ONCOLOGY REPORTS 34: 2011-2021, 2015

NOV inhibits proliferation while promoting apoptosis

and migration in osteosarcoma cell lines through
p38/MAPK and JNK/MAPK pathways
JUAN YAO, YAGUANG WENG, SHUJUAN YAN, MENGYI HOU, HAO WANG, QIONG SHI and GUOWEI ZUO

Key Laboratory of Diagnostic Medicine Designated by the Chinese Ministry of Education,

Chongqing Medical University, Chongqing 400016, P.R. China

Received April 22, 2015; Accepted June 17, 2015

DOI: 10.3892/or.2015.4153

Abstract. The nephroblastoma overexpressed (NOV) gene, a (JNK inhibitor), respectively, the NOV-induced proliferation
member of the CCN gene family that encodes secreted proteins inhibition and cell apoptosis were reversed. In conclusion,
involved in a variety of processes including tumorigenesis, is the results revealed that NOV regulates the tumor growth of
often altered in a variety of tumors, including osteosarcoma. osteosarcoma cells through activation of the MAPK signaling
Recent studies indicated that NOV promotes osteosarcoma pathway and promotes osteosarcoma cell migration in vitro.
metastasis, but its biological functions and molecular
mechanisms on osteosarcoma proliferation have yet to be fully Introduction
elucidated. The aim of the present study was to examine the
role of NOV in osteosarcoma biology. Reverse transcription- Osteosarcoma is the most common primary bone cancer in
polymerase chain reaction (RT-PCR) and western blot analysis adolescents and young adults (1). Although treatment modali-
were performed to characterize the endogenous expression of ties have been improved over the past decades, the survival rate
NOV in osteosarcoma cell lines. Recombinant adenovirus of these patients has limited improvement and the 3-5 years of
expressing NOV/siNOV (AdNOV/AdsiNOV) was used to infect survival rate after amputation is only 5-20% (2,3). Therefore,
osteosarcoma cell lines with a relatively low/high endogenous it is particularly important to understand the pathogenesis of
NOV expression to determine the functional relevance of NOV osteosarcoma in order to develop novel treatment strategies.
expression to osteosarcoma cell growth and migration in vitro, Nephroblastoma overexpressed (NOV) gene maps on chro-
respectively. As a result, osteosarcoma cell proliferation was mosome 8q24.1 and was originally identified as aberrantly
significantly reduced by NOV upregulation, indicated by expressed in avian nephroblastoma induced by myeloblastosis-
3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltrazolium bromide associated virus (4). NOV belongs to the CCN family of
(MTT), colony forming assay and cell cycle analysis. Cell genes, which comprises five additional members: cystein-rich
apoptosis was markedly induced, as indicated by Hoechst 33258 protein 61 (Cyr61), connective tissue growth factor (CTGF),
staining assay and flow cytometry (FCM) detection. Despite Wnt-1-induced secreted protein (WISP)-1, WISP-2 and
the antiproliferative effect, NOV-transfected osteosarcoma WISP-3 (5). The CCN genes encode secreted proteins of
cells exhibited increased migration ability. The possible 35-48 kDa associated with the extracellular matrix (ECM) and
molecular mechanisms underlying the biological role of NOV cell membrane, which are involved in the regulation of various
were also investigated. The results demonstrated that NOV cell functions such as proliferation, differentiation, migration,
increased the phosphorylation of p38 and c-Jun N-terminal attachment, angiogenesis and tumorigenesis (6). These genes
kinase (JNK) mitogen-actived protein kinases (MAPKs) in are expressed in all derivatives of the three embryonic sheets
osteosarcoma cell lines. When the phosphorylation of p38 and and are involved in the development of kidney, nervous system,
JNK were inhibited by SB203580 (p38 inhibitor) or SP600125 muscle, bone marrow, cartilage and bone (7).
Unlike other CCN family members Cyr61 and CTGF,
NOV has been less studied and the functions of NOV protein
among different tissues are inconsistent and sometimes contro-
versial. NOV was originally described as antiproliferative (8)
Correspondence to: Dr Guowei Zuo, Department of Laboratory
and its expression was associated with differentiation and
Medicine, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong,
Chongqing 400016, P.R. China
growth arrest in Wilm's tumor (9), rhabdomyosarcomas (10),
E-mail: 17305105@qq.com cartilaginous tumors (11), renal cell carcinoma (12) and Ewing
sarcoma (13), while more recent data associate NOV expression
Key words: osteosarcoma, nephroblastoma overexpressed gene, with increased proliferative index of 3T3 fibroblast (14,15) and
cell proliferation, apoptosis, migration, tumor metastasis tissue samples of prostate. Furthermore, although NOV reduced
the growth rate of renal cell carcinoma and Ewing sarcoma
transfectants in vitro, NOV expression was associated with
2012 YAO et al: NOV inhibits proliferation and promotes apoptosis of osteosarcoma

poor prognosis and shown to increase cell motility, resulting antibodies were purchased from Immunoway (Immunoway,
in enhanced metastatic potential in these tumors (12,13). It has Newark, DE, USA). Mouse anti‑β-actin monoclonal antibody,
been shown that in osteosarcoma NOV is inversely associated and secondary antibodies including HRP-conjugated goat
with the expression of liver/bone/kidney alkaline phosphatase anti-mouse IgG antibody and anti-rabbit IgG antibody were
isoform (16), an early marker of osteoblastic differentiation, purchased from Zhongshan Goldenbridge Biotechnology
and has prognostic value of in osteosarcoma (17). Nevertheless, (Beijing, China). BeyoECL was purchased from Millipore
the effect of NOV on osteosarcoma cell biological behaviors (Billerica, MA, USA).
and the underlying molecular mechanisms remain unclear.
P38/MAPK and JNK/MAPK signaling pathways, which Adenovirus infection. Οsteosarcoma cells (143B) were infected
have been confirmed to be tightly associated with tumor cell with recombinant adenovirus AdNOV and negative control
apoptosis (18,19), including osteosarcoma (20,21), are two AdGFP, respectively. MG63 osteosarcoma cells were infected
important intracellular signaling pathways. Certain studies with AdsiNOV and negative control AdRFP, respectively.
have suggested that NOV was able to induce cell apoptosis After being infected for 8-12 h, the medium was replaced with
or growth inhibition in various types of cancer through the serum-free or low serum DMEM followed by continued cell
activation of MAPKs signaling, such as choriocarcinoma, culturing for subsequent experiments.
nephroblastoma and Ewing sarcoma (13). Howerover, whether
p38/MPAK and JNK/MAPK signaling pathway activation is Cell viability assay. Cell proliferation was analyzed with the
involved in the NOV-induced effects of osteosarcoma remains MTT assay. Briefly, the cells infected with different adenovirus
to be elucidated. or blank control were incubated in 96-well plates at a density of
Based on the abovementioned studies, we aimed to discuss 1x104 cells/well with DMEM supplemented with 10% FBS. At
the effects of recombinant adenovirus (22,23)‑mediated NOV the indicated time‑points (24, 48, and 72 h), 20 µl of the MTT
overexpression or silencing on osteosarcoma cell lines, as reagent (5 mg/ml) was added to each well and the mixture
well as to investigate the probable molecular mechanisms was incubated for another 4 h. The MTT solution was then
underlying these effects. Our studies found that NOV inhibited removed and formazan was dissolved in DMSO for 10 min
the proliferation while promoting the apoptosis and migration at room temperature and the color reaction was measured
of osteosarcoma cell lines in vitro and the activation of at 492 nm with enzyme immunoassay analyzer (Bio-Rad,
p38/MAPK and JNK/MAPK signal may be involved in these Hercules, CA, USA). The experiment was repeated three times
processes. These results offer a new approach for the treatment and the proliferative activities were calculated for each well.
of osteosarcoma.
Colony‑forming assay. Osteosarcoma cell lines during
Materials and methods the log growth stage were seeded in 6-well culture plates
(4x102 cells/well) and treated with AdNOV/AdsiNOV, respec-
Cell culture and reagents. The 143B, U2OS, MG-63 and tively. After incubation for 2 weeks, the cells were stained with
SaOS2 human osteosarcoma cell lines were purchased from crystal violet and clones were counted. The colony-forming
the American Type Culture Collection (ATCC, Manassas, rate was obtained using the formula: (colony number/seeded
VA, USA). The cells were maintained in Dulbecco's modi- cell number) x 100%. The experiment was repeated three times.
fied Eagle's medium (DMEM; Utah, HyClone, UT, USA)
supplemented with 10% fetal bovine serum (FBS; Gibco Life Cell cycle and apoptosis analysis. Cell cycle and apoptosis
Technologies, Carlsbad, CA, USA) and 100 U/ml strepto- analysis were assessed by FCM. Briefly, the cells were seeded
mycin/penicillin at 37˚C in 5% CO2. Recombinant adenovirus in 6-well plates at a density of 2x105 cells/well and treated with
expressing green fluorescent protein (AdGFP), red fluorescent relevant adenovirus for 48 h. Log-phase cells from each group
protein (AdRFP), NOV (AdNOV) with GFP and recombinant were harvested by centrifugation. After being washed twice
adenovirus expressing siRNA targeted NOV (AdsiNOV) with ice-cold PBS and resuspended, an aliquot of samples
with RFP were donated by Professor T.-C. He, University of were fixed with pre-cold 75% enthanol at 4˚C overnight. The
Chicago Medical Center. 3-(4,5)-Dimethylthiazol(‑z‑yl)-3,5- remaining samples were added into apoptosis analysis solu-
diphenyltetrazolium bromide (MTT) and dimethyl sulphoxide tion and then analyzed by a FACSVantage SE flow cytometer
(DMSO) were obtained from Solarbio Biology (Beijing, (Becton-Dickinson, San Jose, CA, USA). Each experiment was
China). TRIzol reagent was purchased from Invitrogen‑Life performed three times.
Technologies (Carlsbad, CA, USA). Reverse transcription‑poly-
merase chain reaction (RT-PCR) reagents were purchased Hoechst 33258 staining assay. The Hoechst staining assay
from Takara (Otsu, Japan). Reverse transcription‑quantitative was performed according to the manufacturer's instructions
PCR (RT-qPCR) reagents were purchased from Thermo using the Hoechst 33258. The cells seeded in 24-well plates
Fisher Scientific, (Waltham, MA, USA). Hoechst 33258 and were treated with adenovirus for 48 h and then washed twice
western blot detection reagents were purchased from the with cold PBS. The cells were fixed with 4% paraformalde-
Beyotime Institute of Biotechnology (Jiangsu, China). Rabbit hyde for 10 min and continued to be washed twice with PBS.
anti-NOV monoclonal antibody was purchased from Abcam Two hundred mililiters of 1:1,000 Hoechst staining solution
(Abcam, Cambridge, MA, USA). Mouse anti-Bax, anti-Bcl-2 was added to each well and the cells were incubated for 30 min
monoclonal antibodies were purchased from Santa Cruz at room temperature in the dark. The cells were then washed
Biothechnology, Inc. (Santa Cruz, CA, USA). Rabbit anti-p38, twice with PBS and viewed under a UV microscope. Each
anti-phosphor-p38, anti-JNK, anti-phosphor-JNK polyclonal experiment was performed three times.
 ONCOLOGY REPORTS 34: 2011-2021, 2015 2013

Table I. The primer sequences and their product lengths in RT-PCR.

Gene Primer sequences Length of product (bp)

GAPDH F: 5'-CAG CGA CAC CCA CTC CTC-3' 120

R: 5'-TGA GGT CCA CCA CCC TGT-3'
NOV F: 5'-ACC TTC CTG CTT CTC CAT C-3' 490
R: 5'-CTC CCA GTG AAT CCT CCT C-3'
BAX F: 5'-CCC TTT TGC TTC AGG GTT TC-3' 150
R: 5'-TGT TAC TGT CCA GTT CGT CC-3'
BCL-2 F: 5'-GAG ACA GCC AGG AGA AAT CA-3'
R: 5'-CCTGTGGATGACTGAGTACC-3' 128

NOV, nephroblastoma overexpressed; BCL-2, B cell lymphoma-2; BAX, Bcl-2-associated X protein; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.

Transwell chamber migration assay. Cell migration assay and transferred subsequently onto PVDF membranes. After
were performed by 24-well Transwell chambers (8 µm pore blocking with 5% BSA (Bovine Serum Albumin, Solarbio) in
size; Millipore). Briefly, the cells (5x104/400 µl) treated with TBST, the membranes were probed with the primary antibody
different adenovirus in serum-free DMEM were seeded onto and incubated at 4˚C overnight. Horseradish peroxidase‑conju-
the upper chambers and fresh DMEM (600 µl/well) containing gated secondary antibodies were added at a dilution ratio of
20% FBS was added to the bottom chambers. After 24 h, cells 1:5,000 and incubated at 37˚C for 1 h. Protein levels were
that migrated to the underside of the filter were fixed with quantified using the SuperSignal West Pico Chemiluminescent
methanol and stained with crystal violet, and counted under Substrate kit. Three separate experiments were performed for
brightfield microscopy. The experiments were repeated three each group.
times.
Inhibition of p38 and JNK with specific inhibitors and its
RNA extraction, RT-PCR and RT-qPCR analysis. Total RNA effects on the NOV-induced antiproliferative effect and
was extracted from osteosarcoma cells in different groups apoptosis of 143B cells. The cells were treated with 10 µM
using TRIzol reagent, according to the manufacturer's instruc- SB203580 (p38 inhibitor) or 20 µM SP600125 (JNK inhibitor)
tions. First-strand DNA was synthesized using the Reverse for 30 min, followed by treatment with AdGFP or AdNOV for
Transcriptase M-MLV (RNase H) kit with random hexamer the indicated time points. Following treatment for 48 h, the
primers. Touchdown PCR analysis determining the gene phosphorylation of p38 and JNK was detected by western blot
expression level was performed under the following condi- analysis. At the same time, the expression of Bax and Bcl-2
tions: 95˚C x 5 min for one cycle, 12 cycles at 95˚C x 30 sec, was detected by western blot analysis. The experiments were
68˚C x 30 sec (with a decrease of 1 degree/cycle) and repeated three times.
72˚C x 30 sec and 25 cycles at 95˚C x 30 sec, 55˚C x 30 sec,
and 72˚C x 30 sec, 72˚C x 10 min for 1 cycle. PCR products Statistical analysis. Data are presented as the means ± standard
were separated by electrophoresis on a 2% agarose gel. The deviation (SD) from at least three independent experiments.
expression level of mRNA were normalized to GAPDH. One-way analysis of variance (ANOVA) was used to analyze
RT-qPCR was run in the Rotor-Gene 6000 Real-Time PCR the differences between groups and the LSD method of
machine (Corbett Research, Sydney, Australia) using SYBR multiple comparisons was used when the probability for
Premix Ex Taq (Thermo) with the following protocol: initial ANOVA was statistically significant using GraphPad Prism 5
activation of HotStar Taq DNA polymerase at 95˚C for 10 min, and SPSS 17.0. Statistical significance was set at P<0.05.
then 45 cycles of 95˚C for 5 sec and 60˚C for 20 sec. GAPDH
was used as an internal control. The primer efficiencies were Results
confirmed to be high (>90%) and comparable (Table I). Data
were analyzed according to the 2-ΔΔCt method. The expression The endogenous expression of NOV in osteosarcoma cell
level of mRNA were normalized to GAPDH. Three separate lines. In the present study, the endogenous expression of NOV
experiments were performed for each group. in human 143B, U2OS, MG-63 and SaOS2 osteosarcoma
cell lines was evaluated using RT-PCR analysis (Fig. 1A).
Western blot assay. Briefly, osteosarcoma cells with different As shown in Fig. 1A, the mRNA expression level of NOV in
treatments were lysed with RIPA buffer, then centrifuged for these osteosarcoma cell lines was different. The expression
30 min at 4˚C and supernatants were collected. The protein level was significantly higher in MG63 cells although obvi-
concentration was determined by the BCA assay. Cell extracts ously lower in 143B cells as compared to the remaining cells.
were boiled for 10 min in loading buffer and then an equal The result was confirmed by RT-qPCR (Fig. 1B) and western
amount of cell extracts was separated on 10% SDS-PAGE gels blot assays (Fig. 1C). Thus, we selected 143B and MG63
2014 YAO et al: NOV inhibits proliferation and promotes apoptosis of osteosarcoma

Figure 1. The endogenous expression of NOV was quite different in the four osteosarcoma cell lines. (A) NOV mRNA in different types of human osteosarcoma
cell lines was detected by RT-PCR. (B) NOV mRNA was detected by RT-qPCR. Data are reported as mean values ± SD of three individual measurements.
(C) NOV protein was detected by western blot analysis. Data are shown as mean values ± SD of three individual measurements.

Figure 2. NOV was successfully overexpressed in 143B cells and interfered in MG63 cells. (A) Constructed adenovirus vector AdNOV and AdsiNOV was
used to infect the 143B and MG63 cell lines, respectively. The infection efficiency of AdNOV and AdsiNOV (MOI=100) in 143B and MG63 cell lines was
>80% under fluorescence microscopy. (B) RT-PCR, (C) RT-qPCR and (D) western blot assays were performed at 48 h recovery to measure the expression
of NOV, showing that an obvious increase of NOV expression was observed in the AdNOV group compared with the AdGFP and blank groups in 143B cells
(**P<0.01 and *P<0.05 vs. AdGFP group), while a marked decrease of NOV expression was found in the AdsiNOV group compared with the Ad-RFP and
blank groups in MG63 cells (*P<0.05 vs. AdRFP group). No difference was found between the AdGFP/AdRFP and blank groups in 143B and MG63 cell lines
(P>0.05). Data are shown as mean values ± SD of three individual measurements.
 ONCOLOGY REPORTS 34: 2011-2021, 2015 2015

osteosarcoma cell lines for infection with the AdNOV and western blot analysis (Fig. 4E) were performed. In Fig. 4A
AdsiNOV adenovirus, respectively. it is shown that the apoptotic rate of 143B cells was signifi-
cantly increased from (5.23±2.61) to (11.91±2.29)% (P<0.01)
NOV is successfully overexpressed in 143B cells and interfered compared to the AdGFP groups following treatment with
in MG63 cells. To identify the role of NOV in osteosarcoma AdNOV for 48 h, whereas the apoptotic rate of MG63 cells
cell lines, recombinant adenovirus AdNOV and AdsiNOV were was significantly reduced as compared to that of the AdRFP
used to infect 143B and MG63 cell lines, respectively. In the groups after AdsiNOV treatment for 48 h (P<0.05). Staining
present study, the infection efficiency of AdNOV and AdsiNOV with Hoechst 33258 (Fig. 4B) was used to visualise the
in 143B and MG63 cell lines was >60% under fluorescence apoptosis induced by NOV expression, and extensive nuclear
microscopy (Fig. 2A). In addition, RT-PCR  (Fig. 2B), condensation and cell fragmentation were observed. Moreover,
RT-qPCR (Fig. 2C) and western blot assays (Fig. 2D) were a significant increase in Bax expression and a parallel Bcl-2
performed at 48 h recovery to measure the expression level decrease was observed in 143B cells after NOV overexpres-
of NOV after adenovirus infection. An obvious increase of sion, while the contrary result was evident in MG63 cells after
NOV expression was observed in the AdNOV group compared AdsiNOV infection (P<0.05 and P<0.01, Fig. 4C-E).
with the AdGFP and control groups (P<0.01 and P<0.05) in
143B cells, while a marked decrease of NOV expression was NOV promotes osteosarcoma cell migration in vitro. Cell
found in the AdsiNOV group compared with the AdRFP and migration plays a crucial role in the process of tumor metas-
control groups in MG-63 cells (P<0.05) (Fig. 2B and C). These tasis. In the present study, Transwell migration assay was
data indicated that NOV was successfully overexpressed in used to detect the change in cell migration induced by NOV.
143B cells and interfered in MG63 cells. After treatment with adenovirus for 24 h, the number of trans-
membrane cells in the NOV upregulation group increased
NOV inhibits cell viability of osteosarcoma cells. To inves- significantly compared with the control group in 143B and
tigate the effects of NOV on cell growth, osteosarcoma cells MG63 cells (P<0.05 and P<0.01, Fig. 5).
were treated with AdNOV and AdsiNOV, respectively, for
24, 48 and 72 h, and cell viability was examined by MTT NOV-induced activation of the MAPK signaling pathway in
assay (Fig. 3A and B). As shown in Fig. 3A and B, the cell osteosarcoma cell lines. It has been previously reported that
viability of 143B cells treated with AdNOV was significantly activation of p38/MAPK and JNK/MAPK signaling pathway
inhibited at 48 h (P<0.05) and was more significant at 72 h was associated with cell apoptosis. To determine whether NOV
(P<0.01), whereas the cell viability of MG63 cells treated expression is involved in activation of the MAPK signaling
with AdsiNOV was significantly promoted at 48 h (P<0.05) pathway in osteosarcoma cell lines, we detected and analyzed
and 72 h (P<0.05). Thus, we selected 48 h as the time-point the phosphorylation of MAPKs in cell lysates of osteosarcoma
for the remaining experiments. Furthermore, after treatment cells in different groups by western blot analysis (Fig. 6A and B).
with AdNOV for 2 weeks, we found a significant decrease The results showed that NOV overexpression enhanced the
in colony formation in the AdNOV group and the colony- phosphorylation of p38 and JNK/MAPKs in 143B cells,
formation rate decreased by 23% in 143B cells compared with whereas downregulated NOV had an obvious suppressant effect
that of the AdGFP and control groups (P<0.01), while a slight on the phosphorylation of MAPKs in MG63 cells (P<0.05 and
increase in colony formation in the AdsiNOV group in MG63 P<0.01, Fig. 6A and B). These results demonstrated that NOV
cells, compared with that of the AdRFP and control groups enhances the activity of the MAPK signaling pathway (p38 and
(P>0.05, Fig. 3C). JNK) in osteosarcoma cell lines.

NOV induces cell cycle arrest in osteosarcoma cell lines. Impact of the inhibition of MAPK signaling on NOV-induced
Cell cycle arrest plays a crucial role in the process of cell apoptosis of osteosarcoma cells. To investigate whether
proliferation. Flow cytometric assay was used to detect the activation of the MAPK signaling pathway is involved in the
change of the cell cycle induced by NOV expression (Fig. 3D). NOV-induced apoptosis of osteosarcoma cells, the specific
The data showed that NOV overexpression decreased the inhibitors of p38 (SB203580) and JNK (SP600125) were used
percentage of 143B cells in the S phase from (35.14±3.29) to pre-treat the 143B cells. We detected the phosphorylation of
to (23.83±4.66)% (P<0.01) compared to the AdGFP group, MAPKs by western blot analysis (Fig. 7A) and found that the
and increased the percentage of G1 phase from (63.61±4.55) NOV-induced phosphorylation of p38 and JNK was reversed
to (73.92±1.22)% (P<0.05) compared to the AdGFP group, by SB203580 (P<0.05) and SP600125 (P<0.05), respectively.
respectively. Conversely, knockdown of NOV expression Cell viability was measured by MTT assay (Fig. 7B). We found
through RNA interference in MG63 cells, led to the percentage that both SB203580 and SP600125 significantly suppressed
of MG63 cells in the S phase being increased from (19.32±5.16) the NOV-induced antiproliferative effect at 48 h (P<0.05) and
to (39.07±4.16)% (P<0.01) and the percentage of G1 phase 72 h (P<0.05). At the same time, we detected changes in the
was decreased from (58.15±2.33) to (43.29±5.37)% (P<0.05) expression levels of Bax and Bcl-2 in the presence and absence
compared to the AdRFP group, respectively (Fig. 3D). of SB203580 or SP600125 (Fig. 7C). We found that the
NOV-induced upregulated expression of Bax was reversed by
NOV stimulates apoptosis of osteosarcoma cell lines in vitro. SB203580 (P<0.05) and SP600125 (P<0.05), while the down-
To evaluate apoptotic cell death in osteosarcoma cells after regulated expression of Bcl-2 was also reversed by SB203580
AdNOV/AdsiNOV treatment, flow cytometric assay (Fig. 4A), (P<0.05) and SP600125 (P<0.01) in 143B cells. These results
Hoechst staining (Fig. 4B), RT-qPCR (Fig. 4C and D) and suggested that the promotive role of NOV in the apoptosis of
2016 YAO et al: NOV inhibits proliferation and promotes apoptosis of osteosarcoma

Figure 3. NOV inhibits the proliferation and cell cycle of human osteosarcoma cell lines. (A) 143B and (B) MG63 cells were infected with AdNOV or AdsiNOV
for 24, 48 and 72 h. At the end of the indicated time, cell viability was determined by MTT assays. The results show the mean absorbance ± SD of three
independent experiments. (**P<0.01 and *P<0.05 vs. AdGFP group or AdRFP group). (C) Colony forming assay of the osteosarcoma cell lines treated with
AdNOV or AdsiNOV. The number of colonies decreased significantly in the AdNOV group in 143B cells and had a marked increase in the AdsiNOV group in
MG63 cells. Representative images of the colony-forming unit are presented in the upper panel, and colony-forming rates for each group are quantified in the
lower panel. The colony-forming rate was obtained as mentioned in Materials and methods. The experiment was repeated three times. (**P<0.01 vs. AdGFP
group, #P>0.05 vs. AdRFP group). (D) Osteosarcoma cell lines were infected with AdNOV or AdsiNOV for 48 h. Cell cycle distribution was analyzed by flow
cytometry. The data are presented as the means ± SD (n=3). The assay was performed in triplicate. (**P<0.01 and *P<0.05 vs. AdGFP group or AdRFP group).

osteosarcoma cells may be mediated by the phosphorylation NOV, also known as CCN3, is a matricellular protein
of p38 and JNK. encoded by the NOV gene, that can interact with numerous
cell‑surface receptors and participate in various pathological
Discussion physiological activities including tumor (26-28). NOV interacts
with the integrin receptor of cell surface to trigger intracellular
Osteosarcoma is a high‑mortality cancerous tumor localized at signal trans­d uction and activate downstream signaling
the end of metaphysis, which is most prevalent in children and pathways, thus regulate the occurrence and development
young adults. Although early diagnosis and timely treatment of tumor. NOV has been shown to play different roles in
have greatly enhanced the survival rates, the patients have a various types of tumors. A previous study demonstrated
poor prognosis because of the high rate of lung metastasis that NOV expression was associated with the prognosis
and drug resistance (24). Tumor microenvironment, which judgment of osteosarcoma (17). Nonetheless, the biological
contains large amounts of ECM generated by tumor cells foundation of this effect and the exact role of NOV expression
including growth factors, chemotactic factors and matrix- in the progression of osteosarcoma has not been extensively
degrading enzymes, is greatly associated with tumorigenesis, clarified. Therefore, in the present study, we investigated the
development and metastasis by influencing the proliferation, effects of NOV on the proliferation, apoptosis and migration of
apoptosis, migration and invasion of tumor cells (25). As osteosarcoma cells and the possible underlying mechanisms.
mentioned earlier, it is necessary to elucidate a novel strategy In the present study, we firstly evaluated the endogenous
that efficiently inhibits the progression of osteosarcoma. expression of NOV in human osteosarcoma cell lines through
 ONCOLOGY REPORTS 34: 2011-2021, 2015 2017

Figure 4. NOV induces apoptosis of osteosarcoma cell lines in vitro. (A) The 143B and MG63 cells were infected with AdNOV or AdsiNOV for 48 h and were
then analyzed for apoptosis by flow cytometry. The percentage of apoptotic cells in 143B and MG63 cells for each group are quantified in the right panel.
Values are the means ± SD (n=3). (**P<0.01 vs. AdGFP group, *P<0.05 vs. AdRFP group). (B) At the end of the incubation period of 48 h, the 143B and MG63
cells were stained with Hoechst 33258 staining and the cell nucleus was observed under microscopy for apoptosis, showing that the number of apoptotic
cells (strong blue staining) was significantly increased compared with the NOV overexpression group. Magnification, x100 (C) RT-PCR, (D) RT-qPCR and
(E) western blot assays were performed at 48 h recovery to measure the expression of apoptosis-related proteins Bax/Bcl-2 in osteosarcoma cell lines. Data are
shown as mean values ± SD of three individual measurements. (**P<0.01 and *P<0.05 vs. AdGFP or AdRFP group).
2018 YAO et al: NOV inhibits proliferation and promotes apoptosis of osteosarcoma

Figure 5. NOV promotes the migration of osteosarcoma cells. (A) Effects of NOV expression on the migration of 143B and MG63 cells was detected by
Transwell migration assay; magnification, x200. Data are reported as mean values ± SD of three individual measurements. The quantification data are shown
on the right panel. (**P<0.01 vs. AdGFP group, *P<0.05 vs AdGFP group).

Figure 6. NOV activates the p38/MAPK and JNK/MAPK signaling pathway in osteosarcoma cells. (A) The phosphorylation levels of p38/MAPK and
(B) JNK/MAPK in AdNOV-infected 143B cells and AdsiNOV-infected MG63 cells were detected by western blot analysis. Densitometric quantification data
are on the right panel. Data are shown as mean ± SD of three individual measurements. (**P<0.01 and *P<0.05 vs. AdGFP group or AdRFP group).

PCR and western blot analysis and found it was different among cell carcinoma and Ewing sarcoma (12,13), which is consistent
the cell lines. Thus, we selected adenovirus-mediated NOV with previous studies. A possible explanation for the growth
overexpression/downregulation to investigate the effects of inhibition is cell cycle arrest or apoptosis increase. Our results
NOV on different osteosarcoma cell lines. The present results derived from FCM indicated that NOV overexpression may
showed that NOV overexpression had a significant inhibitory induce cell cycle arrest in the G1 phase in 143B cells, while
effect on osteosarcoma 143B cell viability in a time‑dependent its downregulation reversed the effect in MG63 cells, leading
manner, whereas NOV silencing induced opposite effects in to increased cell proliferation ability. Since the G1 phase is
MG63 cells. These results have shown an inhibitory effect of necessary for the material and energy preparation for DNA
NOV overexpression on cancer cell growth including renal replication into the subsequent S period, the abnormity of this
 ONCOLOGY REPORTS 34: 2011-2021, 2015 2019

Figure 7. SB203580 and SP600125 suppresses NOV-induced apoptosis of 143B cells. (A) Effects of SB203580 and SP600125 on the NOV-induced activa-
tion of p38/MAPK and JNK/MAPK in 143B cells were detected by western blot analysis. The phosphorylation of p38 and JNK was reversed by SB203580
and SP600125, respectively; *P<0.05 vs. SB203580 group and SP600125 group. Densitometric quantification data are on the right panel. Data are shown as
mean ± SD of three individual measurements. (B) Effects of SB203580 and SP600125 on NOV-induced proliferation inhibition of 143B cells. Cell viability
was measured by MTT assay. The inhibitory role of NOV in cell proliferation was reversed by SB203580 and SP600125. The results show the mean absor-
bance ± SD of three independent experiments. (*P<0.05 vs. SB203580 and SP600125 groups). (C) The effects of SB203580 and SP600125 on the NOV-induced
protein levels of Bax and Bcl-2 in 143B cells were detected by western blot analysis. The expression levels of Bax and Bcl-2 was reversed by SB203580 and
SP600125, respectively. Densitometric quantification data are shown on the right panel. Data are shown as mean ± SD of three individual measurements.
(*P<0.05 vs. SB203580 group and SP600125 group, **P<0.01 vs. SP600125 group).

phase may inevitably lead to the obstacles of DNA replication that they are involved in mediating apoptosis signals that
and eventually cause the inhibition of cell proliferation (29). cause cell death (32). Integrin receptor-dependent signaling
At the same time, we found that NOV expression induced pathways may activate P38/MAPK and JNK/MAPK, thereby
cell apoptosis of osteosarcoma cell lines. Bax and Bcl-2 are promoting the phosphorylation of Bcl-2, the expression of Bax
two primary proteins that are often used as markers for cell and induce the apoptosis of tumor cells (33,34). Moreover,
apoptosis (30,31). Our results showed a marked increase of Bax previous findings have shown that NOV induced cell apoptosis
and a decrease of Bcl-2 in the AdNOV group compared with the or growth inhibition in many types of cancer through activa-
AdGFP and CON groups in 143B cells, but a decrease of Bax tion of the MAPK signaling pathway (35,36). Therefore, we
and an increase of Bcl-2 in AdsiNOV group compared with the detected the effects of NOV on the MAPK signaling pathway
AdRFP and CON groups in MG63 cells, further comfirming in osteosarcoma cell lines. Our results show that NOV
that NOV may promote the apoptosis of osteosarcoma cells. expression enhanced the phosphorylation of p38 and JNK in
P38/MAPK and JNK/MAPK are two major pathways for osteosarcoma cells in vitro. Furthermore, the phosphoryla-
malignant progression in various tumors. It has been confirmed tion of p38 and JNK was inhibited by the specific inhibitors,
2020 YAO et al: NOV inhibits proliferation and promotes apoptosis of osteosarcoma

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