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Introduction

Hepatocellular carcinoma (HCC) is a common type of cancer, and its incidence is increasing rapidly, causing ~745,000 mortalities in 2012, worldwide (1–4). As a result of the low detection rate, a number of HCC patients are at advanced stages of the disease at the time of diagnosis with limited sensitivity biomarkers (5). Long non-coding RNAs (lncRNAs) are a novel class of non-coding RNAs (ncRNAs) that contribute to the development and progression of cancer and that may serve as novel biomarkers (6). However, the molecular and functional mechanisms of lncRNAs in HCC remain unknown. Therefore, studies on the role of lncRNA in HCC are urgent. LncRNAs are >200 nucleotides in length, and previous studies have demonstrated that lncRNAs exert important effects on diverse cellular functions through gene expression regulation, including chromatin modification, transcription regulation and genomic imprinting (7,8). A number of previous studies have demonstrated that lncRNAs participate in tumor development, including cell cycle (9), differentiation (10), apoptosis (11,12), migration and invasion (13). Therefore, lncRNAs may be important therapeutic targets for diseases.

Fer-1-like family member 4 (FER1L4) is a tumor-associated lncRNA involved in the development of tumors, and it has been demonstrated that FER1L4 expression is downregulated in patients with gastric cancer (14,15). It was reported that FER1L4 inhibits proliferation, migration and invasion of gastrointestinal cancer (16,17). A recent study also revealed that FER1L4 inhibits proliferation and the cell cycle through phosphatase and tensin homolog (PTEN) in endometrial carcinoma (18). Therefore, the interactions between lncRNAs and protein-coding genes are popular topics in cancer biology that provide an important theoretical basis for the diagnosis and treatment of cancers. In the present study, the biological mechanism of FER1L4 for proliferation in regulating PTEN were demonstrated in HCC.

The tumor suppressor gene PTEN is located at chromosome 10q23.31 and is frequently inactivated in cancer (19–22). A recent study also demonstrated that PTEN is closely linked to HCC tumorigenesis (23). As previously noted, downregulated FER1L4 in endometrial carcinoma (EC) tissues was positively correlated with decreased PTEN expression by using RT-qPCR assay; FER1L4 inhibited cell proliferation, promoted cell cycle arrest at G0/G1 phase and cell apoptosis by upregulating PTEN expression with MTT, colony-formation and flow cytometry detection (18). Based on the previous study that FER1L4 serves a potential role in regulating PTEN expression (18), it was hypothesized that FER1L4 may also function as a PTEN regulator in HCC.

Materials and methods

Clinical specimens

HCC cancer tissues and adjacent normal tissues (a distance of at least 5 cm from the tumor) were collected from 35 patients with HCC at the Center Hospital of Cangzhou (Cangzhou, China) between March 2014 and October 2015. These HCC cases without other clinicopathological characteristics included 19 male patients and 16 female patients with a mean age of 67.2 years (age, 36–84 years). All patients had not received preoperative treatment, such as radiotherapy or chemotherapy. The inclusion/exclusion criteria of HCC patients were as follows: i) All enrolled patients were diagnosed with HCC, ii) VEGF evaluation, iii) correlation of VEGF with OS or DFS, iv) all patients were carefully evaluated to identify duplicate patient populations, and v) criteria used to judge duplicate populations, such as treatment information, study period and admission hospital. The present study was approved by the ethics committee of the Center Hospital of Cangzhou, and written informed consent was obtained from all patients prior to enrolment in the study. Histological diagnosis of HCC was evaluated according to the World Health Organization criteria. All collected tissues were immediately stored at −80°C until use.

Cell culture

The normal hepatocyte LO2 cell line was purchased from American Type Culture Collection (Manassas, VA, USA). The HCC cell lines Hep3B and Huh7, and 293T cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). LO2, Hep3B, Huh7 and 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM; High glucose; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich; Merck KGaA, Darmstadt, Germany) and 100 U/ml penicillin/streptomycin (Invitrogen, Thermo Fisher Scientific, Inc.). All cells were cultured in a 37°C, 5% CO2 cell culture incubator.

Lentiviral vector construction, production and transfection

To obtain an FER1L4 expression vector, a full-length FER1L4 DNA fragment was amplified by polymerase chain reaction (PCR) using the Takara Ex Taq Hot Start Version kit (Takara Bio, Inc., Otsu, Japan). The PCR reaction is presented in Table I and the reaction steps were as follows: 94°C for 5 min; 94°C for 45 sec, 60°C for 45 sec, 72°C for 1 min, for a total of 30 cycles and 72°C for 10 min.

Table I. Reaction components used in the RT-qPCR.

Table I.

Reaction components used in the RT-qPCR.

Reagent	Consumption
Premix Ex Taq Hot Start Version	25.0 µl
PCR Forward Primer	1.0 µM
PCR Reverse Primer	1.0 µM
Template	0.2 µl
ddH2O	up to 50 µl

The primer sequences of FER1L4 were: Forward, 5′-GATTCAGGTGGGCGGGCTGGTG-3′ and reverse, 5′-TCAGTGGCTGTGATAGGTTTA-3′. The PCR products were inserted into the mammalian expression vector pCDNA3.1 (Invitrogen; Thermo Fisher Scientific, Inc.). A lentiviral vector expressing Enhanced Green Fluorescent Protein (enhanced green fluorescent protein; EGFP, Gene Bank Accession, no. U57607) was used as an empty vector (pCDNA3.1-expressing EGFP) was selected as a control. 293T cells (1×106 cells/well) were seeded in 10 cm plates and co-transfected with lentiviral packaging vectors [pMD2.G (Addgene; cat. no. 12259), pMDL-G/P-RRE (Addgene; cat. no. 12251) and pRSV-REV (Addgene; cat. no. 12253)] and either the constructed pCDNA3.1-FER1L4 overexpression vector or the Control vector for 48 h. Lentiviral particles were harvested, purified and transfected into cells (1×104 cells/well) with 8 µg/ml polybrene (Sigma-Aldrich; Merck KGaA) in 24-well plates. Finally, the cells with stable expression were screened using 800 µg/ml G418 (Sigma-Aldrich; Merck KGaA).

Small interfering (si)RNA transfection

siRNA sequences were designed to target the human FER1L4 gene and siRNAs were purchased from GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China). The sequences of siRNA-FER1L4 were 5′-CAGGACAGCUUCGAGUUAATT-3′ (sense) and 5′-UUAACUCGAAGCUGUCCUGTT-3′ (antisense). The sequences of the negative control (NC) siRNA were 5′-UUCUCCGAACGUGUCACGU-3′ (sense), 5′-ACGUGACACGUUCGGAGAA-3′ (antisense). Cells (2×105 cells/well) were seeded in 6-well plates and transfected with FER1L4 siRNAs (50 nM) or NC siRNAs (50 nM) for 48 h by using Lipofectamine® 3000 (Invitrogen; Thermo Fisher scientific, Inc.) according to the manufacturer's protocol.

Reverse transcription-quantitative PCR (RT-qPCR)

Total RNA was extracted from 35 HCC tissues or treated cells (1×106 cells) by using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed to cDNA using a cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. RT-qPCR was performed using the standard SYBR Green PCR Master Mix kit (Takara Bio, Inc.) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. The reaction steps were as follows: Initial denaturation at 95°C for 30 sec; followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. Relative expression was determined based on the 2−ΔΔCq method (24). The following primer sequences were used: FER1L4, forward 5′-CCGTGTTGGGTGCTGTTC-3′, reverse 5′-GGCAAGTCCACTGTCAGATG-3′; PTEN, forward 5′-GTTTACCGGCAGCATCAAAT-3′, reverse 5′-CCCCCACTTTAGTGCACAGT-3′; GAPDH, forward 5′-CCTCGTCTCATAGACAAGATGGT-3′, reverse 5′-GGGTAGAGTCATACTGGAACATG-3′; GAPDH was used as an internal control.

Western blot assay

Protein expression levels of PTEN were measured by western blot assay. Briefly, the treated cells (1×106 cells) were lysed in Radioimmunoprecipitation Assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). Protein concentrations were measured using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.). Equal concentrations of protein (30 µg) were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). All the membranes were blocked with 5% skim milk (Shanghai solarbio Bioscience & Technology Co., LTD, Shanghai, China.) for 1.5 h at room temperature and incubated overnight at 4°C with primary antibodies against PTEN (1:500; Abcam, Cambridge, UK, cat. no. ab31392) and beta-actin (1:3,000; Sigma-Aldrich; Merck KGaA; cat. no. A2066)). Subsequently, membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse (1:5,000; cat. no. CW102) and goat anti-rabbit (1:5,000; cat. no. CW103) secondary antibody for 1 h at room temperature. The results were analyzed using an Enhanced Chemiluminescence Detection kit (Amersham Biosciences Inc., Piscataway, NJ, USA) an enhanced chemiluminescence system (Amersham Biosciences Inc.). The results were analyzed by using Image-Pro Plus 6.0 software (National Institutes of Health, Bethesda, MD, USA).

MTT assay

The MTT assay was used to measure the cell viability by using MTT reagent (Cat. number M2128) according to the manufacturer's protocol. Briefly, the treated cells (4,000 cells/well) were seeded in 96-well plates and incubated at 37°C for 0, 12, 24 and 72 h. Subsequently, 20 µl MTT (5 mg/ml) solution was added to each well at the prescribed time and the cells were incubated for an additional 4 h at 37°C. Dimethyl sulfoxide was used to dissolve the formazan crystals and the absorbance was detected at 570 nm under the micro-plate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Colony-formation assay

A colony formation assay was also performed to measure the cell viability. Transfected cells (5×103 cells/plate) were seeded onto 6-cm culture dishes and were maintained in the DMEM (High glucose; Invitrogen; Thermo Fisher Scientific, Inc.) including 10% FBS for 2 weeks; the medium was replaced every 3 days. Colonies were stained with Coomassie blue (cat: B-2025, Sigma-Aldrich; Merck KGaA) at 37°C for 1 h, and colony formation was calculated under the stereomicroscope by counting the number of stained colonies.

Tumor formation in nude mice

Prior to the beginning of the experiments, specific criteria for humane endpoints were established as previously described, including labored breathing, reduced food or drinking water intake, inability to stay upright, unresponsive or unconscious to external stimuli and shaggy coat (25,26); animal reactions were confirmed by an animal specialist. If mice presented one or more of the above reactions, the animals were assumed to have reached the humane endpoint. When mice met humane endpoints, they were anesthetized with sodium pentobarbital (50 mg/kg) by intraperitoneal injection and sacrificed by cervical dislocation immediately.

A total of 14 Female athymic BALB/c mice (age, 4–6 weeks; weight, 20–22 g) were obtained from Yangzhou University Medical Centre (Yangzhou, China). Specific pathogen free (SPF) were provided by the Cangzhou Central Hospital Animal Center, and they were housed in a controlled environment (food and water available ad libitum, 21±1°C, humidity 60%, lights on from 7:00 a.m.-7:00 p.m.). The study was approved by the ethics committee of the Cancer Institute. All the animals were treated humanely in accordance with the guidelines laid down by the Institutional Animal Ethics Committee. siRNA-FER1L4- or NC-siRNA-transfected cells (1×107 cells in 100 µl) were injected into nude mice. Tumor volume and weights were measured and calculated at 0, 5, 10, 15, 20, 25, and 30 days; tumor volume was calculated as the volume of a sphere: (4/3) × (π) × (r3), where radius (r) =½ diameter.

Statistical analysis

Significant differences between two groups were analyzed by Student's t-test. FER1L4 expression was analyzed by using one-way analysis of variance followed by a Dunnett post-hoc test in HCC cell lines. The correlation between FER1L4 and PTEN mRNA expression was analyzed by Pearson's correlation coefficient. All data are presented as the mean ± standard deviation, and analyzed by using IBM SPSS Statistics version 21 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference. All experiments were performed in triplicate.

Results

FER1L4 is expressed at low levels in human HCC

FER1L4 mRNA expression levels were examined in 35 pairs of HCC and paired adjacent normal tissues by RT-qPCR. The results indicated that FER1L4 expression was decreased in HCC tissues (n=34/35) compared with the adjacent non-cancerous tissues (Fig. 1A). Similarly, FER1L4 mRNA expression levels were demonstrated to be significantly lower in Hep3B and Huh7 HCC cell lines compared with expression in LO2 normal hepatocyte cells (P<0.001; Fig. 1B). These results suggested that FER1L4 may function as a tumor suppressor in HCC.

Figure 1. FER1L4 is expressed at a low level in human HCC tissues and cell lines. (A) RT-qPCR was performed to analyze the mRNA expression levels of FER1L4 in 35 pairs of HCC and paired adjacent normal tissues. (B) RT-qPCR was used to compare the mRNA expression levels of FER1L4 in HCC cell lines (Hep3B and Huh7) with expression in the normal hepatocyte LO2 cell line. ***P<0.001 vs. LO2 cells. FER1L4, Fer-1-like family member 4; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

FER1L4 inhibits the proliferative ability of HCC cells

To further identify the effects of FER1L4 on the proliferative ability of HCC cells, Hep3B cells were transfected with lentiviral FER1L4 overexpression vectors or siRNA-FER1L4. RT-qPCR was used to validate the efficiency of lentiviral vector and siRNA transfections. The results demonstrated that Hep3B cells transfected with FER1L4 overexpression vectors expressed significantly higher levels of FER1L4 mRNA, and cells transfected with siRNA-FER1L4 expressed a significantly lower level of FER1L4, compared with the respective empty vector-transfected or siRNA-NC-transfected control cells (P<0.001; Fig. 2A). Results from MTT assays indicated that FER1L4 overexpression significantly inhibited the proliferative ability of Hep3B cells compared with the empty vector-transfected control cells (P<0.001; Fig. 2B). However, the silencing of FER1L4 expression by siRNAs significantly promoted the proliferative ability of Hep3B cells compared with the siRNA-NC-transfected cells (P<0.05; Fig. 2C). In addition, a colony-forming assay was performed to examine the effects of FER1L4 on the long-term proliferative ability of Hep3B cells. It was demonstrated that FER1L4 over expression significantly suppressed the long-term proliferative abilities of Hep3B cells compared with control cells (P<0.001; Fig. 2D), whereas reduced FER1L4 expression significantly increased the long-term proliferative abilities of Hep3B cells compared with control cells (P<0.001; Fig. 2E).

Figure 2. FER1L4 inhibits the proliferative ability of HCC cells. Hep3B HCC cells were transfected with FER1L4-OE or with siRNA-FER1L4. (A) Reverse transcription-quantitative polymerase chain reaction was used to measure the mRNA expression level of FER1L4 in the treated Hep3B cells; ***P<0.001. (B) The proliferative ability was detected by MTT assay in Hep3B cells transfected with FER1L4-OE or with empty vector control; ***P<0.001. (C) The MTT assay was performed to measure the proliferation ability of Hep3B cells transfected with siRNA-FER1L4 or siRNA-NC at 0, 12, 24, 72 h, respectively; *P<0.05. (D and E) Clonal colony-forming experiments were performed to measure the proliferative ability of Hep3B cells that were transfected with (D) FER1L4-OE or the empty vector control or (E) siRNA-FER1L4 or siRNA-NC magnification, ×100; ***P<0.001. FER1L4, Fer-1-like family member 4; NC, negative control; OD, optical density; OE, overexpression; siRNA, small interfering RNA.

Reduced FER1L4 expression promotes the growth of HCC tumors in vivo

Based on the higher efficiency of FER1L4 siRNAs compared with the FER1L4 overexpression vector (data not shown), FER1L4 was chosen to be silenced by siRNAs for the in vivo study. Transfected Hep3B cells were implanted subcutaneously into nude mice. The tumor volume was calculated at 0, 5, 10, 15, 20, 25 and 30 days; tumor volumes and longest diameters are presented in Table II (the values correspond to the tumors at the end of the experiment). The results indicated that the silencing of FER1L4 expression led to a significant increase in tumor volume compared with siRNA-NC (P<0.001; Fig. 3A). Tumor weight was also calculated at 30 days. The results demonstrated that the tumor weights were 2,893±345 and 1,468±321 mg in nude mice implanted with Hep3B cells transfected with siRNA-FER1L4 and siRNA-NC at 30 days, respectively; these data revealed that the silencing of FER1L4 significantly decreased the tumor weight (P<0.001; Fig. 3B).

Figure 3. Reduced FER1L4 expression promotes the growth of HCC tumors in nude mice in vivo. (A) Tumor volume was detected at 0, 5, 10, 15, 20, 25 and 30 days. Arrow indicates that nude mice were injected with Hep3B cells that were transfected with siRNA-FER1L4 or siRNA-NC at 5, 15 and 25 days. ***P<0.001. (B) When animals met humane endpoints, they were sacrificed and tumor weights were measured; ***P<0.001. FER1L4, Fer-1-like family member 4; HCC, hepatocellular carcinoma; NC, negative control; siRNA, small interfering RNA.

Table II. Volume and the longest diameter of subcutaneous tumors in nude mice injected with Hep3B cells transfected with siRNA-FER1L4 or siRNA-NC.

Table II.

Volume and the longest diameter of subcutaneous tumors in nude mice injected with Hep3B cells transfected with siRNA-FER1L4 or siRNA-NC.

	siRNA-NC			siRNA-FER1L4
Day	Tumors (n)	Volume (mm3)	Longest diameter (mm)	Tumors (n)	Volume (mm3)	Longest diameter (mm)
0	2	32.12	1.97	1	42.12	4.32
5	1	64.28	4.97	1	108.30	5.91
10	1	128.32	6.26	2	211.10	3.70
15	2	246.32	3.89	1	444.60	9.47
20	1	503.20	9.87	1	875.32	11.87
25	1	785.40	11.45	2	1,423.40	6.98
30	3	1,132.40	4.31	2	2,552.40	8.48

[i] FER1L4, Fer-1-like family member 4; NC, negative control; siRNA, small interfering RNA.

FER1L4 positively regulates PTEN expression in HCC

To investigate the interaction between FER1L4 and PTEN, RT-qPCR and western blot assays were performed. FER1L4 overexpression significantly upregulated the mRNA expression level of PTEN and the silencing of FER1L4 by siRNAs significantly downregulated the mRNA expression level of PTEN in transfected Hep3B cells, compared with the respective controls (both P<0.001; Fig. 4A). FER1L4 overexpression significantly increased PTEN protein expression whereas the silencing of FER1L4 by siRNAs significantly decreased PTEN protein expression in transfected Hep3B cells compared with the respective controls (both P<0.001; Fig. 4B). In addition, the mRNA expression level of PTEN was measured in HCC tissues with either high FER1L4 (n=21) or low FER1L4 (n=14) expression levels (Cut off=0.435) by using SigmaPlot 10.0 (SigmaPlot Software, La Jolla, CA, USA). The results demonstrated that high PTEN mRNA expression may be associated with high FER1L4 expression in clinical HCC tissues (P<0.001; Fig. 4C) and the expressions were positively correlated (R2=0.6058; P<0.01; Fig. 4D).

Figure 4. FER1L4 positively regulates PTEN expression in HCC. (A) The mRNA expression level of PTEN was measured by RT-qPCR in Hep3B cells that were transfected with lentiviral vectors expressing FER1L4 or control vector and FER1L4 siRNA or NC siRNA. ***P<0.001. (B) Western blotting detected the protein expression level of PTEN in Hep3B cells that were treated as in A; beta-actin was used as a protein-loading control. ***P<0.001. (C) RT-qPCR assay was used to detect PTEN expression in HCC tissues with high FER1L4 expression and low FER1L4 expression. The transcript level was normalized to GAPDH expression (n=35, ***P<0.001). (D) The correlation between FER1L4 and PTEN mRNA expression was analyzed by Pearson's correlation coefficient (R2=0.6058; P<0.01). FER1L4, Fer-1-like family member 4; PTEN, phosphatase and tensin homolog; NC, negative control; siRNA, small interfering RNA; HCC, hepatocellular carcinoma.

Discussion

Previous studies have demonstrated that a number of ncRNAs serve important roles in human diseases by activating or repressing genes (24,27,28). Additional studies have demonstrated that the dysregulated expression or the dysfunction of specific ncRNAs might drive tumorigenesis (29). There are three forms of ncRNAs that are especially important for the regulation of gene expression: MicroRNAs (miRNAs), lncRNAs and circular RNAs (30), of which miRNAs have gained much attention and have been considered to serve important roles in cancer through the transcriptional regulation of specific target mRNAs (31). lncRNAs are one of the emerging fields in ncRNA research that have been reported to participate in various biological processes, including transcription, mRNA splicing, RNA decay and translation (32–34). Previous studies indicated that lncRNAs may be associated with the development and progression of human cancer. For example, one study demonstrated that FER1L4 serves as a potential biomarker of gastric cancer with lymph node invasion (35). FER1L4 was reported to inhibit proliferation in endometrial carcinoma (18) and FER1L4 was expressed at a low level in gastric cancer patients (15). Another study demonstrated that FER1L4 inhibits cancer cell growth by regulating PTEN expression (17). In the preset study, FER1L4 was revealed to be expressed at a low level in human HCC tissues. FER1L4 was demonstrated to inhibit the proliferative ability of HCC cells in vitro, and silencing of FER1L4 promoted the proliferative ability of HCC in vivo. Therefore, the results of the present study strongly supported the potential tumor suppressor role of FER1L4 in cancer. Additionally, it may serve as a target for HCC therapy.

A number of previous studies have demonstrated that PTEN serves crucial roles in the progression of tumors, including cell proliferation, differentiation, migration and apoptosis (36–38). Additional studies suggested that PTEN is associated with miRNA (miR)-21 and affects the prognosis of colorectal cancer (39), whereas PTEN may inhibit the epithelial-mesenchymal transition and invasive ability of tongue cancer cells (40). In addition, low expression of PTEN is also considered to be a potential biomarker for resistance to human epidermal growth factor receptor 2-targeted therapy in advanced gastric cancer (41). Recent work has demonstrated that there is connection between lncRNA and PTEN by the lncRNA-GAS5/PTEN/miR-103 axis in endometrial cancer cells (42). In addition, a previous study reported that the FER1L4/PTEN axis has significant effect in regulating the function of endometrial carcinoma (18). In the present study, it was demonstrated that PTEN was expressed at high levels in HCC tissues with high FER1L4 expression. Additionally, both overexpression and knockdown of FER1L4 may be able to regulate PTEN expression in HCC. Therefore, the results of the present study indicated that FER1L4 may also act as a PTEN regulator in HCC, inhibiting the proliferative ability of HCC cells.

In conclusion, the present study results may be summarized as follows: i) FER1L4 was expressed at a low level in human HCC tissues; ii) FER1L4 inhibited the proliferative ability of HCC cells; iii) PTEN was positively associated with FER1L4 expression in HCC; and iv) FER1L4 silencing by siRNAs promoted the growth of HCC tumors in vivo. Therefore, FER1L4 may be a potential therapeutic target for HCC.

Acknowledgements

Not applicable.

Funding

Not applicable.

Availability of data and materials

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Authors' contributions

XS and GQZ contributed to study design, the majority of the experiments and data collection. CYL and CDL contributed to data analysis and drafted the manuscript. GQZ and CYL were responsible for figure preparation, improving the manuscript and helping to conceive the study. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The present study was approved by the ethics committee of the Center Hospital of Cangzhou, (Cangzhou, China). Written informed consent was obtained from all patients prior to enrolment in the present study.

Patient consent for publication

Not applicable.

Competing interests

The authors declare they have no competing interests.

References