- Email: [email protected]
Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell
Accepted Manuscript Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell Junming Luo, Menglan Zhang, Hong Huang, Yichun Wang, Xin Yuan, Siqing Ma, Jingshi Liu, Shiying Zhou, Shukun Zhang PII:
S0006-291X(16)32066-6
DOI:
10.1016/j.bbrc.2016.12.022
Reference:
YBBRC 36896
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 25 November 2016 Accepted Date: 3 December 2016
Please cite this article as: J. Luo, M. Zhang, H. Huang, Y. Wang, X. Yuan, S. Ma, J. Liu, S. Zhou, S. Zhang, Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2016.12.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell
Junming Luo 1*, Menglan Zhang1*, Hong Huang1, Yichun Wang2, Xin Yuan3, Siqing Ma4,
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Jingshi Liu2, Shiying Zhou1, Shukun Zhang1 #
Department of Pathology, Qinghai People’s Provincial Hospital, Xining 810007, Qinghai
Province, People’s Republic of China
Department of Anesthesiology, Hunan Provincial Tumor Hospital , Central South University,
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2
Changsha 410013,hunan Province , People’s Republic of China
Department of Internal Medicine, Qinghai People’s Provincial Hospital, Xining 810007,
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3
Qinghai Province, People’s Republic of China 4
Department of Critical Care Medicine, Qinghai People’s Provincial Hospital, Xining 810007,
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Qinghai Province, People’s Republic of China
*Equal contribution #Correspondence to: Professor Shukun Zhang, Department of Pathology, Qinghai People’s Provincial Hospital, 2 Gonghe Rd, Xining 810007, Qinghai Province, People’s Republic of China. Phone: +86 971 8066214, email: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Radiation pulmonary injury is related to the accumulation of extracellular matrix proteins in the alveolar interstitial space. Matrilin-2 as a component of extracellular filamentous networks, present higher level in the lung tissue from irradiated mice and
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irradiated pulmonary epithelial cell line, HPAEpiC cells. Knockdown of endogenous matrilin-2 prevents the apoptosis of HPAEpiC cell induced by the irradiation injury. Consistently, over-expression of matrilin-2 reduced the proliferation and induced apoptosis of HPAEpiC cells. Matrilin-2 promotes the expression of p21 via increasing
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the transcriptional activity of p53, by which induces the G1 phase arresting in HPAEpiC cells. In summary, matrilin-2, increased by irradiation, reduced the
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proliferation and induces apoptosis of pulmonary epithelial cells via p53/p21 pathway.
Key words: Matrilin-2, Radiation pulmonary injury, Proliferation, Apoptosis, Cell
Introduction
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cycle.
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Matrilins are non-collagenous glycoproteins implicated in the organization of extracellular matrix (ECM), which form a family of oligomeric extra-cellular adaptor
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proteins that are most strongly expressed in cartilage but also expressed in numerous other types of ECM [1].The matrilin family consists of four members (matrilin-1, -2, -3, -4). Matrilin-2 (encoded by MATN2 gene) is a widely distributed extracellular matrix protein that participates in the formation of both collagen-dependent and collagen-independent filamentous macromolecular network with adaptor functions which are involved in the development and homeostasis of network of extracellular matrix [2-4]. Radiation therapy is a widely used therapy method for cancer, but its side effects usually are unavoidable even when localized radiotherapy is adapted. Radiation pulmonary injury (RPI) is a common delayed side effect of radiotherapy for thoracic 2
ACCEPTED MANUSCRIPT malignancies, which limits the dose delivery to the tumor target and may thus hamper disease course control [5-7]. It has been suggested that the most important pathological basis of radiation-induced lung injury is a metabolic imbalance in the extracellular matrix (ECM) [8-10]. However, the mechanism of radiation-induced
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pulmonary injury has not yet been fully identified. Although the matrilin-2 null mice showed no gross abnormalities during embryonic or adult development [11], the accumulating evidence showed that matrilin-2 has been involved in reorganization of tissue architecture in liver cirrhosis
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and hepatocellular carcinoma, the balance of keratinocyte and fibroblast in response to wounding, peripheral nerve regeneration[2, 12-14]. Furthermore, the recent studies
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reported that matrilin-2 levels were elevated in sporadic pilocytic astrocytoma, human liver cirrhosis and liver cancer, and matrilin-2 functions as a tumor suppressor in hepatocarcinogenesis [15, 16]. Ablation of Matrilin-2 is thought to contribute to hepatocarcinogenesis through activation of the MAPK signaling pathway[15]. Many reports suggested that anti-proliferation effect of matrilin-2 may be related to its G1/S
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arresting effect in cancer cells [17-20]. All of which remind us that matrilin-2 may also mediate RPI via anti-proliferation effect. In this study, we found that the mRNA and protein levels of matrilin-2 were
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increased after irradiation treatment in both lung tissue and HPAEpiC cell. Furthermore, over-expression of matrilin-2 attenuated proliferation and promoted apoptosis of HPAEpiC cell, while knockdown of matrilin-2 protect HPAEpiC from
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apoptosis induced by irradiation treatment. Matrilin-2 promotes the G1 phase arrest via p53/p21 pathway. Together, these data indicate that matrilin-2 might be a potential target to regulate the pathogenesis during RPI. Materials and methods Plasmids and lentivirus. The reporter construct containing promoter region of mouse p21 (pGL3-basic-p21), matrilin-2 expression vector (pcDNA3.1-matrilin-2) and lentivirus expressing shRNA directed against matrilin-2 were purchased from Hanbio Biotechnology Co., Ltd., Shanghai, China. p53 expression vector (pcDNA3 p53 WT) 3
ACCEPTED MANUSCRIPT was a gift from David Meek (Addgene plasmid # 69003). Animals. All animal procedures were approved by the Laboratory Animal Care Committee of Qinghai Province. Male C57BL/6 mice (Experimental Animal Center of Qinghai University, Xining, Qinghai, China) aged 6-8 weeks, with approximate
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body weights of 18-20g, were used in this study. All irradiation was performed on a clinical linear accelerator. For right hemithoracic irradiation, three 8-week old male C57BL/6 mice were anesthetized and thoracic regions corresponding to right lungs were aligned. One centimeter thickness water-density flexible bolus pieces were
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placed on top of the mice for dose buildup and collimated fields that encompassed the right hemithorax were used. For whole thoracic irradiation, 6 mice were
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simultaneously treated. 6 MV photons were used to deliver 16 Gy prescribed at Dmax.
Cell culture. HPAEpiC cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, MD, USA) and
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antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin) in a humidified atmosphere of CO2/air (5%/95%) at 37
(Thermo Fisher Scientific, Nepean, Canada).
Transfection, matrilin-2 silencing by short hairpin RNA (shRNA) and reporter
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gene assays. Matrilin-2 was expressed in HPAEpiC cells by transfecting pcDNA3.1-matrilin-2. After overnight culture, cells were incubated for 4 h in serum-free DMEM containing DNA-polyethylenimine (Sigma, 1 mg/ml) complex
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and then grown in DMEM supplemented with 10% FBS for 60 h. Endogenous human matrilin-2 in HPAEpiC cells was knocked down by infection with a lentivirus expressing shRNA directed against matrilin-2, supplemented with 8 mg/ml Polybrene (Sigma) after overnight attachment. Infection was repeated at intervals of 8 to 12 h. Cells were subjected to experiments 72 h after the second infection. In the luciferase assays, HPAEpiC cells were transfected with expression vectors or luciferase reporter plasmids plus the internal control vector pRL-TK-Renilla for luciferase assays in 24-well plates. Cells got similar transfection process above. Luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega, Madison, WI) 4
ACCEPTED MANUSCRIPT and normalized to Renilla luciferase values. Cell irradiation. HPAEpiC cells were cultured in a 25 cm2 culture flask. Cells were irradiated using a Small Animal Radiation Research Platform, SARRP system (X-ray tube: ISOVOLT 225M2 X-ray source; SARRP system, XStrahl ®, Surrey, UK) at a
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constant rate of 3.45 Gy/min, for 278 seconds, thus receivinga single-fraction of 16.0 Gy (220 kV and 13.0 mA, using an 0.15 mm cupper filter and a 10×10 cm collimator). Cells were positioned at a source-to-surface distance (SSD) of 34 cm.
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Control “sham” samples (0 Gy) received similar handlings except for the irradiation.
Real-time PCR. Total RNA was extracted from lung tissue or cells using RNAiso
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Plus (Takara Bio Inc., Dalian, China) and was reverse-transcribed using M-MMLV Reverse Transcriptase with RNasin Ribonuclease Inhibitors (Promega Biotech Co., Ltd, Beijing, China) according to the manufacturer’s protocols. qPCR was performed using SYBR Premix Master Mix (Thermo Scientific Inc., Shanghai, China). Primer sequences used in the experiments were as follows: Matrilin-2 (mouse) forward
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5’-CTATGTATGCCGTTGGGGTAGG-3’,
5’-AGCTTTTCACTTATTTCGCCCAT-3’;
reverse
Matrilin-2
(human)
5’-GATCCTCGGACAGATCGTCCT-3', 5’-CTGCCCGCTTGTTCTCACA-3’;
reverse p21(human)
forward
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5’-TGTCCGTCAGAACCCATGC-3’,
5’-AAAGTCGAAGTTCCATCGCTC-3’;
reverse GAPDH
(human)
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5’-ACAACTTTGGTATCGTGGAAGG-3’, 5’-GCCATCACGCCACAGTTTC
-3’;
forward
forward reverse
GAPDH
5’-AGGTCGGTGTGAACGGATTTG-3′,
(mouse)
forward reverse
5’-TGTAGACCATGTAGTTGAGGTCA-3’. The expression levels of mRNA were normalized to GAPDH (human) or GAPDH (mouse) mRNA. Western blot analysis and antibodies. Total cell extracts were obtained by lysing the cells in RIPA buffer with protease inhibitors cocktail (MedChem Express, Shanghai, China). Protein concentration was measured by the Bradford assay (Bio-Rad, Hercules,CA, USA). Cellular proteins were extracted and separated in 5
ACCEPTED MANUSCRIPT 4–10% Tris glycine/SDS-polyacrylamide gels and electrotransferred to ECL nitrocellulose membranes (#IPFL00010, Millipore). The membranes were blocked with 5% nonfat milk and incubated with specific antibodies. The β-actin protein was used as the endogenous control. Primary antibodies were used at the dilution of
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1:1000. Anti-matrilin-2 (ab55458), anti-p21 (ab109520) and anti-p53 (ab1101) were purchased from Abcam. β-actin (#3700) and horseradish peroxidase-conjugated anti-mouse or rabbit IgG were purchased from Cell Signaling Technology. Immunocomplexes were visualized by ECL (Pharmacia-Amersham, Freiburg,
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Germany).
MTT proliferation assay. To detect cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium bromide (MTT) colorimetric assay was performed 1 day after transfection. All the cells were washed with phosphate-buffered saline (PBS) and suspended by trypsin, seeded 2000 cells per well in 96-well plate. The plate was incubated for 1 to 5 days in cell culture chamber at 37
and 5% CO2 atmosphere. 50
µL of MTT (10 mg/mL) were added and incubated for 2 h at 37
, discarded the
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MTT and 100 µL Dimethyl Sulphoxide (DMSO, Sigma Inc) was added to each well. The absorbance at 490nm was measured using microplate reader (Bio Tek Instruments, Winooski, VT)
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Flow cytometric analysis. Cell cycle and percentage of apoptotic cells were assessed by FACScalibur flow cytometry (Becton Dickinson). Samples for cell cycle were
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harvested by trypsin and fixed by 70% ethanol in PBS in
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overnight, washed
twice with PBS and stained by propidium iodide (PI) with RNase (Becton Dickinson, San Diego, CA) for 30 min in dark. Fluorescent emissions were collected through FL2 band-pass filter. Cells for apoptosis assay were collected by trypsin without EDTA , washed twice with PBS, stained by 5 µl PI and 5 µl FITC-Annexin V in 100uL binding buffer for 15min in dark (ebioscience). Fluorescent emissions were collected through FL1 band-pass filter for FITC-Annexin V, FL2 for PI. The apoptotic cells were PI and FITC-Annexin V both positive cells. Statistical analysis. Results are expressed as mean ± SE. Data between groups were 6
ACCEPTED MANUSCRIPT analyzed by Student’s t-test or one-way ANOVA followed by Bonferroni–Dunn multiple comparison. Differences were considered significant at P < 0.05. Results Irradiation up-regulates matrilin-2 expression in lung tissues and HPAEpiC cells.
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We first investigated the expression of matrilin-2 in lung tissues after irradiation. Immunohistochemical staining showed significantly increased matrilin-2 level in the lung tissues at 4 weeks p.i. and 8 weeks p.i. compared with the control (Fig. 1A-C).
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Real-time PCR and western blotting indicate the increment of its mRNA and protein levels both (Fig. 1D-E). To make sure that the increased expression of matrilin-2 in
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irradiated lung is from pulmonary epithelial cells, the major cells in lung, we utilized HPAEpiC cell, the cell line of pulmonary epithelial cells and found that matrilin-2 protein levels were significantly up-regulated by exposing to low doses (4 Gy/day for 1–4 days) irradiation in a time-dependent manner (Fig. 1F-G). Taken together, pulmonary epithelial cells got enhanced the expression of matrilin-2 make up the
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main resource of increment of matrilin-2 level in lung with iradiation treatment. Overexpression of matrilin-2 inhibits the growth of HPAEpiC cell in vitro. To mimic
the
increased
expression
of
matrilin-2
after
irradiation
in
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pulmonary epithelial cells and explore the effect of its up-regulation in the development of RPI, we over-expressed matrilin-2 in HPAEpiC cell by transfecting matrilin-2 vectors, which increase the mRNA and protein levels by 4 and 5 fold,
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respectively (Fig. 2A-B). The enhanced expression of matrilin-2 significantly inhibited the proliferation rate of HPAEpiC cells in the MTT assay (Fig. 2C). Consistently, double staining with annexin V-FITC and PI showed an about 4 times increment in the ratio of apoptosis in matrilin-2-overexpressing cells compared with the control cells (Fig. 2D-E). FACs analysis showed higher percentage of cells rested in G1 phase after matrilin-2 upregulation (Fig. 2F). Taken together, these results indicate that Matrilin-2 suppresses cell proliferation of HPAEpiC cells through regulation of G1/S transition. 7
ACCEPTED MANUSCRIPT Matrilin-2 promotes irradiation-induced HPAEpiC cell apoptosis. Since over-expression of matrilin-2 induces apoptosis of HPAEpiC, whether the down-regulation of matrilin-2 has opposite effect and reverses the apoptosis induced by irradiation is deserved to investigate. We utilized the ShRNA to reduce the mRNA
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and protein levels of matrilin-2 to 20% and 25% of control cells (Fig. 3A-B). The down-regulation of matrilin-2 promotes the proliferation of HPAEpiC (Fig. 3C) and prevents the apoptosis induced by irradiation (Fig. 4D-E). Irradiation treatment decreased the percentage of cells in S phase and increased the percentage of cells in
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G1 phase and matrilin-2-knockdown reversed this phase switch (Fig. 2F). Taken together, matrilin-2 mediates irradiation-induced apoptosis via regulating G1/S cell
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cycle transition during radiation pulmonary injury.
Matrilin-2 regulating cell cycle related genes during radiation-induced injury in HPAEpiC cell. The result that matrilin-2 promotes the G1 arrest of HPAEpiC cells remind us that matrilin-2 may interact p21 which has been implicated in the control of the G1 to S phase transition[21]. Thus we examined p21 and found that the mRNA
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and protein levels of p21 were down-regulated in the HPAEpiC cells with matrilin-2 over-expression mediated by matrilin-2 vector transfection or irradiation, while matrilin-2 knockdown reduced p21 expression (Fig. 4A-D). p21 is transcriptional
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regulated by p53[22], the carcinogenesis inhibitor[23] and we found sight increase in p53 protein level after irradiation treatment, but not by matrilin-2 over-expression.
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p53 and matrilin-2 expression vectors and luciferase system containing p21 promoter were co-transfected into
HPAEpiC cells and the luciferse assay result indicated that
matrilin-2 promoted the transcriptinal activity of p53 on p21 (Fig. 4E). These results imply that matrilin-2 amplified the transcriptional activity of p53 on p21, thus promotes the expression of the latter.
Discussion Extracellular matrix (ECM) provides physical support to tissues, anchorage sites for cells and medium for diffusible signaling molecules [24, 25]. ECM is also a major 8
ACCEPTED MANUSCRIPT cytokine reservoir and therefore has pleiotropic effects [26-29]. Matrilin-2, as the component of ECM, should be implicated in the functions of ECM. Our previous study found the distribution of matrilin-2 mRNA in skin of the tail, calvarias, heart, uterus, and brain [30], implying that matrilin-2 may take part in the physiological
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process in those tissues. In the present study, we found that irradiation increased the mRNA and protein levels in lung tissue and pulmonary epithelial cell line, HPAEpiC cells (Fig. 1), reminding us that matrilin-2 may also have functions in the process of RPI.
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To comprehensively understand the effect of matrilin-2 on RPI, we performed in vitro MTT and FACs assays to identify the proliferation and apoptosis of HPAEpiC
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cells with over-expression (Fig. 2A-B) or knockdown (Fig. 3A-B) of matrilin-2. Our results show that matrilin-2 inhibits cell proliferation and promoting apoptosis (Fig. 2 C-E). Consistently, matrilin-2 knockdown prevents the irradiation induced apoptosis of HPAEpiC cells (Fig. 3C-E), implying that matrilin-2 functions as mediator in the RPI. Cell cycle assay revealed that the G1 phase arresting may be the reason for the
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apoptosis-promoting effect of matrilin-2 (Fig. 2F & Fig. 3F). p21 is a potent cyclin-dependent kinase inhibitor. It binds to and inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and thus functions as a repressor
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on cell cycle progression at G1 and S phase [21], by which p21 mediates growth arrest and cellular senescence. Interestingly, the expression of matrilin-2 is positively related to that of p21 (Fig. 4A-D), which indicates that matrilin-2 may promote
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apoptosis via the up-regulation of p21. The expression of p21 is tightly transcriptional controlled by the tumor suppressor protein p53, which is easily activated in response to a variety of stress stimuli, including DNA damage (induced by either UV, IR, or chemical agents such as hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion, and deregulated oncogene expression[31, 32]. As we expected, irradiation increased the p53 protein level in HPAEpiC cells, but neither over-expression nor knockdown of matrilin-2 affect p53 expression (Fig. 4A-B). These data indicates that matrilin-2 does not modulate p21 expression via regulating p53 level. Interestingly, the result of luciferase assay indicates that matrilin-2 9
ACCEPTED MANUSCRIPT promotes the transcription of p21 by p53 (Fig. 4E), i.e. matrilin-2 functions as the co-activator of p53. In conclusion, we report that the extracellular matrix protein matrilin-2, over-expressed in pulmonary epithelial cells after irradiation, functions as cell
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proliferation suppressor via promoting the G1 phase arresting. This effect is based on the up-regulation of p21 by activating its transcriptional factor, p53. Uncovering the underlying molecular mechanisms would develop better preventive measures to RPI
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in the radiotherapy of cancer.
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Acknowledgements
This work was funded partially through Chinese Natural Science and Technology Foundation 81460022 and New Faculty foundation of Qinghai Provincial People's Hospital (Junming Luo), and Chinese Natural Science and Technology Foundation
(Shukun Zhang).
Figure legends
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81560123, Qinghai Provincial Science and Technology Foundation 2014-ZJ-731
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Figure 1. Irradiation up-regulates matrilin-2 expression in lung tissues and HPAEpiC cells. (A-C) Immunohistochemical staining of mouse lung. Three
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representative slides are shown for the control and irradiated mice. (A) Control mice; (B) mice irradiated for 1 weeks p.i.; (C) mice irradiated for 8 weeks p.i. groups. (original magnification, ×200). The mRNA (D) and protein (E) levels of Matrilin-2 in lungs from mice irradiated. The mRNA (F) and protein (G) levels of Matrilin-2 in HPAEpiC cells irradiated. The data are presented as the mean ± S.E. and expressed as fold-change relative to the level of control mice or cells. *p < 0.05.
Figure 2. Overexpression of matrilin-2 inhibits the proliferation and activates the apoptosis of HPAEpiC cells. Matrilin-2 expression vector was transfectedin to 10
ACCEPTED MANUSCRIPT HPAEpiC cells. The mRNA (A) and protein (B) levels the matrilin-2. (C) The cell viability was determined by measuring MTT assay. Cell growth was measured at every 24 hours. (D) Flow cytometry were used to test the cell apoptosis after 48 h post-transfection. Cells staining positive for FITC-Annexin V and negative for PI
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were considered to have undergone apoptosis. (E) Average apoptotic rate. (F) The DNA content of PI-stained cells was analyzed by flow-cytometry for cell cycle determination. The data are presented as the mean ± S.E. and expressed as
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fold-change relative to the level of cells transfected with control vector. *p < 0.05.
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Figure 3. Matrilin-2 knockdown interferes with irradiation-induced apoptosis of HPAEpiC cell. HPAEpiC cells were infected a lentivirus expressing shRNA directed against matrilin-2. The mRNA (A) and protein (B) levels the matrilin-2. (C) The cell viability was determined by measuring MTT assay. Cell growth was measured at every 24 hours. (D) Flow cytometry were used to test the cell apoptosis after 72 h
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post-infection. Cells staining positive for FITC-Annexin V and negative for PI were considered to have undergone apoptosis. (E) Average apoptotic rate. (F) The DNA content of PI-stained cells was analyzed by flow-cytometry for cell cycle
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determination. The data are presented as the mean ± S.E. and expressed as
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fold-change relative to the level of cells infected with Shcontrol. *p < 0.05.
Figure 4. Matrilin-2 promotes the transcriregulating cell cycle related genes during radiation-induced injury in HPAEpiC cell. (A) Matrilin-2 expression vector was transfected into HPAEpiC cells. Cell extractors were blotted with anti-p53, p21 and β-actin antibody. (B) HPAEpiC cells were infected a lentivirus expressing shRNA directed against matrilin-2 and subjected to irradiation. Cell extractors were blotted with anti-p53, p21 and β-actin antibody. (C) mRNA levels of p21 in HPAEpiC cells with matrilin-2 overexpression. (D) mRNA levels of p21 in HPAEpiC cells with matrilin-2 knockdown and irradiation. (E) The expression vectors of p53 and matrilin-2 were co-transfected with luciferase vector containing p21 promoter into 11
ACCEPTED MANUSCRIPT HPAEpiC cells. Luciferase activity was assay and normalized to that of pRL-TK-Renilla. The data are presented as the mean ± S.E. and expressed as fold-change relative to the level of cells transfected with control vector or infected
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with Shcontrol. *p < 0.05.
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[22] S.K. Radhakrishnan, J. Gierut, A.L. Gartel, Multiple alternate p21 transcripts are regulated by p53 in human cells, Oncogene, 25 (2006) 1812-1815. [23] S.P. Hussain, C.C. Harris, p53 Biological Network: At the Crossroads of the Cellular-Stress Response Pathway and Molecular Carcinogenesis, Journal of Nippon Medical School, 73 (2006) 54-64. [24] P. Bainbridge, Wound healing and the role of fibroblasts, Journal of wound care, 22 (2013) 407-408, 410-412.
[25] J.M. Fick, How the structural integrity of the matrix can influence the microstructural response of articular cartilage to compression, Connective tissue research, 54 (2013) 83-93. [26] K. Ikeda, V.P. Michelangeli, T.J. Martin, D.M. Findlay, Type I collagen substrate increases calcitonin and parathyroid hormone receptor-mediated signal transduction in UMR 106-06 osteoblast-like cells, Journal of cellular physiology, 156 (1993) 130-137. [27] G. Ortiz, J.P. Salica, E.H. Chuluyan, J.E. Gallo, Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?, Biological research, 47 (2014) 58. 13
ACCEPTED MANUSCRIPT [28] S. Schwalm, J. Pfeilschifter, A. Huwiler, Sphingosine-1-phosphate: a Janus-faced mediator of fibrotic diseases, Biochimica et biophysica acta, 1831 (2013) 239-250. [29] J. Zhao, N. Zhang, G.D. Prestwich, X. Wen, Recruitment of endogenous stem cells for tissue repair, Macromolecular bioscience, 8 (2008) 836-842. [30] L. Li, L. Zhang, Y. Shao, G. Wang, R. Gong, Z. Wang, J. Peng, S. Wang, D. Genochio, B. Zhao, J. Luo, Distinct roles of two alternative splice variants of matrilin-2 in protein oligomerization and proteolysis, Molecular medicine reports, 6 (2012) 1204-1210. fork stress, Molecular Biology of the Cell, 17 (2006) 402-412.
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[31] R. Rodriguez, M. Meuth, Chk1 and p21 cooperate to prevent apoptosis during DNA replication [32] A. Hirao, Y.Y. Kong, S. Matsuoka, A. Wakeham, J. Ruland, H. Yoshida, D. Liu, S.J. Elledge, T.W. Mak, DNA damage-induced activation of p53 by the checkpoint kinase Chk2, Science, 287
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S0006-291X(16)32066-6
DOI:
10.1016/j.bbrc.2016.12.022
Reference:
YBBRC 36896
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 25 November 2016 Accepted Date: 3 December 2016
Please cite this article as: J. Luo, M. Zhang, H. Huang, Y. Wang, X. Yuan, S. Ma, J. Liu, S. Zhou, S. Zhang, Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2016.12.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Matrilin-2 regulates proliferation, apoptosis and cell cycle during radiation-induced injury in HPAEpiC cell
Junming Luo 1*, Menglan Zhang1*, Hong Huang1, Yichun Wang2, Xin Yuan3, Siqing Ma4,
1
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Jingshi Liu2, Shiying Zhou1, Shukun Zhang1 #
Department of Pathology, Qinghai People’s Provincial Hospital, Xining 810007, Qinghai
Province, People’s Republic of China
Department of Anesthesiology, Hunan Provincial Tumor Hospital , Central South University,
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2
Changsha 410013,hunan Province , People’s Republic of China
Department of Internal Medicine, Qinghai People’s Provincial Hospital, Xining 810007,
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3
Qinghai Province, People’s Republic of China 4
Department of Critical Care Medicine, Qinghai People’s Provincial Hospital, Xining 810007,
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Qinghai Province, People’s Republic of China
*Equal contribution #Correspondence to: Professor Shukun Zhang, Department of Pathology, Qinghai People’s Provincial Hospital, 2 Gonghe Rd, Xining 810007, Qinghai Province, People’s Republic of China. Phone: +86 971 8066214, email: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Radiation pulmonary injury is related to the accumulation of extracellular matrix proteins in the alveolar interstitial space. Matrilin-2 as a component of extracellular filamentous networks, present higher level in the lung tissue from irradiated mice and
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irradiated pulmonary epithelial cell line, HPAEpiC cells. Knockdown of endogenous matrilin-2 prevents the apoptosis of HPAEpiC cell induced by the irradiation injury. Consistently, over-expression of matrilin-2 reduced the proliferation and induced apoptosis of HPAEpiC cells. Matrilin-2 promotes the expression of p21 via increasing
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the transcriptional activity of p53, by which induces the G1 phase arresting in HPAEpiC cells. In summary, matrilin-2, increased by irradiation, reduced the
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proliferation and induces apoptosis of pulmonary epithelial cells via p53/p21 pathway.
Key words: Matrilin-2, Radiation pulmonary injury, Proliferation, Apoptosis, Cell
Introduction
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cycle.
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Matrilins are non-collagenous glycoproteins implicated in the organization of extracellular matrix (ECM), which form a family of oligomeric extra-cellular adaptor
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proteins that are most strongly expressed in cartilage but also expressed in numerous other types of ECM [1].The matrilin family consists of four members (matrilin-1, -2, -3, -4). Matrilin-2 (encoded by MATN2 gene) is a widely distributed extracellular matrix protein that participates in the formation of both collagen-dependent and collagen-independent filamentous macromolecular network with adaptor functions which are involved in the development and homeostasis of network of extracellular matrix [2-4]. Radiation therapy is a widely used therapy method for cancer, but its side effects usually are unavoidable even when localized radiotherapy is adapted. Radiation pulmonary injury (RPI) is a common delayed side effect of radiotherapy for thoracic 2
ACCEPTED MANUSCRIPT malignancies, which limits the dose delivery to the tumor target and may thus hamper disease course control [5-7]. It has been suggested that the most important pathological basis of radiation-induced lung injury is a metabolic imbalance in the extracellular matrix (ECM) [8-10]. However, the mechanism of radiation-induced
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pulmonary injury has not yet been fully identified. Although the matrilin-2 null mice showed no gross abnormalities during embryonic or adult development [11], the accumulating evidence showed that matrilin-2 has been involved in reorganization of tissue architecture in liver cirrhosis
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and hepatocellular carcinoma, the balance of keratinocyte and fibroblast in response to wounding, peripheral nerve regeneration[2, 12-14]. Furthermore, the recent studies
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reported that matrilin-2 levels were elevated in sporadic pilocytic astrocytoma, human liver cirrhosis and liver cancer, and matrilin-2 functions as a tumor suppressor in hepatocarcinogenesis [15, 16]. Ablation of Matrilin-2 is thought to contribute to hepatocarcinogenesis through activation of the MAPK signaling pathway[15]. Many reports suggested that anti-proliferation effect of matrilin-2 may be related to its G1/S
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arresting effect in cancer cells [17-20]. All of which remind us that matrilin-2 may also mediate RPI via anti-proliferation effect. In this study, we found that the mRNA and protein levels of matrilin-2 were
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increased after irradiation treatment in both lung tissue and HPAEpiC cell. Furthermore, over-expression of matrilin-2 attenuated proliferation and promoted apoptosis of HPAEpiC cell, while knockdown of matrilin-2 protect HPAEpiC from
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apoptosis induced by irradiation treatment. Matrilin-2 promotes the G1 phase arrest via p53/p21 pathway. Together, these data indicate that matrilin-2 might be a potential target to regulate the pathogenesis during RPI. Materials and methods Plasmids and lentivirus. The reporter construct containing promoter region of mouse p21 (pGL3-basic-p21), matrilin-2 expression vector (pcDNA3.1-matrilin-2) and lentivirus expressing shRNA directed against matrilin-2 were purchased from Hanbio Biotechnology Co., Ltd., Shanghai, China. p53 expression vector (pcDNA3 p53 WT) 3
ACCEPTED MANUSCRIPT was a gift from David Meek (Addgene plasmid # 69003). Animals. All animal procedures were approved by the Laboratory Animal Care Committee of Qinghai Province. Male C57BL/6 mice (Experimental Animal Center of Qinghai University, Xining, Qinghai, China) aged 6-8 weeks, with approximate
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body weights of 18-20g, were used in this study. All irradiation was performed on a clinical linear accelerator. For right hemithoracic irradiation, three 8-week old male C57BL/6 mice were anesthetized and thoracic regions corresponding to right lungs were aligned. One centimeter thickness water-density flexible bolus pieces were
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placed on top of the mice for dose buildup and collimated fields that encompassed the right hemithorax were used. For whole thoracic irradiation, 6 mice were
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simultaneously treated. 6 MV photons were used to deliver 16 Gy prescribed at Dmax.
Cell culture. HPAEpiC cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, MD, USA) and
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antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin) in a humidified atmosphere of CO2/air (5%/95%) at 37
(Thermo Fisher Scientific, Nepean, Canada).
Transfection, matrilin-2 silencing by short hairpin RNA (shRNA) and reporter
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gene assays. Matrilin-2 was expressed in HPAEpiC cells by transfecting pcDNA3.1-matrilin-2. After overnight culture, cells were incubated for 4 h in serum-free DMEM containing DNA-polyethylenimine (Sigma, 1 mg/ml) complex
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and then grown in DMEM supplemented with 10% FBS for 60 h. Endogenous human matrilin-2 in HPAEpiC cells was knocked down by infection with a lentivirus expressing shRNA directed against matrilin-2, supplemented with 8 mg/ml Polybrene (Sigma) after overnight attachment. Infection was repeated at intervals of 8 to 12 h. Cells were subjected to experiments 72 h after the second infection. In the luciferase assays, HPAEpiC cells were transfected with expression vectors or luciferase reporter plasmids plus the internal control vector pRL-TK-Renilla for luciferase assays in 24-well plates. Cells got similar transfection process above. Luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega, Madison, WI) 4
ACCEPTED MANUSCRIPT and normalized to Renilla luciferase values. Cell irradiation. HPAEpiC cells were cultured in a 25 cm2 culture flask. Cells were irradiated using a Small Animal Radiation Research Platform, SARRP system (X-ray tube: ISOVOLT 225M2 X-ray source; SARRP system, XStrahl ®, Surrey, UK) at a
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constant rate of 3.45 Gy/min, for 278 seconds, thus receivinga single-fraction of 16.0 Gy (220 kV and 13.0 mA, using an 0.15 mm cupper filter and a 10×10 cm collimator). Cells were positioned at a source-to-surface distance (SSD) of 34 cm.
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Control “sham” samples (0 Gy) received similar handlings except for the irradiation.
Real-time PCR. Total RNA was extracted from lung tissue or cells using RNAiso
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Plus (Takara Bio Inc., Dalian, China) and was reverse-transcribed using M-MMLV Reverse Transcriptase with RNasin Ribonuclease Inhibitors (Promega Biotech Co., Ltd, Beijing, China) according to the manufacturer’s protocols. qPCR was performed using SYBR Premix Master Mix (Thermo Scientific Inc., Shanghai, China). Primer sequences used in the experiments were as follows: Matrilin-2 (mouse) forward
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5’-CTATGTATGCCGTTGGGGTAGG-3’,
5’-AGCTTTTCACTTATTTCGCCCAT-3’;
reverse
Matrilin-2
(human)
5’-GATCCTCGGACAGATCGTCCT-3', 5’-CTGCCCGCTTGTTCTCACA-3’;
reverse p21(human)
forward
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5’-TGTCCGTCAGAACCCATGC-3’,
5’-AAAGTCGAAGTTCCATCGCTC-3’;
reverse GAPDH
(human)
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5’-ACAACTTTGGTATCGTGGAAGG-3’, 5’-GCCATCACGCCACAGTTTC
-3’;
forward
forward reverse
GAPDH
5’-AGGTCGGTGTGAACGGATTTG-3′,
(mouse)
forward reverse
5’-TGTAGACCATGTAGTTGAGGTCA-3’. The expression levels of mRNA were normalized to GAPDH (human) or GAPDH (mouse) mRNA. Western blot analysis and antibodies. Total cell extracts were obtained by lysing the cells in RIPA buffer with protease inhibitors cocktail (MedChem Express, Shanghai, China). Protein concentration was measured by the Bradford assay (Bio-Rad, Hercules,CA, USA). Cellular proteins were extracted and separated in 5
ACCEPTED MANUSCRIPT 4–10% Tris glycine/SDS-polyacrylamide gels and electrotransferred to ECL nitrocellulose membranes (#IPFL00010, Millipore). The membranes were blocked with 5% nonfat milk and incubated with specific antibodies. The β-actin protein was used as the endogenous control. Primary antibodies were used at the dilution of
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1:1000. Anti-matrilin-2 (ab55458), anti-p21 (ab109520) and anti-p53 (ab1101) were purchased from Abcam. β-actin (#3700) and horseradish peroxidase-conjugated anti-mouse or rabbit IgG were purchased from Cell Signaling Technology. Immunocomplexes were visualized by ECL (Pharmacia-Amersham, Freiburg,
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Germany).
MTT proliferation assay. To detect cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium bromide (MTT) colorimetric assay was performed 1 day after transfection. All the cells were washed with phosphate-buffered saline (PBS) and suspended by trypsin, seeded 2000 cells per well in 96-well plate. The plate was incubated for 1 to 5 days in cell culture chamber at 37
and 5% CO2 atmosphere. 50
µL of MTT (10 mg/mL) were added and incubated for 2 h at 37
, discarded the
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MTT and 100 µL Dimethyl Sulphoxide (DMSO, Sigma Inc) was added to each well. The absorbance at 490nm was measured using microplate reader (Bio Tek Instruments, Winooski, VT)
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Flow cytometric analysis. Cell cycle and percentage of apoptotic cells were assessed by FACScalibur flow cytometry (Becton Dickinson). Samples for cell cycle were
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harvested by trypsin and fixed by 70% ethanol in PBS in
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overnight, washed
twice with PBS and stained by propidium iodide (PI) with RNase (Becton Dickinson, San Diego, CA) for 30 min in dark. Fluorescent emissions were collected through FL2 band-pass filter. Cells for apoptosis assay were collected by trypsin without EDTA , washed twice with PBS, stained by 5 µl PI and 5 µl FITC-Annexin V in 100uL binding buffer for 15min in dark (ebioscience). Fluorescent emissions were collected through FL1 band-pass filter for FITC-Annexin V, FL2 for PI. The apoptotic cells were PI and FITC-Annexin V both positive cells. Statistical analysis. Results are expressed as mean ± SE. Data between groups were 6
ACCEPTED MANUSCRIPT analyzed by Student’s t-test or one-way ANOVA followed by Bonferroni–Dunn multiple comparison. Differences were considered significant at P < 0.05. Results Irradiation up-regulates matrilin-2 expression in lung tissues and HPAEpiC cells.
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We first investigated the expression of matrilin-2 in lung tissues after irradiation. Immunohistochemical staining showed significantly increased matrilin-2 level in the lung tissues at 4 weeks p.i. and 8 weeks p.i. compared with the control (Fig. 1A-C).
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Real-time PCR and western blotting indicate the increment of its mRNA and protein levels both (Fig. 1D-E). To make sure that the increased expression of matrilin-2 in
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irradiated lung is from pulmonary epithelial cells, the major cells in lung, we utilized HPAEpiC cell, the cell line of pulmonary epithelial cells and found that matrilin-2 protein levels were significantly up-regulated by exposing to low doses (4 Gy/day for 1–4 days) irradiation in a time-dependent manner (Fig. 1F-G). Taken together, pulmonary epithelial cells got enhanced the expression of matrilin-2 make up the
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main resource of increment of matrilin-2 level in lung with iradiation treatment. Overexpression of matrilin-2 inhibits the growth of HPAEpiC cell in vitro. To mimic
the
increased
expression
of
matrilin-2
after
irradiation
in
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pulmonary epithelial cells and explore the effect of its up-regulation in the development of RPI, we over-expressed matrilin-2 in HPAEpiC cell by transfecting matrilin-2 vectors, which increase the mRNA and protein levels by 4 and 5 fold,
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respectively (Fig. 2A-B). The enhanced expression of matrilin-2 significantly inhibited the proliferation rate of HPAEpiC cells in the MTT assay (Fig. 2C). Consistently, double staining with annexin V-FITC and PI showed an about 4 times increment in the ratio of apoptosis in matrilin-2-overexpressing cells compared with the control cells (Fig. 2D-E). FACs analysis showed higher percentage of cells rested in G1 phase after matrilin-2 upregulation (Fig. 2F). Taken together, these results indicate that Matrilin-2 suppresses cell proliferation of HPAEpiC cells through regulation of G1/S transition. 7
ACCEPTED MANUSCRIPT Matrilin-2 promotes irradiation-induced HPAEpiC cell apoptosis. Since over-expression of matrilin-2 induces apoptosis of HPAEpiC, whether the down-regulation of matrilin-2 has opposite effect and reverses the apoptosis induced by irradiation is deserved to investigate. We utilized the ShRNA to reduce the mRNA
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and protein levels of matrilin-2 to 20% and 25% of control cells (Fig. 3A-B). The down-regulation of matrilin-2 promotes the proliferation of HPAEpiC (Fig. 3C) and prevents the apoptosis induced by irradiation (Fig. 4D-E). Irradiation treatment decreased the percentage of cells in S phase and increased the percentage of cells in
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G1 phase and matrilin-2-knockdown reversed this phase switch (Fig. 2F). Taken together, matrilin-2 mediates irradiation-induced apoptosis via regulating G1/S cell
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cycle transition during radiation pulmonary injury.
Matrilin-2 regulating cell cycle related genes during radiation-induced injury in HPAEpiC cell. The result that matrilin-2 promotes the G1 arrest of HPAEpiC cells remind us that matrilin-2 may interact p21 which has been implicated in the control of the G1 to S phase transition[21]. Thus we examined p21 and found that the mRNA
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and protein levels of p21 were down-regulated in the HPAEpiC cells with matrilin-2 over-expression mediated by matrilin-2 vector transfection or irradiation, while matrilin-2 knockdown reduced p21 expression (Fig. 4A-D). p21 is transcriptional
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regulated by p53[22], the carcinogenesis inhibitor[23] and we found sight increase in p53 protein level after irradiation treatment, but not by matrilin-2 over-expression.
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p53 and matrilin-2 expression vectors and luciferase system containing p21 promoter were co-transfected into
HPAEpiC cells and the luciferse assay result indicated that
matrilin-2 promoted the transcriptinal activity of p53 on p21 (Fig. 4E). These results imply that matrilin-2 amplified the transcriptional activity of p53 on p21, thus promotes the expression of the latter.
Discussion Extracellular matrix (ECM) provides physical support to tissues, anchorage sites for cells and medium for diffusible signaling molecules [24, 25]. ECM is also a major 8
ACCEPTED MANUSCRIPT cytokine reservoir and therefore has pleiotropic effects [26-29]. Matrilin-2, as the component of ECM, should be implicated in the functions of ECM. Our previous study found the distribution of matrilin-2 mRNA in skin of the tail, calvarias, heart, uterus, and brain [30], implying that matrilin-2 may take part in the physiological
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process in those tissues. In the present study, we found that irradiation increased the mRNA and protein levels in lung tissue and pulmonary epithelial cell line, HPAEpiC cells (Fig. 1), reminding us that matrilin-2 may also have functions in the process of RPI.
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To comprehensively understand the effect of matrilin-2 on RPI, we performed in vitro MTT and FACs assays to identify the proliferation and apoptosis of HPAEpiC
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cells with over-expression (Fig. 2A-B) or knockdown (Fig. 3A-B) of matrilin-2. Our results show that matrilin-2 inhibits cell proliferation and promoting apoptosis (Fig. 2 C-E). Consistently, matrilin-2 knockdown prevents the irradiation induced apoptosis of HPAEpiC cells (Fig. 3C-E), implying that matrilin-2 functions as mediator in the RPI. Cell cycle assay revealed that the G1 phase arresting may be the reason for the
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apoptosis-promoting effect of matrilin-2 (Fig. 2F & Fig. 3F). p21 is a potent cyclin-dependent kinase inhibitor. It binds to and inhibits the activity of cyclin-CDK2, -CDK1, and -CDK4/6 complexes, and thus functions as a repressor
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on cell cycle progression at G1 and S phase [21], by which p21 mediates growth arrest and cellular senescence. Interestingly, the expression of matrilin-2 is positively related to that of p21 (Fig. 4A-D), which indicates that matrilin-2 may promote
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apoptosis via the up-regulation of p21. The expression of p21 is tightly transcriptional controlled by the tumor suppressor protein p53, which is easily activated in response to a variety of stress stimuli, including DNA damage (induced by either UV, IR, or chemical agents such as hydrogen peroxide), oxidative stress, osmotic shock, ribonucleotide depletion, and deregulated oncogene expression[31, 32]. As we expected, irradiation increased the p53 protein level in HPAEpiC cells, but neither over-expression nor knockdown of matrilin-2 affect p53 expression (Fig. 4A-B). These data indicates that matrilin-2 does not modulate p21 expression via regulating p53 level. Interestingly, the result of luciferase assay indicates that matrilin-2 9
ACCEPTED MANUSCRIPT promotes the transcription of p21 by p53 (Fig. 4E), i.e. matrilin-2 functions as the co-activator of p53. In conclusion, we report that the extracellular matrix protein matrilin-2, over-expressed in pulmonary epithelial cells after irradiation, functions as cell
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proliferation suppressor via promoting the G1 phase arresting. This effect is based on the up-regulation of p21 by activating its transcriptional factor, p53. Uncovering the underlying molecular mechanisms would develop better preventive measures to RPI
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in the radiotherapy of cancer.
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Acknowledgements
This work was funded partially through Chinese Natural Science and Technology Foundation 81460022 and New Faculty foundation of Qinghai Provincial People's Hospital (Junming Luo), and Chinese Natural Science and Technology Foundation
(Shukun Zhang).
Figure legends
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81560123, Qinghai Provincial Science and Technology Foundation 2014-ZJ-731
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Figure 1. Irradiation up-regulates matrilin-2 expression in lung tissues and HPAEpiC cells. (A-C) Immunohistochemical staining of mouse lung. Three
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representative slides are shown for the control and irradiated mice. (A) Control mice; (B) mice irradiated for 1 weeks p.i.; (C) mice irradiated for 8 weeks p.i. groups. (original magnification, ×200). The mRNA (D) and protein (E) levels of Matrilin-2 in lungs from mice irradiated. The mRNA (F) and protein (G) levels of Matrilin-2 in HPAEpiC cells irradiated. The data are presented as the mean ± S.E. and expressed as fold-change relative to the level of control mice or cells. *p < 0.05.
Figure 2. Overexpression of matrilin-2 inhibits the proliferation and activates the apoptosis of HPAEpiC cells. Matrilin-2 expression vector was transfectedin to 10
ACCEPTED MANUSCRIPT HPAEpiC cells. The mRNA (A) and protein (B) levels the matrilin-2. (C) The cell viability was determined by measuring MTT assay. Cell growth was measured at every 24 hours. (D) Flow cytometry were used to test the cell apoptosis after 48 h post-transfection. Cells staining positive for FITC-Annexin V and negative for PI
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were considered to have undergone apoptosis. (E) Average apoptotic rate. (F) The DNA content of PI-stained cells was analyzed by flow-cytometry for cell cycle determination. The data are presented as the mean ± S.E. and expressed as
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fold-change relative to the level of cells transfected with control vector. *p < 0.05.
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Figure 3. Matrilin-2 knockdown interferes with irradiation-induced apoptosis of HPAEpiC cell. HPAEpiC cells were infected a lentivirus expressing shRNA directed against matrilin-2. The mRNA (A) and protein (B) levels the matrilin-2. (C) The cell viability was determined by measuring MTT assay. Cell growth was measured at every 24 hours. (D) Flow cytometry were used to test the cell apoptosis after 72 h
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post-infection. Cells staining positive for FITC-Annexin V and negative for PI were considered to have undergone apoptosis. (E) Average apoptotic rate. (F) The DNA content of PI-stained cells was analyzed by flow-cytometry for cell cycle
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determination. The data are presented as the mean ± S.E. and expressed as
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fold-change relative to the level of cells infected with Shcontrol. *p < 0.05.
Figure 4. Matrilin-2 promotes the transcriregulating cell cycle related genes during radiation-induced injury in HPAEpiC cell. (A) Matrilin-2 expression vector was transfected into HPAEpiC cells. Cell extractors were blotted with anti-p53, p21 and β-actin antibody. (B) HPAEpiC cells were infected a lentivirus expressing shRNA directed against matrilin-2 and subjected to irradiation. Cell extractors were blotted with anti-p53, p21 and β-actin antibody. (C) mRNA levels of p21 in HPAEpiC cells with matrilin-2 overexpression. (D) mRNA levels of p21 in HPAEpiC cells with matrilin-2 knockdown and irradiation. (E) The expression vectors of p53 and matrilin-2 were co-transfected with luciferase vector containing p21 promoter into 11
ACCEPTED MANUSCRIPT HPAEpiC cells. Luciferase activity was assay and normalized to that of pRL-TK-Renilla. The data are presented as the mean ± S.E. and expressed as fold-change relative to the level of cells transfected with control vector or infected
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with Shcontrol. *p < 0.05.
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[31] R. Rodriguez, M. Meuth, Chk1 and p21 cooperate to prevent apoptosis during DNA replication [32] A. Hirao, Y.Y. Kong, S. Matsuoka, A. Wakeham, J. Ruland, H. Yoshida, D. Liu, S.J. Elledge, T.W. Mak, DNA damage-induced activation of p53 by the checkpoint kinase Chk2, Science, 287
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