we were prompted binding proteins, P54, PSF and PSP1, members of the to analyse the functional effects of silencing NEAT1 on the Drosophila melanogaster behaviour, human splicing (DBHS) B-cell lymphoma cell line, BJAB using RNA interference. These family (Bond and Fox, 2009; Novikova, Hennelly and analyses clearly suggest a potential role for NEAT1 in the con- Sanbonmatsu, 2012; Nakagawa and Hirose, 2012). The trol of cell morphology and, consequently, cell function. function of paraspeckles is not fully understood. Prasanth et al. (2005) suggest that paraspeckles retain and store ade- nosine to inosine hyper-edited mRNAs, silencing the corre- Materials and methods sponding gene. Nakagawa and Hirose (2012) note that P54 Materials preferentially recognizes A-to-I edited RNAs. Others suggest that the function of paraspeckles is to localize/sequestrate its NEAT1 siRNA1 (ID: S238175), NEAT1 siRNA2 (ID: protein components, many of which have functions outside S238174) and NEAT1 siRNA3 (ID: S238176) from Life of the nucleus (Nakagawa and Hirose, 2012). Technologies (Ambion), catalogue no.: 4399665 Custom Synthesis (named to differentiate them from each other). Paraspeckle assembly starts with the targeting of newly syn- siRNA1 targets nucleotide 1352, near the centre of the thesized NEAT1 by DBHS dimers (Bond and Fox, 2009). The NEAT1 transcript; siRNA2 targets nucleotide 3373, towards NEAT1–DBHS complexes build up the paraspeckle; the fin - the 5′ end of the transcript and siRNA3 targets nucleotide ished structure consisting of multiple RNA–DBHS complexes, 120 near to the 3′ end of the transcript. Control siRNA from facilitated by DBHS oligomerization. Although both isoforms Life Technologies (Ambion), catalogue no.: AM4635. This is have structural roles, their overlap is unclear. The smaller an RNA molecule designed to have no significant sequence MEN ε transcript may have the ability to form large structural similarity to human sequences used to ensure that observed complexes by itself, as NEAT1 complexes form quickly at the effects from RNAi were not by-products of the transfection transcription site, before PSP1 or P54 are detectable within process (Ambion, Inc., 2007). Cy3-siRNA Labelling Reagent them (Clemson et al., 2009). The MEN ε transcript is self- also from Life Technologies (Ambion), catalogue no.: complementary in many places, which may allow the forma- AM1632. RPMI-1640 medium from Sigma-Aldrich, cata- tion of intramolecular and intermolecular structures (Clemson logue no.: R0883; Phenol Red, 200 mM l-glutamine, 10% et al., 2009). However, MEN β preferentially associates with foetal calf serum, 100 mg streptomycin and 100 units of pen- DBHS proteins in vivo, indicating that MEN β forms the core icillin/ml were pre-added to indicate pH, provide growth- of the paraspeckle and then recruits MEN ε (Sasaki et al., encouraging factors and prevent bacterial contamination, 2009). Souquere et al. (2010) show that MEN ε and the 3′ end respectively. OptiMEM and GlutaMAX solution from Life of MEN β are localized to the periphery of the paraspeckle, Technologies (Invitrogen), catalogue no.: 51985-026 (Lot: and the central sequence of MEN β runs through the centre of 905272). Iscove’s Modified Dulbecco’s Medium from Sigma- the paraspeckle. They also show that while the MEN ε overex- Aldrich, catalogue no.: I3390. Solution V from Lonza, as pression increases the number of paraspeckles, and transient part of Amaxa Nucleofector transfection kit. CellTiter 96 overexpression does not rescue a total NEAT1 knockdown, solution for MTS assay from Promega, catalogue no.: G3580 the MEN β knockdown causes only residual paraspeckles to (Lot: 326240). Trypan blue powder from Sigma-Aldrich, remain. At present, it is currently unclear how the structural catalogue no.: T6146. Acridine orange powder from Sigma- roles of each isoform overlap. Aldrich, catalogue no.: A6014. Various non-coding RNA molecules, along with subnuclear disorganization, have been implicated in a number of diseases Methods (Mehler and Mattick, 2007; O’Rourke and Swanson, 2009; Cell culture Qureshi, Mattick and Mehler, 2010; Gibb et al., 2011; BJAB cell lines were maintained in the RPMI-1640 medium Wapinski and Chang, 2011; Johnson, 2012). This suggests (sigma) in a 1:9 ratio. Cells were incubated at 37°C and 5% that aberrant NEAT1 function may be associated with disease. CO . NEAT1 is down-regulated in lung, liver, oesophageal and reti- 2 nal cancers (Gibb et al., 2011) and is a novel target of P53 Preparation of Cy3 labelling reagent (Lipovich, Johnson and Lin, 2010). The MEN ε isoform is up- Hundred microlitres of reconstitution solution was added to regulated in the Huntington’s caudate nucleus and the nucleus dry Cy3 labelling reagent (Ambion). Solution was vortexed accumbens of heroin users (Johnson, 2012). NEAT1 is also 2 Bioscience Horizons • Volume 6 2013 Research article and after 5 min was re-vortexed to ensure full suspension. carded and the pellet washed with 1 ml of phosphate-buff- Labelling reagent was then stored in the dark at −20°C. ered saline (PBS). The solution was centrifuged (settings above) and washed with 1 ml PBS. The whole sample was Labelling siRNA pipetted onto a microscope slide, a cover slip applied and To eppendorf tubes, 18.3 µ l nuclease-free water was added, viewed under a microscope, with normal light and fluores - followed by 5 µ l 10× labelling buffer, 19.2 µ l siRNA (20 µ M) cence to estimate the transfection efficiency. and finally 7.5 µ l labelling reagent. Finally, the solution was MTS assay incubated at 37°C in the dark for 1 h. The reagent was then stored at −20°C. CellTiter 96 solution was removed from freezer and allowed to thaw. BJAB suspensions in a 6-well plate were gently agitated to Cell count mix. In a 96-well plate, 100 µ l of transfected BJAB suspension Cell suspension was agitated gently to ensure mixing. Twenty was added to wells from each siRNA used. This was repeated microlitres of suspension was transferred to a 96-well plate. three times. Twenty microlitres of the CellTiter 96 solution was Twenty microlitres of trypan blue was added and mixed gen- added to each 96-well plate suspension. The 96-well plate was tly. Cells were counted under microscopy. incubated at 37°C for 2 h. Absorbance readings were taken at 490 nm wavelength on a Victor Wallac 1420 machine. Electroporation Acridine orange (% apoptosis) Cells were counted, and the volume of suspension required for electroporation was calculated (10–20 × 10 cells/ml minimum). One millilitre samples of transfected BJAB suspensions, one Suspension was centrifuged at 1200 rpm (220 g) and room tem- from each siRNA used, were centrifuged at 2000 rpm (600 g) perature (23–25°C) for 8 min. Supernatant was discarded and the for 5 min. Most of the supernatant was discarded and the pellet pellet suspended in 5 ml Optimem (Invitrogen). The solution was resuspended in the remaining solution. Twenty microlitres of centrifuged again (settings above). Supernatant was then dis- the solution was added to a slide to which 5 µ l of acridine carded and the pellet resuspended in 1.2 ml Optimem (400 µ l for orange (50 µ g/ml) was added. Acridine orange is fluorescent, each electroporation). Four hundred microlitres of the solution nucleic-acid selective dye that interacts with DNA by intercala- was added to one electroporation cuvette. siRNA of 1.56 µ l to be tion, emitting green fluorescence. A cover slip was placed on the used was added to the cuvette. The electroporation cuvettes were slide and was viewed under a microscope with fluorescence. allowed to rest for 10 min, and 5 ml of Iscove’s Modified Preparation of cells for transmission electron microscopy Dulbecco’s Medium (Sigma-Aldrich) was added to wells in a (TEM) 6-well plate. After 10 min, the cuvettes were placed in the BioRad Gene Pulser II machine and electroporated at 1050 µ f capacitance The cells were counted and centrifuged (settings as above). The and 292 V. Ten minutes after electroporation, the solutions from supernatant was discarded and the pellet resuspended in 2.5% each cuvette were pipetted into separate wells in the 6-well plate. glutaraldehyde tannic acid (GTA) and incubated at 37°C for The plate was then incubated at 37°C and 5% CO for 48 h. 1 h. Glutaraldehyde is a popular fixing agent for electron microscopy but alone does not provide adequate preservation Nucleofection of cellular structures. In combination with tannic acid and Cells were counted and the volume required for nucleofection heavy metal treatment, the cellular structures are protected was calculated (2 × 10 cells/ml minimum). This solution was from shrinkage by dehydration and drying (Svitkina and centrifuged at 1500 rpm (350 g) and room temperature for Borisy, 1998). The cells were span down, supernatant dis- 8 min and 1.5 ml of Iscove’s Modified Dulbecco’s Medium was carded and the pellet resuspended in 0.5–1% GTA to store as added to wells in a 6-well plate, and the plate was incubated at needed. For use: the 0.5–1% GTA supernatant was discarded 37°C. After 8 min, the supernatant was discarded and the pel- and 1 ml of sodium cacodylate buffer (pH 7.4) with 2 mM let suspended in 0.3 ml Solution V (Lonza) (100 µ l for each calcium chloride was added. The solutions were vortexed to nucleofection). Hundred microlitres of solution was trans- disperse the cells and were centrifuged. Osmium tetroxide, ferred to an eppendorf tube, where 0.39 µ l of the siRNA to be which acts as a fixing agent and stain ( Bozzola and Russell, transfected was added and mixed. The mixture was pipetted 1999), was added to the solutions, and then vortexed, to dis- into nucleofection cuvettes. These cuvettes were placed into perse the cells. The solutions were left to stand for 1 h. After the Lonza Nucleofector Device II machine, and nucleofected 1 h, the solutions were centrifuged to remove the excess using program T-001. A small amount of the incubated Iscove’s osmium. One millilitre of sodium cacodylate buffer was added Modified Dulbecco’s Medium was pipetted into each cuvette. and the solutions were centrifuged. The supernatant was dis- Using a fine pipette, the entire solution in the cuvettes was carded and liquid agar was added to the eppendorf tubes. transferred to separate wells in the 6-well plate. The plate was Finally, they were dehydrated using 70% ethanol. then incubated at 37°C and 5% CO for 48 h. Statistical analysis Fluorescent microscopy (transfection efficiency) All statistical analyses were carried out using a two-sample 1 ml of Cy3-labelled negative control siRNA was centrifuged t-test at a 95% confidence level (unless stated otherwise). at 2000 rpm (600 g) for 8 min. The supernatant was dis- All results are ±standard error of the mean. 3 Research article Bioscience Horizons • Volume 6 2013 conductive pore produced by the field exceeds a certain, crit - Results ical size (dependent on local factors such as mechanical stress and lipid bilayer edge energy) it can rapture the bilayer, lead- To determine the most efficient method of siRNA transfec - ing to cell death (Joshi and Schoenbach, 2000). The effect of tion, two techniques were investigated. Electroporation and each transfection method on cell viability was, therefore, also nucleofection both move charged molecules into the cell, via investigated. water-filled membranous pores induced by an applied electri - cal field ( Neumann et al., 1982; Melikov et al., 2001). Figure 1 shows that the average transfection efficiency for Electroporation is a general technique that can be used to electroporation was lower than that for nucleofection. transfect cells with any serum-free cell medium. Nucleofection, Transfection by nucleofection had a higher efficiency and was on the other hand, uses cell-type specific reagents and electri - more consistent than transfection by electroporation. Cell cal parameters and transfers the molecules directly into the viability was determined by vital dye exclusion, 72 and 96 h nucleus. These are ‘harsh’ techniques and have some detri- post-transfection, as shown in Fig. 2. At 72 h cell viability mental effects regarding cell survival, more specifically, if the Figure 1. The average transfection efficiency (%) for the methods of electroporation (blue) and nucleofection (red). * P < 0.05. Figure 2. The average cell viability for the methods of electroporation (yellow) and nucleofection (green), at 72 and 96 h post-transfection, allowing 48 h for RNA knockdown to occur. 4 Bioscience Horizons • Volume 6 2013 Research article was higher for the nucleofected cells. At 96 h, cell viability was also higher for the nucleofected cells. Nucleofection was chosen as the method for siRNA transfection for its higher efficiency and higher cell viability rates. Transfection with the NEAT1 siRNAs caused down-regu- lation of NEAT1 by 65–75% as was confirmed by qRT-PCR, performed in a subsequent study using the same methodol- ogy (Dr Mourtada-Maarabouni, personal communication). It is unclear how the silencing affected the different isoforms of the NEAT1 transcript. Cell viability was measured to determine the number of living cells present in suspension. This indicates whether transfection of NEAT1 siRNAs has any direct consequences for cell survival. Figure 3 shows cell viability measured by vital dye exclusion using trypan blue. Non-viable cells cannot remove the dye, appearing blue under microscopy. Overall, the samples transfected with NEAT1 siRNAs showed a decreased cell viability. At 72 h, the samples trans- fected with the NEAT1 siRNAs had a lower viability than the respective controls. At 96 h, NEAT siRNAs 2 and 3 had a lower viability than their controls, and NEAT1 siRNA1 had an equal viability with the negative control siRNA. These results indicate that NEAT1 knockdown may adversely affect cell viability. Cell viability was also measured by an MTS assay. The MTS dye (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth- oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) is reduced by mitochondrial enzymes to a chromogenic formazan dye. As mitochondrial components are involved, the MTS assay is also an indicator of metabolic activity. A higher the rate of activity produces more formazan dye; therefore the activity of the cell can be measured colourimetrically. The results of the MTS assays (Fig. 4) were similar to the vital dye exclusion. At 72 h, NEAT1 siRNAs 1 and 3 had lower absorbance readings than the respective controls, indi- cating less viability. NEAT1 siRNA2 had a higher absorbance reading compared with its control sample; however, the error bar for the control exceeds the result and error bar of the siRNA sample, indicating the possibility that the control sample had a higher viability. At 96 h, all NEAT1 siRNAs had lower absorbance values than the respective controls. Overall, these results similarly indicate that cells transfected with NEAT1 siRNA had decreased viability. BJAB cells were stained with acridine orange and their mor- phology was viewed under microscopy. The morphology of a cell is a good indicator of its health, and any abnormal features can indicate a pathological process. The number of cells under- Figure 3. T he viable cell number for BJAB suspensions transfected going apoptosis and the number of giant/multinucleated cells with NEAT1 siRNAs, 72 and 96 h post-transfection, as measured by vital were counted (Figs 5 and 6) and interesting morphological dye exclusion. The respective negative controls (blue) are shown with features (such as the appearance of cytoplasmic vacuoles, giant (A) NEAT1 siRNA1 (red), (B) NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). *P < 0.05. cells and multinucleated cells) were observed (Fig. 7). At 72 and 96 h post-transfection, all samples transfected undergoing apoptosis, compared with controls. These results with NEAT1 siRNA contained a higher number of cells clearly demonstrate that samples transfected with NEAT1 5 Research article Bioscience Horizons • Volume 6 2013 Figure 4. The cell viability for BJAB suspensions transfected with NEAT1 siRNAs, 72 and 96 h post-transfection, as measured by the MTS assay. Higher absorbance readings correspond with increased viability. The respective negative controls (blue) are shown with (A) NEAT1 siRNA1 (red), (B) NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). Figure 5. T he percentage of apoptosis present in BJAB suspensions transfected with NEAT1 siRNAs, 72 and 96 h post- siRNAs, contain a higher percentage of apoptotic cells. transfection, as determined by acrid orange morphology staining. The At 72 h, samples transfected with NEAT siRNAs 1 and 2 respective negative controls (blue) are shown with (A) NEAT1 siRNA1 clearly contain a higher number of giant cells with aberrant (red), (B) NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). morphology, compared with the respective control samples. *P < 0.05. 6 Bioscience Horizons • Volume 6 2013 Research article For cells transfected with NEAT1 siRNA3, at 72 h there were fewer cells with aberrant morphology than the control. At 96 h, the NEAT1 siRNA1 sample still clearly has a higher number of giant cells than its control. At 96 h, the samples transfected with siRNAs 2 and 3 contained fewer giant cells compared with the controls. Overall, samples transfected with the NEAT1 siRNAs all showed a marked increase in the percentage of apoptotic cells compared with controls. Although not as clear, NEAT1 knockdown also appears to influence the appearance of giant cells and aberrant morphology. Figures 7 and 8 clearly show atypical cells with irregular morphology. Figure 8B and D indicate the formation of ‘spaces’ inside the cytoplasm, bordered by a membrane-like structure on at least one side. In Figure 8B, these ‘spaces’ almost appear like swollen mitochondria, the entities inside could be the cristae of the inner-membrane. Some of these ‘spaces’, however, are bordered completely and have no inner ‘artefacts’ and appear like vacuoles (Fig. 8D). Another fea- ture of Fig. 8 is the aberrant nuclear morphology. Figure 8D illustrates this well, with a large ‘U-shaped’ nucleus with a smaller nuclear body that may have ‘budded off’ from the main nucleus. Although the cell in Fig. 8C also shows irregu- lar nuclei, the shape and position suggest that the cell is sim- ply undergoing mitosis. Another prominent example of irregular nuclear morphology is Fig. 7D, a giant cell with a huge number of nuclear entities. Figure 9 shows confocal microscopy images of BJAB cells transfected with (A) the negative control siRNA and (B) NEAT1 siRNA2. In the control sample, the cell nuclei are regular: large, mostly round, consistent densities and no obvious cytoplasmic aberrations. In the NEAT1 siRNA2 sample, cells display nuclei with irregular shape and inconsis- tent densities, cytoplasmic vacuole-like structures and a num- ber of the cells are multinucleate. These results suggest that aberrant morphology occurs more often in cells transfected with the NEAT1 siRNAs, compared with the negative control siRNA. This suggests that NEAT1 knockdown could influence the processes that cause this morphology to appear and, consequently, cell func- tion. Discussion We were prompted to analyse the functional effects of silenc- ing NEAT1 on the B-cell lymphoma cell line BJAB. The results show that cells in which NEAT1 was silenced, had lower viability, increased levels of apoptosis and more cells displayed atypical morphology. While the atypical morphol- Figure 6. The percentage of giant, multinucleate or otherwise ogy of the cells can be explained as a facet of cancer physiol- morphologically aberrant cells, present in BJAB suspensions ogy (see below), the increased number of cells cannot. This transfected with NEAT1 siRNAs, 72 and 96 h post-transfection, as may be explained by two general mechanisms: first, NEAT1 determined by acrid orange morphology staining. The respective knockdown may directly affect the malignancy of the cells. negative controls (blue) are shown with (A) NEAT1 siRNA1 (red), (B) This implies that paraspeckles loss may release mRNA that NEAT1 siRNA2 (yellow) and (C) NEAT1 siRNA3 (green). 7 Research article Bioscience Horizons • Volume 6 2013 Figure 7. A selection of nucleofected BJAB cells stained with acridine orange and viewed under fluorescent microscopy. Magnification ×400. Transfected with (A and B) NEAT1 siRNA1, (C) NEAT1 siRNA2 and (D) NEAT1 siRNA3 and (E and F) negative control siRNA. (A) and (D) both show a huge cell with numerous subnuclear entities and (B) and (C) clearly show numerous vacuoles in the cytoplasm. The majority of cells in (E) and (F) display regular morphology. Figure 8. (A) Selection of nucleofected BJAB cells under electron microscopy (scale bar included). Transfected with the (A) negative control siRNA, (B) and (C) NEAT1 siRNA3 and (D) NEAT1 siRNA1. Aberrant morphology is apparent in (B) and (D), with ‘spaces’ forming in the cytoplasm and in (D), the nucleus appears disorganized. The cytoplasmic ‘spaces’ may represent swollen or burst mitochondria. 8 Bioscience Horizons • Volume 6 2013 Research article Figure 9. Confocal microscopy images of cells transfected with the (A) negative control siRNA and (B) NEAT1 siRNA2. In (A), there are very few cytoplasmic vacuole-like structures whereas in (B), many of these structures appear in a number of cells. In (B), it can also be seen that the nuclear morphology is irregular for many cells. codes for a mutated/oncogenic protein. Secondly, can revert back to normal parental cells. Apoptosis, mitotic NEAT1 knockdown could activate normal cellular pathways catastrophe and necrosis have been shown to follow the for- (e.g. cell-cycle progression), where the aberrant morphology mation of giant cells; however, some studies have shown that arises simply because the BJAB cell line is cancerous. This giant cells can escape cell death and give rise to new cancer implies that paraspeckle loss would release mRNA that is cells (Rengaswami et al., 2005, 2006). The physical cell produced by cells as part of their normal function. This latter enlargement can be induced by anti-mitotic/cancer agents mechanism is supported by the fact that NEAT1 has been that interfere with microtubule organization [(such as vin- found to be down-regulated in various cancers but not up- blastine (Horbay and Stoika, 2011) and docetaxel (Morse regulated (Gibb et al., 2011). et al., 2005)] and occurs during cell-cycle arrest. This is often preceded by mitotic catastrophe due to spindle disorganiza- It is significant to note that a number of cell-cycle compo - tion. Horbay and Stoika, 2011, speculate that the cell cycle of nents (cdk10, cyclin F, E3 ubiquitin-protein ligase) have alu cancer cells is halted in this giant state to survive unfavour- sequences in their mRNA transcript (Levy, Sela and Ast, able conditions. 2007), which could represent a target for A-I editing and sub- sequent nuclear retention (Chen, DeCerbo and Carmichael, Frequently, cancer cells are poly/aneuploid, associated 2008). In particular, cyclin F has an alu sequence in its 3′UTR with their genomic instability (Geddis, 2008). This is caused and is known to regulate the timing of the G /M transition by endomitosis, where mitosis occurs, but the cell fails to (Bai, Richman and Elledge, 1994; Tetzlaff et al., 2004). While undergo teleophase or cytokinesis, resulting in DNA repli- purely speculative, it is not entirely unreasonable to suggest cation but no cell division (Lee, Davidson and Duronio, that the mRNA of cell-cycle components/regulators is 2009). This explains the altered nuclear morphology in the retained in a paraspeckle and that NEAT1 knockdown dis- above figures. While endomitosis can give rise to the poly - rupts the cell cycle by allowing inappropriate mRNA export ploidization of giant cells, Vakifahmetoglu, Olsson and from the nucleus. This may accelerate cells through their cell Zhivotovsky (2008) state that it is endocycling (the uncou- cycle, giving rise to an increased number of cells displaying pling of DNA synthesis from cell division) which is the atypical morphology, as observed. cause of polyploidy. Another hypothesis for giant cell for- mation, is simply formation via cell fusion, which could Perhaps the most interesting phenomenon observed dur- occur spontaneously (Woodgett and Jin, 2005). There is ing this study was the presence of ‘giant cells’. These gigantic evidence for and against both mechanisms of giant cell for- cells had irregular nuclear morphology and size and were mation; however, endocycling seems to be more likely for often multinucleated (see Fig. 7). Shown by the figures above, two main reasons: First, endocycling has been observed giant cells occur more readily in samples transfected with the widely in tumours/cancer cells, particularly in those NEAT1 siRNAs, suggesting that NEAT1 knockdown may with dysfunctional P53 (Vakifahmetoglu, Olsson and influence their appearance. Without further analysis, it is Zhivotovsky, 2008) and secondly, reports of spontaneous hard to define the etiology of these cells; however, several cell fusion in the BJAB cell line do not exist in the literature. hypotheses (below) have been put forward in the literature In light of its relation to mitotic catastrophe (see below) and which may be relevant. the consequences of aberrant cell-cycle progression, endo- Giant cells appear in most cancers (Horbay and Stoika, cycling seems a more likely explanation for multinucleate 2011). It has been suggested that they are terminal cells, but giant cell formation. 9 Research article Bioscience Horizons • Volume 6 2013 Implicated in the formation and fate of giant cells is a pro- Translocation of c-myc to immunoglobulin loci and its subse- cess known as mitotic catastrophe. While there is no clear defi - quent massive increase in expression drive excessive prolifera- nition of mitotic catastrophe, it is a form of cell death that tion through the E2F1 transcription factor. E2F1 expression shares some hallmarks of apoptosis (Castedo et al., 2004). It is stimulates concomitant expression of tumour suppressor ARF unclear whether mitotic catastrophe is a special case of apopto- P14 , which promotes P53 activation by binding MDM2 sis or pre-process that leads to cell death by apoptosis or necro- (Bates et al., 1998). Because oncogene expression seemingly sis. Some studies have even shown mitotic catastrophe to be a drives activation of P53, Burkitt’s lymphoma cell lines must survival mechanism for tumour cells and a way of switching have a mutation in P53 (Hollstein et al., 1991) or at least the ARF from an abnormal cell cycle to mitosis (Castedo et al., 2004). P14 -MDM2-P53 pathway (Smardova et al., 2008). Cells cannot undergo mitotic catastrophe without prematurely Although P53 is considered the ‘guardian of the genome’, entering mitosis, making the abrogation of the G or G check- there are other ways to halt the cell cycle and initiate apopto- 1 2 points in the cell-cycle essential (Vakifahmetoglu, Olsson and sis (Vakifahmetoglu, Olsson and Zhivotovsky, 2008), includ- Zhivotovsky, 2008). In tumour cells, where cell-cycle check- ing activation of the proapoptotic proteins APAF-1, P73 [both points are often faulty or absent, cells can die by apoptosis after are driven by E2F1 expression (Castedo et al., 2004)] and repeated mitotic divisions and so this does not imply that abro- PML, and activation of the CDC25A and CDC25C phospha- gated checkpoints will automatically lead to mitotic catastro- tases to halt the cell cycle (Castedo et al., 2004). It seems more phe (Vakifahmetoglu, Olsson and Zhivotovsky, 2008). likely that apoptosis has occurred through one of these mech- anisms than through the P53 pathway. The ultimate fate for the majority of giant cells is death. Mitotic catastrophe and apoptosis share some features and As the above discussion indicates, aberrant morphology differ in others (Vakifahmetoglu, Olsson and Zhivotovsky, and giant cells seem to be the normal product of cancer phys- 2008). Previous studies show that caspases are not required iology. The reduced viability and increased apoptosis levels for initiation of mitotic catastrophe, but are required for its could be considered an extension of this. The results suggest termination, suggesting it is a ‘pre-stage’ of apoptosis that NEAT1 knockdown increases the appearance of this (Vakifahmetoglu, Olsson and Zhivotovsky, 2008). Castedo aberrant morphology and the number of giant cells, possibly et al. (2004) indicate that although cell death by apoptosis by causing the premature release of paraspeckle-retained can follow mitotic catastrophe, death by necrosis can also components that, for example, may progress the cell through occur. Similar morphology has been observed and could be a its cell cycle. product of aneuploid instability (Vakifahmetoglu, Olsson In conclusion, the present study provides evidence that and Zhivotovsky, 2008; Horbay and Stoika, 2011). NEAT1 knockdown causes an increase in the number of cells In the present study, a prominent feature of these giant displaying atypical morphology in the BJAB cell line, which cells is the presence of cytoplasmic bodies that appear to be ultimately may affect cell fate and survival. These observa- vacuoles (Figs 7B and C, 8B and D and 9B). While vacuoles tions should stimulate further investigation into the potential classically appear as part of autophagy (Mansilla, Bataller involvement of NEAT1 dysregulation in leukaemias and lym- and Portugal, 2006), other studies have shown that they also phomas, and other cancers. occur in certain Burkitt’s lymphomas (Liu et al., 2007) and more readily in Epstein-Bar Virus-negative Burkitt’s lym- Acknowledgements phoma cells (Ishii et al., 1997). Without further analysis, it is The author would like to acknowledge the following people hard to define the etiology of these vacuoles, it appears that for their advice and guidance through the course of the proj- NEAT1 knockdown may have influenced their appearance. ect: Dr Mirna Mourtada-Maarabouni (project supervisor Cell viability results show that NEAT1 siRNA transfected and for performing the confocal microscopy), Dr Mark cells were consistently less viable than the control sample. Prichard (lab procedure guidance), Prof Gwyn Williams, This indicates that NEAT1 knockdown may adversely effect Karen Walker (for performing the electron microscopy and cell survival. This contrasts with studies by Nakagawa et al. imaging) and fellow students Dave Lambley, Jay Coombes (2011), who show that NEAT1 − /− mice are still viable and and Elizabeth Clipson. fertile under laboratory growth conditions concluding that paraspeckles are non-essential nuclear bodies. In light of the Funding above discussion concerning giant cells and their fate, it may be the case that paraspeckle loss in combination with other This project was supported by the School of Life Sciences, factors is the cause of the reduced viability. Keele University, Keele, Staffs, UK. The results also indicate that there is a consistently higher amount of apoptosis in the NEAT1 siRNA transfected cells. Author biography Considering the cell viability results and the above discussion regarding giant cells, this may not come as a surprise; C.H. studied BSc (Hons) Biomedical Science at Keele however, it is significant to note that P53 is often dysfunc - University, achieving a first class degree and graduating top tional in Burkitt-type lymphomas (Smardova et al., 2008). of the class. The project was completed as a contribution to 10 Bioscience Horizons • Volume 6 2013 Research article Hutchinson, J. N., Ensminger, A. W., Clemson, C. M. et al. (2007) A screen the degree and was chosen due to an interest in blood malig- for nuclear transcripts identifies two linked noncoding RNAs associ - nancies and the techniques of RNA interference. Other fields ated with SC35 splicing domains, BMC Genomics, 8 (39). of particular interest include endocrinology, immunology and the potential applications of stem cell engineering and Ishii, H., Gobe, G., Joshita, T. et al. 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Journal

Bioscience HorizonsOxford University Press

Published: Jun 7, 2013

Keywords: lncRNA NEAT1 MEN BJAB lymphoma cancer

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