Background
MicroRNAs (miRNA) are endogenous, non-coding RNAs that direct gene repression by inhibiting the mRNA stability or translation [
1]. Evolutionarily conserved, they frequently target 3′ untranslated regions (UTRs) of target transcripts [
2]. A variety of cellular events such as proliferation, differentiation and apoptosis are controlled by miRNAs- thus implicating them in tumorigenesis [
3,
4]. Moreover, altered expression levels of miRNAs have been associated with myriad kinds of cancers [
4]. However, elucidation of molecular pathogenic pathways involving these miRNAs has not kept pace with the fast-increasing number of association studies between altered miRNA expressions and different types of cancers.
Meanwhile, a vast, complex network of signalling pathways mediate tumour progression and all such signals have to enter the nucleus through the only gateway- the nuclear pore complex (NPC). The NPCs regulate the nucleocytoplasmic trafficking of macromolecules and are massive protein complexes within the cell, composed of multiple copies of about 30 different nucleoporins (Nups) [
5,
6]. Cell division demands exquisite control over the arrangement and location of events and failure to observe this control has dire consequences, including chromosome mis-segregation and aneuploidy [
7]. The activation or inactivation of a series of mitotic regulators determines the timing, but the completion of one event [for example, nuclear envelope breakdown (NEBD)] might also provide a signal for the beginning of the next, maintaining a chronological sequence through mitosis. Unveiling of surprising links between the nuclear transport machinery, kinetochore and the spindle assembly checkpoint (SAC) besides canonical mitotic proteins [
8] support this idea [
9,
10]. Certain proteins that localize to the NPC in interphase can relocalize to the kinetochores during mitosis, wherein they are required for the alignment of chromosomes to the metaphase plate. The sequestration of kinetochore proteins at the NPC might therefore ensure that the kinetochores cannot function until NEBD has been completed [
11]. For instance, the checkpoint proteins Mad1, Mad2 and Mps1 are all associated with the NPCs in vertebrate cells [
12]. Remarkably, this ‘dual citizenship’ is reciprocal: Nups also localize to both nuclear pores and kinetochores, and seem to perform distinct functions in each location [
11]. Hence, the mayhem that can occur from the disruption of even a single Nup function is fathomable. Moreover, the dynamics of NPCs are tightly regulated during the cell cycle [
13]. Although it was assumed that Nups were just structural components of the NPC, this view was transformed by the surprising discovery that Nup107 and Nup133 localize to the spindle poles and to the kinetochores during mitosis [
14]. In this context, several Nups such as Nup153, Nup 107–160 complex, Tpr, Rae1 and Nup88 have been implicated in mitotic processes such as chromosome condensation, sister-chromatid cohesion, kinetochore assembly and spindle formation. Naturally, altered cellular levels of Nups can thus contribute to chromosomal instability (CIN), aneuploidy and tumorigenesis [
15]. In this regard, it is well-documented that CIN and aneuploidy are hallmarks of many different malignancies [
16,
17]. Furthermore, deregulation of the cell cycle in general and mitosis in particular is known to be frequently causal to CIN [
18,
19].
Our earlier data predicted a large number of mitotic targets of miRNAs deregulated in head and neck squamous cell carcinoma (HNSCC) [
20]. Of these, NUP214 was observed to be a potential target of hsa-miR-133b, a downregulated miRNA in HNSCC. The human proto-oncogene NUP214 is reported to have a dual role in nuclear protein import and mRNA export. The fact that mice embryos homozygous for the disrupted NUP214 allele die
in utero mirrors the essentiality of Nup214 for cell survival [
21]. Moreover, Nup214 localizes to both the cytoplasmic and nucleoplasmic sides of the NPC in over-expressing cells [
22]. Additionally, it is also reported to be recruited to the spindles during mitosis [
23,
24]. However, the exact function of Nup214 in mitosis remains elusive till date. Moreover, to the best of our knowledge, the role of miRNAs in the regulation of Nups has not been elucidated yet. Given the fact that both Nups and miRNAs are crucial to genome integrity, here we aim to elucidate how the regulation of one (Nup) by the other (miRNA) modulates this very genome integrity as well as its possible implications in tumorigenesis.
In the current study, we present NUP214 as a novel target of miR-133b. We also show that downregulation of Nup214 by miR-133b perturbs the normal mitotic progression. This perturbation subsequently gives rise to chromosomal abnormalities and leads to cell death by apoptosis. Through our study, we also raise some interesting questions about the possible locations and functions of Nup214 during mitosis.
Discussion
We have validated here that the human miR-133b can specifically repress the nucleoporin proto-oncogene NUP214. miR-133b remains downregulated in head and neck, bladder, colorectal, gastric, lung and prostate cancers while it is upregulated in cervical cancer [
26]. This miRNA is also reported to be tumour suppressive in esophageal squamous cell carcinoma [
27]. Furthermore, miR-133b has been shown to target anti-apoptotic genes to mediate death-receptor-induced apoptosis [
28]. The present study further illustrates that the miR-133b-mediated Nup214 downregulation delays cells in the early mitotic phase and thus leads to aberrant chromosome segregation and cell death. Importantly, from our observations we make an intriguing hypothesis that the import of one or more mitotic proteins (that is/are cell cycle regulated) may be dependent on the levels of Nup214 and hence, on the levels of miR-133b. Our study highlights this possibility through a hitherto unknown miRNA-regulated association of NUP214 with proper mitotic progression.
It was previously reported that NUP214-depleted embryo cells arrested in the G2 phase of the cell cycle [
21] while over-expressing cells arrested in G0 [
22]. In our attempt to uncover the downstream functional effects of Nup214 downregulation by miR-133b, we also observed a similar G2-M arrest in cells overproducing miR-133b by flow cytometric analysis. The accumulation of cyclinA in miR-133b treated synchronised cells was also able to prove an early mitotic arrest. Furthermore, co-transfection with Nup214 expression-plasmid was able to rescue the degradation of both cyclinA and cyclinB1, dephosphorylation of p-H3 and the MI thus ascertaining the specificity of this interaction. Moreover, the accumulation of cyclinB1 and p-H3 as well as increased MI endorsed the involvement of one or more SAC proteins in this pathway. Notably, because Nup214 lacks its 3′UTR in the expression-vector, co-expression with miR-133b did not alter Nup214’s ability to change the cellular phenotype. This also indicates that other known targets of miR-133b (like CXCR4 [
29]) might not play any role in the NUP214-mediated phenotype observed here. To the best of our knowledge, Mad1 is the only SAC protein with known interactions with different Nups across species other than humans [
30,
31]. Nup88 and hCRM1 also have been previously identified as Nup214 interacting proteins [
32]. Interestingly a study of substrate transport through the NPC upon NUP214 depletion revealed that the import of nuclear localization sequence (NLS)-containing proteins into the nucleus was impaired [
21]. However, in the absence of concrete evidence for known associations of Nup214 with any of the SAC proteins, for the time being we need to leave it to experimental investigations for further elucidation and explanation of our observations.
To be able to direct the mitotic perturbations to logical explanations, it is incumbent on us to question the sub-cellular localization and distinct functions of Nup214 at mitosis. As mentioned in the
Background section, Nup214 exhibits dual citizenship – being relocated from the nuclear pores at interphase to the spindles at mitosis. Not much is known about this, however, in a study by Lussi
et al. [
30], it has been shown that another member of the Nup family, Nup153, can associate with Mad1 (a mitotic protein). However, this role of Nup153 in mitosis was found to be independent of its role in nucleocytoplasmic transport. Because the localization of Nup214 at the spindles during mitosis is as yet unexplained, we suggest a similar situation wherein there may arise two possibilities at the initiation of mitosis: (1) the early mitotic arrest might occur due to defective nuclear transport of some protein/factor (discussed in the
Results section) as a result of Nup214 downregulation or (2) Nup214 downregulation may cause mitotic arrest as a result of some altered transport-independent event, for example, defective NEBD, etc. (we mention NEBD because destruction of cyclinA is known to start minutes after NEBD takes place [
33]). According to our observations, the former seems to be more plausible, stemming from the fact that cyclinA, cyclinB1 and p-H3 are stabilized upon Nup214 repression by miR-133b. Nevertheless, the latter possibility also demands to be probed by valid experiments.
An earlier report has shown that abolishment of Nup214 from the spindles during mitosis results in chromosome separation defects and aneuploidy, multinucleate structures being the most dramatic [
23]. In this context, altered levels of another Nup, Nup153, has also been implicated in the formation of multipolarization and multilobulation of cells [
30]. Similarly, our study too, has shown chromosome defects upon Nup214 downregulation by miR-133b- particularly some which appear like “flower cells” with greatly lobulated nuclei (Figure
5a, 2
nd row, 12 h time-point) as described by the same authors [
30]. miR-133b is reported in death-receptor-induced apoptosis in HeLa cells and
ex vivo models of pancreatic cancer [
28]. We too, observed ectopic miR-133b as well as Nup214 siRNA-mediated induction of apoptotic cell death in UPCI:SCC084 cells. Importantly, incidence of both chromosomal defects as well as apoptosis was greatly reduced upon co-expression of Nup214 along with miR-133b or the siRNA. Hence, we speculate that the late mitotic exit leads to erroneous chromosome segregation and abnormal nuclear structures further leading to cell death, the phenomena being specific to Nup214 in our study.
While the functions of NPCs in transport are well established, coupling of the nuclear transport machinery to processes that control chromosome segregation during mitosis, including the SAC, is still an emerging area of investigation. Our study contributes by involving miRNA-mediated regulation in this process. These findings might also help to understand the molecular mechanisms that contribute towards the high degree of CIN associated with various cancers. In fact, given that miR-133b is downregulated in HNSCC (and many other cancers), our study has significant therapeutic implications- (1) miR-133b mimics could potentially be used to alleviate the oncogenic effects of endogenous Nup214 that lead to malignancies, (2) molecules other than miRNA mimics such as small molecules, nanoparticles or other silencing RNAs could also be used against Nup214. More importantly, the preliminary observation that ectopic miR-133b does not alter the fate of non-tumoral cells also makes it a good therapeutic candidate. Moreover, the fact that our observations have been validated in cell lines other than UPCI:SCC084 (like HCT116 and HeLa) underscores a broader functionality of these mechanisms in cancers. It should be mentioned though that in colorectal cancer, miR-133b has been shown to block cell proliferation by directly targeting the c-MET protein [
34]. However, here we have defined Nup214 as a novel direct target of the miRNA and extended our study to more than one cell system.
Methods
Cell culture, synchronization, drug treatment and transfection
HCT116, SCC25, SW480 and HEK293 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) and HepG2 cell line was gifted by Dr. Samit Adhya (Indian Institute of Chemical Biology, India). Human oral cancer cell line UPCI:SCC084 was a kind gift from Dr. Susanne M. Gollin (University of Pittsburgh, USA). HeLa-H4-pEGFP stable cell line was developed as reported previously [
35]. All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum and antibiotics (1% Pen Strep Glutamine and 0.006% Gentamicin, Invitrogen) at 37°C in a 5% CO
2 incubator. For synchronization, cells were treated with thymidine (2.5 mM; USB, Cleveland, USA) for 16 h followed by an 8 h release. A second thymidine treatment was given for another 22 h. Cells were released in thymidine free complete medium and harvested at various time-points following this double thymidine block. Transient transfections were done with various plasmids, miRNA inhibitors and siRNAs in different cell lines using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. All transient transfections were performed for 48 h, except in case of synchronization experiments. Transfection of synchronized cells was done 6 h before first thymidine addition. For induction of apoptosis by etoposide (Sigma) and camptothecin (Sigma), cells were treated with the drug (20 μg/ml for 72 h and 3 μM for 24 h, respectively) for the time-periods mentioned.
Plasmids, miRNA inhibitors and siRNAs
The cloning of pSB-miR-133b (human precursor miR-133b; chromosome 6: 52011721–52015839, + strand) is described by Bhattacharjya
et al. [
20]. The NUP214 3′ UTR (NM_005085.2, +6355 to +6475) was amplified from human genomic DNA with the primers listed in Additional file
8. This region was cloned into the linearized pMIR-REPORT vector (Applied Biosystems, Foster City, USA) downstream of the luciferase gene using the MluI and HindIII restriction sites (New England Biolabs, Beverly, MA). It is referred to as pSB-NUP214/3′UTRLuc in the text. The mutant version of this clone was constructed using the primers listed in Additional file
8 and Quikchange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s protocol. It is referred to as pSB-NUP214/3′UTRMutLuc in the text. Full length Myc-tagged Nup214 expression-plasmid pCMV-Myc-CAN/Nup214 was a kind gift from Dr. Toshimi Michigami (Osaka Medical Center for Maternal and Child Health, Japan). Anti miR-133b (Ambion, Austin, TX, USA) was used at a final concentration of 20 nM. siRNAs directed against Nup214 (Santa Cruz Biotechnology) as well as scrambled control (Ambion) were used at a final concentration of 80 nM.
Quantitative Real-Time PCR
Total RNA from different cell lines, oral swab and tissue specimens was isolated using TRIZOL (Invitrogen) according to the manufacturer’s protocol. 5 μg isolated RNA was treated with DNase (Promega, Madison, USA) and 1 μg of this DNase treated RNA was used for cDNA preparation using random hexamer (Invitrogen) and MMLV-RT (Promega). For miRNAs, 200 ng isolated RNA was used for cDNA preparation in a master mix containing stem-loop primers specific for the desired miRNAs (Sigma) as given in Additional file
9, dNTPs (Invitrogen) and MMLV-RT (Promega). Real time PCR was performed in the 7500 Fast Real-Time PCR System (Applied Biosystems) using power SYBR Green PCR Master Mix (Applied Biosystems). The comparative threshold cycle method (ΔΔCt) was used to quantify relative amounts of product transcripts with GAPDH or 18S rRNA (for mRNAs) and hsa-miR-17-5p or U6 snRNA (for miRNAs) as endogenous reference controls. Primer sets are listed in Additional file
9. Fold activation values were calculated as mean of three independent experiments.
Western blotting and antibodies
Whole cell lysates having equal protein concentrations were resolved by SDS/PAGE (6%–12% gel) and transferred onto a PVDF membrane (Millipore, Billerica, USA). Various primary antibodies used are goat polyclonal NUP214, rabbit polyclonal p-H3, rabbit polyclonal cyclinA, goat monoclonal Bub1, rabbit polyclonal Nup98 (Santa Cruz Biotechnology, CA, USA), mouse monoclonal cyclinB1 (Cell Signaling Technology, Beverly, MA, USA) and mouse monoclonal β-actin antibody (Sigma). Bands were detected using Super Signal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA) after treating with HRP-conjugated secondary antibody (Sigma).
Luciferase assay
Cells were lysed with luciferase cell culture lysis reagent supplied with the luciferase assay kit (Promega). Following a short vortex, whole cell lysates were centrifuged at 4°C at 13,000 rpm for 2 min and 15–30 μl of supernatants was mixed with 30–60 μl of luciferase assay substrate. Luminescence was measured as relative luciferase unit (RLU) in GLOMAX luminometer (Promega). Total protein concentration in each lysate was measured by Bio-Rad protein assay reagent (Bio-Rad Laboratories, CA, USA) and then used to normalize the luciferase activity of each lysate. Fold activation values were calculated as mean of three independent experiments.
Cell cycle and apoptotic assays
UPCI:SCC084 cells were synchronized and after release, approximately 106 cells were harvested at respective time-points and resuspended in 0.25 ml of cold phosphate buffered saline (PBS). Cells were fixed by adding cold 70% ethanol dropwise into the samples while vortexing gently and then incubated at 4°C for minimum 24 h. After fixation, cells were resuspended in 1 ml PBS containing 100 μg/ml PI (Sigma) and 20 μg/ml RNase A (Invitrogen). Fixed cells were kept at room temperature for 40 min and then analysed by FACS (BD FACSARIA™ III, Becton-Dickinson, San Jose, CA, USA).
For annexin V-PI binding assay, UPCI:SCC084 or HEK293 cells were seeded at a density of 105 cells and transfected as mentioned. Apoptosis was measured at the indicated time-points using FITC AnnexinV Apoptosis Detection Kit I (BD Pharmingen) according to the manufacturer’s protocol. Analysis was done by LSRFortesa (Becton-Dickinson).
To measure caspase3/PARP cleavage, 105 UPCI:SCC084 cells were seeded and transiently transfected as mentioned. At indicated time-points, cells were fixed with 4% paraformaldehyde, permeabilized by fluorescence-activated cell sorter permeabilizing solution (BD Biosciences, San Jose, CA, USA). Before staining, permeabilized cells were treated with heat-inactivated 2% normal goat serum to block non-specific staining. Cells were then stained with normal rabbit sera or rabbit anti-human cleaved caspase3/PARP antibody (Cell Signaling Technology). After washing, cells were incubated with multiple adsorbed FITC-conjugated secondary antibody (goat anti-rabbit immunoglobulin; BD Biosciences), washed and analysed by LSRFortesa (Becton-Dickinson).
For colony formation assay, 103 UPCI:SCC084 or HEK293 cells were seeded and transiently transfected with indicated concentrations of plasmid. After 7 days, cells were stained with 0.2% Methylene Blue [Sisco Research laboratories (SRL), Mumbai, India] and washed with distilled water. The colonies were then counted and the number of colonies was calculated as mean of three independent experiments.
Immuno-fluorescence
Synchronized HCT116 cells were harvested at different time-points (as mentioned) after thymidine release. They were then fixed with ice-cold acetomethanol (1:1) and permeabilized with 0.03% saponin (Calbiochem, Darmstadt, Germany). After blocking in 3% BSA, rabbit polyclonal antibody against p-H3 (Santa Cruz Biotechnology) was added. Secondary antibody conjugated with FITC (Sigma) was added and the nuclei were stained with (DAPI) (Invitrogen). Slides were observed under a fluorescence microscope (Leica DM 3000, IL, USA). Abnormal chromosomes were counted among 100–200 cells each time from three independent experiments and plotted as percent abnormality.
Live cell microscopy
HeLa-H4-pEGFP cells were grown in 35 mm dishes with glass bottoms, transiently transfected (as mentioned) and synchronized. DMEM medium was replaced with phenol red-free DMEM medium (Invitrogen). Cells were monitored for duration in mitosis starting from 8 h post thymidine release under an inverted fluorescent microscope with live cell chamber (Leica DMI6000) at 37°C and 5% CO2. Images with a z step size of 500 nm were captured after every 10 minutes with both DIC and green channels using a 63× oil immersion objective.
Tumour samples
Matched oral tumour (n = 10) and normal (n = 10) tissues were obtained from the hospital section, Chittaranjan National Cancer Institute (CNCI), Kolkata, India. Prior to sample collection, written informed consent was taken from each individual and approved by the Research Ethics Committee of CNCI. The pathological history of the tumours is provided in Additional file
4.
Target prediction of miR-133b was done by miRBase (version 17). Complementarity between NUP214-3′UTR and the seed sequence of miR-133b was obtained from RNAhybrid (version 2.1) [
36] (
http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/). Oncomine 4.4 research edition database [
37] (
http://www.oncomine.org/resource/login.html) was used for the dataset of Nup214 over-expression in primary tumours. Cancer versus normal datasets of Nup214 over-expression with fold change ≥1.5 and p-value ≤0.05 were selected. Densitometric scanning for Western blots was done by ImageJ software (
http://rsb.info.nih.gov/ij/index.html). Students
t test was used to determine statistically significant differences which were defined by 2-sided p < 0.05. GraphPad Prism 5 was used to perform Mann Whitney
t-test in order to determine significant differences in miR-133b and NUP214 expressions between individual groups (normal and tumour).
Acknowledgements
We thank Dr. Toshimi Michigami of Osaka Medical Center for Maternal and Child Health, Japan for the pCMV-Myc-CAN/Nup214 clone as a kind gift. We also thank Diptadeep Sarkar (Application Support Engineer, Andor Spinning Disc Confocal Microscope, Olympus IX81) and Anushila Gangopadhyay (Flow Cytometer Technologist, BD Biosciences) for technical assistance. This work was supported in part by Department of Biotechnology Grant BT/01/COE/05/04 and Council of Scientific and Industrial Research Grant IAP 001 and HCP 004 awarded to Dr. Susanta Roychoudhury. SB and KSR are supported by pre-doctoral fellowships from the University Grants Commission (New Delhi, India), AG and SS are supported by pre-doctoral fellowships from ACTREC-DAE (Navi Mumbai, India) and Council of Scientific and Industrial Research (New Delhi, India), respectively.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SB conceived, designed and performed the experiments, analysed the data and drafted the manuscript. KSR and AG designed and performed the experiments and analysed the data. SS performed the experiments. DB, NPB and CKP designed the experiments and analysed the data. SR designed the experiments, analysed the data and drafted the manuscript. All authors read and approved the final manuscript.