alexa Cyclin D2 Promotes the Proliferation of Human Mesenchymal Stem Cells | OMICS International
ISSN: 2572-4916
Journal of Bone Research
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Cyclin D2 Promotes the Proliferation of Human Mesenchymal Stem Cells

Ken Kono, Shingo Niimi and Rumi Sawada*

Division of Medical Devices, National Institute of Health Sciences, Japan

Corresponding Author:
Rumi Sawada
Division of Medical Devices
National Institute of Health Sciences
1-18-1 Kamiyoga, Setagaya-ku
Tokyo 158-8501, Japan
Tel: 81-3-3700-1487
Fax: 81-3-3700-9196
E-mail: [email protected]

Received date: October 24, 2013; Accepted date: December 26, 2013; Published date: December 28, 2013

Citation: Kono K, Niimi S, Sawada R (2013) Cyclin D2 Promotes the Proliferation of Human Mesenchymal Stem Cells. J Bone Marrow Res 2:136. doi:10.4172/2329-8820.1000136

Copyright: © 2013 Kono K, 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.

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Abstract

Background: Human mesenchymal stem cells (hMSCs) hold promise for use in cell-based therapies and tissue engineering. Although hMSCs are thought to be stable ex vivo, it is possible that they undergo an undesirable transformation to a phenotype of unlimited proliferation during ex vivo. In this study, we searched for the factor required for unlimited proliferation of hMSCs. Methods: Changes in gene expression were evaluated between hMSCs and Ewing’s sarcoma cell lines, which may be derived from hMSCs, using GeneChip Human Genome U133 plus 2.0 Array. A gene up-regulated by at least 10-fold in Ewing’s sarcoma cell lines, Cyclin D2, was overexpressed in hMSCs by a lentiviral vector. Results: Overexpression of Cyclin D2 in hMSCs altered cell morphology and promoted cell proliferation. Expression of transforming growth factor-b2 (TGF-b2), which induces senescence in hMSCs, was down-regulated in Cyclin D2- overexpressing hMSCs. Furthermore, Gene Ontology analysis revealed that Cyclin D2 overexpression activated expression of genes associated with proliferation and interphase. Conclusions: Cyclin D2 promotes hMSC proliferation and is a candidate biomaker for hMSC transformation.

Keywords

hMSCs; Ewing’s sarcoma; Cyclin D2; Cell proliferation

Introduction

Mesenchymal stem cells (MSCs) self-replicate and differentiate into a variety of cell types such as osteoblasts, chondrocytes, adipocytes, and smooth muscle cells [1-5]. These capacities have made MSCs useful in studies of bone and cartilage regeneration [6-8]. One of the sources of human MSCs (hMSCs) is adult bone marrow, although they occur at a rate of one per one-hundred-thousand nucleated cells [6], and the available volume of bone marrow is limited. To secure the numbers of hMSCs required for tissue regeneration, the cell must be expanded ex vivo. Although hMSCs are stable ex vivo, it is possible that they undergo transformation to an unlimited proliferation phenotype during expansion.

Previous studies have demonstrated that Ewing’s sarcoma is derived from MSCs [9-12]. Ewing’s sarcoma is a malignancy that primarily affects children and young adults, with a peak incidence between the ages of 14 and 20 years. It arises mainly in bone and less commonly in soft tissues. The t(11;22)(q24;q12) chromosomal translocation generating EWS-FLI-1 fusion gene is found in 85% of cases [13]. EWS-FLI-1 knockdown inhibits cell proliferation in Ewing’s sarcoma cells [14,15]. Thus, EWS-FLI-1 expression is believed to play a key role in Ewing’s sarcoma development. However, EWS-FLI-1 expression does not transform normal murine and human fibroblasts [16,17], suggesting EWS-FLI-1 promotes malignant transformation in selective cells.

Several reports have demonstrated that EWS-FLI-1 expression transforms murine MSCs; indeed, tumors form when these cells are injected into immunodeficient mice [9,12]. In contrast, EWSFLI- 1 expression in hMSCs does not accelerate cell proliferation and transformation (10). EWS-FLI-1 expression in hMSCs induces a gene expression profile that closely mimics that of Ewing’s sarcoma [9-11] without affecting proliferation. Therefore, MSCs are thought to be the origin of Ewing’s sarcoma, but because EWS-FLI-1 alone cannot transform hMSCs, we believe other factors are required for transformation.

The most important safety concern when using hMSCs in cell- based therapies and tissue engineering is the occurrence of unlimited proliferation during ex vivo culture. To identify the factors required for unlimited hMSC proliferation, we compared the gene expression profiles of hMSCs and Ewing’s sarcoma cell lines and found that Cyclin D2 expression was extremely high in the Ewing’s sarcoma cell lines. Overexpression of Cyclin D2 promotes proliferation of hMSCs, suggesting that Cyclin D2 is a candidate biomaker for hMSC transformation.

Materials and Methods

Cell culture

hMSCs derived from bone marrow were purchased from Lonza (Walkersville, MD) and cultured in MSCGM BulletKit, a mesenchymal stem cell basal medium with mesenchymal cell growth supplement, L-glutamine, and gentamycin/amphotericin-B (Lonza Walkersville, MD). Ewing’s sarcoma cell lines (Hs 822.T, Hs 863.T, RD-ES, and SK-ES-1) were purchased from American Type Culture Collection (ATCC; Manassas, VA). Hs 822.T and Hs 863.T were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Intergene). RDES was cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS. SK-ES-1 was cultured in McCoy’s 5a medium modified (Gibco) supplemented with 15% FBS. 293T (human kidney; ATCC) was cultured in DMEM supplemented with 10% FBS.

Microarray analysis

Total RNA was extracted from hMSCs and Ewing’s sarcoma cell lines with the RNeasy Mini Kit (QIAGEN, Valencia, CA). Total RNA quantity and quality were assessed on an Agilent 2100 Bio-analyzer (Agilent, Santa Clara, CA); 100 ng total RNA was used to generate biotin-modified amplified RNA (aRNA) with GeneChip 3′IVT Express Kit (Affymetrix, Santa Clara, CA). Reverse transcription (RT) of firststrand complementary DNA (cDNA) with the T7 promoter sequence was performed with the T7 oligi(dT) primer. Second-strand cDNA synthesis was used to convert the single-stranded cDNA into a doublestranded DNA template by using DNA polymerase and RNase H to simultaneously degrade the RNA and synthesize second-strand cDNA. In vitro transcription of biotin-modified aRNA with IVT Labeling Master Mix generated multiple copies of biotin-modified aRNA from the double-stranded cDNA templates. The aRNA was purified and quantified; after fragmentation, it was hybridized to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix). The arrays were stained with phycoerythrin and washed at the GeneChip Fluidics station 450 (Affymetrix). The microarrays were scanned and data extracted using GeneChip scanner 3000 7G (Affymetrix); image analysis was performed using the Affymetrix GeneChip Command Console Software and digitized with the Affymetrix Expression Console.

Data processing and pathway analysis

Data analysis was performed with GeneSpring GX 11.0 software (Agilent Technologies, Santa Clara, CA). Raw data were normalized to the 50th percentile per chip and the median per gene. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis (IPA) 9.0 (Ingenuity Systems, Redwood City, CA). Fisher’s exact test was used to calculate a P-value. Activation z-score was calculated as a measure of functional and translational activation in Networks and Upstream regulators analysis. An absolute z-score >2 was considered significant.

Real-time RT-PCR

Total RNA was reverse-transcribed with SuperScript III First- Strand Synthesis System for RT-PCR (Life Technologies Co., Carlsbad, CA). Real-time RT-PCR was performed with LightCycler Fast Start DNA Master SYBR Green I (Roche Applied Science, Basel, Switzerland) in a Roche LightCycler instrument (software version 4.0). mRNA expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers for Cyclin D2 and p16 were 5´-TACTTCAAGTGCGTGCAGAAGGAC-3´ and 5´-TCCCACACTTCCAGTTGCGATCAT- 3´ (Cyclin D2) and 5´-CACTCACGCCCTAAGC- 3´ and 5´-GCAGTGTGACTCAAGAGAA-3´ (p16). The primers for transforming growth factor-b2 (TGF-b2) and GAPDH were from Light Cycler Primer Sets (Search LC GmbH, Heidelberg, Germany).

Cloning and expression of Cyclin D2

Cyclin D2 cDNA was amplified by RT-PCR of mRNA extracted from SK-ES-1 using 5´-GAATTCGCCACCATGGAGCTGCTGTGCCACGAGG-3´ (forward; EcoR I site underlined) and 5´-CTCGAGTCACAGGTCGATATCCCGCACG-3´ (reverse; Xho I site underlined).The amplified products were cloned into pTA2 (ToYoBo, Osaka, Japan) and verified by sequencing. The verified Cyclin D2 cDNA was cloned into the EcoR I and Xho I sites of pLVSIN-CMV Pur (TaKaRa, Shiga, Japan). Lentiviral vector was prepared with the Lenti-XTM Packaging System (TaKaRa) according to manufacturer protocols.

Viral infection

hMSCs were infected with the lentiviral vector containing Cyclin D2 (hMSCs/CyclinD2) or empty vector (hMSCs/Empty) at 37°C for 24 h. Infected cells were selected with 1 mg/mL puromycin for 14 days and the bulk of the resistant cells was used in subsequent experiments.

Western blotting

hMSCs/CyclinD2 and hMSCs/Empty were lysed in RIPA buffer (Wako, Osaka, Japan). Cyclin D2 was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a membrane (Immunobilon-PSQ; Millipore, Billerica, MA). Blots were blocked and probed overnight at 4°C with a mouse monoclonal antibody against Cyclin D2 (MBL, Nagoya, Japan). Blots were incubated with peroxidase-conjugated anti-mouse IgG (Abcam) and bound antibodies were visualized with Chemi-Lumi One Super Chemiluminescence (Nacalai Tesque, Kyoto, Japan).

Cell proliferation

Proliferation of hMSCs/CyclinD2 and hMSCs/Empty was measured with the TetraColor ONE reagent (Seikagaku Co., Tokyo, Japan). Cultures were incubated for 2 h in medium containing the reagent. Absorbance was read at 450 nm (reference at 600 nm) on a plate reader (SH-9000, Corona Electric Co., Ibaraki, Japan).

Results

Cyclin D2 expression in Ewing’s sarcoma cell lines versus hMSCs

To identify the factors required for proliferation of hMSCs, we compared the gene expression profiles of hMSCs and four Ewing’s sarcoma cell lines (Hs 822.T, Hs 863.T, RD-ES, and SK-ES-1) (Figure 1a). Hs 822.T and Hs 863.T had similar expression profiles, as did RD-ES and SK-ES-1. The expression profiles of Hs 822.T and Hs 863.T were more similar to those of hMSCs than to those of the other Ewing’s sarcoma (RD-ES and SK-ES-1). Therefore, we first compared the expression profiles of hMSCs, Hs 822.T, and Hs 863.T. We identified 44 genes that differed by at least 10-fold between hMSCs and Ewing’s sarcoma cell lines (data not shown). These were narrowed to 9 genes by selecting genes that also differed from hMSCs by more than 10-fold in RD-ES and SK-ES-1 (Table 1). CCND2 (Cyclin D2) stood out in this group of 9 genes, because it represents a family of key cell-cycle regulators. Indeed, aberrant expression of Cyclin D2 has been associated with tumor progression in many tumor types [18-21]. We measured Cyclin D2 mRNA expression in hMSCs and Ewing’s sarcoma cell lines by real-time PCR and confirmed its extreme induction in Ewing’s sarcoma (Figure 1b). Hs 822.T and Hs 863.T exhibited similar Cyclin D2 expression levels; expression was higher in RD-ES and SK-ES-1.

Gene Symbol Entrez Gene Name Fold Change
Hs822.1                   Hs863 T       RD-ES   SK-ES-1
BIEND5 BEN domain containing 5 -16.720 -14.819 27.302 26.690
CCND2 Cyclin D2 38.078 37.702 67.745 247.690
FBLN1 Fibulin1 -18.992 -24.363 -36.755 -198.523
HAPLN 1 Hyaluronan and and proteo glycan link protein 1 -22.460 -11.738 -26.674 -110.084
1GF2BP1 Insulin-Re growth factor 2 mRNA binding protein 1 22.847 22.358 288.588 207.475
SLC24A3 Solute carrier may 24 (sodium/potassium calcum exchanger), member 3 38.867 75.637 324.331 173.842
SSTR1 Somatostain receptor 1 79.594 14.840 22.406 28.783
STEAP4 STEAP  family member 4 -24.490 -18.837 -21.747 -33.153
TMEFF2 Transmembrane protein with EGF-like and two follistatin-ike domain 2 37.577 10.463 34.418 197.834

Table 1: Expression of Cyclin D2 was higher in Ewing’s sarcoma cell lines than in hMSCs. The nine genes differentially expressed by at least 10-fold between hMSCs and the four Ewing’s sarcoma cell lines.

business-management-review-Ewing-sarcoma-cell

Figure 1a: Expression of Cyclin D2 was higher in Ewing’s sarcoma cell lines than in hMSCs. Global gene expression was measured in hMSCs and Ewing’s sarcoma cell lines. The whole-genome expression pattern is displayed in hierarchical cluster format. The color key shown at the bottom is in Log2 scale.

business-management-review-Cyclin-D2-expression

Figure 1b: Expression of Cyclin D2 was higher in Ewing’s sarcoma cell lines than in hMSCs. Cyclin D2 expression in hMSCs and Ewing’s sarcoma cell lines was determined by real-time RT-PCR with values normalized to GAPDH. Results are plotted relative to hMSCs. Data from triplicate samples (means ± SD) are shown.

Overexpression of Cyclin D2 promoted hMSC proliferation

We transduced the Cyclin D2 gene into hMSCs by using a lentiviral vector; an empty vector served as a negative control. At 14 days after infection when puromycin selection was completed, Cyclin D2 mRNA expression in hMSCs infected with lentiviral vector containing Cyclin D2 (hMSCs/CyclinD2) was about 10,000-fold higher than in hMSCs infected with empty vector (hMSCs/Empty) (Figure 2a). Cyclin D2 was stably expressed in hMSCs at least 56 days after infection (Figure 2b). Next, we observed the cell morphology of the hMSCs/CyclinD2. Phase contrast microscopy revealed the normal fibroblast-like morphology of hMSCs/Empty (Figure 2c) and smaller spread areas in hMSCs/ CyclinD2 (Figures 2c and 2d). Furthermore, proliferation of hMSCs/ CyclinD2 was faster than that of hMSCs/Empty, indicating that overexpression of Cyclin D2 promoted hMSC proliferation. However, the proliferation slowed over time and did not result in unlimited proliferation (data not shown).

business-management-review-RT-PCR-normalized

Figure 2a: Over expression of Cyclin D2 promoted hMSC proliferation. At 14 days after infection when puromycin selection was completed, Cyclin D2 mRNA in hMSCs/CyclinD2 and hMSCs/Empty was determined by real-time RT-PCR and normalized to GAPDH. Results are plotted relative to hMSCs/ Empty.

business-management-review-hMSC-proliferation

Figure 2b: Overexpression of Cyclin D2 promoted hMSC proliferation. Cyclin D2 protein expression was visualized by western blotting at the indicated days after infection. Equal amounts of protein were loaded in each lane.

business-management-review-14-days-after-infection

Figure 2c: Overexpression of Cyclin D2 promoted hMSC proliferation. At 14 days after infection, cells were observed by phase contrast microscopy.

business-management-review-Fifteen-random-hMSCs

Figure 2d: Overexpression of Cyclin D2 promoted hMSC proliferation. Fifteen random hMSCs/Empty and hMSCs/CyclinD2 cells were measured with Image J 1.43t software (NIH, USA). Values were plotted relative to the averaged area of hMSCs/Empty.

business-management-review-Tetra-Color-One

Figure 2e: Overexpression of Cyclin D2 promoted hMSC proliferation. At 14 days after infection, equal numbers of cells were seeded in 24-well plates and proliferation was determined with Tetra Color One 1, 6, and 12 days after seeding. The optical density (Cyclin D2/Empty) of triplicate samples is shown.

TGF-b2 expression was down-regulated in hMSCs/CyclinD2, but p16 expression was not

To investigate the effects of overexpression of Cyclin D2 on the cell cycle, we examined the change in cell cycle-associated gene expression over time (p16, p21, Bmi1, TGF-b1, and TGF-b2). We did not detect significant differences in expression of p21, Bmi1, and TGF-b1 between hMSCs/CyclinD2 and hMSCs/Empty (data not shown). However, TGF-b2 expression was lower in hMSCs/CyclinD2 than in hMSCs/ Empty (Figure 3a). In addition, the increase in TGF-b2 expression during culture was suppressed in hMSCs/CyclinD2 compared with hMSCs/Empty. In contrast, the increasing rate of p16 expression in hMSCs/CyclinD2 was higher than in hMSCs/Empty, although expression in both cell types was comparable 14 days after infection (Figure 3b).

business-management-review-TGF-b2-mRNA

Figure 3a: TGF-b2 expression over time. TGF-b2 mRNA expression was determined by real-time RT-PCR at 14, 28, and 42 days after infection. Results are plotted relative to hMSCs/Empty at 14 days after infection. A representative of three independent experiments is shown

business-management-review-real-time-RT-PCR

Figure 3b: p16 expression over time. p16 mRNA expression was determined by real-time RT-PCR at 14, 28, and 42 days after infection. Results are plotted relative to hMSCs/Empty at 14 days after infection. A representative of three independent experiments is shown.

Overexpression of Cyclin D2 altered the expression of genes associated with cell proliferation and interphase

Total RNA was extracted from hMSCs/CyclinD2 and hMSCs/ Empty 14 days after infection and analyzed by DNA microarray, which identified 690 genes that were differentially expressed by at least 2-fold between hMSCs/CyclinD2 and hMSCs/Empty (Figure 4a). Gene ontology (GO) analysis revealed these genes are associated with movement, development, growth and proliferation, cell cycle, and intercellular signaling and interactions (Table 2). Specific predictions indicated that proliferation and interphase are activated in hMSCs/CyclinD2. The induced genes that are associated with proliferation and interphase are listed in tables 3 and 4; in summary, 94 of 186 genes and 19 of 50 genes exhibited expression shifts consistent with increased in proliferation and interphase, respectively.

business-management-review-Global-gene-expression

Figure 4a: Global gene expression in hMSCs/CyclinD2. Global gene expression patterns were compared in hMSCs/CyclinD2 and hMSCs/ Empty. Genes differentially expressed by at least 2-fold are shown (690 genes). The color key shown at the bottom is in Log2 scale

Top 5 functional category Function annotation p-Value Activation z-score No. of genes
Cellular Movement cell movement of prostate cancer 7.94E-06 0.918 14
migration of prostate cancer cells 1.54E-05 1.339 12
recruitment of cells 6.57E-05 1.55 28
Cellular Development proliferation of tumor cell lines 9.04E-06 1.15 92
proliferation of cancer cells 1.96E-05 1.302 30
differentiation of connective tissues 4.35E-05 -0.379 41
proliferation of tumor cells 4.61E-05 1.036 37
Cellular Growth and Proliferation proliferation of tumor cell lines 9.04E-06 1.15 92
proliferation of cancer cells 1.96E-05 1.302 30
proliferation of tumor cells 4.61E-05 1.036 37
proliferation of cells 9.27E-05 3.142 186
Cell Cycle interphase 1.34E-05 2.19 50
Cell-To-Cell Signaling and Interaction recruitment leukocytes 7.13E-05 1.159 26

Table 2: Global gene expression in hMSCs/CyclinD2. Gene ontology (GO) analysis of the 690 genes was performed with Ingenuity Pathway Analysis (IPA) 9.0. Top five functional categories and the specified categories are listed. An absolute z-score >2 was considered as significant.

     
ID   Genes in dataset   Fold Change   Prediction (based on expression direction) ID   Genes in dataset   Fold Change   Prediction (based on expression ID Genes in dataset Fold Change Prediction (based on Expression direction)
209292_at ID4 3.609 Increased 201195_s_at SLC7A5 2.083 direction) 203083_at THBS2 2.096 Decreased
206271_at TLR3 2.023 Increased 222749_at SUFU -3.251 Increased 203468_at CDK10 2.228 Decreased
203373_at SOCS2 3.513 Increased 204508_s_at CA12 3.237 Increased 1552721_a_at FGF1 -2.054 Decreased
206649_s_at TFE3 2.069 Increased 203764_at DLGAP5 2.188 Increased 204052_s_at SFRP4 2.249 Decreased
201292_at TOP2A 2.434 Increased 214012_at ERAP1 5.288 Increased 205548_s_at BTG3 2.073 Decreased
204766_s_at NUDT1 2.158 Increased 204614_at SERPINB2 -2.048 Increased 242989_at STRN 2.145 Decreased
209321_s_at ADCY3 2.329 Increased 201416_at SOX4 3.11 Increased 213905_x_at BGN 2.09 Decreased
206693_at IL7 2.399 Increased 214581_x_at TNFRSF21 -2.049 Increased 228780_at POU3F3 -2.721 Decreased
205345_at BARD1 2.029 Increased 223570_at MCM10 2.545 Increased 205016_at TGFA -3.032 Decreased
226377_at NFIC 2.026 Increased 219743_at HEY2 2.13 Increased 1569791_at STK4 2.396 Decreased
212148_at PBX1 2.514 Increased 209970_x_at CASP1 3.111 Increased 216008_s_at ARIH2 2.331 Decreased
202213_s_at CUL4B 2.093 Increased 202684_s_at RNMT 2.503 Increased 221577_x_at GDF15 2.143 Decreased
224954_at SHMT1 2.105 Increased 242979_at IRS1 2.329 Increased 202153_s_at NUP62 2.009 Decreased
209960_at HGF 2.732 Increased 225141_at NFATC3 2.553 Increased 202556_s_at MCRS1 2.165 Decreased
217371_s_at IL15 2.953 Increased 218030_at GIT1 3.032 Increased 203395_s_at HES1 2.898 Decreased
205887_x_at MSH3 2.1 Increased 218750_at TAF1D 2.304 Increased 203184_at FBN2 2.145 Decreased
1568865_at FNTB 2.556 Increased 201795_at LBR 2.383 Increased 203904_x_at CD82 2.382 Decreased
208296_x_at TNFAIP8 2.118 Increased 210045_at IDH2 2.018 Increased 206558_at SIM2 2.745 Decreased
226534_at KITLG 2.22 Increased 1553810_a_at KIAA1524 2.457 Increased 203665_at HMOX1 2.358 Decreased
214981_at POSTN 2.095 Increased 209160_at AKR1C3 2.281 Increased 203543_s_at KLF9 2.577 Decreased
214981_at POSTN 2.095 Increased 209160_at AKR1C3 2.281 Increased 203543_s_at KLF9 2.577 Decreased
206026_s_at TNFAIP6 2.008 Increased 232424_at PRDM16 2.334 Increased 1557729_at GRK5 2.098 Decreased
210135_s_at SHOX2 2.194 Increased 1554509_a_at FAM188A -2.494 Increased 236028_at IBSP -3.459 Decreased
204457_s_at GAS1 2.134 Increased 217494_s_at PTENP1 -2.849 Increased 221539_at EIF4EBP1 2.487 Decreased
221884_at MECOM 2.004 Increased 216205_s_at MFN2 -2.34 Increased 202430_s_at PLSCR1 2.324 Decreased
209919_x_at GGT1 2.63 Increased 200644_at MARCKSL1 3.121 Increased 204054_at PTEN 2.145 Decreased
229468_at CDK3 2.022 Increased 217991_x_at SSBP3 3.21 Increased 209617_s_at CTNND2 -2.01 Decreased
1563182_at ACVR1C -2.241 Increased 211653_x_at AKR1C1/AKR1C2 2.791 Increased 231697_s_at VMP1 2.741 Decreased
227404_s_at EGR1 2.543 Increased 228302_x_at CAMK2N1 -3.3 Increased 210143_at ANXA10 2.124 Decreased
210933_s_at FSCN1 2.227 Increased 234040_at HELLS 2.476 Increased 206233_at B4GALT6 -2.767 Decreased
205357_s_at AGTR1 2.987 Increased 218413_s_at ZNF639 2.019 Increased 209705_at MTF2 2.504 Decreased
209925_at OCLN -2.024 Increased 242907_at GBP2 2.488 Increased 209802_at PHLDA2 2.17 Decreased
205732_s_at NCOA2 2.484 Increased 219377_at GAREM 2.237 Increased 219685_at TMEM35 -2.404 Decreased
215404_x_at FGFR1 2.421 Increased 212865_s_at COL14A1 2.516 Increased 219047_s_at ZNF668 2.244 Decreased
235521_at HOXA3 2.05 Increased 203572_s_at TAF6 2.79 Decreased 64474_g_at DGCR8 2.201 Decreased
218807_at VAV3 2.555 Increased 232231_at RUNX2 2.12 Decreased 205159_at CSF2RB -4.605 Decreased
202202_s_at LAMA4 3.771 Increased 202931_x_at BIN1 2.056 Decreased 236012_at PSMF1 2.172 Affected
205500_at C5 2.375 Increased 214433_s_at SELENBP1 2.103 Decreased 235833_at PPAT 3.266 Affected
204128_s_at RFC3 2.06 Increased 228766_at CD36 -4.325 Decreased 221261_x_at MAGED4/MAGED4B 3.294 Affected
200951_s_at CCND2 18.529 Increased 235300_x_at RCHY1 2.569 Decreased 204983_s_at GPC4 -2.902 Affected
222073_at COL4A3 -2.278 Increased 222999_s_at CCNL2 2.019 Decreased 231837_at USP28 2.365 Affected
226731_at ITGA1 2.816 Increased 208791_at CLU -2.492 Decreased 1554606_at CEP120 2.141 Affected
227125_at IFNAR2 2.053 Increased 203973_s_at CEBPD 2.099 Decreased 224325_at FZD8 2.488 Affected
222036_s_at MCM4 2.31 Increased 209383_at DDIT3 2.065 Decreased 217650_x_at ST3GAL2 2.003 Affected
204061_at PRKX 2.226 Increased 219266_at ZNF350 2.143 Decreased 224022_x_at WNT16 -3.268 Affected
228962_at PDE4D 2.562 Increased 206825_at OXTR -2.396 Decreased 208962_s_at FADS1 3.311 Affected
212672_at ATM 2.024 Increased 205027_s_at MAP3K8 2.11 Decreased 202948_at IL1R1 2.512 Affected
225572_at CREB1 2.243 Increased 205891_at ADORA2B 2.166 Decreased 50277_at GGA1 2.292 Affected
203046_s_at TIMELESS 2.156 Increased 231947_at MYCT1 -2.144 Decreased 1555843_at HNRNPM 2.611 Affected
213943_at TWIST1 2.021 Increased 212430_at RBM38 2.254 Decreased 214157_at GNAS -2.307 Affected
209465_x_at PTN 2.357 Increased 204159_at CDKN2C 2.004 Decreased 215987_at RAPGEF2 2.046 Affected
213506_at F2RL1 4.829 Increased 212401_s_at CDK11A/CDK11B 2.086 Decreased 216237_s_at MCM5 2.753 Affected
231559_at NNMT 2.208 Increased 205080_at RARB 2.058 Decreased 206086_x_at HFE 2.218 Affected
225740_x_at MDM4 2.283 Increased 214727_at BRCA2 2.489 Decreased 229807_s_at MAZ 2 Affected
226636_at PLD1 2.022 Increased 202718_at IGFBP2 -2.099 Decreased 231002_s_at RABEP1 2.266 Affected
227048_at LAMA1 2.296 Increased 213811_x_at TCF3 2.395 Decreased 209753_s_at TMPO 2.116 Affected
230462_at NUMB 2.763 Increased 202526_at SMAD4 2.16 Decreased 201627_s_at INSIG1 3.156 Affected
201727_s_at ELAVL1 2.216 Increased 1567013_at NFE2L2 3.005 Decreased 218019_s_at PDXK 2.152 Affected
205204_at NMB 2.09 Increased 234339_s_at GLTSCR2 2.573 Decreased 204639_at ADA 2.051 Affected
232044_at RBBP6 2.324 Increased 206332_s_at IFI16 2.24 Decreased 236223_s_at RIT1 2.823 Affected
205394_at CHEK1 2.071 Increased 228967_at EIF1 2.161 Decreased 208913_at GGA2 2.062 Affected
57532_at DVL2 2.03 Increased 235593_at ZEB2 2.106 Decreased 201286_at SDC1 2.164 Affected
208937_s_at ID1 2.256 Increased 1556583_a_at SLC8A1 -2.156 Decreased 201106_at GPX4 2.087 Affected

Table 3: ‘Proliferation of cells’ genes differentially expressed by at least 2-fold between hMSCs/CyclinD2 and hMSCs/Empty.

ID Genes in dataset Fold Change Prediction (based on expression direction)
206649_s_at TFE3 2.069 Increased
209960_at HGF 2.732 Increased
217371_s_at IL15 2.953 Increased
232231_at RUNX2 2.12 Increased
235300_x_at RCHY1 2.569 Increased
226534_at KITLG 2.22 Increased
235423_at ORC2 3.145 Increased
203973_s_at CEBPD 2.099 Increased
227404_s_at EGR1 2.543 Increased
205027_s_at MAP3K8 2.11 Increased
212401_s_at CDK11A/CDK11B 2.086 Increased
213811_x_at TCF3 2.395 Increased
206332_s_at IFI16 2.24 Increased
212672_at ATM 2.024 Increased
235593_at ZEB2 2.106 Increased
205394_at CHEK1 2.071 Increased
208937_s_at ID1 2.256 Increased
223570_at MCM10 2.545 Increased
242979_at IRS1 2.329 Increased
212148_at PBX1 2.514 Decreased
204159_at CDKN2C 2.004 Decreased
1552721_a_a FGF1 -2.054 Decreased
205016_at TGFA -3.032 Decreased
201727_s_at ELAVL1 2.216 Decreased
203395_s_at HES1 2.898 Decreased
203665_at HMOX1 2.358 Decreased
204054_at PTEN 2.145 Decreased
219312_s_at ZBTB10 2.171 Affected
229861_at RFFL 2.017 Affected
228302_x_at CAMK2N1 -3.3 Affected
235764_at PRDM5 2.205 Affected
1554509_a_a FAM188A -2.494 Affected
232424_at PRDM16 2.334 Affected
221539_at EIF4EBP1 2.487 Affected
204986_s_at TAOK2 -2.03 Affected
221577_x_at GDF15 2.143 Affected
225740_x_at MDM4 2.283 Affected
203046_s_at TIMELESS 2.156 Affected
200951_s_at CCND2 18.529 Affected
1567013_at NFE2L2 3.005 Affected
215404_x_at FGFR1 2.421 Affected
209753_s_at TMPO 2.116 Affected
209383_at DDIT3 2.065 Affected
229468_at CDK3 2.022 Affected
204457_s_at GAS1 2.134 Affected
218833_at ZAK 2.125 Affected
214048_at MBD4 2.383 Affected
205345_at BARD1 2.029 Affected
206693_at IL7 2.399 Affected
209292_at ID4 3.609 Affected

Table 4: ‘Interphase’ genes differentially expressed by at least 2-fold between hMSCs/CyclinD2 and hMSCs/Empty

Discussion

Although EWS-FLI-1 expression transformed murine MSCs, expression in hMSCs did not promote cell proliferation. In this study, we found that Cyclin D2 expression was extremely high in the Ewing’s sarcoma cell lines and overexpression of Cyclin D2 in hMSCs promoted cell proliferation. GO analysis also predicted that cell proliferation and interphase were activated by overexpression of Cyclin D2.

Cyclin D2 is a member of the family of D-type cyclins that mediate cell cycle regulation, differentiation, and oncogenic transformation [22,23]. D-type cyclins inactivate retinoblastoma (Rb) by phosphorylation, inducing release of E2F. Free E2F activates genes involved in the activation and maintenance of DNA synthesis. Thus, overexpression of Cyclin D2 generally has growth-promoting effects. Consistent with this notion, overexpression of Cyclin D2 in HeLa cells, in which Rb is inactivated by human papillomavirus E6 and E7 proteins [24], did not promote cell proliferation (data not shown). On the other hand, increased expression of Cyclin D2 inhibits proliferation of primary human fibroblasts [25], indicating that Cyclin D2 has both positive and negative roles in the cell cycle, depending on cell type. We found that Cyclin D2 in hMSCs has a positive role in the cell cycle (Figure 2e).

TGF-b2 expression was suppressed in hMSCs/CyclinD2 compared with hMSC/Empty during culture (Figure 3a). We previously demonstrated that hMSC growth is reduced and TGF-b2 expression increases during long-term culture [26]. We also reported that fibroblast growth factor-2 (FGF-2) stimulates hMSC growth by suppressing the up-regulation of TGF-b2 [27]. It is unclear how overexpression of Cyclin D2 suppresses the TGF-b2 increase, but this suppression may be involved in the acceleration of hMSCs/CyclinD2. In contrast, the expression of p16, which is up-regulated with aging [28], was increased in both cell types during culture, indicating that not only hMSCs/Empty but also hMSCs/CyclinD2 were aging normally. The rate of increase in hMSCs/Cyclin D2 was higher than in hMSCs/Empty (Figure 3b), suggesting that the promotion of cell proliferation in hMSCs/CyclinD2 induced cellular senescence and enhanced p16 expression. p16 is a tumor suppressor gene [29]; thus, this increase in p16 expression probably prevented unlimited proliferation. Consistent with this notion, some Ewing’s sarcomas contain a homozygous deletion of the p16 locus [16], possibly facilitating subsequent transformation.

In this study, overexpression of Cyclin D2 promoted proliferation of hMSCs but did not lead to unlimited proliferation. Other factors are required for the unlimited proliferation of hMSCs. IGF2BP1 was aberrantly expressed in Ewing’s sarcoma (Table 1), consistent with a previous report of an association between increased IGF2BP1 expression and tumor progression in patients with lung cancer [30]. Thus, we attempted to transduce the IGF2BP1 gene into hMSCs, but IGF2BP1 expression was up-regulated by only 2-fold and transduction efficiency was low (data not shown). The cause for this inefficiency is unclear. Because the growth kinetics of IGF2BP1-transformed E. coli is quite slow (data not shown), it is likely that overexpression of IGF2BP1 is deleterious for hMSCs.

We did not tested whether the other genes listed in Table 1 affect proliferation of hMSCs, because these genes were not thought to directly affect the proliferation. Furthermore, not all Ewing’s sarcomas express EWS-FLI-1: indeed, EWS-FLI-1 mRNA was not detected in Hs 822.T and Hs 863.T (data not shown). Thus, we did not transduce the EWSFLI- 1 gene into hMSCs. However, it is possible that the cooperation of these proteins is important for the development of Ewing’s sarcoma. Thus, it would be interesting to transduce these genes into hMSCs in addition to Cyclin D2.

Conclusion

Cyclin D2 promotes hMSC proliferation and is a candidate biomaker for hMSC transformation.

Acknowledgements

The authors would like to thank Atsuko Matsuoka for helpful discussions. This work was supported by the Health and Labor Sciences Research Grants for Research on Regulatory Science of Pharmaceuticals and Medical Devices (H23- IYAKU-SHITEI-027) from the Ministry of Health, Labor and Welfare of Japan.

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