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RESEARCH ARTICLE
Genome Wide Expression Profiling of Cancer
Cell Lines Cultured in Microgravity Reveals
Significant Dysregulation of Cell Cycle and
MicroRNA Gene Networks
Prasanna Vidyasekar
1
, Pavithra Shyamsunder
1
, Rajpranap Arun
1
,
Rajalakshmi Santhakumar
1
, Nand Kishore Kapadia
2
, Ravi Kumar
3
, Rama
Shanker Verma
1
*
1Stem cell and Molecular Biology laboratory, Department of Biotechnology, Indian Institute of Technology
Madras, Chennai, India, 2Department of cardiothoracic Surgery, Global Hospital, Perumbakkam, Chennai,
India, 3Department of cardiology, Fortis Malar Hospital, Adyar, Chennai, India
*vermars@iitm.ac.in
Abstract
Zero gravity causes several changes in metabolic and functional aspects of the human
body and experiments in space flight have demonstrated alterations in cancer growth and
progression. This study reports the genome wide expression profiling of a colorectal cancer
cell line-DLD-1, and a lymphoblast leukemic cell line-MOLT-4, under simulated microgravity
in an effort to understand central processes and cellular functions that are dysregulated
among both cell lines. Altered cell morphology, reduced cell viability and an aberrant cell
cycle profile in comparison to their static controls were observed in both cell lines under
microgravity. The process of cell cycle in DLD-1 cells was markedly affected with reduced
viability, reduced colony forming ability, an apoptotic population and dysregulation of cell
cycle genes, oncogenes, and cancer progression and prognostic markers. DNA microarray
analysis revealed 1801 (upregulated) and 2542 (downregulated) genes (>2 fold) in DLD-1
cultures under microgravity while MOLT-4 cultures differentially expressed 349 (upregu-
lated) and 444 (downregulated) genes (>2 fold) under microgravity. The loss in cell prolifer-
ative capacity was corroborated with the downregulation of the cell cycle process as
demonstrated by functional clustering of DNA microarray data using gene ontology terms.
The genome wide expression profile also showed significant dysregulation of post transcrip-
tional gene silencing machinery and multiple microRNA host genes that are potential tumor
suppressors and proto-oncogenes including MIR22HG,MIR17HG and MIR21HG. The
MIR22HG, a tumor-suppressor gene was one of the highest upregulated genes in the micro-
array data showing a 4.4 log fold upregulation under microgravity. Real time PCR validated
the dysregulation in the host gene by demonstrating a 4.18 log fold upregulation of the miR-
22 microRNA. Microarray data also showed dysregulation of direct targets of miR-22, SP1,
CDK6 and CCNA2.
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 1/20
OPEN ACCESS
Citation: Vidyasekar P, Shyamsunder P, Arun R,
Santhakumar R, Kapadia NK, Kumar R, et al. (2015)
Genome Wide Expression Profiling of Cancer Cell
Lines Cultured in Microgravity Reveals Significant
Dysregulation of Cell Cycle and MicroRNA Gene
Networks. PLoS ONE 10(8): e0135958. doi:10.1371/
journal.pone.0135958
Editor: Zheng Li, Peking Union Medical College
Hospital, CHINA
Received: February 26, 2015
Accepted: July 28, 2015
Published: August 21, 2015
Copyright: © 2015 Vidyasekar et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by Defense
Research Development Organization (DLS/81/48222/
LSRB-189/ID/2009 and DLS/81/48222/LSRB- 273/
SH&DD/2013) to RSV. url: http://www.drdo.gov.in/
drdo/boards/lsrb/fplsrb.htm
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
Microgravity on space flights has been shown to affect the physiology of a cell considerably [1].
Normal gravity (1 g) affects 2-Dimensional culture by depositing cells on the surface of the tis-
sue culture plate (TCP) where anchorage-dependent cells adhere and proliferate as a mono-
layer with very limited cell–cell interactions. The weightlessness and reduced acceleration (less
than 1 g) in space, removes the effect of gravity, allowing cell cultures in space to have unhin-
dered movement of the culture medium, a shear free environment and, as cells are not bound
by any directional force, unrestricted movement of cells within the medium. Under such condi-
tions cells tend to coalesce and form aggregates creating three dimensional (3D) environments
where they interact on multiple planes [2]. The effect of reduced gravity is not restricted to
changes in culture conditions as the unique environment can produce changes in the funda-
mental physiology of the cell. While the mechanism of action of how gravity, or the lack of it,
affects molecular and cellular functions is still unclear, it has been established that microgravity
or zero gravity affects vital processes of the cell and importantly, microgravity has been shown
to alter cancer growth and progression [3–5]. However, different cancers respond differently to
microgravity by losing or enhancing cellular processes and functions. In this study we cultured
cell lines representative of solid and hematological tumors—DLD-1, MOLT-4 and HL-60 in a
rotating cell culture system (RCCS) that simulated microgravity. The RCCS is a mechanical
system that simulates reduced gravity on earth by canceling the directional vector through con-
stant rotation of a High Aspect Ratio Vessel (HARV). This maintains cells in a constant free
fall and a shear free environment allowing cells to coalesce and form 3D aggregates [2]. These
aggregates are maintained in free fall and experience conditions of reduced gravity for the
remainder of the culture period. We hypothesized that physiological changes to the cell func-
tions such as cell proliferation and viability could be corroborated with changes in fundamental
processes of the cell such as gene expression. To relate physiological changes such as an altered
cell cycle profile with dysregulation of gene expression, real time PCR analysis for cell cycle
genes, oncogenes and cancer development and progression markers was carried out. Genome
wide expression profiling by DNA microarray of these cell lines cultured under microgravity
revealed the dysregulation of several pathways in cancer and importantly, corroborated with
observed physiological changes to the cell. We also used the gene expression profile to investi-
gate dysregulation in pathways central to cancer such as the Notch signaling system and dysre-
gulation in post transcriptional gene silencing machinery. The gene expression profile also
revealed dysregulation of microRNA host genes in microgravity including the significant
tumor suppressor, miR-22 in DLD-1.
Materials and Methods
Cell culture
DLD-1 is an epithelial, adherent cell line derived from a colorectal adenocarcinoma (Dukes
type C). MOLT-4 is a T lymphoblast, suspension cell line derived from an acute lymphoblastic
leukemia while the HL-60 cell line is a promyeloblast derived from acute promyelocytic leuke-
mia. Cell lines were procured from the National centre for cell science, Pune, India and were
maintained in DMEM-F12 (DLD-1) or RPMI1640 (MOLT-4, HL-60) medium supplemented
with 10% fetal bovine serum (Life Technologies, USA) at 37°C in a humidified 5% CO2 incuba-
tor in 25mm
3
tissue culture plates (TCP) and in 10ml
3
high aspect ratio vessels (HARV) within
a rotating cell culture system (RCCS). The cell lines were introduced into the 10ml HARV
through 5ml syringes and a rotating speed of 27 revolutions per minute (RPM) was standard-
ized based on the aggregation of DLD-1 cells loaded at 0.5 x 10
6
cells within 24 to 48 hours.
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 2/20
Cells were grown in HARV for a maximum of 72 hours with additional medium injected into
the HARV every 16 to 24 hrs to prevent foaming or air bubbles. Contents of the HARV were
transferred to 60mm TCP, without dissociating cell aggregates, for routine micrographic obser-
vation and other cell based assays by using 10ml pipettes. 0.25% Trypsin-EDTA was used for
dissociation of cell aggregates and monolayer cultures when required.
Total RNA extraction and cDNA conversion
Total RNA was extracted from cells using RNeasy kit (Qiagen, Germany). 2 × 10
6
cells were
centrifuged and washed twice with PBS. RNA was isolated from these cells as per the manufac-
turer's instructions. 1.5 μg of total RNA was converted to cDNA using MMLV-RT (Thermo
scientific, USA) and Oligo-dT primers (NEB, USA). miRNA conversion to cDNA was carried
out using stem-loop reverse transcriptase (RT) primers without dithiothreitol (DTT) and the
RNA denaturation step to maintain integrity of the stem-loop primer.
Microarray analysis
Microarray analysis was performed with RNA samples from DLD-1 and MOLT-4 cell lines
grown under microgravity and under static conditions in replicates. Expression data for each
sample was obtained on the Affymetrix GeneChip Human Primeview Array. Hybridization
was carried out for a duration of 16 hours at 60 rpm at 48°C and scanned on the GeneChip
microarray Scanner 3000 7G. Raw data was extracted after scanning of slides and raw data sets
were analyzed using GeneSpring GX 12.6 software followed by differential gene expression
(DE), fold change & cluster analysis.
Gene ontology analysis
The DE genes were studied for their overabundance in different Gene ontology (GO) terms as
well as pathways using the microarray analysis software DAVID (The Database for Annota-
tion, Visualization and Integrated Discovery) [6]. Two tools in the DAVID program were used,
gene functional classification tool and the functional annotation clustering tool. The DAVID
functional annotation tool was used to highlight relevant GO terms associated with the submit-
ted gene list by grouping similar, redundant, and heterogeneous annotation contents from the
same or different resources into annotation groups based on the hypothesis that similar anno-
tations should have similar gene members. Gene enrichment is based on set of submitted genes
that are highly associated with certain terms, which is statistically measured by Fisher Exact in
DAVID system. In this study, we used the group enrichment score which is the geometric
mean of all p-values of individual members in a corresponding annotation cluster. A higher
score indicates a highly enriched cluster which in turn indicates the biological significance of
the cluster in the list.
Real time PCR amplification
Real time PCR was carried out using SYBR green Real time PCR kit from Qiagen on an Eppen-
dorf mastercycler, ep realplex (Eppendorf, Germany). Relative mRNA expression was deter-
mined by normalization to the expression of a housekeeping gene, beta-actin and U6 for
microRNA gene expression. Primer list is provided in S1 Table.
Western blotting
Cells were lysed with Radio Immuno Precipitation Assay (RIPA) buffer, mixed with Laemmli
sample buffer (1×) and boiled. Proteins were subjected to 12% SDS-PAGE and electroblotted
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 3/20
onto BioRad, 0.22 μM nitrocellulose membrane (BioRad Laboratories, USA). Membrane was
blocked with Tris-buffered saline plus 0.2% Tween 20 (TBS-T) containing 3% BSA (Sigma
Aldrich, USA) followed by primary antibody incubation overnight and washing with TBS-T
buffer. Secondary antibody (anti-mouse, HRP conjugate, 1:10000 Sigma Aldrich USA) diluted
in blocking buffer was incubated for 1 h at room temperature and washed again with TBS-T.
Antibody-reactive proteins were detected by means of enhanced chemiluminescence, Amer-
sham ECL Plus western blotting detection reagents (GE health care, UK). Antibody details are
provided in S1 Table.
Flow cytometry for cell cycle analysis
The cells were harvested and washed in PBS before fixation in cold 70% ethanol which was
added drop wise to the pellet while vortexing. Cells were fixed for 30 min at 4°C. Fixed cells
were washed twice in PBS and spun at 250g in a centrifuge. Cells were incubated with 50 μlofa
100 μg/ml stock of RNase and 200 μl Propidium Iodide (from 50 μg/ml stock solution). A BD
FACSCalibur (USA) flow cytometer was used to analyze the cell population for cell cycle
changes.
CFU- assay
Cells were cultured in methyl cellulose at 10X of final concentration (0.3 mL of cells to 3 mL of
Methyl cellulose). Tubes were vortexed to ensure cells and components were thoroughly
mixed. Methyl cellulose was dispensed using a 3cc syringe and evenly spread in the dish by
gentle swirling. Cultures were incubated at 37°C, 5% CO2 in air and 95% humidity. Colonies
were then stained with crystal violet (0.5mg/ml in 1% methanol) for 20 minutes and air dried
after washing in distilled water. Stained colonies were visualized under a Nikon eclipse Ti
phase contrast microscope.
Statistical analysis
All experiments were carried out in replicates and results were expressed as mean ± S.D. Statis-
tical significance was calculated by using student t-test with the Prism 5 program (GraphPad
software, USA).
Results and Discussion
Culture of cells in microgravity
Availability of a surface to adhere for cells on the TCP is a stimulus for growth for adherent
cells such as DLD-1 and therefore, static cultures in TCP were confluent (Fig 1A). Slow rota-
tions per minute (RPM) of the HARV (16 RPM, Fig 1B) did not allow the cells to coalesce but
cells could form aggregates at 27 RPM (Fig 1C). Staining with AO/EB demonstrated no cell
death in static TCP cultures (Fig 1D) but revealed cell death in microgravity cultures of DLD1
at 16 RPM (Fig 1E). Cell viability was improved when cells aggregated at 27 RPM (Fig 1F).
When these cell aggregates were enzymatically dissociated and cultured in a TCP after 48
hours in microgravity, cells took 48 hours to adhere (Fig 1G, Day 2) while cells from static cul-
tures adhered within 24 hours (Fig 1G, Day 1) and produced confluent cultures by day 4.
Microgravity has been shown to affect several cell functions [7] and the expression or function-
ing of cell adhesion molecules could be affected [8] reducing the number of DLD-1 cells that
could adhere to the TCP. When such cell aggregates were plated in TCP without enzymatic dis-
sociation, the morphology of DLD-1 cells was altered (Fig 1H and 1I). Crystal violet staining of
cells grown in static monolayer culture shows the typical morphology of DLD-1 cells (Fig 1H)
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 4/20
Fig 1. Effect of microgravity on cell morphology and cell viability of DLD-1 cell cultures. A DLD-1 cell
cultures; Static culture (control) BDLD-1 Microgravity culture at 16 RPM CDLD-1 Microgravity culture at
27RPM Differential staining to detect apoptotic population D DLD-1Static monolayer cultures E
Microgravity cultures of DLD1 at 16 RPM FMicrogravity cultures of DLD1 at 27 RPM G Cell adhesion and
proliferation assay Top panel—static cultures, bottom panel—microgravity cultures shifted to static TCP H
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 5/20
while cell aggregates assume a growth pattern similar to an explant culture and peripheral cells
contained a large cytoplasm and nucleus (Fig 1I). The enlarged cell maybe due to the cytoskele-
tal changes during growth in microgravity [9] or cells, having lost control over the cell cycle,
may accumulate pre-mitotic protein in the cytoplasm with polyploidy in the nucleus [9]
increasing in size. A colony forming assay (CFA) on DLD-1 cultures in microgravity shifted to
static TCP after enzymatic dissociation (Fig 1J and 1K) revealed the reduced potential of DLD-
1 cells to form colonies (Fig 1K) as compared to static cells (Fig 1J). The reduced proliferative
capacity of cultures in microgravity was confirmed by a cell viability assay using MTT. Static
cultures were 41% more viable than 27 RPM cultures and 75% more viable than 16 RPM cul-
tures in microgravity (Fig 2A). When these results are taken together, microgravity appears to
significantly affect the viability and proliferation of DLD-1 cells. Flow cytometry analysis of PI
loaded DLD-1 cells cultured under microgravity was compared to the cell cycle profile of cul-
tures in static TCP and static suspension on agar underlay (Fig 2B, 2C and 2D). From lack of
anchorage, static suspension cell cultures on agar also aggregate similar to the RCCS, however
the simulation of microgravity is absent. DLD-1 cultures in microgravity had a substantial pop-
ulation of cells in the sub G0 phase although a large population of cells was still viable in micro-
gravity (Fig 2C). Static monolayer, TCP cultures were completely viable (Fig 2B) and
significantly, the static suspension cultures did not demonstrate a sub G0 phase and had profile
similar to the static control (Fig 2D). Fig 2E shows the average sub G0 phase population across
the three culture conditions in replicates of cell cycle analysis, which confirmed that reduced
Morphological changes in DLD-1; Crystal violet staining of DLD-1 cells in static monolayer culture ICrystal
violet staining of DLD-1 cells after transfer of cell aggregates from microgravity to TCP JColony forming
ability assay; Static cultures KColony forming ability assay; DLD-1 cells after transfer of cell aggregates from
microgravity to TCP
doi:10.1371/journal.pone.0135958.g001
Fig 2. Effect of microgravity on cell viability and cell cycle of DLD-1 and MOLT-4 cell lines. A Cell viability assay for DLD-1 cells; Viability measured for
microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT BCell cycle analysis for DLD-1 cells; Static CCell cycle analysis; Microgravity D
Cell cycle analysis; Static suspensions on agar underlays EThe average sub G0 population in replicates of cell cycle analysis for microgravity, static and
static suspension cultures of DLD-1 cells F MOLT-4 cell culture Static and Microgravity cultures of MOLT-4 G Cell viability assay Viability measured for
microgravity cultures (16 RPM and 27 RPM) and static cultures using MTT HCell cycle analysis; Static ICell cycle analysis; Microgravity cultures of MOLT-4.
doi:10.1371/journal.pone.0135958.g002
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 6/20
viability was an exclusive effect of microgravity in DLD-1 cell aggregates. The MOLT-4 cell
line grows as a suspension culture and it did not aggregate favorably in microgravity at high or
low RPMs, showing individual cells in the HARV (Fig 2F, 16 RPM HARV). However, cell via-
bility was reduced by 20% as compared to the static control in both RPM at 48 hours (Fig 2G)
which correlated with reduced cell numbers under microscopic observation after 48 hours (Fig
2F, 16 RPM HARV). Flow cytometry detected a small apoptotic population (Sub G0 stage) in
cell cycle analysis of MOLT-4 cultures in microgravity at 16 RPM (Fig 2H and 2I).
Real Time PCR for gene expression analysis
To affect central processes of cancer such as cell proliferation and cell cycle, microgravity must
significantly influence fundamental functions of the cell such as gene expression. We measured
the mRNA levels of significant genes involved in cell cycle and cancer progression to check for
their dysregulation under microgravity. Cyclin gene expression levels significantly influence
cancer progression and metastasis as they can direct cell proliferation or apoptosis. Cdk are
essential for G1/S and G2/M phase transitions of the cell cycle and their dysregulated gene
expression can affect the progression of the cell cycle. The transcription of CDK1 is regulated
such that it functions during the mitotic prophase and metaphase [10]. CDK1 expression was
down regulated in MOLT-4 and upregulated in DLD-1 (5-fold over static control) (Fig 3A).
The expression of genes fundamental to cancer development and progression, which include
oncogenes and potential cancer stem cell markers, were dysregulated in microgravity. CD117
(receptor tyrosine kinase—c-kit) expression was upregulated by 11.2 fold in MOLT-4 and
downregulated by 0.2 fold in DLD-1 under microgravity (Fig 3A). High c-kit expression pro-
tects colon carcinoma cells against apoptosis and enhances their invasive potential [11]; there-
fore, c-kit downregulation in DLD-1 under microgravity may be significant. DLD-1
constitutively over expresses the MYC gene [12] under normal conditions. Overexpression of
MYC sensitizes cells to apoptosis and under microgravity MYC gene expression was further
increased in DLD-1 by 3 fold (Fig 3A). MOLT4 expressed lowered levels of MYC (0.4 fold) in
microgravity (Fig 3A). JUNB encodes a transcriptional regulator of cell proliferation genes and
is part of the immediate early gene family [13]. One of the most significant genes to be dysregu-
lated in both cell lines in microgravity, JUNB is upregulated in microgravity by 2.1 and 1.2 fold
in MOLT-4 and DLD-1 respectively (Fig 3A).
Gene expression analysis in HL-60, a promyelocytic leukemia cell line
As an additional control for blood tumor (and suspension) cultures, we checked the gene
expression levels of the cell cycle genes and oncogenes in a promyelocytic leukemia cell line,
HL-60. Real time PCR revealed the up regulation of CCNE1 and CDK2 in HARV cultures with
CDK2 being significantly up-regulated (1.1 and 1.8 fold respectively) (Fig 3B). CCNB1 and
CDK1 gene expression was dysregulated with CDK1 being up regulated 1.5 fold and CCNB1
down regulated by 0.8 fold (Fig 3B). Significantly, the proto oncogenes CD117 and MYC were
highly up regulated in microgravity by 4.7 fold and 10.8 fold respectively (Fig 3B). Similar to
the DLD-1 cell line, HL-60 also over expresses the MYC gene constitutively under standard
conditions. The prognostic markers CD71,CD105 and CD90 were dysregulated under micro-
gravity by 0.75 fold (downregulated), 1.4 fold (upregulated) and 2.1 fold (upregulated) respec-
tively (Fig 3B). Endoglin (CD105) aids neovascularization in cancer [14] and CD90 expression
indicates a positive prognosis as leukemic progenitor cells in AML that are capable of main-
taining the disease in vitro and in vivo do not express CD90 [15]. Real time PCR analysis of a
candidate cell cycle, oncogene, transcription factor and cancer progression marker showed
both upregulation and downregulation. As the cell cycle is regulated by a multitude of factors
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 7/20
many of which could be affected by microgravity, a ‘collective’downregulation or dysregula-
tion of processes associated with cell cycle could validate the observed physiological halt or
reduction in cell proliferation under microgravity. Towards this aim, a genome wide expression
profiling using DNA microarray was carried out. The genomic profiling also allowed us to
speculate on the effect of microgravity on central pathways in cancer such as the Notch signal-
ing system, and expression levels of novel regulators such as microRNA.
Microarray analysis of DLD-1 and MOLT-4 cells cultured in microgravity
Microarray analysis revealed 1801 and 2542 genes up and down regulated more than 2 fold in
DLD-1 cells cultured in microgravity compared to static control. MOLT-4 cultures under
microgravity differentially expressed a total of 349 and 444 genes up and down regulated over
2 fold, respectively. Table 1 represents a short list of common genes deregulated among both
cell lines. A complete list of highly deregulated genes among both cell lines is provided in S2,
Fig 3. Quantitative PCR analysis for changes in mRNA expression of significant, candidate genes involved in cell proliferation and cancer. A CDK1
—Cell cycle kinase gene, CD117—proto-oncogene, JUNB—transcription factor and immediate early gene, MYC—proto-oncogene expression in DLD-1 and
MOLT-4 BReal time PCR analysis in HL-60 CCNE1 and CDK2,CCNB1 and CDK1, Oncogenes: CD117 and MYC, Cancer prognostic markers CD105,CD90
and CD71.
doi:10.1371/journal.pone.0135958.g003
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 8/20
Table 1. Commonly deregulated genes in DLD-1 and MOLT-4 cells under microgravity.
UPREGULATED GENES
GENE SYMBOL GENE NAME Log FC DLD-1 Log FC MOLT-4
ARRDC3 arrestin domain containing 3 3.6286426 2.5424619
ATF3 activating transcription factor 3 2.6872826 1.0043197
CCPG1 cell cycle progression 1 1.5802538 1.8111625
CDKN2AIP CDKN2A interacting protein 1.6052608 1.0670295
CDKN2D cyclin-dependent kinase inhibitor 2D (p19, inhibits CDK4) 1.3287859 1.0733795
CREBBP CREB binding protein 1.6003945 1.1786065
CREBRF CREB3 regulatory factor 1.7348738 1.2481394
CXCL3 chemokine (C-X-C motif) ligand 3 1.6632779 1.1129959
DDIT3 DNA-damage-inducible transcript 3 2.0701203 1.280817
EGR2 early growth response 2 1.3966465 3.9244506
ETS1 v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) 1.3279192 1.0439773
ETV5 ets variant 5 1.2021747 2.488372
FGF7 fibroblast growth factor 7 2.2669797 1.1106136
GORAB golgin, RAB6-interacting 1.1204505 1.0041857
HDAC9 histone deacetylase 9 1.0828898 1.4470437
HINT3 histidine triad nucleotide binding protein 3 1.7376733 1.7083709
HIVEP2 human immunodeficiency virus type I enhancer binding protein 2 1.2518463 1.2009206
IRS2 insulin receptor substrate 2 2.5675535 1.0355048
JUN jun proto-oncogene 2.8459454 3.4484391
MIR1304 microRNA 1304 1.694859 1.0175457
NCOA7 nuclear receptor coactivator 7 1.3721471 1.0397696
NDFIP2 Nedd4 family interacting protein 2 1.0812235 1.1697233
PIBF1 progesterone immunomodulatory binding factor 1 1.6272297 1.2976668
PLEKHF2 pleckstrin homology domain containing, family F (with FYVE domain) 2.5150435 1.105514
PTEN phosphatase and tensin homolog 1.2177935 1.0146773
RAB30 RAB30, member RAS oncogene family 1.8227141 1.6890364
SKIL SKI-like oncogene 1.7823422 1.4903564
SMAD7 SMAD family member 7 1.0359683 1.338062
TNFAIP3 tumor necrosis factor, alpha-induced protein 3 1.2988296 1.3440051
XIAP X-linked inhibitor of apoptosis 1.3545609 1.2860351
ZFAND2A zinc finger, AN1-type domain 2A 1.9506464 1.2355728
ZFY zinc finger protein, Y-linked 2.238248 1.2044845
ZMYM5 zinc finger, MYM-type 5 1.7800057 1.4686751
DOWNREGULATED GENES
GENE SYMBOL GENE NAME Log FC DLD-1 Log FC MOLT-4
ASIC1 acid-sensing (proton-gated) ion channel 1 -1.1945584 -1.4278164
CD24 CD24 molecule -2.1321063 -1.3863628
CDCA7L cell division cycle associated 7-like -1.6832285 -1.0647273
CDKN1C cyclin-dependent kinase inhibitor 1C (p57, Kip2) -1.2500896 -1.0963018
DHFR dihydrofolate reductase -1.8050942 -1.0276904
DNHD1 dynein heavy chain domain 1 -1.464865 -1.0485542
DUT deoxyuridine triphosphatase -1.0620422 -1.175415
EEF1A1 eukaryotic translation elongation factor 1 alpha 1 -1.0099685 -1.0678835
EIF4A1 eukaryotic translation initiation factor 4A1 -1.0106502 -1.0274181
ENSA endosulfine alpha -1.0821726 -1.1114485
FANCL Fanconi anemia, complementation group L -1.2304835 -1.0152798
(Continued)
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 9/20
S3 and S4 Tables. The highly dysregulated genes represented in the supporting information
tables contain interesting candidate genes such as the ribonucleotide reductase M2 (RRM2)
subunit which is the most down regulated gene in DLD-1 under microgravity. RRM2 overex-
pression may be associated with Colo Rectal cancer (CRC) progression and may play an impor-
tant role in the infiltration and metastasis of CRC [16]. It serves as a prognostic biomarker and
predicts poor survival of colorectal cancers [17]. While both cell lines exhibited changes in cell
cycle and cell viability, microgravity elicited a greater response from the solid tumor cell line
DLD-1. Sub lethal stress can push a cell into a state that is similar to replicative senescence
[18]. Stress-induced premature senescence (SIPS) can occur after DNA damage, oxidative
stress and treatment with histone deacetylase inhibitors [18]. The phenomenon of SIPS can
explain the loss in cell viability in both cell lines. Microarray analysis revealed the downregula-
tion of the retinoblastoma gene (RB1; -0.42 log fold change) in DLD-1 cells under microgravity
and as the presence of the RB1 protein is necessary for SIPS [19], the response of DLD-1 cells
to microgravity may not be through SIPS and its related pathways. Conversely MOLT-4 cells
show upregulated RB1 expression (0.47 log fold change) under microgravity and the loss in cell
viability may be attributed to SIPS. This could also explain the relatively lesser number of genes
that were differentially expressed in MOLT-4 cells compared to the gene expression profile of
DLD-1 cells under microgravity. Other biomarkers of SIPS, apolipoprotein J and fibronectin,
which are overexpressed in replicative senescence and SIPS [19], were not differentially
expressed in DLD-1 or MOLT-4 under microgravity. The mechanism of SIPS is not clearly
understood as yet and whether microgravity can be a trigger for SIPS pathways needs to be
Table 1. (Continued)
FAR1 fatty acyl CoA reductase 1 -1.0487332 -1.0339952
FGFR3 fibroblast growth factor receptor 3 -1.6760249 -1.3410914
GSPT1 G1 to S phase transition 1 -1.3995273 -1.0519781
GSTA4 glutathione S-transferase alpha 4 -1.1619577 -1.0276983
HES4 hairy and enhancer of split 4 (Drosophila) -1.1826415 -2.0193136
HMGB1 high mobility group box 1 -1.002748 -1.2596858
HMGB3 high mobility group box 3 -1.2634602 -1.1885982
HSPA4 heat shock 70kDa protein 4 -1.5835454 -1.2644181
IFI30 interferon, gamma-inducible protein 30 -1.5613956 -1.5471194
IFRD2 interferon-related developmental regulator 2 -1.1622665 -1.0979714
JPH1 junctophilin 1 -1.1266716 -1.6309524
LOC100653301 /// NRBP2 nuclear receptor-binding protein 2-like /// nuclear receptor binding protein 2 -1.5101182 -1.0871661
MTPAP mitochondrial poly(A) polymerase -2.2048273 -1.046771
NEURL1B neuralized homolog 1B (Drosophila) -1.2790649 -1.0592682
NFIA nuclear factor I/A -1.2247176 -1.1426408
PARP1 poly (ADP-ribose) polymerase 1 -1.6084354 -1.0683279
PHKA1 phosphorylase kinase, alpha 1 (muscle) -1.4648097 -1.3255553
PLXNA1 plexin A1 -1.1962962 -1.3295693
PNPT1 polyribonucleotide nucleotidyltransferase 1 -1.3692455 -1.0695791
POLR3H polymerase (RNA) III (DNA directed) polypeptide H (22.9kD) -1.0398519 -1.1780653
RBBP4 retinoblastoma binding protein 4 -1.0829744 -1.1924767
TUBB tubulin, beta class I -1.1920624 -1.0390439
Table shows a section of commonly deregulated genes under microgravity in both cell lines as revealed by microarray analysis. The complete list of
genes is provided in S5 Table.
doi:10.1371/journal.pone.0135958.t001
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 10 / 20
confirmed. The data discussed in this publication have been deposited in NCBI's Gene Expres-
sion Omnibus and are accessible through GEO Series accession number GSE69271 (http://
www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69271). The differentially expressed genes
from the microarray data were studied for their overabundance in different Gene Ontology
(GO) terms as well as pathways using the microarray analysis software DAVID. The enrich-
ment score of individual GO terms that the genes associated with was used to identify processes
that were significantly dysregulated. GO or functional enrichment analysis was performed with
over 2 fold-differentially expressed genes in both cell lines. Functional classification of genes
based on similarity in function or family was carried out.
Validation of candidate genes selected from microarray data
Microarray analysis revealed the upregulation of CCNB1 and the down regulation of ROMO1
and HES1 genes when MOLT-4 cells were cultured under microgravity (Fig 4A). Similarly,
microarray analysis demonstrated the down regulation of CDK2 gene and the upregulation of
STAT3 and HEY1 genes in DLD-1 cells cultured under microgravity (Fig 4B). Microarray analy-
sis revealed that both cell lines commonly showed the down regulation of CCNE1 and the upre-
gulation of CD71 and CD44 genes (Fig 4C,Fig 5A). Significantly, the deregulation of the
microRNA-22 host gene and its targets was also under microgravity (Fig 5B). mRNA expression
of these candidate genes representative of the cell cycle, transcriptional regulation and cancer
progression were validated by quantitative real time PCR. Cyclin B1 which has been reported as
constitutively overexpressed in human colorectal cancers [20] is over expressed in MOLT-4 cells
which showed a 7.5 fold increase of CCNB1 mRNA expression under microgravity (Fig 4A).
Lowered expression of ROMO1 leads to inhibition of cell growth [21] and MOLT-4 cells
expressed 0.5 fold less ROMO1 than the static control (Fig 4A). Transcriptional and replication
controllers regulate oncogenes and prognostic markers and influence cell cycle events. HES1 is a
transcriptional repressor and is involved in DNA repair [22] and HES1 gene expression is con-
trolled by the Notch and Jun signaling system [23]. HES1 gene expression is down regulated by
0.7 fold in MOLT-4 (Fig 4A) under microgravity. CDK2 gene expression in DLD-1 cells was 0.5
fold lower compared to static control (Fig 4B)whileHEY1, a transcriptional regulator was highly
up regulated by 10.3 fold (Fig 4B). Signal transducer and activator of transcription 3 (STAT3)is
an oncogenic transcription factor which is activated and aberrantly expressed in many colorectal
cancers [24] and the genes upregulation is validated by RT-PCR (Fig 4B). Cyclin E1 gene expres-
sion is down regulated in both cell lines under microgravity (Fig 4C). Cyclin E1 controls the pro-
gression of the cell cycle through the G1 phase by its interaction with cyclin dependent kinase 2
[25]. Similarly, both cell lines expressed lowered levels of CD71, which encodes a transmembrane
glycoprotein that is responsible for cellular iron uptake (Fig 4C). DLD-1 expressed significantly
lower levels (0.42 Fold) in microgravity. Higher expression of CD71 is associated with negative
prognosis for many solid tumors and some lymphomas [26,27] and numerous studies have
found a positive correlation between iron storage and the risk of tumors such as in colorectal car-
cinoma [28]. Significantly, the CD44 gene was upregulated in both cell lines (Fig 4C,Fig 5A).
While most isoforms of CD44 are associated with the malignant form of the disease, some forms
of CD44 prevent the tumor cells from spreading out of the primary site [29]. As CD44 is
expressed in both colon and lymphoid cancers and as mRNA analysis would involve multiple
variants, we investigated its protein levels in DLD-1 and MOLT-4 cultures in microgravity.
Western blotting for standard isoform of CD44 shows higher levels of the protein in both cell
lines over static control (Fig 5A). Densitometric analysis of the bands and normalization with β-
actin values demonstrates significant up regulation of CD44 protein in microgravity. microRNA
have been identified as potential oncogenes or tumor suppressors [30]. The miR-22 host gene,
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 11 / 20
Fig 4. Effect of microgravity on gene expression levels in DLD-1 and MOLT-4 cell cultures; Validation of microarray analysis by real time PCR and
western blotting. A Log fold change of CCNB1,ROMO1 and HES1 deregulation in MOLT-4 cells under microgravity as observed by microarray analysis
validated by real time PCR BLog fold change of CDK2,HEY1 and STAT3 deregulation in DLD-1 cells under microgravity as observed by microarray analysis
validated by real time PCR CLog fold change of commonly up and downregulated genes CCNE1,TFRC (CD71) and CD44 as observed by microarray
analysis validated by real time PCR forCCNE1 and TFRC (CD71).
doi:10.1371/journal.pone.0135958.g004
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 12 / 20
Fig 5. Dysregulation of stem cell marker CD44 and tumor suppressor microRNA under microgravity.
AValidation of microarray data for CD44 by Western blotting for CD44 protein and beta-actin in static and
Microgravity (μG) cultures of DLD1 and MOLT-4 and Densitometric analysis of western blots. BMIR22HG
expression under microgravity; Overexpressed MIR22HG, host gene of miR-22 microRNA in DLD-1 cells
under microgravity, MOLT-4 cells show no differential expression; Levels of dysregulation of direct targetsof
miR-22 microRNA CDK6,CCNA2,SP1 and CDKN1A in microarray data; RT-PCR validation of microRNA
miR-22 levels and target genes in DLD-1 shows over expressed microRNA miR-22 in DLD-1 cells under
microgravity confirming upregulation in microarray data. No significant dysregulation of direct targets
CDKN1A (similar to expression levels in microarray data) and CCND1A CGO analysis (by DAVID) of
microarray data to depict other dysregulated microRNA host genes in DLD-1 and MOLT-4 cells under
microgravity.
doi:10.1371/journal.pone.0135958.g005
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 13 / 20
MIR22HG was highly upregulated in DLD-1 (Log fold 4.4) but not differentially expressed in
MOLT-4 (Fig 5B). miR-22 functions as a tumor suppressor through post-transcriptional regula-
tion of p21 to determine cell fate [31]. It represses cancer progression by inducing cellular senes-
cence [32] and controls EVI-1 oncogene expression in metastatic breast cancer cells [33]. Some
targets of miR-22 such as SP1,CDK6 and CCNA2 were also significantly downregulated (Fig 5B)
while others such as p21 (CDKN1A) were not significantly dysregulated. The farnesoid X recep-
tor regulates miR-22 which targets CCNA2 in colon and liver cancer cells [34]. Real time PCR
for miR-22 microRNA also showed a 4.18 log fold upregulation in DLD-1 cells under micro-
gravity (Fig 5B) confirming the fold change observed in microarray analysis. Real Time PCR for
miR-22 targets—CCND1 and CDKN1A however, did not show significant dysregulation with
-0.09 log fold (down regulation) and 0.11 log fold (up regulation) change, respectively (Fig 5B).
Other micro RNA host genes including MIR17HG, was significantly downregulated in MOLT-4
while not differentially expressed in DLD-1 (Fig 5B). The MIR21HG is significantly downregu-
lated in DLD-1 while it is not differentially expressed in MOLT-4 (Fig 5B). The MIR17HG or
MiR-17-92 Cluster Host Gene encodes for six miRNAs that influence cell survival, proliferation,
differentiation, and angiogenesis [35]. miR-21 downregulates tumor suppressor Pdcd4 and stim-
ulates invasion, intravasation and metastasis in colorectal cancer [36]. The downregulation of
miR-21 also induces differentiation of chemoresistant colon cancer cells enhancing their suscep-
tibility to therapy [37].
Dysregulation of genes involved with the Notch signaling system and
microRNA processing
The Notch signaling system plays an important role in the mechanical unloading of bone in
microgravity, and changes to the mesenchymal and hematopoietic stem cell compartments. The
Notch pathway is also significant in cancer progression and microRNA processing. As one of
the highly deregulated genes observed in the microarray analysis is a tumor suppressor micro-
RNA, we looked at other genes and pathways in the Notch signaling system that could be dereg-
ulated. GO analysis revealed multiple genes that were functionally clustered under processes
associated with the Notch system (Fig 6A, 6B and 6C). Significantly dysregulated genes involved
in transcriptional regulation include HDAC1,HDAC3,HEY1,MAML2,MESP1,SPEN,TBL1X
and TLE1 (Fig 6A). The HDAC1 and HDAC3 genes are potential tumor suppressors that inter-
act with retinoblastoma 1 and p53 proteins respectively [38] while MAML2 is a potential onco-
gene [39] that is down regulated in DLD-1 by more than 2 log fold and upregulated in MOLT-4
(Fig 6A). Major genes involved in post transcriptional gene silencing were dysregulated under
microgravity (Fig 6B)suchasEIF2C2 which was dysregulated only in DLD-1 while EIF2C1 and
EIF2C3 were significantly downregulated and upregulated in both cell lines (Fig 6B). The
human eukaryotic initiation factor 2C1 (EIF2C1) and EIF2C2 are components of the core RNA-
induced silencing complex (RISC) and are members of the argonaute protein family [40] that
are potential biomarkers for human colon cancer [41]. DLL1 was significantly up regulated in
the process of Notch signaling pathway by 2.46 log fold among other genes in DLD-1 (Fig 6C)
but the gene was not differentially expressed in MOLT-4. DLL4 and JAG1 were differentially
expressed in DLD-1 while JAG2 was expressed only in MOLT-4 (Fig 6C). Epigenetic regulation
of DLL1 controls Notch1 activation in gastric cancer and along with DLL4;DLL1 is required for
homeostasis of intestinal stem cells [42,43]. Significantly, all the NOTCH genes were up regu-
lated in both cell lines (Fig 6C) and these genes encode receptors for membrane-bound ligands
Jagged1/2 and Delta1. JAG1 is involved in cell-fate decisions during hematopoiesis while JAG2
is overexpressed in malignant myeloma plasma cells [44,45]. TLE1,TLE2 and FBXW7 genes,
which have the conserved WD40 repeat domain, were dysregulated (S1A Fig) in both cell lines
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 14 / 20
while TLE3 is highly upregulated in DLD-1 and not differentially expressed in MOLT-4.
FBXW7 is a potential tumor suppressor gene [46] involved in ubiquitination and subsequent
degradation of cyclin E and MYC. The ribosomal Protein S27a (RPS27A) promotes prolifera-
tion, regulates cell cycle progression and inhibits apoptosis of leukemia cells. RPS27A was signif-
icantly dysregulated only in DLD-1 and not in MOLT-4 (S1B Fig). APH1B and the presenilin
protein encoding genes, PSEN2 and PSENEN were dysregulated only in the DLD-1 cell line
(S1B Fig). APH1B encodes a subunit of the gammasecretase complex that catalyzes the cleavage
of proteins such as Notch receptors and APP (beta-amyloid precursor protein) while PSEN2
and PSENEN regulate APP processing through gamma-secretase [46] and are involved in Alz-
heimer’s disease [47]. A substantial number of genes involved in microRNA processing and reg-
ulation were dysregulated (Fig 6D)suchasDROSHA, which is the core nuclease that executes
the initiation step of miRNA processing in the nucleus [48] and DICER, the endoribonuclease
that cleaves naturally occurring long dsRNAs and short hairpin pre-microRNAs (miRNA) into
short interfering RNAs (siRNA) and mature microRNAs [49]. The SMAD proteins control
DROSHA-mediated microRNA maturation [50] and the SMAD1 gene is upregulated in DLD-1
while SMAD2 is downregulated in MOLT-4 (Fig 6D). SMAD3 is downregulated in both cell
lines (Fig 6D). Micro RNA host genes including MIR17HG, was significantly downregulated in
MOLT-4 while not differentially expressed in DLD-1 (Fig 6D). The MIR21HG is significantly
downregulated in DLD-1 while it is not differentially expressed in MOLT-4 (Fig 6D). The
MIR17HG or MiR-17-92 Cluster Host Gene encodes for six miRNAs that influence cell survival,
proliferation, differentiation, and angiogenesis [35]. miR-21, miR-17 and miR-19a are directly
involved in the proliferation and metastasis of colon cancer [51]. miR-21 downregulates tumor
suppressor Pdcd4 and stimulates invasion, intravasation and metastasis in colorectal cancer
Fig 6. Analysis of microarray data using Gene Functional Classification and Functional Annotation; Dysregulation of genes involved in the Notch
signaling system and microRNA processing and regulation. A Regulation of transcription BRNA mediated gene silencing (PTGS) CNotch signaling
pathway DDysregulated microRNA processors and regulators.
doi:10.1371/journal.pone.0135958.g006
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 15 / 20
[36]. The downregulation of miR-21 also induces differentiation of chemoresistant colon cancer
cells enhancing their susceptibility to therapy [37].
Overview of cellular processes and functions with deregulated genes
under microgravity
Fig 7 shows a summary view of the functional annotation clustering of GO terms using DAVID,
demonstrating processes or functions which the highly dysregulated genes (>2 log fold) in the
DNA microarray list associated with. Upregulated genes were involved in functions associated
with transcriptional regulation, proteolysis and negative regulation of cell growth (Fig 7A).
Prominently, correlating with the observations in gene expression analysis by real time PCR and
experimental procedures on the cell cycle progression of DLD-1 cells, the most significant cluster
of genes with the highest enrichment score were involved in cell cycle (Fig 7A). All genes that
were associated with these GO terms were downregulated. Other clusters that were associated
with downregulated genes included cytoskeleton, nucleoplasm and DNA repair; biological pro-
cesses that are vital to cell cycle and proliferation. MOLT-4 cells showed similar results with
lower enrichment scores. Upregulated genes again clustered in transcriptional regulation and
programmed cell death (Fig 7B) and significantly, in pathways in cancer. DNA replication was
an enriched GO term in the downregulated gene cluster with many associated processes such as
Fig 7. Functional annotation of microarray data using DAVID. A Functional annotation of upregulated and downregulated genes of DLD-1 cells under
microgravity BFunctional annotation of upregulated and downregulated genes of MOLT-4 cells under microgravity.
doi:10.1371/journal.pone.0135958.g007
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 16 / 20
DNA repair and nucleotide binding also being enriched. From the functional annotation cluster-
ing of the deregulated list of genes, it can be surmised that microgravity has a suppressive effect
on the progression of cancer, particularly in the colorectal cancer cell line- DLD-1.
Conclusion
Although the mechanism of action is still unclear, normal cells, stem cells and cancer cells have
altered physiological properties under microgravity. The cytoskeleton and the plasma mem-
brane of the cell may be sensitive to changes in gravity [52] and respond by altering vital intra-
cellular signaling networks or the modulation of proto oncogene expression through
intracellular signaling pathways could be a mechanism of action [53]. It is of significance that
cancer cell proliferation and progression can be altered by microgravity in DLD-1 cells and to a
lesser extent, in MOLT-4 cells, as demonstrated by this study. Simulated microgravity may
affect solid tumor cell lines markedly such as DLD-1, which showed a higher percentage of dys-
regulated genes compared to the hematological tumor cell line, MOLT-4. The process of cell
cycle in DLD-1 cells was markedly affected with reduced viability, reduced colony forming abil-
ity, an apoptotic population and dysregulation in cell cycle genes. Real time PCR and western
blotting also demonstrated dysregulation of significant oncogenes and cancer progression
markers such as JUNB,CD44,MYC and CD117. This was corroborated with the downregula-
tion of the process of cell cycle as demonstrated by the functional clustering of DNA microar-
ray data using GO terms by DAVID. This study also demonstrated for the first time, the
dysregulation of the microRNA host genome, miR-22 in a colorectal cancer cell line, DLD-1.
Due to the significant tumor suppressive role of microRNA-22, its upregulation under micro-
gravity may contribute to the anti-proliferative effect of microgravity. Identifying mechanisms
by which microgravity influences miR-22 expression and the other dysregulated microRNA
host genes identified in this study, may provide potential candidates for cancer therapy.
Supporting Information
S1 Fig. Analysis of microarray data using Gene Functional Classification and Functional
Annotation- Dysregulation of genes involved in the Notch signaling system. A Conserved
WD-40 domain (proteins involved in signal transduction, pre-mRNA processing and cytoskel-
eton assembly) BRegulators of ubiquitination of proteins and Membrane protein proteolysis
involved in notch signaling.
(TIF)
S1 Table. List of Primers and antibodies.
(DOCX)
S2 Table. >2 log fold down regulated genes in microarray of DLD-1 cells under micrograv-
ity.
(DOCX)
S3 Table. >2 log fold upregulated genes in microarray of DLD-1 cells under microgravity.
(DOCX)
S4 Table. >2 log fold Up and down regulated genes in microarray of MOLT-4 cells under
microgravity.
(DOCX)
S5 Table. Microarray analysis reveals commonly deregulated genes under Microgravity in
both cell lines.
(DOCX)
Microgravity Dysregulates Cell Cycle and MiRNA Gene Networks in Cancer
PLOS ONE | DOI:10.1371/journal.pone.0135958 August 21, 2015 17 / 20
Acknowledgments
This work was supported by a grant to R.S.V by the Defense Research Development Organiza-
tion (DLS/81/48222/LSRB-189/ID/2009 and DLS/81/48222/LSRB-273/SH&DD/2013). P.V., P.
S. wish to thank the Council of Scientific and Industrial Research, India for senior research fel-
lowship, R.A., and R.S., wish to thank Indian Institute of Technology Madras for their
fellowship.
Author Contributions
Conceived and designed the experiments: PV PS RSV. Performed the experiments: PV PS RS
RA. Analyzed the data: PV PS RSV NK RK. Contributed reagents/materials/analysis tools:
RSV. Wrote the paper: PV PS RSV.
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