Tre2 (USP6NL) promotes colorectal cancer cell proliferation via Wnt/β-catenin pathway
Most colorectal cancer (CRC) patients are diagnosed at an advanced or metastatic stage with poor prognosis. Ubiquitin-specific protease 6 N-terminal-like protein (USP6NL) with high expression in CRC tissues regulates CRC cell proliferation via Wnt/β-catenin pathway. We hypothesized that USP6NL impacts CRC growth and inhibition of USP6NL may be a novel treatment strategy to improve CRC therapy.
USP6NL level in human CRC tissues and its association with tumor growth and metastasis were examined. Its roles and potential mechanisms in regulating tumor growth were studied by genetic and pharmacological manipulation of CRC cells in vitro and in vivo.
Herein, we found that USP6NL was up-regulated in tumorous tissues of CRC patients. Our data suggested that knockdown of USP6NL in human CRC cell lines (HCT116 and LOVO cells) inhibited cell proliferation, induced G0/G1 cell cycle arrest, and prevented the tumorigenicity of HCT116 cells in nude mice, and which was associated with the prevention of Wnt/β-catenin pathway. On the contrary, USP6NL overexpression in human CRC cells (SW480) showed the opposite result. Our data suggested that the promoted cell proliferation, G1/S cell cycle progression, and the enhanced expression of β-catenin Cyclin D1 and C-myc while reduced P27 induced by the overexpression of USP6NL were significantly reversed by additional treatment of XAV939, indicating that activating Wnt/β-catenin pathway was the mechanism, by which USP6NL exerted carcinogenesis in CRC in vitro. Besides, our data suggested that knockdown of USP6NL increased the ubiquitination of β-catenin, indicating that USP6NL may serve as a deubiquitinase that regulated β-catenin accumulation in this process. Furthermore, 10058-F4 down-regulated USP6NL, inhibited CRC cell proliferation and induced cell cycle arrest. The result demonstrated a possible feedback loop between USP6NL, β-catenin and C-myc in regulating CRC cell growth.
USP6NL was an oncogene in CRC, and it may be a potential target for the treatment of CRC.
KeywordsUSP6NL Colorectal cancers Wnt/β-catenin pathway
GTPase-activating proteins (GAPs) family proteins can bind to activated G proteins and motivate their GTPase activity, impairing the binding with downstream signaling targets . GAP activity is essential for its oncogenic function . Ubiquitin-specific protease 6 N-terminal-like protein (USP6NL), or RN-tre, is a GTPase-activating protein for Rabs, thereby is involved in control of cell migration, endocytosis of PM receptors and retrograde transport of Golgi [3, 4]. As a member of RabGAPs, USP6NL harbors an TBC (Tre-2/Bub2/Cdc16) domain , which exerts oncogenic activity in many cancers . USP6NL is up-regulated and predicts a poor prognosis in human breast cancer (BC) . However, the roles of USP6NL in human colon carcinoma (CRC) remained largely unknown.
Wnt/β-catenin signaling is essential in carcinogenesis, embryonic development and tissue homeostasis . Activating Wnt signaling cascade contributes to the etiology of CRC during intestinal epithelium homeostasis and differentiation . These primarily consist of recruitment of the transcription factor β-catenin in the nucleus and the stimulation many downstream oncogenes, for example c-Myc and Cyclin D-1 . Increased expression of β-catenin has been consistently found in tumorous colon tissue, and predicts poor survival of CRC patients . However, the mechanisms of the upregulation of β-catenin in CRC need to be fully understood.
Ubiquitin-specific proteases (USPs), acting as deubiquitinating enzymes (DUBs), is emerging as master regulator of Wnt/β-catenin signaling . We found that USPs members such as USP4, USP7 and USP47 have been identified as a β-catenin-specific deubiquitinylating enzyme, and involved in the increased stability of β-catenin protein [10, 11, 12]. USPs member USP6 positively regulates WNT/β-catenin pathway and facilitates β-catenin nuclear accumulation . Homologous to USP6, USP6NL contains the domain that encodes a deubiquitinating enzyme . However, the mechanism of USP6NL acted on Wnt/β-catenin signaling pathway in CRC remained unclear.
Here, we found that USP6NL was up-regulated and functioned as an oncogene in human CRC cells. USP6NL favored β-catenin accumulation in CRC cells and regulated its downstream oncogenes (P27, Cyclin D1 and C-myc). As a result, the Wnt/β-catenin signal pathway is activated and promoted pro-proliferation and normal cell cycle progression of CRC cells.
Materials and methods
To study the role of USP6NL in CRC, mRNA expression of USP6NL in 32 pairs of tumorous colorectal tissues and adjacent non-tumorous tissue were assessed, using RT-PCT, in CRC patients recruited at the Affiliated Hospital of Jiangsu University. The researches were supported by the Independent Ethics Committee (IEC) of the Affiliated Hospital of Jiangsu University and all patients were provided written informed consent. Besides, mRNA expression data of USP6NL in 260 tumorous colorectal tissues and 41 none-tumorous adjacent tissues were downloaded from The Cancer Genome Atlas database (TCGA) (https://cancergenome.nih.gov/researchhighlights/researchbriefs/ColorectalCancerInsights). Furthermore, mRNA expression data of USP6NL in 70 colorectal tissues and 12 adjacent tissues were deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with accessible number GES9348. Immunohistochemistry (IHC) analysis was performed to assess USP6NL expression in 10 tumorous tissues samples and 5 precancerous tissue from patients and with CRC in the Affiliated Hospital of Jiangsu University. The use of CRC patients was permitted by the ethics committee of the Affiliated Hospital of Jiangsu University, and written informed consent of each participate were acquired.
Immunohistochemistry (IHC) assay
Expression pattern of USP6NL in normal or tumorous colorectal tissues was assessed using IHC method. Colorectal tissue sections (4–7 μm thick) were incubated with USP6NL antibody (ab233414, Abcam) at 4 °C overnight followed by secondary antibodies (D-3004, Long Island Biotech, China) at 25 °C for 30 min. DAB substrate (Long Island Biotech) and hematoxylin (714094, BASO) was used for coloration. A light microscope (Eclipse Ni-E/Ni-U, NIKON) with an image analysis system (DS-Ri2, NIKON) were used for visualization and calculation of USP6NL-positive cell areas.
Cell culture and treatment
HT29, SW480, LOVO, HCT116, CACO2 and FHC cells were bought from ATCC (Manassas, VA, USA). Culture medium for HCT116, FHC and CaCO2 was DMEM (Hyclone, Logan, UT) while feed medium for LOVO, SW480 and HT-29 was RPMI-1640 (Hyclone), and both of which were supplemented with penicillin (100 U/ml, Solarbio, Beijing, China), streptomycin (100 µg/ml, Solarbio) and heat-inactivated fetal bovine serum (FBS) (10%, Gibco Laboratories)). CRC cells were cultured at 37 °C under 5% CO2 with the confluency above 80%.
To verify how Wnt/β-catenin pathway played in USP6NL-induced SW480 cell growth, after USP6NL overexpression transfection, SW480 cells were treated with 10 μmol/l of XAV939 (S1180, Selleck) and then cultured as mentioned above. To verify the roles of C-myc in regulating USP6NL and SW480 cell growth, after transfection, cells were treated with 100 μmol/l of 10058-F4 (S7153, Selleck).
Production and transfection of USP6NL overexpression vectors
The primers of human USP6NL gene (NCBI NM_AB449916.1) were: 5′-CGGAATTCATGAATTCAGACCAGGATGTAGC-3′ (forward) and 5′-CGGGATCCTCACAGCAACACTGACTCTTGG-3′ (reverse), which were implanted into pLVX-Puro vector (Clontech). Plasmids (pLVX-Puro-USP6NL), as well as psPAX2 and pMD2G (Addgen) were co-transfected into 293T cells (ATCC) using Lipofectamine™ 2000 (Invitrogen, CA, USA). After 4–6 h, cells were feed in a complete medium, and lentiviruses with USP6NL expression were collected at 48 h and 72 h. 1.5 μg of lentiviruses with USP6NL expression was transfected into SW480 cells using Lipofectamine 2000 reagent. Meanwhile, cells transfected with pLVX-Puro without USP6NL expression were thought as the corresponding control.
siRNA- USP6NL sequences
5′-GGAGCGAGCTGAAATAGTT-3′, start point 39
5′-CCGGTGGAGCGAGCTGAAATAGTTCTCGAGAACTATTTCAGCTCGCTCCTTTTTG-3′ (forward); 5′-AATTCAAAAAGGAGCGAGCTGAAATAGTTCTCGAGAACTATTTCAGCTCGCTCCA-3′ (reverse)
5′-GCACATTTCGGGACCACAT-3′, start point 449
5′-CCGGTGCACATTTCGGGACCACATCTCGAGATGTGGTCCCGAAATGTGCTTTTTG-3′ (forward); 5′-AATTCAAAAAGCACATTTCGGGACCACATCTCGAGATGTGGTCCCGAAATGTGCA-3′ (reverse)
5′-GCTTTACTCCTCATGTATA-3′, start point 580
5′-CCGGTGCTTTACTCCTCATGTATACTCGAGTATACATGAGGAGTAAAGCTTTTTG-3′ (Forward); 5′-AATTCAAAAAGCTTTACTCCTCATGTATACTCGAGTATACATGAGGAGTAAAGCA-3′ (reverse)
Cell counting kit-8 (CCK-8) assay
Proliferation of HCT116, LOVO and SW480 cells was assessed using CCK8. Briefly, after treatment at 0, 24, 48, 72 h, 3 × 103 cell/well in cultured medium (90 μl/well) were hatched with CCK-8 working solution (10 μl/well) at 37 °C for 1 h. Optical density (OD) value was read by a DNM-9602 microplate reader (PerLong, Beijing, China) at 450 nm.
Flow cytometry for cell cycle detection
After knockdown of USP6NL, CRC cells were fixed in ethanol, rinsed in PBS, remove RNA using 1 mg/ml of RNase A (R8020-25, Solarbio, Beijing, China), and then incubated with 50 μg/ml of propidium iodide solution (C001-200, 7sea Biotech, Shanghai, China) for 10 min in darkness at 25 °C. DNA content was monitored via flow cytometry (Accuri C6, BD Biosciences, San Jose, CA, USA) with FlowJo v10 (Tree Star, Ashland, OR).
Dual luciferase report gene assay
USP6NL promoter was amplified with the primers: 5′-CCCTCGAGTATCATTTAATGGGTTCTGGAGGTC-3′ (forward with XhoI site as indicated by the underline) and 5′-CCAAGCTTATAGAGTAACTTATCTATTCCAAGATTTC-3′ (reversed, HindIII site as indicated by the underline), and inserted into pGL3-Enhance to construct pGL3-Enhancer-p USP6NL. SW480 cells (5 × 105 cells/well) on a 6-well plate were cultured at 37 °C overnight for adherent growth, and then transfected with plasmid pGL3-Enhancer-p USP6NL (1.5 μg) and internal control plasmid pRL-TK (0.05 μg) using Lip 2000 (11668-019, Invitrogen) according to a provided instruction. Besides, pGL3-Enhancer was used as a negative control.
6 h later, SW480 cells with USP6NL overexpression were maintain in fresh culture medium with or without 10058-F4 (100 μmol/l) at 37 °C for 24 h. After fully lysis, Dual-Luciferase® Reporter Assay System (E1910, Promega) was used to detect the activity of firefly luciferase in plasmid pGL3-Enhancer and pGL3-Enhancer-p USP6NL, as well as the activity of renilla luciferase in internal control plasmid pRL-TK. The activity of USP6NL promoter was presented as the ratio of firefly/renilla. Each group set 3 wells, and the experiment was repeated 3 times.
Total RNA from colorectal tissue, CRC cell lines (HT29, SW480, LOVO, HCT116, CACO2), or human normal colon FHC cells was extracted by Trizol Reagent (1596-026, Invitrogen) and reverse transcribed using cDNA synthesis kit (Fermentas). The primers targeted USP6NL (NCBI NM_AB449916.1) were: 5′-TACTCAGCCTTTCAACTC-3′ (USP6NL-forward) and 5′-GCAAGTACACGTCAAATC-3′ (USP6NL-reverse); Pos: 2389-2604; size: 216 bps; primers targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NCBI NM_001256799.2) were: 5′-AATCCCATCACCATCTTC-3′ (GAPDH-forward) and 5′-AGGCTGTTG∆∆∆TCATACTTC-3′ (GAPDH-reverse). mRNA levels of USP6NL were determined using SYBR Green PCR Kit (Thermo Fisher) on an ABI7300 system (Applied Biosystem). Amplification condition was: 95 °C, 10 min (95 °C, 15 s; 60 °C, 45 s) × 40; 95 °C, 15 s; 60 °C, 1 min; 95 °C, 15 s; 60 °C, 15 s. USP6NL was normalized by GAPDH and quantified using the 2−∆∆Ct method according to a reported study .
Western blot analysis
Colorectal tissue (20 mg) and CRC cell lines samples were lysed in RIPA (JRDUN, Shanghai, China). Total protein content in supernatant was determined using BCA protein assay kit (PICPI23223, Thermo, MA, USA). Total protein (25 μg) was separated on 15% SDS-PAGE, and electrophoretically pure were sent onto PVDF membranes (Millipore, USA) and incubated with anti-USP6NL antibody (Ab233414, Abcam), anti-β-catenin antibody (#8480, CST), anti-P27 antibody (#3686, CST), anti-Cyclin D1 antibody (#2922, CST), antibody against C-myc (ab32072, Abcam), and antibody against GAPDH (#5174, CST) at 4 °C overnight followed by horseradish peroxidase-conjugated antibodies (Beyotime, Shanghai, China) for 1 h at 25 °C. USP6NL, β-catenin, P27, Cyclin D1 and C-myc were normalized by GAPDH and quantified using ECL system (GE Healthcare/Amersham Biosciences).
In vitro co-immunoprecipitation (Co-IP) and ubiquitination assay
To study whether USP6NL was associated with β-catenin, 100 μg of total protein in lysis supernatant of HCT116 cells transfected with siUSP6NL was added into protein G-Agarose beads (Roche), and then immune-precipitated with anti-USP6NL (Abcam), anti-β-catenin (Abcam) or control IgG antibody overnight at 4 °C. USP6NL and β-catenin in the immune complex were immunoblotted using anti-USP6NL (NBPI-47264), anti-β-catenin (ab6301, Abcam), respectively according the western blot method. Same amount of protein in each group was reserved for input control.
To study the effect of USP6NL on β-catenin ubiquitination, after fully lysis of HCT116 cells transfected with siUSP6NL, 100 μg of total protein in supernatant was added into Protein A/G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003), and then immune-precipitated with 1 μg of IgG (Santa Cruz Biotechnology, sc-2027) or Anti-β-catenin antibody (ab227499) overnight at 4 °C. Ubiquitination levels of β-catenin in precipitated immune-complexes were separated and quantified using Anti-ubiquitin antibody (ab7780) according the western blot.
12 nude mice, 4–6-week-old, were obtained from Shanghai Laboratory Animal Company, and randomly divided into siNC and siUSP6NL groups. Mice in siNC group were subcutaneously injected with HCT116 cells transfected with siNC (7 × 106 cells, 100 μl). Mice in siUSP6NL group were subcutaneously injected with HCT116 cells transfected with siUSP6NL (7 × 106 cells, 100 μl). Then, mice in different groups were separately fed and the bedding was changed regularly. After tumors formation (12 day later), tumor volume (mm3) and weight (g) were calculated every 3 days from 12th day to 33th day. Tumor volume were calculated as length × (width2/2). We confirmed that the use of mice were abided by the relevant agreements of animal ethics committee in The Affiliated Hospital of Jiangsu University.
In Situ Cell Death Detection Kit (Roche) was used for detection of cell apoptosis in mouse colorectal tissue. Briefly, 4–7 μm of colorectal tissue section on slides was maintained with 50 μl of TUNEL working solution (11684817910, Roche) at 37 °C for 1 h, and then rinsed twice with PBS. We then dyed the cell nucleus with DBA substrate kit (FL-6001, Long Island, Shanghai, China) followed by hematoxylin (714094, BASO). Individual apoptotic thyroid cells were analyzed using IMS image system (JRDUN, Shanghai, China).
Statistics and data analysis
Dates were described as mean ± standard error of the mean (SEM), which was calculated by triplicate determinations in each experiment. Comparisons between two groups were assessed by Student’s t test with P value < 0.05 being statistically significance.
USP6NL was enhanced in CRC
Furthermore, mRNA expression of USP6NL in five CRC cell lines (HT29, SW480, LOVO, HCT116 and CACO2) and one human normal colon FHC cells were assessed. Our data suggested that USP6NL was obviously enhanced in CRC cell lines when compared with FHC cells, substantiating the amplification of USP6NL in CRC in vitro (Fig. 1e). Besides, among CRC cell lines, USP6NL was highly expressed in HCT116 and LOVO cells while lowly expressed in SW480 cells. Therefore, we chose HCT116, LOVO and SW480 for the following study.
Knockdown of USP6NL suppressed CRC cell proliferation and induced cell cycle arrest
The proliferation of CRC cells transfected with siUSP6NL was examined using CCK-8 assay. Figure 2b, f noted that USP6NL silencing time-dependently reduced CRC cell proliferation with the suppressed rates being 13%, 24% and 35% at 24, 48 and 72 h, respectively in HCT116 cells and 13%, 25% and 38% at 24, 48 and 72 h, respectively in LOVO cells. Figure 2c, g showed that knockdown of USP6NL evidently enhanced the percentage of HCT116 and LOVO cells in G0–G1 phase and inhibited the percentage of HCT116 and LOVO cells in S and G2–M phases, demonstrating an obvious cycle arrest in G0–G1 phase in those two cells.
The transition from G0/G1 to S phase is regulated by the P27, Cyclin D1 and C-myc, which are targeted genes of β-catenin (the core element of Wnt/β-catenin pathway) . To investigate the molecular mechanism of USP6NL in the growth inhibition of HCT116 and LOVO cells, protein expression of β-catenin, as well as cell cycle associated P27 (a negative effector of cell cycle progression), Cyclin D1 and C-myc (positive regulator in cell cycle progression) were assessed. Figure 2d, h showed that siUSP6NL dramatically decreased β-catenin, Cyclin D1 and C-myc while increased P27 when compared with siNC within HCT116 and LOVO cells (all P < 0.01).
Wnt/β-catenin pathway was the mechanism, by which USP6NL regulated CRC cell growth
To confirm that Wnt/β-catenin pathway was the mechanism, by which USP6NL regulated cell growth, SW480 cells with USP6NL overexpression were treated with XAV939 (a specific inhibitor for Wnt/β-catenin pathway). Then, cell proliferation, cell cycle, and protein levels of β-catenin, P27, Cyclin D1 and C-myc were assessed. Our data suggested that USP6NL significantly enhanced cell proliferation in a time-depended manner (Fig. 3b), promoted cell cycle progression, by remarkably reducing cell accounts in G0–G1 phase while enhancing cell accounts in S and G2–M phases (Fig. 3c), and up-regulated protein expression of β-catenin, Cyclin D1 and C-myc while down-regulated P27 (Fig. 3d) when compared with vector. On the contrary, cells treated with XAV939 exhibited opposite results. Moreover, the phenotype USP6NL induced was significantly reversed by additional XAV939 treatment. Those results suggested that Wnt/β-catenin pathway is essential for USP6NL to regulate CRC cell growth.
USP6NL directly interacted with β-catenin and regulated β-catenin ubiquitination
Without Wnt, β-catenin can be degraded through the ubiquitination/proteasome pathway . Presently, we examined if USP6NL controls β-catenin ubiquitination in HCT116 cells transfected with siUSP6NL. Our data suggested that the levels of ubiquitinated β-catenin were significantly enhanced in siUSP6NL when compared with siNC, (Fig. 4c), demonstrating that USP6NL may be a deubiquitinase that prevents β-catenin ubiquitination.
C-myc was the mechanism, by which USP6NL regulated CRC cell growth
Knockdown of USP6NL suppressed tumorigenicity of HCT116 cells in nude mice
Ubiquitin-specific proteases (USPs), acting as deubiquitinating enzymes (DUBs), is emerging as the master regulator of tumor [10, 18, 19, 20, 21]. In recent years, more and more evidence that the USPs family in oral malignant tumor, liver cancer, gastric cancer, breast cancer, colorectal cancer, and other abnormal expression in tumor . In recent years, studies have found that USPs members USP4, USP7 and USP47 have been identified as β-catenin-specific deubiquitinylating enzyme [10, 11, 12]. As a member of the USP family, USP6NL plays an important role in the physiological process of the body [23, 24], and is closely related to Wnt/β-catenin signaling. Therefore, we propose whether USP6NL is involved in colorectal cancer cell proliferation and metastasis through Wnt/β-catenin signaling. At present, there are few studies on the role of USP6NL in CRC. Whether and how USP6NL acted on Wnt/β-catenin signaling pathway in CRC remained extremely poor requires further exploration.
Previous studies have shown that the cell cycle of tumor cells is significantly different from that of normal tissue cells, and its S and G2–M phase is significantly higher than that of ordinary cells, while the percentage of G0–G1 phase representing cell division stagnation is lower . In this study, we found that knockdown of USP6NL significantly enhanced the percentage of CRC cells in the G0–G1 phase and inhibited the percentage of CRC cells in the S and G2–M phases. Therefore, we proved that the overexpression of USP6NL in CRC cells enhanced cell proliferation and promoted cell cycle progression from G0/G1 phase to S phase. Knockdown of USP6NL has the opposite effect, inhibiting CRC cell proliferation and inducing cell cycle arrest at G0/G1 phase. The transition from G0/G1 to S is mediated by P27, Cyclin D1, and c-myc, which are target genes for β-catenin (the core component of the Wnt/β-catenin pathway) .
We then investigated whether and how USP6NL acts on Wnt/β-catenin to regulate CRC cell growth. By treating USP6NL overexpressing CRC cells with XAV939, a specific inhibitor of the Wnt/β-catenin pathway, the results suggest that CRC cell proliferation, G1/S cell cycle progression, and β-catenin Cyclin D1 and C-myc are promoted. Enhanced expression, while a decrease in P27 induced by overexpression of USP6NL, was significantly reversed, suggesting that activation of the Wnt/β-catenin pathway is a mechanism by which USP6NL is carcinogenic to CRC in vitro. Our data suggested that 10058-F4 (C-myc inhibitor) significantly reduced USP6NL mRNA and protein levels, and suppressed the activity of USP6NL promoter when compared with vector, demonstrating the inhibited effect of 10058-F4 on USP6NL expression.
Furthermore, our data suggest that knockdown of USP6NL increases ubiquitination of β-catenin, suggesting that USP6NL may act as a deubiquitinating enzyme that regulates β-catenin accumulation during this process. In addition, 10058-F4 downregulated USP6NL, inhibited CRC cell proliferation and induced cell cycle arrest, demonstrating that USP6NL//β-catenin/C-myc may have a feedback loop in regulating CRC cell growth.
Our study reveals the new highlight is that USP6NL activated Wnt/β-catenin pathway probably by the inhibition of β-catenin ubiquitination, revealing other pathways that have not been prioritized, including the Wnt/β-catenin pathway, and has filled a gap in the mechanism of USP6NL’s impact on CRC.
The tumor growth rate and size of the mouse model siRNA-USP6NL group were significantly lower than that of the siRNA-NC group, which provided a strong basis for the purpose of this study, and provided a possibility for the subsequent human targeted therapy research of USP6NL.
However, there are some limitations in our study. The specific mechanism of how USP6NL may activate the Wnt/beta-catenin pathway by inhibiting ubiquitination of beta-catenin is still not clear enough. The treatment of USP6NL inhibitor has not been added in animal experiments, which brings it closer to clinical trials and provides more background information for later experiments.
USP6NL was an oncogene in the tumorigenesis of CRC both in vitro and in vivo, and activation of Wnt/β-catenin pathway was the underlying mechanism. USP6NL activated Wnt/β-catenin pathway probably by the inhibition of β-catenin ubiquitination. Our research elucidated that targeting USP6NL may represent a novel therapeutic target in CRC treatment.
JXC designed the study; KS and SBH performed the experiment and made data entry; YZY, JGQ, RX and MHZ made data collection/entry/analysis, KS, SB, YQM and JXC drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Consent for publication
Ethics approval and consent to participate
The study was approved by the Ethics Committee of The Affiliated Hospital of Jiangsu University (SWYXLL20183205).
This study was supported by Grant No. jdfyrc2017006 from Doctor foundation of the affiliated hospital of Jiangsu university, Grant No. SH2018082 from Zhenjiang Science and Technology Program, Grant No. 81672348 from Project of Nature Science Foundation of China, the Special Clinical Research Fund from Wu Jie Ping Medical Foundation (320.6750.17276), Six Major Talent Peak Project of Jiangsu Province of China (2015-WSW-014), Six One Project for Advanced Medical Talent of Jiangsu Province of China (LGY2016031), and Jiangsu Provincial Medical Youth Talent of China (QNRC2016735).
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