Effects of DTX3L on the cell proliferation,adhesion,and drug resistance of multiple myeloma cells

Abstract

Cell adhesion–mediated drug resistance is an important factor that influences the effects of chemotherapy in multiple myeloma. DTX3L, a ubiquitin ligase, plays a key role in cell-cycle-related process. Here, we found that the expression of DTX3L gradually increased during the proliferation of myeloma cells, which resulted in arrest of the cell cycle in the G1 phase and promoted the adherence of myeloma cells to fibronectin or bone marrow stromal cells. In addition, silencing of DTX3L improved sensitivity to chemotherapy drugs in multiple myeloma cell lines adherent to bone marrow stromal cells and increased the expression of caspase-3 and poly-adenosine diphosphate-ribose polymerase, two markers of apoptosis. Finally, we also found that DTX3L expression was regulated by focal adhesion kinase. Taken together, the results of this study show that DTX3L plays an important role in the proliferation and cell adhesion–mediated drug resistance of multiple myeloma cells and as such may play a key role in the development of multiple myeloma.

Keywords

DTX3L proliferation adhesion drug resistance multiple myeloma

Introduction

Multiple myeloma (MM), which is characterized by the clonal proliferation of malignant plasma cells,¹ is an incurable malignant hematologic malignancy. Patients with this disease have increased risk of infection, anemia, thrombocytopenia, and bone disease.² Although some targeted drugs can prolong the survival of patients with MM, the patient eventually relapses and often shows multiple drug resistance.³ Metastasis in this disease is facilitated by interactions between tumor cells and adhesion molecules on the surface of MM and bone marrow (BM) cells in the extracellular matrix (ECM) of the BM microenvironment. This interaction leads to the emergence of anti-apoptotic and cell-cycle arrest signals, which can result in drug resistance,^4–6 a phenomenon known as “cell adhesion–mediated drug resistance” (CAM-DR). Understanding the possible molecular mechanisms of this resistance may improve the treatment effects in patients with MM.

DTX3L, a member of the DTX family, functions as an E3 ubiquitin ligase through auto-phosphorylation.^7,8 DTX3L and its ligand BAL are highly expressed in lymphoma cells and are resistant to proteasome inhibitors. DTX3L promotes 53BP1 accumulation and histone H4K20 methylation through the ubiquitination of histone H4,⁹ leading to cell-cycle arrest and repair of DNA damage. Therefore, the high expression of DTX3L in lymphoma, which is associated with chemotherapy drugs, blocks the resistance and cell cycle. Abnormal DTX3L expression is mediated by the focal adhesion kinase (FAK) signaling pathway in melanoma.^10,11 DTX3L is closely related to the growth and adhesion of tumor cells. For example, it inhibits the growth of tumor cells by regulating the signal transducer and activator of transcription 1 (STAT1), STAT3, interferon regulatory factor-1, and poly-adenosine diphosphate-ribose polymerase 9 (PARP-9) in prostate cancer.⁸ In addition, in endometrial carcinoma, the role of DTX3L is related to the adhesion and migration of tumor cells.¹² However, the effects of DTX3L on the proliferation and CAM-DR of MM cells remain unclear.

In this study, we determined the effects of DTX3L on the proliferation, cell-cycle control, and CAM-DR of MM cells. We found that DTX3L promoted MM cell proliferation, and its high expression resulted in cell-cycle arrest and enhanced the adhesion of MM cells. In addition, activation of the FAK signaling pathway in MM cells adherent to BMSCs led to high DTX3L expression. The results of this study may provide a better understanding of the role of DTX3L in the CAM-DR of MM cells.

Materials and methods

Cell lines and culture

The human MM cell lines RPMI 8226 and NCI-H929 and the BM stromal cell line HS-5 were obtained from the Cell Library, Chinese Academy of Sciences (Shanghai, China). RPMI 8226 and NCI-H929 cells were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA), and HS-5 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum (Gibco BRL) at 37°C in a 5% CO₂ incubator. To study apoptosis, cells were seeded in a 60-mm dish and incubated in a low concentration of serum (1% fetal bovine serum) for 24 h prior to treatment with different concentrations of bortezomib.

Cell co-culture and detection of adhesion rate

We used adhesion assays to test the specificity of adhesion of RPMI 8226 and NCI-H929 cells to soluble fibronectin (FN) or HS-5 cells. The cells were incubated overnight at 37°C with or without 40 µg/mL FN (Sigma-Aldrich, Rehovot, Israel) in a final volume of 1 mL phosphate-buffered saline (PBS) or HS-5 cells. Then, MM cells (100,000 cells/mL) were adhered to pre-established monolayers of FN or HS-5 for 2–4 h. Finally, adherent cells were carefully removed for subsequent experiments, with the HS-5 monolayer kept intact. The cell adhesion rate was detected by staining MM cells with Calcein-AM (Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min according to the manufacturer’s instructions. Then, the cells were incubated in 96-well plates with pre-established monolayers of HS-5 or an FN-coated surface in RPMI 1640 medium. After 2 h of co-culture, the non-adherent cells were washed off twice in PBS, and the number of adherent cells was monitored with a fluorometer (CytoFluor; Applied Biosystems, Foster City, CA, USA) at 490 nm.

Western blot analysis

Western blot experiments were conducted as previously described.¹³ Briefly, the proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA) after being resolved by sodium dodecyl sulfate–polyacrylamide gels electrophoresis. After blocking in 0.1% Tween 20/PBS with 5% (w/v) nonfat milk, the membranes were incubated with primary antibody overnight at 4°C. Then, they were washed three times, 5 min each, with PBS containing 0.1% Tween 20, each for 5 min, incubated with horseradish peroxidase–conjugated secondary antibody for 2 h at room temperature. The membranes were developed using the enhanced chemiluminescence (ECL) detection system. The band density was quantified by the ImageJ analysis system (Wayne Rasband; National Institutes of Health, USA). The primary antibodies included the following: anti-DTX3L antibody (1:500), anti-cyclin E antibody (1:500), anti–proliferating cell nuclear antigen (PCNA) antibody (1:500), anti–cyclin-dependent kinase 2 (CDK2) antibody (1:500), anti-FAK antibody (1:1000), anti-caspase3 antibody (cleaved1:1000), anti-AKT antibody (1:1000), anti-p-AKT antibody (1:1000), anti-PARP antibody (cleaved1:1000), anti-β-actin antibody (1:1000), anti–extracellular signal-regulated kinase (ERK)1/2 (1:1000), and anti-P-ERK1/2 (1:1000). All antibodies used were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), except anti-caspase3 antibody, anti-PARP antibody, anti-AKT antibody, anti-p-AKT antibody, anti-ERK1/2 antibody, and anti-P-ERK1/2 antibody (Cell Signaling Technology, Danvers, MA, USA). The experiments were carried out on three separate occasions.

Preparation of shRNA and transient transfection

DTX3L shRNA was designed and synthesized by Genechem (Shanghai, China). The short hairpin RNA (shRNA) oligos targeting on DTX3L gene were designed and synthesized by Genechem. The sequences are as follows: shDTX3L-1: 5′-GATGGACATTGATAGCGAT-3′; shDTX3L-2: 5′-CGTATTAGGAGTCTCAGAT-3′; shDTX3L-3: 5′-TGATTTAATGCCAGTCTAA-3′; and shDTX3L-4: 5′-CAATTACATGATGAATGTA-3′. The negative control (NC) shRNA was also designed and provided by Genechem. According to the manufacturer’s instructions, transfections were performed by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Cells were cultured at 37°C in RPMI 1640 medium without serum for 6 h, and then, the medium was replaced with RPMI 1640 containing 10% fetal bovine serum. Transfected cells were harvested 48 h after transfection for following experiments.

Cell-cycle analysis

The cell-cycle distribution flow instrument analysis. First, the cold cell was fixed in 70% ethanol at −20°C, collected after at least 24 h, and then the cells were washed twice with PBS with 1 mg/mL RNase A for 20 min at 37°C and incubated. Second, these cells were stained with propidium iodide (PI; 50 µg/mL; Becton–Dickinson, San Jose, CA, USA) in PBS and 1% Triton-X 100 for an additional 20 min at 4°C in dark. These data were analyzed by Becton–Dickinson flow cytometer BD FACScan.

Cell viability assay

First, the MM cells were inoculated into a 96-well plate (Corning Inc., Corning, NY, USA) at the density of 10⁵ cells/well in a volume of 90 µL and grew 48 h with or without chemotherapy drugs. Then, the 10 µL Cell Counting Kit-8 (CCK-8) reagents (Dojindo, Kumamoto, Japan) were added into every well and incubated for an additional 1 h at 37°C in dark. Finally, the absorbance was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at a test wavelength of 450 nm and a reference wavelength of 630 nm.

Flow cytometry analysis of cell apoptosis

The drug-induced apoptosis was tested following exposure to bortezomib and mitoxantrone (Sigma–Aldrich, St. Louis, MO, USA) in MM cells. Flow cytometry analysis was conducted as previously described.¹⁴ After the manufacturer’s protocol following 48-h exposure to chemotherapy agents, the apoptotic cells were detected with Annexin-V-FLUOS Staining Kit (Roche). The cells were washed three times by cold PBS and resuspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/mL. Then, 5 µL of 7-aminoactinomycin D (7-ADD) solution and 5 µL of Annexin V were added to each tube. Another 400 µL 1× binding buffer was added to each tube after incubating for 15 min at room temperature in the dark. The apoptosis assay was performed with Becton–Dickinson flow cytometer BD FACScan (San Jose, CA, USA) according to the manufacturer’s instructions.

Statistical analysis

Each experiment was repeated at least three times. All values are reported as mean ± standard error of mean (SEM). The calculations were analyzed with the Statistical Package for the Social Science (SPSS) 19.0 software (SPSS Inc. (an IBM Company), Chicago, IL, USA). The probability of 0.05 or less was considered statistically significant.

Results

DTX3L was highly expressed in proliferating MM cells

DTX3L, a ubiquitin ligase, plays a key role in the cell-cycle-related processes and cell-cycle regulation. Thus, we determined whether it participates in regulation of the cell cycle and proliferation of myeloma cells. We found that the expression of DTX3L gradually increased during the proliferation of MM cells. RPMI 8226 cells were cultured in serum-free medium for 72 h, which led to cell-cycle arrest in the G1 phase. The cells were released from G1 phase and re-entered the S phase after serum re-feeding (Figure 1(c) and (d)). Western blot analysis showed that DTX3L expression increased after serum stimulation, as did the percentage of cells in the S phase, consistent with the upregulation of cyclin E, cyclin-dependent kinase 2 (CDK2), and proliferating cell nuclear antigen (PCNA; Figure 1(a) and (b)). These results suggest that DTX3L plays an important role in promoting the proliferation of MM cells.

Figure 1.

Expression of DTX3L in proliferating MM cells. (a) Western blot analysis of DTX3L, PCNA, cyclin E, and CDK2 expression in serum-starved and serum-refed RPMI 8226 cells. (b) The bar chart shows the ratio of DTX3L/PCNA/cyclin E/CDK2 to β-actin by densitometry at different times. Data are presented as the mean ± SEM of three independent measurements (*p < 0.05, ^#p < 0.05, ^{^}p < 0.05, compared with S72 h). (c) and (d) Flow cytometry was used to detect the cell-cycle distribution of RPMI 8226 cells, which were serum starved (S) for 72 h and then refed (R) with RPMI 1640 medium containing 10% fetal bovine serum for 0, 6, 12, 24, and 48 h. Data are presented as the mean ± SD of three independent experiments (*p < 0.05 and ^#p < 0.05 compared with S72h).

Knockdown of DTX3L inhibited the proliferation of MM cells

In order to perform further research on the influence of DTX3L on cell proliferation of MM cells, NCI-H929 cells and RPMI 8226 cells were transfected with DTX3L shRNA and their respective control. Western blot analysis confirmed the transfection efficiency. As shown in Figure 2(a) and (b), the expression of DTX3L is maximally decreased in shDTX3L-1 in transfected RPMI 8226 cells and NCI-H929 cells. So, shDTX3L-1 is selected in the following experiments. The CCK-8 test showed that proliferation of RPMI 8226 cells and NCI-H929 cells was significantly suppressed after the DTX3L knockdown, and it was consistent with the remarkably downregulated expression of PCNA (Figure 2(c) and (d)). To explore whether DTX3L is associated with the regulation of cell cycle of MM cells, we next assessed the effect of DTX3L shRNA and control shRNA on cell-cycle distribution of RPMI 8226 cells and NCI-H929 cells with the FACS. It suggests that the cells silencing DTX3L expression were arrested in G1 phase (Figure 2(e)), and the expression of CDK2 and cyclin E is also reduced (Figure 2(d)). This also proves that the cells were suppressed in the G1–S transition. Together, these data proved that DTX3L may promote the proliferation of MM cells through regulating the cell cycle.

Figure 2.

Knockdown of DTX3L inhibited the proliferation of MM cells. (a) RPMI 8226 cells and (b) NCI-H929 cells were transfected with DTX3L shRNA or control shRNA. The interference efficiencies of DTX3L shRNA were measured by western blot analysis. β-actin was used as a control for protein loading and integrity. The bar chart demonstrates the ratio of DTX3L protein to β-actin after transfection with shNC or DTX3L shRNA. Data are presented as mean ± SEM of three independent measurements. (c) Cell growth was tested with the CCK-8 assay (*p < 0.05 and ^#p < 0.05 compared with shNC). (d) RPMI 8226 and NCI-H929 cells transfected with shDTX3L-1 or shNC were lysed, followed by western blot analysis of DTX3L, PCNA, cyclin E, and CDK2 expression in two conditions. The bar chart demonstrates the ratio of DTX3L/PCNA/cyclin E/CDK2 to β-actin (*p < 0.05 and ^#p < 0.05 compared with shNC). (e) RPMI 8226 cells and NCI-H929 cells were transfected with shDTX3L-1 or shNC. Then, flow cytometry was used to detect the cell-cycle distribution of the cells. Data are presented as mean ± SD of three independent experiments (*p < 0.05 and ^#p < 0.05 compared with shNC).

The expression of DTX3L was related to cell adhesion of MM cells

In order to determine whether DTX3L participated in CAM-DR MM cells, cell adhesion assay was performed to investigate the expression of DTX3L. Western blot analysis showed that compared with the expression of DTX3L in suspension both in RPMI 8226 and NCI-H929 cells, the expression of DTX3L is significantly increased in adherent cells (Figure 3(a) and (b)). So, we explored whether the suppression of DTX3L is influential on myeloma cell adhesion. Cell adhesion tests showed that knockdown of DTX3L led to a significant reduction in cell adhesion rate (Figure 3(c) and (d)). In conclusion, DTX3L may participate in the regulation of cell adhesion.

Figure 3.

The expression of DTX3L was related to cell adhesion in MM cells. (a) RPMI 8226 cells and (b) NCI-H929 cells were adhered to fibronectin (FN) or HS-5 cells or in suspension during western blot analysis of the expression of DTX3L in these conditions. The bar chart demonstrates the ratio of DTX3L protein to β-actin by densitometry (*p < 0.05 and ^#p < 0.05 compared with the suspension cells). The cell adhesion rate of (c) RPMI 8226 and (d) NCI-H929 cells transfected with shDTX3L-1 or shRNA was detected as described above. The mean ± SEM of three independent experiments are shown (*p < 0.05 and ^#p < 0.05 compared with shRNC). MM cells cultured in suspension; FN and MM cells adhered to fibronectin; and HS-5 and MM cells adhered to HS-5 cells.

Knockdown of DTX3L reduced CAM-DR

An increasing amount of evidence showed that CAM-DR is one of the main obstacles of myeloma treatment, which can prevent tumor cells from apoptosis induced by chemotherapy drugs.^15,16 In order to verify this conclusion, we deal with cells in suspension or adherent to FN or HS-5 by chemotherapy drugs and then use CCK-8 to detect cell viability. As previously reported, compared with the adhesion of the MM cell, the suspension of MM cells is more sensitive to chemotherapy drugs (Figure 4(d) and (e)). However, we still do not know the role of DTX3L in this process. To study the role of DTX3L in CAM-DR, we treated MM cells with chemotherapy drugs after the expression of DTX3L was interfered by DTX3L shRNA and adhered to FN or HS-5 for 24 h. It showed that the cells whose DTX3L expression was interfered are more sensitive to chemotherapy drugs (Figure 4(f) and (g)). Flow cytometry analysis also showed that when the expression of DTX3L was decreased, adherent cells become more sensitive to drug-induced apoptosis (Figure 4(b) and (c)). To verify the role of DTX3L in CAM-DR, we use the western blot analysis to detect the expression of apoptosis-related proteins. Consistent with previous studies, the expression of cleaved caspase 3 and cleaved PARP, two kinds of apoptotic marker proteins, was significantly increased in DTX3L-depleted cells in the case of chemotherapy treatment (Figure 4(a)). On the whole, these data confirm that DTX3L has an important role in the CAM-DR of the MM cells.

Figure 4.

Knockdown of DTX3L reduced cell adhesion–mediated drug resistance. (a) Western blot analysis of cleaved caspase-3 and cleaved PARP expression in the above condition. The bar chart demonstrated the ratio of DTX3L/caspase3/PARP to β-actin (*p < 0.05 and ^#p < 0.05 compared with shRNC). Sus and MM cells cultured in suspension; FN and MM cells adhered to fibronectin; and HS-5 and MM cells adhered to HS-5 cells. (b) RPMI 8226 cells and (c) NCI-H929 cells were adhered to FN and then transfected with shDTX3L-1 or shRNC. Flow cytometry assay was used to detect the number of apoptotic cells after chemotherapy drug stimulation. The bar chart demonstrates the number of Annexin V+ cells after drugs treatment (*p < 0.05 and ^#p < 0.05 compared with the shNC group). (d) RPMI 8226 and (e) NCI-H929 cells were adhered to FN or HS-5 for 24 h. Then, the cells and suspension cells were each treated with 10 µM bortezomib (Bort) or 2 µM mitoxantrone (Mito) or no chemotherapy drugs and cultured in 1640 medium without fetal bovine serum for 48 h. Then, CCK-8 reagents were added to the different wells and incubated for an additional 1 h in dark at 37°C. Cell viability was analyzed by fluorometer. Data were from three independent experiments (*,^#p < 0.05 compared with the suspension cells). (f) RPMI 8226 and (g) NCI-H929 cells were first transfected with shDTX3L-1 or shNC, and then, the cells were adhered to FN or HS-5 for 24 h. After treated with the two chemotherapy drugs, cell viability was analyzed as described above.

DTX3L is regulated by the FAK signal pathway in the MM cells

Previous research suggested that DTX3L was regulated by the FAK signal pathway in melanoma cells.¹⁷ The ERK and AKT signaling pathways are implicated in proliferation and survival of MM cells.^13,18,19 So, we suspected that DTX3L affected the CAM-DR by regulating the activity of ERK signaling pathway or FAK signaling pathway. In order to solve the above-mentioned problem, we compared the expression of FAK and ERK in suspension cells with the expression of adherent cell and found that FAK, AKT, and ERK signaling pathways were increased significantly in adhesion cells (Figure 5(a) and (b)). It showed that the DTX3L was regulated by the FAK signaling pathways in MM cells. In order to further explore the role of DTX3L in CAM-DR, we transfected shDTX3L-1 in RPMI 8226 cells and NCI-H929 cells, which adhered to FN with the treatment of the bortezomib. Transfection efficiency was confirmed by western blot analysis (Figure 5(c)). It shows that the FAK and p-ERK signal did not change significantly, but the p-AKT was reduced after interfering with the DTX3L. These data suggest that DTX3L is regulated by the FAK signaling pathways and plays a role through the AKT in MM cells.

Figure 5.

DTX3L is regulated by the FAK signal pathway in the MM cells. (a) RPMI 8226 and (b) NCI-H929 cells were adhered to FN or HS-5 24 h later. Western blot analysis was performed to detect the expression of FAK, DTX3L, AKT, p-AKT, ERK1/2, and p-ERK1/2. Bar chart demonstrated the ratio of FAK, DTX3L, AKT, p-AKT, ERK1/2, and p-ERK1/2 to β-actin (*p < 0.05 and ^#p < 0.05 compared with the suspension cells). (c) RPMI 8226 and NCI-H929 cells were transfected with shDTX3L-1 or shNC, adhered to FN, and treated with the Bort. Western blot analysis was performed to detect the expression of FAK, DTX3L, p-AKT, and P-ERK1/2. The bar chart demonstrates the ratio of FAK, DTX3L, and p-AKT to β-actin by densitometry (*p < 0.05 and ^#p < 0.05 compared with the shNC).

Discussion

MM is characterized by clonal proliferation of malignant plasma cells and production of large amounts of monoclonal immunoglobulin in the whole BM. The chemotherapy treatment was obvious in the initial stage of myeloma, but the chemotherapy drug resistance often occurs in the later stages. The interaction between MM cells and BM stromal microenvironment (BMSM) is an important factor which leads to drug resistance. It has been shown that the adhesion of MM cells can prevent the apoptosis induced by chemotherapy drugs. This phenomenon is called the CAM-DR.^14,15 In order to solve this problem, we need to have a further knowledge of the molecular mechanism of CAM-DR.

According to the report, DTX3L can function as E3 by phosphorylation of its own, closely related to the growth and adhesion of other tumor cells, such as prostate cancer, endometrial cancer and lymphoma.^8,9,12 But the role of DTX3L in the proliferation and CAM-DR of MM cells is still not clear. In this study, we found that DTX3L could promote the proliferation of myeloma cells by CCK-8 experiments. The proliferation of cells usually is related to cell cycle.^20–22 Therefore, we further study that the function of DTX3L promoting the proliferation of myeloma cells was associated with the cell cycle. Flow cytometry instrument analysis showed that the cells were accumulated in G1 phase is consistent with the expression of cell cycle E and CDK2 after silencing DTX3L. Our results also showed that the expression of DTX3L was significantly increased after RPMI 8226 and NCI-H929 cells had an adhesion of FN or HS-5 cells. The expression of cell adhesion test showed that the cell adhesion rate was significantly reduced after interfering with DTX3L. Then, we check the role of DTX3L in CAM-DR. It suggests that the MM cells are more sensitive to chemotherapy drugs after silencing DTX3L. In addition, we also proved that DTX3L was regulated by the FAK signal pathways in MM. In short, these data suggest that DTX3L plays a important role in mediating the CAM-DR of MM cells by regulating the cell cycle and cell adhesion, which was regulated by the FAK signaling pathways.

The specific mechanism of DTX3L promoting proliferation of MM cells by regulating the cell cycle is still unknown. The high expression of DTX3L is related to the chemotherapy drug resistance and the blocking of the cell cycle in lymphoma. DTX3L causes the cell-cycle arrest through the promotion of histone H4 of ubiquitin 53 BP1 with methylation of histone H4K20.⁹ This may explain the phenomenon that cells were blocked in G1 phase after interfering with DTX3L. However, the specific mechanisms need to be further discussed.

In conclusion, our study shows that DTX3L plays an important role in the proliferation and CAM-DR of MM cells. Interfering with DTX3L could inhibit the proliferation of cells, reduce the rate of cell adhesion, and reduce CAM-DR of MM cells. In short, our research contributes to a better understanding of the role of DTX3L in CAM-DR of MM cells.

Footnotes

Acknowledgements

The authors thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. The authors thank New Cell & Molecular Biotech Co, Ltd () for its products (Antibody Diluent, Fast PAGE Plus) are a great help to this experiment. H.L. and Y.S. designed and performed the experiments. Y.S. participated in the statistical analysis. L.Z. participated in culturing the cells and performed the flow cytometry experiments. Ya.S. and Yu.S. contributed equally to this work.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical approval

This article does not contain any studies with human participants or animals.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the National Natural Science Foundation of China Grants (No. 81070400), Medical Innovation Team and the Leading Talent Project of Jiangsu (No. LJ201136), and Social Science and Technology Innovation and Demonstration in Nantong-Clinical Medical Science and Technology (No. HS2013067).

Informed consent

Informed consent was obtained from all of the participants of the study.

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