Differential Effects of WRAP53 Transcript Variants on the Biological Behaviours of Human Non-Small Cell Lung Cancer Cells


 Background: The WD40-encoding RNA antisense to p53 (WRAP53) gene, an antisense gene of TP53, has 3 different transcriptional start sites that yield 3 transcript variants. One of these variants WRAP53-1β encodes a WD repeat-containing protein WRAP53β, whereas WRAP53-1α is a noncoding RNA that regulates p53 mRNA levels. These variants are involved in the progression of non-small cell lung cancer (NSCLC). However, how the different transcript variants regulate NSCLC cell behaviours is to be elucidated.Methods: Wild-type p53 NSCLC A549 cells and p53-mutated H1975 cells were transfected with WRAP53-1α and WRAP53-1β siRNAs, and their behaviours were examined colony formation, cell viability, apoptosis, cell cycle, wound healing, and cell invasion assays.Results: WRAP53-1α knockdown increased the mRNA and protein levels of p53, whereas depletion of WRAP53-1β had no effect on p53 expression. WRAP53-1α knockdown suppressed colony formation and proliferation of A549 cells, but had the opposite effects on H1975 cells. However, WRAP53-1β knockdown promoted A549 cell growth. Depletion of WRAP53-1α and WRAP53-1β promoted apoptosis in A549 but not H1975 cells. WRAP53-1α knockdown increased the proportion of A549 but not H1975 cells at the G0/G1 phase. However, WRAP53-1β knockdown decreased the proportion of cells at the G0/G1 phase in A549 cells. Depletion of WRAP53-1α suppressed A549 cell migration and invasion, and promoted H1975 cell migration and invasion. However, depletion of WRAP53-1β had the opposite effects.Conclusions: The 2 WRAP53 transcript variants exerted opposite functions in NSCLC cells and regulated NSCLC cell behaviours in a p53-dependent manner.

variant corresponds to the rst exon of p53 in a cis-antisense manner. WRAP53-1α is an antisense transcript that stabilizes TP53 [8]. The WRAP53-1β transcript is not complementary to TP53, but encodes the WD repeat-containing protein WRAP53β, also known as WD repeat domain 79 and telomerase Cajal body protein 1, which is involved in multiple cellular processes. WRAP53β was reported to regulate the maintenance of the nuclear organelles known as Cajal bodies via recruiting motor neuron proteins, small Cajal body-speci c RNAs, and telomerases [9][10][11]. WRAP53β also targeted telomerase to telomeres, promoting their elongation [9]. In addition, WRAP53β was reported to facilitate DNA damage repair via recruiting the ubiquitin ligase ring nger protein 8 to DNA breaks during both homologous recombination and non-homologous end joining. The effects of WRAP53β on DNA damage repair were p53-independent, as it also functioned in p53-de cient cells [12,13].
The effects of WRAP53 on cancer progression are controversial. WRAP53 is believed to be associated with cancer pathogenesis, because WRAP53-1α regulates the expression of TP53. Loss of WRAP53β impairs telomere maintenance and DNA repair, increasing genomic instability and the probability of carcinogenesis. It is conceivable that WRAP53-1α and WRAP53-1β play different roles in NSCLC cells. In order to test this hypothesis, we examined the distinct biological functions of these 2 WRAP53 transcript variants in cell function assays in vitro.

Methods
Cell culture NSCLC cell lines, A549 and H1975, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI-1640 medium (Hyclone, USA) supplemented with 10% foetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 mg/mL streptomycin (Sangon, China). All cells were maintained in a humidi ed incubator (Sanyo, Japan) at 37°C and 5% CO 2 .
RNA extraction and quantitative real-time polymerase chain reaction Total RNA was isolated using TRIzol (Vazyme, China). The RNA concentrations and the A260/A280 ratios were assessed with a Nanodrop spectrophotometer (Thermo, USA). Total RNA (500 ng) was reversetranscribed using the HiScript® Q RT SuperMix for qPCR kit (Vazyme) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ChamQ TM SYBR ® qPCR Master Mix (Vazyme) on the CFX Connect TM RealTime PCR Detection System (Bio-Rad, USA). Glyceraldehyde-3-phosphate dehydrogenase was used as the reference for normalization.
The relative fold change of mRNAs was calculated using the 2 −ΔΔCt method. The primers used for qRT-PCR are presented in Table 1.

RNA interference
Small interfering RNAs (siRNAs) targeting WRAP53-1α and WRAP53-1β [8] and negative control (NC) siRNAs were purchased from Genepharm Technologies (GenePharma, China). Transient transfection was performed using jetPRIME transfection reagent (Polyplus-transfection® SA, France) following the manufacturer's recommendations. The cells were transfected with NC or target siRNAs at 30-50% con uence. Immunoblotting Cells were lysed in RIPA cell lysis buffer (Beyotime, China) supplemented with phosphatase inhibitors and protease inhibitors (Sigma, USA). The lysates were then centrifuged (13300 rpm) at 4 °C for 15 min. The supernatants were collected, and protein concentration was measured using a bicinchoninic acid kit (Beyotime). Equal amount of proteins were applied to 10-12% sodium dodecyl sulphate-polyacrylamide separating gels and transferred to polyvinylidene uoride membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies at 4 °C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. An enhanced chemiluminescence kit (Bio-Rad) was used to detect the proteins under a gel imaging analyser (Bio-Rad). Antibodies used for immunoblotting are listed in Table 2.

Colony formation assay
Tumour cells were plated in 6-well plates (500 cells/well) and cultured for 2 weeks. Cell colonies were xed with 4% paraformaldehyde (Sangon) for 20 min and stained with 0.5% crystal violet (Beyotime) at room temperature for 30 min. After washing with water, the numbers of colonies in each well were counted under a microscope. This assay was repeated 3 times and performed in triplicates. Wound healing assay Cells were seeded in 6-well plates and transfected with NC or WRAP53 siRNAs. When the cells were con uent, they were scratched with a 10-μL pipette tip and washed with PBS gently to remove cell debris.

Cell viability assay
After that, 2 mL serum-free medium was added into each well, and pictures were taken using a phase contrast microscope (Nikon, Japan) at 0, 24, and 48 h. The migration rate was calculated as: Wound closure (%) = (distance of initial scratch -distance of closed scratch) / distance of initial scratch.

Cell invasion assays
Cells (1 × 10 5 cells/well) transfected with NC or WRAP53 siRNAs were seeded in the upper chambers of transwell units pre-coated with 100 μL Matrigel (Becton-Dickinson, USA, dilution 1:40). After incubation for 24 h, the lters were xed with 4% paraformaldehyde for 20 min and stained with 0.5% crystal violet for 20 min. The cells on the upper surface of the lter were completely removed using a cotton swab. The cells that invaded through the Matrigel and reached the lower surface of the lter were counted.
Experiments were independently repeated 3 times.

Statistical analysis
All statistical analyses were performed with GraphPad software. All data are presented as the mean ± standard deviation. Unpaired 2-tailed student's t-test was used to compare 2 groups. P < 0.05 was considered statistically signi cant.

Results
Knockdown of WRAP53-1α, but not WRAP53-1β, induced p53 expression Transcript-speci c siRNAs were designed to speci cally target WRAP53-1α and WRAP53-1β. There was no marked difference in WRAP53-1α mRNA levels between cells transfected with si-Control and si-WRAP531β, whereas there was signi cantly less WRAP53-1α mRNA in A549 and H1975 cells transfected with si-WRAP531α (P < 0.05; Figure 1A). Similarly, si-WRAP53-1β reduced WRAP53-1β RNA levels in A549 and H1975 cells (P < 0.01 and P < 0.05, respectively; Figure 1A). Knockdown of WRAP53-1α signi cantly increased TP53 mRNA levels in both A549 and H1975 cells, whereas knockdown of WRAP53-1β had no effect on TP53 levels ( Figure 1B). Immunoblotting showed that there was no difference in WRAP53β protein levels between cells transfected with si-Control and si-WRAP531α, whereas the protein levels of WRAP53β were decreased in cells transfected with si-WRAP531β. Moreover, knockdown of WRAP53-1α increased p53 protein levels in both A549 and H1975 cells, whereas knockdown of WRAP53-1β had no effect ( Figure 1C).
WRAP53-1α and WRAP53-1β had opposite effects on NSCLC cell growth and proliferation To investigate the functions of WRAP53-1α and WRAP53-1β in NSCLC cell growth, both A549 and H1975 cells were transfected with si-WRAP531α or si-WRAP531β. The colony formation assay revealed that knockdown of WRAP53-1α signi cantly decreased colony formation in A549 cells, whereas knockdown of WRAP53-1β signi cantly increased colony formation ( Figure 2A). However, in H1975 cells, knockdown of WRAP53-1α promoted colony formation, and knockdown of WRAP53-1β had no signi cant effect ( Figure 2B). The results of the cell viability assay indicated a signi cant decrease in A549 cell proliferation with WRAP53-1α knockdown, whereas WRAP53-1β de ciency signi cantly promoted A549 cell proliferation ( Figure 2C). In H1975 cells, knockdown of WRAP53-1α induced cell proliferation, and depletion of WRAP53-1β had no signi cant effect ( Figure 2C). These results suggested that WRAP53-1α and WRAP53-1β had opposite effects on NSCLC cell proliferation and function in a p53-dependent manner.
WRAP53-1α and WRAP53-1β de ciency induced apoptosis in NSCLC cells Flow cytometry was used to measure apoptosis in NSCLC cells transfected with si-WRAP531α or si-WRAP531β. Knockdown of either WRAP53-1α or WRAP53-1β induced A549 cells apoptosis (P < 0.05; Figure 3A), but had no signi cant effect on H1975 cells ( Figure 3B). Immunoblotting results indicated that knockdown of either WRAP53-1α or WRAP53-1β upregulated Bcl-2 associated X protein and downregulated B cell lymphoma 2 in A549 cells. However, these changes were mild in H1975 cells transfected with si-WRAP531α or si-WRAP531β ( Figure 3C). These results suggested that WRAP53-1α and WRAP53-1β negatively regulated apoptosis in cells with wild-type p53 (A549) but not in cells with mutant p53 (H1975).
WRAP53-1α and WRAP53-1β had opposite effects on cell cycle progression in NSCLC cells Cell cycle distribution was assessed by ow cytometry. In A549 cells, knockdown of WRAP53-1α induced a signi cant G0/G1 arrest, whereas knockdown of WRAP53-1β decreased the proportion of cells at the G0/G1 phase ( Figure 4A). However, in H1975 cells, knockdown of WRAP53-1α decreased the proportion of cells at the G0/G1 phase (P < 0.05), and depletion of WRAP53-1β had no signi cant effect on the cell cycle ( Figure 4B). To verify the impacts of WRAP53-1α and WRAP53-1β on the cell cycle, the levels of cell cycle regulation and checkpoint proteins were measured by immunoblotting. Cyclin-dependent kinase 4 (CDK4) was downregulated by knockdown of WRAP53-1α and upregulated by depletion of WRAP53-1β in A549 cells. However, in H1975 cells, the levels of CDK4 were increased by knockdown of WRAP53-1α, but not affected by depletion of WRAP53-1β ( Figure 4C). These results suggested that WRAP53-1α and WRAP53-1β had opposite effects on NSCLC cell cycle arrest, and the regulation was p53-dependent.
WRAP53-1α and WRAP53-1β had opposite effects on migration and invasion in NSCLC cells The impacts of WRAP53-1α and WRAP53-1β on NSCLC cells migration and invasion were examined by wound healing and modi ed Boyden chamber assays. Knockdown of WRAP53-1α signi cantly suppressed A549 cell migration, whereas WRAP53-1β de ciency promoted A549 cell migration ( Figure  5A). In H1975 cells, knockdown of WRAP53-1α induced cell migration (P < 0.05), and depletion of WRAP53-1β had no signi cant effect ( Figure 5A). The results of the modi ed Boyden chamber assay demonstrated that knockdown of WRAP53-1α reduced A549 cell invasion, whereas WRAP53-1β de ciency increased A549 cell invasion ( Figure 5B). In H1975 cells, knockdown of WRAP53-1α promoted cell invasion (P < 0.05), whereas depletion of WRAP53-1β had no signi cant effect ( Figure 5B). In addition, the results of immunoblotting indicated that matrix metalloproteinase 9 (MMP9) was downregulated by knockdown of WRAP53-1α and upregulated by depletion of WRAP53-1β in A549 cells. However, in H1975 cells, MMP9 was upregulated by knockdown of WRAP53-1α and not affected by depletion of WRAP53-1β ( Figure 5C). These results suggested that WRAP53-1α and WRAP53-1β had opposite effects on migration and invasion in NSCLC cells in a p53-dependent manner.

Discussion
In the present study, the 2 WRAP53 transcript variants, WRAP53-1α and WRAP53-1β, were knocked down in A549 cells with wild-type p53 and H1975 cells with mutated p53. WRAP53-1α not only regulates wildtype p53 expression, but also regulates mutant p53 expression. Knockdown of WRAP53-1α had antitumor effects in A549 cells, but had the opposite effects on H1975 cells. WRAP53-1α regulates NSCLC cell behaviours in a p53-dependent manner. WRAP53-1β does not regulate expression of either wild-type or mutant p53. WRAP53-1β acts as a tumour suppressor in A549 cells, but has no effect in H1975 cells.
Our results suggest that the 2 WRAP53 transcript variants have distinct effects on p53 and NSCLC cells.
Natural antisense transcripts (NATs), a type of long non-coding RNAs (lncRNAs), occur naturally and play important roles in carcinogenesis, invasion, and metastasis [14]. The lncRNA WRAP53-1α is a naturally occurring p53 antisense transcript that acts as a crucial effector in several cancers [15,16]. WRAP53-1α is upregulated by anti-cancer drugs, and miR-4732-5p has a binding site in the 5'-untranslated region of WRAP53 [17][18][19]. In addition, WRAP53-1α methylation is signi cantly associated with worse survival in NSCLC. It is worth noting that WRAP53-1α stabilizes TP53 mRNA to increase the tumour suppressor activity of wild-type p53, leading to better prognosis. However, downregulation of WRAP53-1α by promoter methylation does not affect survival in p53-mutated tumours [20]. These results suggest that WRAP53-1α regulates p53 signalling.
WRAP53β also acts as a tumour suppressor to regulate various cancer cellular activities [21]. The signi cance of WRAP53β in tissue homeostasis is demonstrated by the nding that inherited mutations in WRAP53β lead to telomere dysfunction and dyskeratosis congenita, increasing the risk of tumorigenesis [22]. Moreover, single nucleotide polymorphisms and downregulation of WRAP53β are associated with various sporadic forms of cancer, including breast and ovarian cancer [23][24][25][26]. In addition, WRAP53β downregulation is correlated with resistance of head and neck cancer to radiotherapy [27], as well as disruption of the DNA damage response in ovarian tumours [28].
Previous studies indicated that WRAP53β was a potential oncoprotein, whose overexpression led to transformation and promoted cancer cell survival, and whose downregulation induced massive cell death [29][30][31][32]. In addition, overexpression of WRAP53β was related to NSCLC progression. Knockdown of WRAP53β signi cantly inhibited NSCLC cell proliferation both in vitro and in vivo via inducing cell cycle arrest and apoptosis. WRAP53β induced cell cycle arrest at the G0/G1 phase and regulated the expression of G0/G1-related cyclins and cyclin-dependent kinase complexes. WRAP53β de ciency was also reported to induce apoptosis through the mitochondrial pathways [33]. WRAP53β colocalized and interacted with USP7, which reduced the ubiquitination of MDM2 and p53, thereby extending the half-life of these proteins and increasing their stability [34]. Moreover, WRAP53β exerted proliferative effects on NSCLC cells via stabilizing UHRF1 [35]. Recent studies revealed that WRAP53β might be related to p53 mutations and acted as an independent biomarker to predict poor prognosis of patients with surgically resected NSCLC[36].
The involvement of WRAP53 in disease progression is evident in lung cancer, but whether different targets of WRAP53 variants exert different biological functions still requires further investigation. In the present study, we used different variant-speci c siRNAs to knockdown WRAP53-1α and WRAP53-1β and evaluated the functions of these variants in NSCLC cells. First, signi cant associations were found between WRAP53-1α and p53 expression, whereas WRAP531β expression was not associated with p53 expression, which was consistent with previous reports [8]. However, knockdown of WRAP531α upregulated p53 in A549 and H1975 cells, which con icted with a previous study wherein WRAP53-1α stabilized p53 [8,20]. At the molecular level, NATs can have a concordant or discordant relationship with the sense transcript, and this interaction may result in higher stability or translational repression of the sense RNA [37]. Therefore, we believe that WRAP53-1α might play different roles based on the p53 mutation status.
We further demonstrated that WRAP53-1α had different effects on NSCLC cell behaviours. It is highly probable that the different mutation statuses of p53 led to these contradictory biological functions. The tumour suppressor p53 is a cellular gatekeeper that guards against genetic abnormality and instability via sensing multiple stress signals, including DNA damage and oncogene activation [38]. In addition, mutations in p53 usually result in increased half-life and nuclear accumulation, which can promote cancer progression via subverting multiple tumour suppression pathways [39]. In A549 cells, knockdown of WRAP53-1α induced upregulation of wild-type p53 and activation of cell cycle arrest and apoptosis. However, in H1975 cells with p53 mutations, the knockdown of WRAP53-1α exerted the opposite effects on cell cycle arrest and had no effect on apoptosis.
Cell function assays indicated that WRAP53-1β acted as a tumour suppressor in A549 cells. Knockdown of WRAP53-1β induced slight apoptosis and promoted the G1/S phase transition in A549 cells, but had no effect on H1975 cells. These ndings concerning the contribution of WRAP53β to cancer might occur in a p53-dependent manner, which was consistent with the results of animal models [36]. Inactivation of WRAP53-1β could help initiate tumour development by impairing telomere maintenance and DNA repair, leading to genomic instability. Our data suggested that WRAP53-1β was a putative tumour suppressor during the progression and metastasis of NSCLC, whereas WRAP53-1α might have a dual function. These ndings suggest potential interactions between the 2 transcript variants. This study provides a foundation for further examining the mechanisms by which WRAP53-1α and WRAP53-1β exert their functions in NSCLC, but more in vivo validation is needed.

Conclusions
In summary, our results indicated that different transcript variants of WRAP53 played different and even opposite roles in NSCLC cells. Speci cally targeting WRAP53 variants may contribute to NSCLC therapeutic strategies.  Table 2 Antibodies used in this study.

Supplementary Files
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