Abnormal level of CUL4B-mediated histone H2A ubiquitination causes disruptive HOX gene expression
Neural tube defects (NTDs) are common birth defects involving the central nervous system. Recent studies on the etiology of human NTDs have raised the possibility that epigenetic regulation could be involved in determining susceptibility to them.
Here, we show that the H2AK119ub1 E3 ligase CUL4B is required for the activation of retinoic acid (RA)-inducible developmentally critical homeobox (HOX) genes in NT2/D1 embryonal carcinoma cells. RA treatment led to attenuation of H2AK119ub1 due to decrease in CUL4B, further affecting HOX gene regulation. Furthermore, we found that CUL4B interacted directly with RORγ and negatively regulated its transcriptional activity. Interestingly, knockdown of RORγ decreased the expression of HOX genes along with increased H2AK119ub1 occupancy levels, at HOX gene sites in N2/D1 cells. In addition, upregulation of HOX genes was observed along with lower levels of CUL4B-mediated H2AK119ub1 in both mouse and human anencephaly NTD cases. Notably, the expression of HOXA10 genes was negatively correlated with CUL4B levels in human anencephaly NTD cases.
Our results indicate that abnormal HOX gene expression induced by aberrant CUL4B-mediated H2AK119ub1 levels may be a risk factor for NTDs, and highlight the need for further analysis of genome-wide epigenetic modifications in NTDs.
KeywordsNeural tube defect Histone ubiquitination RA CUL4B RORγ HOX genes
neural tube defects
polycomb protein complexes
retinoic acid-related orphan nuclear receptor gamma
NT2 human embryonic carcinoma cells
Neural tube defects (NTDs) are a group of severe congenital malformations caused by the failure of neural tubes to close completely. Human NTDs are a complex condition impacted by multiple factors, including both genetic and environmental. Recently, a study showed that the formation of the neural tube is under precise spatiotemporal control by cooperative actions between environmental factors and intrinsic signal transduction . In humans, the development of NTDs is thought to involve the interplay of genes in the fetus and the effects of environmental factors. However, the molecular mechanisms in the etiology of NTDs through which environmental factors affect epigenetic regulation, thereby influencing the expression of susceptibility genes, are poorly understood.
Retinoic acid (RA), a derivative of vitamin A (retinol), is an extrinsic signal essential for neuronal differentiation in embryonic development [2, 3]. Paraxial mesoderm surrounding the neural tube expresses the retinaldehyde dehydrogenase-2 (Raldh2) enzyme, which converts retinaldehyde to RA, serving as a ligand for nuclear RA receptors that directly regulate gene expression at the transcriptional level . It has been demonstrated that RA plays an essential role in anteroposterior patterning of neuroectoderm in the central nervous system (CNS) [5, 6, 7, 8]. It is found that epigenetic change occurs in response to RA and is related to cell differentiation. The observation of histone enzymes involved in transcription of response genes, and many of these enzymes are modulated by RA treatment. Currently, some of these transcription complexes regulate histone modification to influence stem cell fate. Polycomb group complexes (PRC) play critical regulatory roles that change the expression of numerous genes during neuronal cell specification. Two main polycomb group complexes exist in mammals: PRC1 and PRC2. PRC1 catalyzes histone H2AK119 monoubiquitylation (H2AK119ub1). More recent studies have suggested that PRC1 plays an important role in H2AK119ub1 and Hox gene silencing. CUL4B is an important member of the Cullin–Ring ligase (CRL) complex, which has E3 ubiquitin ligase activity. It participates in many developmental processes, such as cell cycle progression, replication, and the DNA damage response and involves in the proliferation and organization of neuronal cells. Moreover, CUL4B might also be involved in the regulation of appropriate brain development. Recently, it was indicated that CUL4B possesses an intrinsic transcription repressive activity by promoting H2AK119ub1. CUL4B regulates transcription via H2AK119ub1 in coordination with H3K27me3 leading to derepression of target genes that are critically involved in cell growth and migration. Nuclear receptors constitute a superfamily of ligand-dependent transcription factors that includes receptors for retinoids and orphan receptors. In this study, screening of CUL4B targets by IP/mas identified the ROR γ as a specific substrate of CUL4B. The retinoid-related orphan receptors RORα, RORβ, and RORγ are a distinct subfamily of nuclear receptors. RORγ is an orphan nuclear receptor related to retinoic acid. RA can bind to RORγ as a ligand to produce biological effects. RA and synthetic retinoic acid ALRT1550 can bind to RORγ, thereby stimulating RORγ-mediated transcriptional activation . Recent studies have demonstrated that RORs function as ligand-dependent transcription factors. Transcriptional regulation by RORs is mediated by the recruitment of corepressor and coactivator complexes modulating the response genes. It has been showed RORγ played major roles in neural stem cells development and neurological diseases. Although several studies have indicated that RA signaling is involved in neuron specification and neurogenesis, the molecular mechanisms of action associated with the target genes of RA-bound RORs during neural development have remained elusive.
Recently, more than 200 candidate genes have been identified as potentially being related to NTDs in humans. A prominent focus has been on the PCP, BMP, and Wnt pathways and on homeobox genes (Hox). Hox genes play an important role in brain and spinal cord development during establishment of the anteroposterior body axis in embryogenesis. In mammals, 39 Hox genes have been identified, which are divided into four clusters: A, B, C, and D. During embryonic development, the conserved Hox genes are clustered and expressed linearly, resulting in the formation of the body shape. Hox genes also play important roles in specifying the morphology of the vertebrae . Hoxa1 plays an important role in regulating nerve ridge function, hindbrain formation, and early development . Hoxa1 is also necessary for the RA-induced differentiation of embryonic stem cells (ESC) into neurons . In addition, Hoxa7 participates in regulating cell differentiation  and the process of embryogenesis . Moreover, Hoxa9 plays a role in regulating the differentiation of pluripotent progenitor cells , while Hoxa10 can regulate the expression of region-specific genes . Hoxb1 regulates the proliferation of neural stem cells and progenitor cells . The regulation of Hoxb7 over the course of development is time dependent and tissue specific . In addition, it has recently been shown that some of the Hox genes play roles in global patterning in vertebral development. For example, several studies have indicated that RA induces the expression of Hox genes needed for rhombomeric segmentation of neuroectoderm during neural tube formation and spinal cord development . It has been shown that RA signaling is necessary to erase polycomb repressive (PRC) activated Hox genes during embryonic stem cell differentiation . However, the molecular mechanism by which CUL4B catalyzes H2AK119ub1 and contributes to RA-mediated HOX gene regulation in NTDs remains unclear.
To obtain a better understanding of the molecular mechanisms underlying CUL4B’s involvement in RA-mediated HOX gene regulation in NTDs, we here show that CUL4B is required for the activation of retinoic acid (RA)-inducible developmentally critical homeobox (HOX) genes in NT2/D1 cells. RA treatment led to attenuation of H2AK119ub1 due to a decrease in CUL4B, further affecting HOX gene regulation. Our study also identifies CUL4B as a novel repressor of RORγ transcriptional regulation. Knockdown of RORγ decreased the expression of HOX genes, along with increased H2AK119ub1 occupancy levels at HOX gene sites in N2/D1 cells under RA treatment. In addition, upregulation of HOX genes was observed, along with lower levels of H2AK119ub1, in both mouse and human anencephaly NTD cases. Together, our findings highlight how RA affects CUL4B and cofactor RORγ through histone modification, leading to the regulation of HOX gene transcription during the early stage of development. Our work also reveals the need for further genome-wide analysis of epigenetic modifications in NTDs.
CUL4B represses transcription of HOX genes in RA-induced human NT2 cells
RA-induced Hox genes expression correlates with a decreased level of promoter H2AK119ub1 in mouse ESCs
CUL4B interacts with RORγ and alterations in HOX genes expression in human NT2/D1 cells
RORγ is required for activation of HOX genes expression during RA-induced differentiation human NT2/D1 cells
Cul4b level downregulated in RA-induced mouse NTD model
HOXA10 gene transcription is altered with decreased CUL4B level in NTD fetuses
Recent studies showed that two polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental in embryonic stem cells (ESCs). Within PRC1, CUL4B act as E3 ubiquitin ligases for H2AK119ub1 that mediate transcriptional repression. In this study, we found that RA treatment led to a decrease in CUL4B and attenuation of H2AK119ub1, resulting in the upregulation of Hox gene expression. We also showed that CUL4B interacted directly with RORγ and involved in the regulation of HOX genes via adjustment of the H2AK119ub1 occupancy level at HOX genes sites. In addition, upregulation of HOX genes was observed along with aberrant levels of CUL4B-mediated H2AK119ub1 in both mouse and human anencephaly NTD cases.
The epigenetic regulation of gene expression involves several interconnecting layers, such as histone modification. CUL4B can mediate the ubiquitination of histone H2AK119 and has transcriptional inhibitory activity. Deletion of CUL4B can lead to a lack of H2AK119ub and H3K27me3, thus enhancing cell differentiation . In this study, we showed that CUL4B knockdown results in alterations in HOX genes expression. Furthermore, the results revealed that binding of H2AK119ub1 was decreased in HOX genes upon RA treatment. Taken together with the previous finding that CUL4B is found in a complex with PRC, these results indicate that RA-induced differentiation results in the dissociation of a complex containing H2AK119ub1 E3 ligase activity that mediates transcriptional activation of the HOX promoters.
NTDs are a group of congenital malformations of the brain and/or spinal cord caused by failure of the morphogenesis associated with neural tube closure in early embryonic development [29, 30, 31, 32, 33, 34]. Research on the factors behind the pathogenesis of NTDs has focused on environmental and genetic factors [35, 36, 37, 38, 39, 40, 41, 42]. More than 200 candidate gene mutations potentially causative of NTDs in mice have been identified. However, very few of these candidates identified in mice have been confirmed to be involved in human NTDs [43, 44, 45, 46, 47, 48]. Hox genes play an important role in the differentiation of the nervous system, especially in the determination of the anteroposterior axis. In this study, we investigated the role of the abnormal expression of HOX genes in human NTD cases and mouse embryos with RA-induced NTDs. The abnormal upregulation of HOX genes was detected in both human NTD cases and mouse embryos with RA-induced NTDs (Figs. 5f, 6a). Interestingly, aberrant HOX gene expression was detected in anencephalic but not spina bifida fetuses. The occurrence of NTD phenotype involved in region-specific mechanisms and precisely gene expression, such as Hox genes, which are activated in a temporally collinear manner to drive the progressive specification of different segments. Furthermore, to analyze the expression of HOX genes, there is a need to further expand the sample size and increase the number of studied phenotypes to provide a theoretical basis for the development of useful clinical markers.
Some studies have addressed the possible role of altered histone modification in the development of NTDs. The histone deacetylases SIRT1 and HDAC4-mediated histone deacetylation have also been implicated in NTDs [49, 50]. Moreover, loss of function of the GCN5 and P300-mediated histone acetylation also has been reported to cause development defects [51, 52]. Mice with Cul4b gene knockout died at an early stage of embryonic development, which showed the importance of Cul4b [53, 54]. Environmental factors also play a role in NTDs, such as folic acid and RA. Our findings also indicated that the downregulation of CUL4B resulted in a loss of H2AK119ub1 both in RA-induced mouse anencephaly and in human anencephaly samples. This study suggests that CUL4B might be related to the mechanism behind the development of human NTDs under RA dysmetabolism.
RA participates in the regulation of many functions in mammals, such as cell differentiation and apoptosis [40, 55, 56, 57, 58, 59, 60]. The binding of RA to retinoic acid receptor results in covalent modification of the N-end and tail of nucleosome histone, resulting in the formation of an active transcription complex that regulates gene expression [61, 62, 63]. RORs participate in the regulation of various physiological processes, which include the pathway to maintain energy homeostasis and the regulation of biological clock-related components . Recent studies have demonstrated that RORs function as ligand-dependent transcription factors . Transcriptional regulation by RORs is mediated through interaction with corepressors and coactivators, including histone acetylases P300 and CBP . In this study, we found that CUL4B interacts with and negatively regulates the transcriptional activity of RORγ, suggesting that it functions as a novel corepressor of RORγ. RORγ is required for RA-induced activation of HOX gene expression in human NT2/D1 cells. ChIP analysis showed that the level of H2AK119ub1 associated with these HOX genes regulatory sites was considerably lower in cells in which RORγ was downregulated. These observations suggest that the dissociation of RORγ to regulatory regions may correlate with a less closed chromatin structure, consistent with the functional activities of RORγ.
In summary, our study showed that epigenetic modifications of H2AK119ub1 could cause abnormal Hox gene expression after exposure to RA, which may significantly contribute to development and etiology of NTDs. The present study provided novel insight into the dysregulation of CUL4B in NTDs, which was involved in the upregulation of Hox gene transcription. Therefore, study of the pathogenesis of NTDs can provide a new approach to uncovering the complexity of NTDs and for the early prevention of NTDs.
In this study, we show that the H2AK119ub1 E3 ligase CUL4B is required for the activation of retinoic acid (RA)-inducible developmentally critical homeobox (HOX) genes in NT2/D1 embryonal carcinoma cells. RA treatment led to the attenuation of H2AK119ub1 due to a decrease in CUL4B, further affecting HOX gene regulation. Furthermore, we found that CUL4B interacted directly with RORγ and knockdown of RORγ decreased the expression of HOX genes along with increased H2AK119ub1 occupancy levels, at HOX gene sites in N2/D1 cells. In addition, upregulation of HOX genes was observed along with lower levels of CUL4B-mediated H2AK119ub1 in both mouse and human anencephaly NTD cases. Notably, the expression of HOXA7, HOXA10, and HOXB7 genes was negatively correlated with CUL4B levels in human anencephaly NTD cases. Our results indicate that abnormal HOX gene expression induced by lower H2AK119ub1 levels may be a risk factor for NTDs. It also highlights the need for further analysis of genome-wide epigenetic modifications in NTDs.
C57BL/6 mice (44007200007011, 9–10 weeks, 18–23 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in SPF cage, approved facility on a 12-h light/dark cycle. Mature male and female C57BL/6 mice were mated overnight. The vaginal plug was detected at 8:00 am on the following morning, which designated as E0.5 if the presence of a vaginal plug. NTDs mouse embryos were induced by gavage with 28 mg/kg (body weight) of RA (Sigma, USA) on E7.5 (RA is dissolved in sesame oil). On E10.5, pregnant mice were euthanized by cervical dislocation and embryos were dissected from decidual tissue and placed in ice-cold, DEPC-treated PBS. All procedures involving animal handling were in compliance with institutional guidelines on the care of experimental animals.
Cell culture and RA treatment
SV/129 mouse embryonic stem cells (ESCs), maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA), were supplemented with 0.1 mM β-mercaptoethanol (Invitrogen, Carlsbad, USA), nonessential amino acids (Invitrogen, Carlsbad, USA), 2 mM glutamate (Invitrogen, Carlsbad, USA), 15% fetal bovine serum (Gibco, USA), and 1000 U/ml leukemia inhibitory factor (Millipore, Billerica, USA), cultured in the culture dishes coated with 0.2% gelatin (Invitrogen, Carlsbad, USA). Cells were placed in atmosphere with 37 °C, 5% CO2 and passaged every 2 days. NT2 cells were developed in Dulbecco’s modified Eagle medium with 15% FBS. ESCs were treated with 1 μM RA for 24 h. Cells were incubated at 37 °C/5% CO2 and passaged every 2 days. ESCs were treated with 1 μM RA for 24 h.
Transfection was performed using Lipofectamine 2000 (Invitrogen). Cells were harvested 24–48 h after transfection and lysed in Beyotime lysis buffer (0.5% NP-40, 50 mM Tris, pH 7.6, 120 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, and 1 mM β-mercaptoethanol) supplemented with protease inhibitor cocktail (Roche). For immunoprecipitation, 800 μg lysates were incubated with the CUL4B antibody 2 μg for 3–4 h at 4 °C followed by 1-h incubation with Protein A/G Sepharose beads (Thermo Fisher Scientific). The resulting immunoprecipitates were washed three times in HEPES lysis buffer (20 mM HEPES pH 7.2, 50 mM NaCl, 0.5% Triton X-100, 1 mM NaF, 1 mM dithiothreitol) before being resolved by SDS-PAGE and immunoblotted with indicated antibodies.
CUL4B-siRNA interference (5′–3′): GCAGCAGGUGGAUCGAAUAUTTAUAUUCGAUCCACUGCUGCTT
RORγ-siRNA interference (5′–3′): CCCGAGAUGCUGUCAAGUUTT.
The blots were incubated with the primary antibody, mouse anti-H2AK119ub monoclonal antibody (1:1000, CST, USA) and mouse anti-H3 monoclonal antibody (1:1,500,000, CST, USA) overnight at 4 °C, and then incubated with secondary anti-mouse HRP-conjugated antibody (1:5000, Santa, USA) for 1 h at room temperature. The blots were developed with SuperSignal West Pico Chemiluminescence Substrate (Thermo, USA) and quantitated on densitometer (Bio-Rad, Universal Hood II, USA) using Quantity One software.
Chromatin immunoprecipitation (ChIP) analysis
ChIP assays were performed using the SimpleChIP enzymatic chromatin IP system (Cell Signaling, California, USA) following the manufacturer’s protocols. Chromatin was prepared, sonicated to DNA segments between 200 and 1000 bp, and then immunoprecipitated with anti-H2AK119ub (CST, USA). The immunoprecipitated DNA was analyzed by qPCR, which was performed using QuantStudio 7 Flex with SYBR Green detection. The primers used for ChIP assays are shown in Additional file 5: Table S4. Mouse IgG antibodies were used as negative controls in the immunoprecipitations. The following equation was used to calculate percent input = 2% × 2^ (CT) 2% input sample − (CT) IP sample.
Total RNA was extracted using the Trizol method (Ambion, USA); first-strand synthesis was done with RevertAid First-Strand cDNA Synthesis Kit (Thermo, USA). Maxima SYBR Green/ROX qPCR Master Mix (Abm, Canada) was used for qPCR, and the procedure was as follows: (50 °C, 2 min) × 1 cycle; (95 °C, 10 min) × 1 cycle; (95 °C, 15 s; 60 °C, 30 s; 72 °C, 30 s) × 40 cycles; collect fluorescence at 72 °C. Primer sequences are shown in Additional file 6: Table S5.
Luciferase reporter assay
RORE luciferase reporter assay was carried out as described previously . Human NT2 cells were co-transfected as indicated with pCMV-Flag, pCMV-Flag-CUL4B, siRNA-CUL4B, and a pGL4.27-(RORE)5 reporter plasmid containing 5-RORE, using Lipofectamine 2000 (Invitrogen). After 24-h incubation, the luciferase activities were measured by a dual-luciferase assay system (Promega) and luciferase detection kit (YuanPingHao). All transfections were performed in triplicate and repeated at least twice.
Extraction of nucleoprotein
Core histone proteins of cells were extracted using acid extraction. Briefly, cells were first homogenized in lysis buffer (10 ml solution containing 10 mM Tris–HCl with pH 8.0, 1 mM KCl, 1.5 mM MgCl2, and 1 mM dithiothreitol (DTT)) and chilled on ice. 5% of protease inhibitors were added immediately before lysis of cells and chilled on ice for 30 min, and nuclei were isolated by centrifugation (1500g for 5 min). For the preparation of histones, nuclei were incubated with four volumes of 0.2 M sulfuric acid (H2SO4) for overnight at 4 °C. The supernatant was precipitated with 33% trichloroacetic acid (final concentration) and followed by centrifugation (12,000g for 5 min at 4 °C). The obtained pellet was washed with cold acetone and subsequently dissolved in distilled water. Nucleprotein extraction was extracted from mouse and human brain samples using kit (Sangon Biotech) according to the manufacturer’s protocols.
The digested peptides were separated using a Thermo Scientific EASY-nLC 1000 System. Peptide mixtures were loaded onto a self-made C18 trap column (Acclaim Pepmap100 column, 2 cm × 100 μm, C18, 5 μm) in solution A (0.1% formic acid) and then separated with a self-made capillary column (EASY-Spray column, 12 cm × 75 μm, C18, 3 μm) with gradient solution B (100% acetonitrile and 0.1% formic acid) at a flow rate of 350 nL/min. The separated peptides were examined in an Orbitrap Fusion mass spectrometer (Thermo Scientific). The spray voltage of the ion source was set to 2.1 kV. Full-scan mass spectra were acquired in the MS over 35–1800 m/z with a resolution of 70,000. The HCD spectra resolution was 17,5000. The normalization collision energy was set to 29%.
The mouse brain tissue was soaked in 4% paraformaldehyde to make the tissue fully infiltrated. Forty-eight hours after washing with PBS, ethanol is added to dehydrate, paraffin-embedded, and sliced. After washing, it was dissolved in ethanol and then placed in double evaporated water for 10 min. After rinsing the slices, the tissue antigen was repaired. We performed immunohistochemical staining for CUL4B and H2AK119ub on the same paraffin-embedded tissue blocks that were used for clinical diagnosis. Immunohistochemistry was performed using the avidin–biotin complex (ABC) method (Vector Laboratories), including heat-induced antigen-retrieval procedures. Incubation with polyclonal antibodies against CUL4B (dilution 1:100; OriGene) and H2AK119ub (dilution 1:100; CST) was performed at 4 °C for 18 h. Quality assessment was performed on each batch of slides by including a negative control in which the primary antibody was replaced by 5% BSA to preclude nonspecific signals. Pathologists who were blinded to the sample origins and the patient outcomes assessed staining. The final immunoreactivity score was determined by the Bioinformatics analysis software.
For the detection of subcellular localization by immunofluorescence, after fixed with 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 (PBS), cells were incubated with the indicated CUL4B and H2AK119ub antibodies (dilution 1:50; CST) for 8 h at 4 °C, followed by incubation with TRITC-conjugated or FITC-conjugated secondary antibody (dilution 1:200; Zsbio Commerce Store) for 1 h at 25 °C. The nuclei were stained with DAPI (Sigma), and images were visualized with a Zeiss LSM 510 Meta inverted confocal microscope.
All clinical samples were from the Lvliang area of Shanxi Province in northern China with informed consent from the patients or their families. The enrolled pregnant women were diagnosed by trained local clinicians using ultrasonography and then registered (Additional file 7: Table S6). The surgical procedures were performed as previously described . The epidemiological method was described in detail in our previous publication .
The NanoString nCounter was used to detect the number of transcripts in human brain tissues. Total RNA was extracted following the manufacturer’s instructions (miRNeasy Mini Kit, Qiagen), and gene-specific probes were designed by the manufacturer (NanoString Technologies). Hybridizations were carried out according to the nCounter Element 24-plex Assay Manual. Approximately 100 ng of each RNA sample was mixed with 20 μl of nCounter Reporter probes in hybridization buffer and 5 μl of nCounter Capture probes for a total reaction volume of 30 μl. The hybridizations were incubated at 65 °C for approximately 16 h, then eluted, and immobilized in the cartridge for data collection, which was performed on the nCounter Digital Analyzer. Gene expression data were filtered using quality control (QC) criteria according to the manufacturer’s recommendations. Raw counts of QC-passed samples were normalized using three reference genes as internal controls (GAPDH, CLTC, and GUSB). All QC and normalization procedures were performed using nSolver Analysis Software v2.0; all data were log2-transformed before further analysis. The Student’s t test was used to compare normalized expression values between normal and NTDs.
The experimental data were analyzed by SPSS 22 statistical software. Three independent experimental data are collected. First, the normality of the experimental data is analyzed. In the case of normal distribution of the data, the statistical description is carried out by the mean ± SD, and the independent sample t test is used for statistical analysis. If the experimental data do not conform to normal distribution, one-way ANOVA is used. When P < 0.05, it was statistically significant.
TZ conceived the project and performed the project planning. YL performed the main experimental work. JY participated in mouse culture. YL and SW designed the experiments, analyzed the data, and wrote the manuscript. JY participated in data discussion. All authors read and approved the final manuscript.
We thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
The authors declare no competing interests related to this work.
Availability of data and materials
All the data generated or analyzed during this study are included in this article and its supplementary information files.
Consent for publication
Ethics approval and consent to participate
We are grateful to all participating hospitals for their assistance in sample collection and recording of clinical information. This work was supported by the National Natural Science Foundation Projects (81771584), CAMS initiative for Innovative Medicine (CAMS-12M-1-008), National Natural Science Foundation Projects (31571324), and Beijing Municipal Program of Medical Research (Grant No. 2016-04).
Details of other procedures are provided in Additional files.
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