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Introduction

Pancreatic cancer (PC) is one of the leading causes of cancer-associated mortality in developed and developing countries and one of the most lethal malignant neoplasms in the world (1–4). Based upon the estimates of GLOBOCAN 2012, >331,000 mortalities per year were caused by PC, accounting for 4% of all mortalities, and PC was considered to be the seventh leading cause of cancer-associated mortality in males and females (1). With regards to treatment, surgery is the only option with the potential to cure PC; however, as a result of the concealed pathogenesis, rapid progress and high metastatic rate, a limited number patients with PC (15–20%) are candidates for radical surgery at the time of disease diagnosis (5). Less than 30% of the patients who undergo surgery and adjuvant chemotherapy survive >5 years following treatment (5). Life expectancy is decreased by up to 98% in patients with PC compared with healthy patients (6).

The causes of PC remain unknown, but certain risk factors have been reported, including cigarette smoking, a family history of PC, excessive alcohol consumption, diabetes mellitus, obesity, dietary factors and a lack of physical exercise (7–10). The present study focused on the Jumonji AT-rich interactive domain 1B (JARID1B) gene, which is a member of JmjC domain-containing protein family. JARID1B specifically removes the methyl residues from tri-, di- and mono-methylated lysine 4 on histone H3 (H3K4) that are associated with gene activation (11,12). JARID1B acts as a transcriptional repressor due to its intrinsic histone demethylase activity. Trimethylation at H3K4 is an important histone modification associated with the activation of transcribed genes, including PTEN (13), and JARID1B specifically demethylates H3K4me3 (tri-methylated histone H3 at lysine 4) to a transcriptionally inactive state that will repress the activation of target genes (14).

Expression of JARID1B has been reported be elevated in a number of different types of cancer, including breast cancer, bladder cancer, lung cancer, colorectal cancer, prostate cancer and malignant melanoma and is required for the proliferation of cancer cells and tumor growth (11,15–22). It has been reported that the depletion of JARID1B inhibited the proliferation of breast cancer cells and restrained tumor growth in xenografts (23) and a syngeneic mouse mammary tumor model (24). Similar results were obtained in lung, bladder and colorectal tumors (19–21). To the best of our knowledge, the present study was the first to reveal that the expression of JARID1B was elevated in PC, and that this was responsible for the inhibition of cell proliferation and tumor growth. Furthermore, it was also previously revealed that JARID1B is associated with the inactivation of phosphatase and tensin homolog (PTEN) in hepatocellular carcinoma (13) which was verified in the present study. The results of the present study reveal a novel function of JARID1B in PC and may provide perspective in order to develop novel therapeutic interventions for PC.

Materials and methods

Clinical specimens and cell culture

The present study was conducted with the approval of the Institutional Ethical Review Board of the Second Affiliated Hospital of Soochow University (Suzhou, Jiangsu, China). Between June 2014 and April 2015, a total of 42 paired (25 male and 17 female, from 37 to 62 years old, mean 50.6 years old) PC specimens and adjacent tissue samples, frozen in liquid nitrogen, were obtained from the pathology laboratory at the Second Affiliated Hospital of Soochow University. None of the patients had received any antitumor treatments prior to biopsy. PC cell lines H6c7, MIA PaCa-2, AsPC-1, BxPC-3, Su86.86, UACC462 were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in adaptive culture medium (Sigma-Aldrich; Merck KgaA, Darmstadt, Germany) according to ATCC and cultured at 37°C in 5% CO2.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted from freshly-frozen samples or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total RNA was reverse transcribed at 42°C for 60 min using a First Strand cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR reactions were conducted using Platinum SYBR Green qPCR SuperMix-UDG reagents (Invitrogen; Thermo Fisher Scientific, Inc.) on the PRISM 7900HT system (Applied Biosystems; Thermo Fisher Scientific, Inc.) followed the following primers: JARID1B forward primer 5′-AGAGGCTGAATGAGCTGGAG-3′ and reverse primer 5′-TGGCAATTTTGGTCCATTTT-3′; GAPDH forward primer 5′-ATCACTGCCACCCAGAAGAC-3′ and reverse primer 5′-ATGAGGTCCACCACCCTGTT-3′. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 50°C for 30 sec and 72°C for 20 sec. All reactions were performed in triplicate and reactions without reverse transcriptase were used as negative controls. GAPDH was used as an endogenous control and the 2−ΔΔCq method was used to calculate the relative expression levels (25).

Western blot analysis

Western-blot assay was used to analyze the expressions of candidate proteins in indicated cell lines. Cells were cultured in 100 mm dishes and radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing protein inhibitor cocktail (cat. no. P9599; Sigma-Aldrich; Merck KGaA) was input at 70–90% density. Whole protein was extracted by centrifugation (13,000 × g) for 20 min at 4°C. And the concentration of proteins was measured by NanoDrop (NanoDrop2000; Thermo Fisher Scientific, Inc.). Samples containing 30–50 ug of protein were separated by 10% SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes (Merck KGaA) which was blocked in 5% bovine serum albumin (Beijing Seasky Bio Technology, Co., Ltd., Beijing, China) or 5% skim milk at room temperature for 1 h. Then the membrane was probed overnight at 4°C in 5% bovine serum albumin (Beijing Seasky Bio Technology, Co., Ltd.) with primary antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA) against Actin (1:2,000; cat. no. CST 3700S), anti-JARID1B (1:1,000; cat. no. CST 3273T), anti-PTEN (1:1,000; cat. no. CST 9188T), anti-protein kinase B (Akt) (1:1,000; cat. no. CST 2920S), anti-p-Akt (1:1,000; cat. no. CST 4060T), anti-P13K (1:1,000; cat. no. CST 4249T), anti-p-P13K (1:1,000; cat. no. CST 4228T), anti-p53 (1:1,000; cat. no. CST 2527T), anti-p21 (1:1,000; cat. no. CST 2947T), anti-p27 (1:1,000; cat. no. CST 3686T), anti-H3K4me3 (1:1,000; cat. no. CST 9751T), anti-K3K9me3 (1:1,000; cat. no. CST 4658T), anti-H3K27me3 (1:1,000; cat. no. CST 9728T) followed by washing in Tris-buffered saline with Tween-20 (TBST; 0.02M Tris PH 7.6, 0.8% NaCl, 01% Tween-20) and incubated in TBST containing HRP secondary antibodies (1:10,000; cat. no. ZB2301; ZSGB-BIO) for 1 h at room temperature. Following washing in TBST again, the Chemiluminescent HRP substrate (cat. no. P90720; Merck KGaA) was added, according to the manufacturer's protocol, and the fluorescence was assessed using FluorChem E (version 4.1.3; Proteinsimple, FE0444). All antibodies were purchased from Cell Signaling Technology.

Establishment of cell lines

Human JARID1B shRNA was cloned into a pSuper-puro vector (GENEWIZ, South Plainfield, NJ, USA) while JARID1B mRNA was cloned into a pBabe-puro vector (GENEWIZ). A total of 10 µg plasmid was transfected into Phoenix packaging cells by X-tremeGENE HP (Roche Applied Science, Penzberg, Germany) and retrovirus supernatants containing pSuper and pSuper-sh.JARID1B, pBabe and pBabe-JARID1B were collected and filtered. AsPC-1 was incubated in retrovirus supernatants contaning pBabe and pBabe-JARID1B, but UACC-462 was incubated in retrovirus supernatants containing pSuper and pSuper-shJARID1B 1 or 2. A total of 4 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) was added to accelerate the transfection. Then AsPC-1-pBabe-JARID1B, UACC-462-pSuper-shJARID1B 1 or 2 and control cells were subsequently selected by puromycin (2 µg/ml) as previously described (26).

Proliferation assay

An MTT assay was used to detect the proliferative rate of AsPC-1-pBabe-JARID1B, UACC-462-pSuper-shJARID1B 1 or 2 and control cell lines. A total of 1,000 cells were plated into a 96-well plate and were cultured at 37°C. MTT was added at 70% density and the 96-well plate was incubated for 4 h, followed by the addition of 150 µl dimethyl sulfoxide in order to dissolve the purple formazan. The optical density values at 492 nm were measured using Multiskan 3 at 12, 24, 48 and 72 h.

In vivo tumor growth model

A total of 20 Male BALB/c nude mice (weight ~15 g) aged 4–6 weeks were purchased from the Hunan Slac Jingda Laboratory Animal Company, Ltd. (Changsha, China) and provided with sterile food and water in the specific pathogen free (SPF) laboratory animal room (22°C, 40% humidity, noise ≤60 dB, clean pass box). All animals were used in accordance with institutional guidelines and the current experiments were approved by the Animal Care and Use Ethics Committe. For the tumor growth assay, AsPC-1-pBabe-JARID1B, UACC-462-pSuper-shJARID1B 2 and control cells were resuspended in phosphate-buffered saline and 4×106 cells (100 µl) were subcutaneously injected into the axilla of the nude mice. Six weeks later, the mice were sacrificed using 1 mg/kg chloral hydrate, and the tumors were dissected and weighed. Animal handling and research protocols were approved by the Animal Care and Use Ethics Committee.

Chromatin immunoprecipitation (ChIP)-qPCR

A ChIP kit was purchased from EMD Millipore (Billerica, MA, USA) and ChIP experiments were performed as previously described (27). Immunoprecipitated DNA was analyzed on the ABI PRISM 7900HT sequence detection system. The PTEN promoter primers of −1,235 to −1,072 bp (forward primer, 5′-CGCCCAGCTCCTTTTCCC-3′; reverse primer, 5′-CTGCCGCCGATTCTTAC-3′) and 260 to 512 bp (forward primer, 5′-GCTCGCACCCAGAGCTAC-3′; reverse primer, 5′-GGAGAGAACTGAGC-3′) used for detection of promoters after ChIP are available upon request.

Statistical analysis

Statistical analysis data are presented as the mean ± standard deviation Associations between JAEID1B and PTEN, Akt, P13K, p53, p21 and p27 expression in PC tissues was assessed using Spearman's rank correlation. Comparisons between different groups were undertaken using one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS 11.0 software for Windows (SPSS, Inc., Chicago, IL, USA).

Results

JARID1B is highly expressed in PC

In order to investigate whether JARID1B was associated with the progression of PC, the expression levels of JARID1B in 42 patient PC samples RT-qPCR. As demonstrated in Fig. 1A and B, JARID1B was significantly overexpressed in 42 PC tissues compared with the normal adjacent tissues. To further confirm the elevated expression of JARID1B in PC, the expression levels of JARID1B in distinct PC cell lines were determined. The results revealed that JARID1B was significantly overexpressed in PC cell lines including MIA PaCa-2, AsPC-1, BxPC-3, Su86.86 and UACC-462 compared with expression in the control cell line H6c7 (Fig. 1C). Furthermore, the expression levels of JARID1B in these cell lines described above were verified using western blot analysis (Fig. 1D). The results revealed that JARID1B was overexpressed in PC.

Figure 1. The expression of JARID1B is increased in PC. (A) Quantification of JARID1B in pancreatic cancer and adjacent normal tissues. (B) Analysis of JARID1B levels in PC and adjacent normal tissues. (C) Reverse transcription-quantitative polymerase chain reaction of JARID1B levels in PC cell lines (D) Western blot analysis of JARID1B levels in PC cell lines. **P<0.01 compared with control. Error bars represent the standard deviation. PC, pancreatic cancer; JARID1B, Jumonji AT-rich interactive domain 1B.

JARID1B promotes the proliferative capacity of PC cells in vitro and in vivo

In order to further identify the role of JARID1B in PC cells, the pancreatic cell lines exhibiting overexpression or silencing of JARID1B were established using a lentiviral vector and the expression levels of JARID1B were examined by western blot analysis and RT-qPCR. As demonstrated in Fig. 2A, the expression levels of JARID1B in UACC-462 cells with three different shRNAs were examined by western blot analysis and JARID1B shRNA2 was the most effective. Similar results were observed in RT-qPCR (Fig. 2B). Overexpression of JARID1B in the AsPC-1 cell line was observed in western blot analysis (Fig. 2C) and RT-qPCR (Fig. 2D).

Figure 2. Detection of JARID1B levels in established PC cell lines. (A) Western blot analysis of JARID1B levels in UACC-462 cells with silencing of JARID1B. (B) Reverse transcription-quantitative polymerase chain reaction analysis of JARID1B levels in UACC-462 cells with silencing JARID1B. (C) Western blot analysis of JARID1B levels in AsPC-1 cells with overexpression of JARID1B. (D) JARID1B levels in AsPC-1 cells with overexpression of JARID1B. **P<0.01 compared with control. Error bars represent the standard deviation. PC, pancreatic cancer; JARID1B, Jumonji AT-rich interactive domain 1B.

Based on the established cell lines, the effect of JARID1B silencing on the proliferation of PC cancer cells was subsequently determined using an MTT assay. The results revealed that silencing of JARID1B significantly inhibited the proliferation of UACC-462 cells (Fig. 3A), while overexpression of JARID1B promoted the proliferation of AsPC-1 cells (Fig. 3B).

Figure 3. JARID1B promotes the proliferation of pancreatic cancer cells. (A) MTT assay of the UACC-462 cells with silencing of JARID1B. (B) MTT assay of the AsPC-1 cells with overexpression of JARID1B. **P<0.01 compared with control. Error bars represent the standard deviation and each experiment was repeated 3 times. JARID1B, Jumonji AT-rich interactive domain 1B; OD, optical density.

In order to further confirm the effect of JARID1B on the proliferation of PC cells, xenografts were used to reveal whether JARID1B affected the proliferation rate of pancreatic tumors. AsPC-1-pBabe-JARID1B, UACC-462-pSuper-shJARID1B 2 and control cells were injected subcutaneously into the axilla of nude mice. As demonstrated in Fig. 4, silencing of JARID1B significantly inhibited the proliferation of pancreatic tumors formed in nude mice (Fig. 4A), which were smaller in size (Fig. 4B) and lighter in weight (Fig. 4C). However, overexpression of JARID1B significantly promoted the proliferation of pancreatic tumors formed by the injection of AsPC-1 cells (Fig. 5A), in terms of tumor size (Fig. 5B) and weight (Fig. 5C). The aforementioned results demonstrated that JARID1B may promote the proliferation of PC cells in vitro and tumorigenesis in vivo.

Figure 4. Silencing of JARID1B inhibits the tumor growth of pancreatic cancer. (A) Tumors formed by UACC-462 cells with silencing of JARID1B or the control vector. (B) Growth curve of the tumors formed by UACC-462 cells with silencing of JARID1B or the control vector. (C) The weight analysis of tumors formed by UACC-462 cells with silencing of JARID1B or the control vector. **P<0.01 compared with control. Error bars represent the standard deviation. JARID1B, Jumonji AT-rich interactive domain 1B.

Figure 5. Overexpression of JARID1B promotes the tumor growth of pancreatic cancer. (A) Tumors formed by AsPC-1 cells with overexpression of JARID1B or the control vector. (B) Growth curve of the tumors formed by AsPC-1 cells with overexpression of JARID1B or the control vector. (C) The weight analysis of tumors formed by AsPC-1 cells with overexpression of JARID1B or the control vector. **P<0.01 compared with control. Error bars represent the standard deviation. JARID1B, Jumonji AT-rich interactive domain 1B.

JARID1B regulates PTEN expression through the demethylation of H3K4me3

It has been demonstrated that JARID1B may affect the demethylation of H3K4me3 and that the activation of PTEN is regulated by H3K4me3 (13). In order to determine whether JARID1B regulates the tumorigenesis of PC through the PTEN/Akt signaling pathway, the expression levels of proteins participating in the PTEN/Akt signaling pathway were examined. As demonstrated in Fig. 6, silencing of JARID1B significantly elevated the expression of PTEN in UACC-462 cells (Fig. 6A), while overexpression of JARID1B inhibited the expression of PTEN in AsPC-1 cells (Fig. 6B). It has been reported that PTEN, as well as p53, p27 and p21, is able to regulate the phosphoinositide 3-kinase (PI3K)/Akt pathway in a number of types of tumor cell (28,29). Therefore, whether or not JARID1B-mediated inhibition of PTEN may affect the expression of the aforementioned proteins was subsequently investigated. Notably, silencing of JARID1B followed by ectopic expression of PTEN significantly inhibited the level of phosphorylated PI3K and Akt but increased the level of p53, p27 and p21 (Fig. 6A), while overexpression of JARID1B induced the opposite effect (Fig. 6B). In further research, silencing of JARID1B significantly increased the mRNA level of PTEN and p53 (Fig. 6C), while overexpression of JARID1B decreased the mRNA level of PTEN and p53 (Fig. 6D). Western blot analysis and RT-qPCR revealed that JARID1B did not affect the level of P13K and Akt (Fig. 6A-D). These data revealed that JARID1B may inhibit the transcription of PTEN and p53. In order to further confirm the correlation between JARID1B and PTEN or p53, Spearman's rank correlation analysis was performed. As demonstrated in Fig. 7, the mRNA level of JARID1B was inversely correlated with the mRNA levels of PTEN (Fig. 7A) and p53 (Fig. 7D), while no association was observed between JARID1B and P13K, Akt, p27 or p21 (Fig. 7B, C, E and F). Based upon these aforementioned data, JARID1B inhibited the expression of PTEN and p53 at the RNA level.

Figure 6. JARID1B activates the PTEN signaling pathway. (A) Western blot analysis of the proteins involved in the PTEN signaling pathway, and p53, p27 and p21 in UACC-462 cells with silencing of JARID1B. (B) Western blot analysis of the proteins involved in the PTEN signaling pathway, and p53, p27 and p21 in AsPC-1 cells overexpressing JARID1B. (C) RT-qPCR analysis of the proteins involved in the PTEN signaling pathway, and p53, p27 and p21 in UACC-462 cells with silencing of JARID1B. (D) RT-qPCR analysis of the proteins involved in the PTEN signaling pathway, and p53, p27 and p21 in AsPC-1 cells overexpressing JARID1B. **P<0.01 compared with control. Error bars represent the standard deviation. JARID1B, Jumonji AT-rich interactive domain 1B; PTEN, phosphatase and tensin homolog; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 7. Correlation analysis of JARID1B and components in the PTEN signaling pathway. (A) Correlation analysis between JARID1B and PTEN mRNA. (B) Correlation analysis between JARID1B and P13K mRNA. (C) Correlation analysis between JARID1B and Akt mRNA. (D) Correlation analysis between JARID1B and p53 mRNA. (E) Correlation analysis between JARID1B and p27 mRNA. (F) Correlation analysis between JARID1B and p21 mRNA. JARID1B, Jumonji AT-rich interactive domain 1B; PTEN, phosphatase and tensin homolog; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B.

Subsequently, the manner in which JARID1B inhibited the expression of PTEN was investigated. It has previously been demonstrated that JARID1B regulates the methylation of H3K4 (11). To begin with, the expression levels of H3K4me3 with silencing or overexpression of JARID1B were investigated. Silencing of JARID1B significantly increased the level of H3K4me3 (Fig. 8A), while overexpression of JARID1B decreased the level of H3K4me3 (Fig. 8B). However, silencing and overexpression of JARID1B did not affect the levels of H3K9me3 or H3K27me3 (Fig. 8A and B). Due to the fact that H3K4me3 is associated with the active transcription of a number of genes, including PTEN, whether or not JARID1B expression was associated with the H3K4me3 modification at the promoter of PTEN in PC cells was investigated. Quantitative chromatin immunoprecipitation (qChIP) assays were performed in AsPC-1-pBabe-JARID1B, UACC-462-pSuper-shJARID1B and their corresponding control cells. Silencing of JARID1B was revealed to be associated with increased H3K4me3 levels at the region −1,235 to −1,072 bp (Fig. 8C1) and 260 to 512 bp (Fig. 8C2) in the PTEN gene regions (Fig. 8D). Furthermore, less occupancy of those PTEN gene regions by H3K4me3 was detected in AsPC-1 cells with overexpression of JARID1B (Fig. 8E). Taken together, these results indicated that JARID1B induced inactive transcription of PTEN due to decreased levels of H3K4me3 in the PTEN gene.

Figure 8. JARID1B activates PTEN transcription by decreasing H3K4me3. (A) Western blot analysis of the proteins, H3K4me3, H3K9me3 and H3K27me3, in UACC-462 cells with silencing of JARID1B. (B) Western blot analysis of the proteins, H3K4me3, H3K9me3 and H3K27me3, in AsPC-1 cells with overexpression of JARID1B. (C) The predicted binding region of H3K4me3 to the PTEN promoter. (D) ChIP-qPCR was performed to assess H3K4me3 occupancy in UACC-462 cells with silent expression of JARID1B. (E) qChIP was performed in order to assess H3K4me3 occupancy in AsPC-1 cells overexpressing JARID1B. **P<0.01. Error bars represent the standard deviation. JARID1B, Jumonji AT-rich interactive domain 1B; PTEN, phosphatase and tensin homolog; qChIP, quantitative chromatin immunoprecipitation.

Discussion

PC has been reported to be the most lethal malignant neoplasm, with a difficult diagnosis and a high rate of relapse. The results of the present study demonstrated that JARID1B was aberrantly expressed in patients with PC and that elevated JARID1B significantly promoted the cell and tumor proliferation of PC, while silencing of JARID1B had the opposite effect. With regards to the mechanism of JARID1B-promoted cell and tumor proliferation, it was revealed that PTEN serves an essential role in these processes. JARID1B may regulate the methylation of H3K4 in order to affect the activation of PTEN, which ultimately promotes the progression of PC. Therefore, JARID1B may be a novel target for the diagnosis of PC.

In recent years, a number of novel insights into the pathogenesis of PC have been reported. Extracellular signal-regulated kinase promoted the degradation of FBXW7 through phosphorylation, which resulted in the inhibition of the tumor suppressor function of FBXW7 and eventually promoted the occurrence of PC (30). Ataxia telangiectasia group D complementing gene (gene-ATDC), which serves an essential role from the early development of invasive cancer to metastatic cancer, was also reported to be associated with 90% of the proliferation of PC (31). Patients with high gene-ATDC levels often experience very early metastasis, resulting in a <30% survival rate in patients with early PC (32). Another study reported that >95% of the patients with PC exhibited KRAS mutations (32), while a different study revealed that miRNA-21 serves a key role in the drug resistance of PC (33). These data may aid doctors in better understanding the pathogenesis of PC. The present study revealed the novel role of JARID1B in the proliferation of PC, which may further improve the pathogenesis of PC and may serve as a novel therapeutic target for PC.

Overexpression of JARID1B occurs in a wide variety of cancer types, but the function of JARID1B is not fully understood (15–22). It was recently observed that the demethylation of H3K4 regulated by JARID1B contributed to the silencing of retinoblastoma target genes in senescent cells, presumably through closing the chromatin in which the silencing of retinoblastoma trigger genes was involved (34). In esophageal cancer, JARID1B promoted cell proliferation and tumor growth following an effect of JARID1B on the activation of PTEN (35). The association between JARID1B and PTEN has also been demonstrated in previous studies (13,36,37). The present study also revealed that JARID1B promoted the cell proliferation and tumor growth of PC by regulating the activation of the PTEN gene through demethylation of H3K4me3, which further confirmed the regulatory association between JARID1B and PTEN.

In recent years, good diagnostic markers, drug targets and therapeutic strategies remain insufficient for the successful treatment of PC. The results of the present study identified the role and mechanism of JARID1B in the cell proliferation and tumor growth of PC, which may contribute toward the diagnosis and therapeutic strategy of PC patients.

Acknowledgements

Not applicable.

Funding

No funding received.

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' Contributions

PD was responsible for the conception and design of the present study. XS, GC and PD developed the methodology. XS, GC, LX, WW and ZC undertook the acquisition of data. XS, GC, LX, WW and ZC were responsible for the analysis and interpretation of data. XS and GC wrote and undertook any manuscript revisions and PD was responsible for study supervision and any administrative, technical, or material support.

Ethics approval and consent to participate

The present study was conducted with the approval of the Institutional Ethical Review Board of the Second Affiliated Hospital of Soochow University (Suzhou, Jiangsu, China).

Consent to publish

The study participants provided their consent for the publication of this data.

Competing interests

The authors declare that they have no competing interests.

References