- Open Access
EZH2 promotes DNA replication by stabilizing interaction of POLδ and PCNA via methylation-mediated PCNA trimerization
© The Author(s) 2018
- Received: 24 April 2018
- Accepted: 25 July 2018
- Published: 2 August 2018
Proliferating cell nuclear antigen (PCNA), a ring-shaped homotrimer complex, promotes DNA replication via binding to DNA polymerase. Trimerized PCNA is critical for DNA replication. Enhancer of zeste homologue 2 (EZH2), which primarily acts as a histone methyltransferase, is essential for proliferation. However, how EZH2 promotes proliferation by controlling DNA replication through PCNA remains elusive.
Here, we showed that low EZH2 levels repressed the proliferation of human dental pulp cells (hDPCs). The EZH2 protein level was dramatically upregulated in hDPCs at S phase in the absence of H3K27 trimethylation. Molecularly, EZH2 interacted with PCNA via the PIP box and dimethylated PCNA at lysine 110. Dimethylation of PCNA is essential for stabilization of the PCNA trimer and the binding of DNA polymerase δ to PCNA.
Our data reveal the direct interaction between PCNA and EZH2 and a novel mechanism by which EZH2 orchestrates genome duplication.
- DNA replication
Replication of the genome is an essential step in cell cycle progression and proliferation. Failure in this process will lead to abnormal cell proliferation, cell cycle arrest, and genomic instability [1–3]. At the heart of DNA replication, proliferating cell nuclear antigen (PCNA) has an essential role in orchestrating normal DNA synthesis by providing a platform to which DNA polymerases and other factors bind via the PIP box . PCNA, a ring-shaped sliding clamp, encircles DNA by the dimerization (prokaryotes) or trimerization (eukaryotes) of monomers [5–7]. Post-translational modifications (PTMs) of PCNA facilitate these protein interactions and are essential for the high processivity and accuracy of DNA synthesis. Modifications on PCNA, carried by “writers,” profoundly regulate the interaction between PCNA and its partners. Phosphorylation, acetylation, ubiquitination, and sumoylation on PCNA have been strongly suggested to regulate DNA replication by affecting the binding affinity of PCNA partners or the stability of PCNA [8–11]. Additionally, SETD8, a methyltransferase, has been reported to interact with, and methylate PCNA [12, 13].
Protein lysine methylation has been widely elucidated in histone modification [14, 15]. However, accumulating non-histone proteins have been reported to be methylated by histone methyltransferases [16, 17]. Enhancer of zeste homologue 2 (EZH2), a methyltransferase, is primarily responsible for the trimethylation of histone H3 lysine 27 (H3K27me3) [18–21]. In addition, EZH2 has been reported to methylate non-histone proteins, such as cardiac transcription factor 4 (GATA4), retinoic acid-related orphan nuclear receptor α (RORα), and signal transducer and activator of transcription 3 (STAT3) [22–24]. EZH2 is positively related to proliferation, and its role as a transcriptional silencer via repression of INK4A/ARF is well established [25–27]. Beyond its role as the transcriptional repressor, EZH2 was also reported to localize at the replication fork by interacting with histone in response to DNA damage [28, 29]. Moreover, a recent study suggests that Polycomb (PcG) repressive complexes (PRCs) control proliferation independent of transcriptional repression of cell cycle-regulating genes . However, there is still a lack of evidence indicating whether EZH2 directly binds to PCNA, and information regarding whether EZH2 participates in DNA replication remains elusive.
In this study, we demonstrated, for the first time, that EZH2 directly binds to PCNA via the PIP box and promotes methylation at lysine 110. Then, we showed that K110me2 is critical for the stabilization of the PCNA trimer. Finally, we showed that EZH2 promotes the interaction between DNA polymerase δ (POLδ) and PCNA.
EZH2 regulates HDPC proliferation independent of its role as a gene repressor
To experimentally validate these observations, the expression of cell cycle-related genes was measured using a cell cycle RNA-sequencing assay in EZH2-knockdown hDPCs from three different donors. EZH2 has been reported to function as a transcriptional repressor  and represses the expression of INK4A, INK4B, and ARF [27, 33]. In the RNA-sequencing analysis of EZH2-knockdown hDPCs, eight genes were transcriptionally altered (Fig. 1e); however, there was no transcriptional difference in INK4A and ARF. Among the eight genes, cyclin-dependent kinase 2 (CDK2), cyclin E2 (CCNE2), and cyclin-dependent kinase inhibitor 2B (CDKN2B, also known as INK4B) are involved in controlling cell cycle phase transition [26, 34]. CDK2 and CCNE2, which also regulate the DNA replication process, were downregulated, and CDKN2B was upregulated. Interestingly, when verifying the differentially expressed genes in the RNA-Seq data by qPCR in hDPCs from a fourth donor, we found that only ANAPC1, CDKN2B, and SMAD2 exhibited the same changes in gene expression (Fig. 1f). These findings are consistent with a previous study showing that Polycomb (PcG) controls the proliferation of mouse embryonic fibroblasts (MEFs) independent of Ink4a/Arf suppression .
Combined with the observation that the defect in proliferation upon EZH2 knockdown did not elicit significant cell cycle arrest at G1 or G2/M phase, our data suggest that instead of canonically controlling transcription at cell cycle phase transitions , EZH2 may regulate the proliferation of hDPCs with an alternative mechanism (Additional file 1: Figure S1).
EZH2 elevates at S phase in hDPCs and interacts with PCNA
Because cells duplicate their genome during S phase, we hypothesized that EZH2 might participate in the DNA replication of hDPCs in S phase. PCNA, a sliding clamp, is at the heart of DNA synthesis by providing the platform for factors to orchestrate DNA replication [4, 37]. Additionally, EZH2 has been reported to be present at the replication fork in human cells [29, 38]. Thus, we investigated whether EZH2 directly interacts with PCNA in hDPCs. We performed co-immunoprecipitation (co-IP) of endogenous EZH2 and PCNA (Fig. 2c, upper panel). Moreover, by using a proximity ligation assay (PLA) with EZH2- and PCNA-specific antibodies, we visualized the interaction of EZH2 and PCNA at the molecular level (Fig. 2c, lower panel). Additionally, the interaction of EZH2 and PCNA was verified in 293T cells (data not shown). These results indicate that EZH2 directly interacts with PCNA and strongly indicates the participation of EZH2 in DNA replication.
The presence of a PIP box in EZH2
EZH2 methylates PCNA
Stabilized trimerization of PCNA by EZH2 is essential for the binding of DNA polymerase δ to PCNA
In normal DNA replication, enhancing the processivity of DNA polymerase δ (POLδ) is the primary function of PCNA [46, 48, 49]. Thus, we explored whether EZH2 could affect the binding of POLδ to PCNA. First, we immunoprecipitated endogenous PCNA protein using a PCNA antibody to assess the interaction between POLδ (represented by the catalytic subunit, POLD1) and PCNA in hDPCs and 293T cells with EZH2 knockdown. We found decreased binding of POLD1 to PCNA when EZH2 was knocked down (Fig. 5d). We next investigated whether the binding of POLδ to PCNA depends on the interaction between EZH2 and PCNA by examining the interaction between PCNA and POLδ in EZH2∆PIP-transfected 293T cells. We found that the binding of POLD1 to PCNA was dramatically reduced upon mutation of the PIP box (Fig. 5d). Moreover, we also investigated the effect of the methyltransferase activity of EZH2 on the interaction between PCNA and POLδ. Compared to the cells expressing wild-type EZH2, cells transfected with EZH2 ∆SET showed decreased levels of POLD1 precipitated with PCNA (Fig. 5d). Taken together, these results indicate that the methyltransferase activity of EZH2 is necessary for the interaction between PCNA and POLδ.
As our data demonstrated that PCNA can undergo EZH2-mediated dimethylation at lysine 110, we hypothesized that K110me2 may affect the stabilization of the PCNA trimer. We introduced a point mutation at lysine 110 on PCNA (Fig. 5e), expressed this mutant in 293T cells, and then conducted the protein crosslink assay. As demonstrated, the trimer form of PCNA was dramatically impaired upon mutating PCNA at this residue (Fig. 5e). Moreover, the binding of POLδ to PCNA was dramatically impaired (Fig. 5e). Based on the fact that K110A-mutated PCNA exhibited impaired binding to POLδ, we next tested whether this mutant PCNA could affect DNA synthesis. By using a DNA fibre assay, we observed that the DNA length dramatically decreased in cells expressing PCNA with the K110A mutation (Fig. 5f).
Taken together, our data suggested that the K110me2 catalysed by EZH2 is important for the trimerization of PCNA and the binding of POLδ to PCNA.
Epigenetic modulators such as methyltransferases have been widely studied in the histone lysine methylation field with regard to their modulation of chromatin properties and regulation of gene expression [21, 50–52]. However, owing to growing interest in the post-translational modifications of proteins, the importance of methylation on non-histone proteins has frequently been indicated, and non-histone proteins have been reported to be potential substrates of histone lysine methyltransferases [16, 17]. In the present study, we demonstrated that PCNA is a novel substrate of EZH2 that can be dimethylated at K110. To date, only three non-histone proteins, RORα, GATA4, and STAT3, have been reported to be substrates of EZH2 [22–24]. In addition, lysine methylation is reversible, and H3K27 has been shown to be demethylated by KDM6B [15, 53]. Thus, the occurrence of demethylation on PCNA and the verification of de-methyltransferase activity on PCNA need to be elucidated in future. Recently, studies have reported a lower accumulation of H3K27me3 following DNA replication [35, 36], which is consistent with our observation of decreased H3K27me3 in hDPCs in S phase.
In the present study, we found that knockdown of EZH2 did not induce a significant cell cycle arrest at G1 or G2/M phase, but the proliferation activity of hDPCs was continuously repressed. This was also supported by the observation that the transcript levels of most cell cycle regulatory genes did not change when EZH2 was knocked down in hDPCs. These findings are consistent with previous studies showing that Polycomb (PcG) controls the proliferation of MEFs independent of INK4A/ARF suppression .
PCNA, a sliding clamp, plays a key role in duplicating the genome by providing a scaffold for relevant factors and DNA polymerases. PCNA interacts with DNA replication cofactors during DNA replication via the evolutionarily conserved PIP box or APIM motif [4, 43, 54]. Previous studies reported that the SET-domain proteins ATXR5 and ATXR6, which have primarily been demonstrated to be H3K27 mono-methyltransferases, interacted with PCNA in Arabidopsis [44, 55]. In addition, other epigenetic modifiers, such as DNMT1, HDAC1, p300, and SETD8, were reported to interact with PCNA in humans [11, 13, 56, 57]. So far, SETD8 is the only histone methyltransferase that has been reported to methylate PCNA . Furthermore, previous studies suggested that in response to DNA damage, EZH2 was recruited to stalled replication forks to function as an H3K27me3 methyltransferase [28, 29]. However, there was no evidence indicating a direct interaction between EZH2 and PCNA. Here, we provide the first evidence that EZH2 directly interacts with PCNA via the conserved PIP box motif to promote the methylation of PCNA. In the study by Andrea Piunti et al., the association between RING1B and PCNA is reduced in the absence of EZH2, suggesting the possible hierarchical recruitment of PRC1 and PRC2 [30, 58]. In our study, we confirmed the interaction between EZH2 and PCNA; thus, we hypothesized that the reduced association between RING1B and PCNA was possibly influenced by the diminished interaction between EZH2 and PCNA when EZH2 was depleted. However, this mechanism needs to be verified in future.
PCNA, a ring-shaped complex, consists of three homo-monomers that assemble in a “head-to-tail” manner in eukaryotes . Maintenance of the PCNA trimer is a prerequisite for promoting the processivity of DNA polymerase δ (POLδ). Here, we uncovered that PCNA K110me2 catalysed by EZH2 is essential for stabilizing the trimer form of PCNA. Moreover, consistent with previous studies [30, 59], we observed an interaction between SUZ12 and PCNA (data not shown). This suggested that other PRC2 components might be involved in the regulation of PCNA methylation. The trimerization of PCNA is established through the binding of β-sheets of each monomer at the interfaces between the subunits [5, 6, 60]. Three types of interactions between the monomer interfaces of PCNA have been proposed to stabilize the PCNA trimer: hydrogen bonds, hydrophobic interactions, and ion pairs. Among these, ion pairs between the β-sheets and the interface were considered the main contributor . Based on the investigation of PCNA among various species, a positive polar amino acid, lysine, was found at position 110, which is located within the β-sheet . Here, we report that K110me2 is critical for the trimerization of PCNA. Methylation does not perturb the charge of the lysine residue and induces a small change in its size; thus, K110me2 is more likely to enhance the hydrophobic interaction between the monomers to stabilize the PCNA trimer by changing the hydrogen property and hydrophobic force of lysine 110 [17, 60, 61]. To verify this hypothesis, further structural studies are needed. Moreover, in normal DNA replication, PCNA interacts with POLδ and stimulates its processing activity [4, 47, 62, 63]. Here, we claimed that EZH2-mediated dimethylation of PCNA K110 is essential for the interaction of PCNA with POLδ, leading to the control of DNA synthesis. Because EZH2 regulates the trimerization of PCNA, it is evident that the binding of POLδ to PCNA decreases in the absence of EZH2.
hDPCs were harvested from normal impacted third molars extracted from adult humans (19–22 years old) in West China Hospital of Stomatology as described previously . The procedures were approved by the Ethical Committee of the West China School of Stomatology, Sichuan University, and performed in accordance with approved guidelines; all subjects provided informed consent. HEK293T (ATCC) cells were obtained from Ohio (Shanghai). In all experiments, cells were grown in DMEM (GIBCO) supplemented with 10% FBS (GIBCO) and 1% antibiotics (GIBCO). HEK293T cells were cultured in growth medium on plates coated with 0.01% poly-l-lysine (Sigma-Aldrich). All cells were grown at 37 °C in a humidified atmosphere containing 5% CO2.
For G0/G1 synchronization, hDPCs were grown in 0.2% FBS-containing medium for 48 h, after which they were cultured in normal growth medium and harvested at the indicated time points.
Lentivirus production and transduction
Lentivirus expressing shEZH2 (forward oligo: 5′-CCGGGAGGGAAAGTGTATGATAACTCGAGTTATCATACACTTTCCCTCTTTTTG-3′, reverse oligo: 5′-AATTCAAAAAGAGGGAAAGTGTATGATAACTCGAGTTATCATACACTTTCCCTC-3′) was generated by inserting shRNA oligos against EZH2 into the pLKO.1-TRC cloning vector (gifts from Zhipeng Fan, Capital Medical University) following the Addgene protocol. Oligos were synthesized by Invitrogen. The control vector was also a gift from Zhipeng Fan. Constructs containing wild-type EZH2, EZH2∆SET, PIP box point mutations of EZH2 (EZH2∆PIP), wild-type PCNA, or point mutations of PCNA (PCNA∆) were acquired from Ohio (Shanghai). Lentiviruses containing shRNA were produced in 293T cells with the following plasmids: 9 µg of pLKO.1 shRNA vector, 6 µg of ∆8.2 packaging plasmid, and 3 µg of VSV-G envelope plasmid. After 60 h, the supernatant containing viral particles was harvested and filtered at 0.45 μm and added to either hDPCs or 293T cells in the presence of 5 μg/ml polybrene (Sigma-Aldrich) for 48 h. Then, positive cells were selected with 2 μg/ml puromycin (Sigma-Aldrich) for 3 days, and the cells were maintained in growth medium containing 1 μg/ml puromycin.
Western blot analysis
Antibodies used in this study
WB, IF, PLA, IP
DNA Polymerase delta (607)
Ubiquityl-PCNA (Lys164) (D5C7P)
Goat anti-rabbit IgG (HRP)
Goat anti-mouse IgG (HRP)
Rabbit anti-goat IgG-FITC
Rabbit anti-goat IgG-Cy3
Goat anti-mouse IgG-Cy3
Goat anti-mouse IgG-FITC
Goat anti-rabbit IgG-Cy3
Donkey anti-mouse IgG (H + L)(Alexa Fluor 555)
Cell proliferation assays
hDPCs were seeded in 96-well plates at a density of 3 × 103 cells per well in triplicate for each time point, and the medium was changed every 2 days. To determine the cell viability, a 7-day time course was implemented, and a 10-μl CCK-8 solution (Dojindo) was added every 2 days and incubated for 1.5 h according to the manufacturer’s protocol. The absorbance at 450 nm was measured by a microplate reader (BioTek) and normalized to the background readings.
Colony formation assay
hDPCs were seeded in a six-well plate at a density of 500 cells/well and cultured in growth medium for 3 wks. Cells were washed with PBS, fixed in a 4% paraformaldehyde for 5 min, and then stained with crystal violet (Beyotime) for 5 min.
Flow cytometry analysis
To analyse the cell cycle distribution, trypsinized cells were washed with PBS and fixed in ice-cold 70% ethanol overnight. Cells were then washed 2X with PBS and incubated with RNase for 30 min at 37 °C followed by incubation with propidium iodide (PI) (KeyGEN Biotech) for 30 min at 4 °C. The PI-stained cells were examined on a Guava EasyCyte HT flow cytometer (Millipore) and analysed with InCyte2.7 software (Millipore).
hDPCs from three different donors were collected, treated with TRIzol (Invitrogen) and subjected to cell cycle RNA sequencing (RiboBio) to detect the transcripts of cell cycle-related genes. Array and data were performed and analysed by RiboBio Inc. (Guangzhou). Genes with an expression fold change ≥ 1.5 and P ≤ 0.05 were considered differentially expressed. RNA-Seq data are available upon demand from the corresponding author.
GAPDH: 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′;
EZH2: 5′-GCCAGACTGGGAAGAAATCTG-3′ and 5′-TGTGCTGGAAAATCCAAGTCA-3′;
CDK2: 5′-GGCACGTACGGAGTTGTGTA-3′ and 5′-CTCAGTCTCAGTGTCCAGGC-3′;
ANAPC1: 5′-AGGCCTGCGAAGGAAACTTA-3′ and 5′-ACGTTGACACGGACAGGATG-3′;
CDKN2B: 5′-GAATGCGCGAGGAGAACAAG-3′ and 5′-CATCATCATGACCTGGATCGC-3′;
SMAD2: 5′-GCTTCCCTCGTGCTGATTGG-3′ and 5′-GTATGGAAGACGGAGGGAGC-3′;
CCNE2: 5′-GGCCTATATATTGGGTTGGCG-3′ and 5′-ACGGCTACTTCGTCTTGACA-3′;
MCM2: 5′-ATCTACGCCAAGGAGAGGGT-3′ and 5′-GTAATGGGGATGCTGCCTGT-3′;
BUB1B: 5′-GGATGGGTCCTTCTGGAAAC-3′ and 5′-AAGCTCCCCAAGAACAGACA-3′;
CDC27: 5′-GACAGCTCCACAACCAAGGA-3′ and 5′-TCAAACCTTCTGCTGCTGCT-3′.
hDPCs were seeded on coverslips overnight before serum deprivation. Cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min and then permeabilized with 0.5% Triton X-100 in PBS for 20 min at room temperature. Cells were blocked for 30 min with blocking buffer (Zhongshanjinqiao) and incubated overnight at 4 °C with primary antibodies in blocking solution. Coverslips were washed 5X with PBST (0.5% Tween-20) and incubated for 1 h at room temperature with fluorescent secondary antibodies (Table 1). Next, the coverslips were washed 5X with PBST (0.5% Tween-20) and then stained with 4′6-diamidino-2-phenylindole (DAPI) to identify nuclear DNA. Images were captured on a Nikon Eclipse 300 fluorescence microscope (CompixInc). ImageJ was used to calculate Pearson’s correlation coefficient and Mender’s coefficient of colocalization.
5-Ethynyl-2′-deoxyuridine (EdU) staining
EdU staining was conducted using an EdU imaging kit (RiboBio) according to the manufacturer’s protocol. Briefly, hDPCs were seeded on coverslips overnight followed by serum starvation. Then, cells were released from deprivation in complete growth medium for 24 h, during which an EdU pulse was applied for 3 h at a concentration of 50 μM. After labelling, cells were stained with EdU. For double staining with other antigens, additional immunohistochemical staining was performed following EdU staining before the nuclei were stained with DAPI. Images were captured with a Nikon Eclipse 300 fluorescence microscope (CompixInc).
Proximity ligation assay
Cells were prepared as described in the immunofluorescence section and incubated with anti-EZH2 (Goat) and anti-PCNA (mouse) primary antibodies following the manufacturer’s instructions (Sigma-Aldrich). Briefly, cells were incubated for 60 min at 37 °C with anti-mouse PLUS (Sigma-Aldrich, DUO92001) and anti-Goat MINUS (Sigma-Aldrich, DUO92006), washed twice with Buffer A (Sigma-Aldrich, DUO82047), and then incubated with the ligation solution (Sigma-Aldrich, DUO92014) for 30 min at 37 °C. After ligation, cells were washed twice with Buffer A and then incubated for 100 min at 37 °C with amplification reagents (Sigma-Aldrich, DUO92014). Finally, the cells were washed three times with Buffer B (Sigma-Aldrich, DUO82048), stained, and mounted with mounting medium (Sigma-Aldrich, DUO82040) to visualize the nuclei. Images were captured with a Nikon Eclipse 300 fluorescence microscope (CompixInc).
Cells were harvested at the indicated times, lysed in lysis buffer supplemented with a protease inhibitor cocktail, incubated on ice for 20 min, and cleared by centrifugation at 14,000 rpm at 4 °C for 20 min. Total protein lysate (500 μg) was incubated with agarose-conjugated protein A/G beads for 2 h at 4 °C; the lysate was washed 5X with PBS at 4 °C and centrifuged at 14,000 rpm at 4 °C for 20 min. Then, the lysate was subjected to immunoprecipitation with agarose (Beyotime)-immobilized antibodies or isotype control antibodies overnight at 4 °C. The mix was washed 3X with lysis buffer at 4 °C. The precipitated proteins were subjected to SDS-PAGE and detected by western blot.
Formaldehyde crosslinking assays were performed as previously described . Trypsinized cells were resuspended and incubated in a 1.0–1.5% formaldehyde solution at room temperature for 30 min. Then, the reaction was quenched by the addition of a 1.5 M glycine solution to a final concentration of 0.15 M and incubated at room temperature for 5 min. Cells were pelleted by centrifugation at 4000 rpm at room temperature for 5 min and resuspended and washed twice in PBS. Products were analysed by western blot using PCNA (PC10) or Myc-tagged antibodies.
DNA fibre assay
A DNA fibre assay was performed according to a previous study . Briefly, 293T cells were labelled with 25 μM IdU (Sigma-Aldrich, I17125) for 2 h. A 2-μl cell suspension was spotted at the end of the microscope slide and incubated with 7 μl of lysis buffer (0.5% SDS, 200 mM Tris–HCl (pH 7.4), and 50 mM EDTA) for 2 min. Slides were tilted 15° to allow the DNA fibres to stretch along the slide and then allowed to air-dry. After 10 min of fixation in methanol:acetic acid (3:1), the DNA was denatured with 2.5 M HCl for 60 min and blocked in 5% BSA in PBS (Table 1).
Slides were incubated with primary antibodies (anti-IdU), washed three times in PBS, incubated with secondary antibodies, and mounted.
The statistical analysis of the RNA sequencing was calculated with ANOVA, whereas the statistical analysis of other experiments was carried out with Student’s t test (two-tailed) using Prism 5 (GraphPad Software). Error bars represent the standard deviation (SD). P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001. All experiments were repeated three times biologically unless specifically indicated.
PA formed the conception, designed the experiments, drafted, and revised this article. XX performed experiments, interpreted data, and revised this article. CW, JY, SW, and JD revised the manuscript. LY revised the work critically. All authors read and approved the final manuscript.
We would like to thank Dr. Zhipeng Fan from Capital Medical University for kindly providing pLKO.1 plasmids. We would like to thank Dr. Wantae Kim, Dr. Prem Swaroop Yadav from Department of Developmental Biology, Harvard School of Dental Medicine for support and advice.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets in this study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation West China School of Stomatology, Sichuan University. Informed consent was obtained from all patients for being included in the study.
This work was supported by the National High-tech R&D Program (863 Program) (No. 2015AA020306), the Sichuan Province Science and Technology Support Program (Grant Numbers. 2016JY0022), the National Natural Science Foundation of China (Grant Numbers. 81271127, 81322013), and the Program of International Science and Technology Cooperation (Grant Number. 2014DFA31990).
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