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Introduction

Cholangiocarcinoma (CCA) is a malignant tumor of the extrahepatic bile duct, extending from the hilar area to the lower end of the common bile duct (1). The etiology of CCA may be associated with cholelithiasis, primary sclerosing cholangitis and other types of disease (2). Surgical treatment, radiotherapy and chemotherapy can be used clinically; however, the prognosis is poor (3,4). Consequently, it is crucial to investigate efficient therapies and identify sensitive biomarkers for early diagnosis of CCA. Molecular-targeted therapy has exhibited significant effects against cell proliferation, recurrence and metastasis in CCA, with few adverse effects (5). Therefore, it is important to understand the molecular mechanisms of carcinogenesis and progression in CCA, which may contribute to identifying novel diagnostic markers and sensitive therapeutic targets.

Long non-coding RNAs (lncRNAs) are a class of RNA molecules that are >200 nucleotides and exhibit no protein coding function (6). Recent studies have demonstrated that lncRNAs are closely associated with the development of diverse human diseases, including cancer (7,8). lncRNAs are involved in the regulation of gene expression at the epigenetic, transcriptional and post-transcriptional levels and affect the occurrence, development and invasion of tumors (9,10). Investigations regarding the association between lncRNAs and digestive system tumors may provide a new strategy for the prevention, diagnosis and treatment of these tumors (11,12). Recently, the biological roles and mechanisms of several lncRNAs, including AFAP1 antisense RNA 1 (AS1), ASAP1 intronic transcript 1, colon cancer associated transcript 1, nuclear enriched abundant transcript 1 and sprouty4-intron transcript 1, have been reported to be involved in CCA occurrence and metastasis (13–17). A previous study demonstrated that the expression of lncRNA FLVCR1-AS1 is significantly increased in hepatocellular carcinoma. Additionally, it was identified to promote cancer cell proliferation, migration and invasion by sponging microRNA (miR)-513c in hepatocellular carcinoma (18). However, to the best of our knowledge, the role and function of FLVCR1-AS1 in CCA remains unclear.

The present study identified an increased expression level of FLVCR1-AS1 in CCA tumor tissues and cell lines. The biological effect of FLVCR1-AS1 on cell proliferation, migration and invasion was investigated. It was revealed that downregulation of FLVCR1-AS1 inhibited these processes. Furthermore, bioinformatics analysis and luciferase reporter assay demonstrated that FLVCR1-AS1 serves an oncogenic role in CCA by sponging miR-485-5p.

Materials and methods

Patients and tissue samples

In total, 22 paired CCA and normal tissue samples were collected from patients (age range, 42–77 years; mean age, 59 years; 13 males and 9 females) who underwent surgical treatment at Shanghai Seventh People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Shanghai, China) between January 2010 and December 2016. The clinical characteristics of patients with CCA are presented in Table I; the Tumor-Node-Metastasis (TNM) staging system was used as previously described (19). Tissue samples were snap frozen in liquid nitrogen immediately after surgical resection and stored at −80°C. Patients who received radiotherapy and/or immunotherapy before or following surgery were excluded from the study. Samples were collected after written informed consent was obtained from all patients. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee of Shanghai Seventh People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Shanghai, China).

Table I. Clinical characteristics of patients with cholangiocarcinoma.

Table I.

Clinical characteristics of patients with cholangiocarcinoma.

Characteristic	Patients (n=22)
Sex
Male	13
Female	9
Age, years
>60	12
≤60	10
Tumor size, cm
>3	13
≤3	9
Lymph node metastasis
No	15
Yes	7
TNM stage
I/II	14
III/IV	8

[i] TNM, Tumor-Node-Metastasis (19).

Cell culture and CCA cell transfection

Human cell lines, including the noncancerous cholangiocyte cell line HIBEC and the CCA cell lines RBE, CCLP1, HuCCT1 and HCCC-9810 were purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). All cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and grown in humidified 5% CO2 at 37°C. Short hairpin RNA (shRNA) targeting FLVCR1-AS1 (shFLVCR1-AS1) and the relative control shRNA (shNC) were obtained from Shanghai Genepharma Co., Ltd., (Shanghai, China). The sequence of shFLVCR1-AS1 was 5′-GGTAAGCAGTGGCTCCTCTAA-3′ and the sequence of shNC was 5′-AATTCTCCGAACGTGTCACGT-3′. The transfection was performed using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in 5×106 cells at a final concentration of 50 nM, according to the manufacturer's protocol. After transfection for 24 h, expression of FLVCR1-AS1 was validated by reverse transcription-quantitative PCR (RT-qPCR).

RT-qPCR

Total RNA was extracted from tissues and cells using TRIzol® reagent (Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocol. Following quantification using Nanodrop 2000 (Thermo Fisher Scientific, Inc.), the extracted total RNA was reverse-transcribed using Reverse Transcription kit (Takara Biotechnology Co., Ltd., Dalian, China). qPCR with SYBR® Green RT-PCR Master mix (Takara Biotechnology, Co., Ltd., Dalian, China) was performed on an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Primer specific sequences (FLVCR1-AS1, forward, 5′-GTGGCTCTCTCGTTCCC-3′ and reverse, 5′-CCGTCCTTCGGTAGTGTC-3′; miR-485-5p, forward, 5′-AGAGGCTGGCCGTGAT-3′ and reverse, 5′-ATGTGTTGCTGTGTTTGTCG-3′) were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The PCR conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 40 sec and extension at 72°C for 10 sec. Universal miRNA RT-qPCR primer, 5′-AACGAGACGACGACAGAC-3′; 5′-GCAAATTCGTGAAGCGTTCCATA-3′ for U6 was used as an endogenous control to normalize miR-485-5p expression level. GAPDH (forward, 5′-TCCTCTGACTTCAACAGCGACAC-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAATTC-3′) was used as an endogenous control to normalize lncRNA FLVCR1-AS1 expression level. The relative expression level was calculated using the 2−ΔΔCq method (20). All the experiments were performed in triplicate.

Cell proliferation (MTT) assay

CCA cells transfected either with shNC or shFLVCR1-AS1 were seeded into 96 well plates at 5,000 cells/well. At 12, 24, 48 and 72 h after transfection, the medium was removed by suction and replaced with serum-free RPMI-1640 medium containing 1 mg/ml MTT. The cells were then incubated at 37°C for 4 h. Following removal of the MTT solution, the formazan precipitate was dissolved with 100 µl DMSO and the absorbance was measured at 570 and 600 nm in a microplate reader. shNC cells were used as the normal controls.

Colony formation assay

CCA cells were trypsinized, counted and seeded into 12-well plates at 100 cells/well. The medium was replaced every 3 days during colony growth. Following incubation in Dulbecco's modified Eagle's medium (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 12 days, the colonies were fixed with methanol for 20 min at room temperature and stained with 0.2% crystal violet for 15 min (Sigma-Aldrich; Merck KGaA) at room temperature. The colonies were counted under an inverted microscope (Nikon Corporation, Tokyo, Japan) at magnification, ×100.

In vivo xenograft experiments

All animal experiments were performed in accordance with institutional and international animal regulations. The animal protocols were approved by the Animal Care and Use Committee at Shanghai Seventh People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (Shanghai, China). Male 6-week old BALB/c nude mice (N=8) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). All mice were kept in pathogen-free conditions in 26–28°C and 50% humidity on a 12 h light/dark cycle. Mice had ad libitum access to food and water. For tumor propagation analysis, HuCCT1 cells, transfected either with shNC or shFLVCR1-AS1, were subcutaneously injected into the flanks of BALB/c nude mice at 1×106 cells/mouse. The length and width of tumors were measured using a caliper every 7 days for a period of 28 days. All mice were euthanized at day 28 post-injection, and the tumor nodules of the mice were removed and weighed. The tumor volume was calculated using the following formula: Tumor volume (mm3)=length (mm) × width (mm)2/2.

Wound-healing assay

A wound-healing assay was performed to detect cell migration. In brief, 48 h after transfection, CCA cells were cultured in 6-well plates (5×104 cells/well). The monolayer of cells was scratched using a sterile plastic micropipette tip and cells were cultured under standard conditions for 24 h. Following several washes, recovery of the wound was observed and images were obtained using a light microscope (magnification, ×100; Olympus Corporation, Tokyo, Japan).

Cell invasion assay

After transfection for 48 h, CCA cells (1×105) transfected with shNC or shFLVCR1-AS1 were seeded into the upper chamber of Matrigel-coated inserts with serum-free medium. RPMI-1640 medium with 10% FBS was added to the lower chamber as a chemoattractant. The cells were allowed to invade for 48 h at 37°C with 5% CO2. Migrating cells were fixed in 70% ethanol for 30 min and stained with 0.1% crystal violet for 10 min at 25°C. The number of cells that migrated to the lower side were counted in five randomly selected fields under an inverted microscope (Olympus Corporation) at ×100 magnification.

Luciferase reporter assay

Wild-type FLVCR1-AS1 (5′-AUACACGGCCCUC-3′) and mutant FLVCR1-AS1 (5′-AUACUGCCGCCUC-3′) sequences were cloned into a pmirGLO reporter vector (Promega Corporation, Madison, WI, USA). HuCCT1 cells were co-transfected with NC mimic (5′-GCAUGAUGGUCAUAGGUCC-3′) or miR-485-5p mimic (5′-AGAGGCUGGCCGUGAUGAAUUC-3′), and wild-type FLVCR1-AS1 or mutant FLVCR1-AS1 (1 µg per well) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Relative luciferase activity was measured on a dual-luciferase reporter assay system (Promega Corporation) at 48 h post-transfection. Data are presented as the ratio of Renilla luciferase activity to firefly luciferase activity.

Western blot analysis

CCA cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and sonicated. Lysates containing soluble proteins were collected and stored at 80°C. Protein concentration was determined using a Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total protein lysates (10 µl per lane) were resolved on 10% SDS-PAGE gels and transferred to polyvinyldifluoride membranes (EMD Millipore, Billerica, MA, USA). Following blocking in TBS containing 0.1% Tween-20 (TBS-T) with 5% non-fat dry milk for 30 min at room temperature, membranes were washed four times in TBS-T and incubated with primary antibodies overnight at 4°C. All primary antibodies were obtained from Abcam (Cambridge, MA, USA) and used at the following dilutions: Anti-Twist (1:500; catalog no. ab49254), anti-matrix metalloproteinase (MMP)-2 (1:500; catalog no. ab92536), anti-MMP-9 (1:500; ab38898), anti-β-actin (1:1,000; catalog no. ab8226). Following extensive washing, membranes were incubated with horseradish peroxidase-linked goat polyclonal anti-rabbit IgG secondary antibodies at a dilution of 1:2,000 for 1 h at room temperature. Immunoreactivity was detected by enhanced chemiluminescence using an ECL kit (Pierce; Thermo Fisher Scientific, Inc.) and exposure to radiography film. GAPDH served as the loading control. Western blots were quantified by densitometry with Labworks Software (version 4.0; UVP BioImaging Systems, Upland, CA, USA).

Statistical analysis

Data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Two-tailed Student's t-test was applied to compare the differences between two groups and one-way analysis of variance followed by Dunnett's multiple comparison test was used to compare the differences among three independent groups. Correlation between FLVCR1-AS1 and miR-485-5p expression in CCA tissues was identified using Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated in triplicate.

Results

Expression of FLVCR1-AS1 in tissues from patients with CCA and cells

To investigate the role of FLVCR1-AS1 in CCA, the expression of FLVCR1-AS1 in 22 CCA and normal tissue samples was analyzed by RT-qPCR. The clinical characteristics of patients with CCA are presented in Table I. It was identified that the expression level of FLVCR1-AS1 was significantly increased in CCA tissues compared with normal tissues (P<0.001; Fig. 1A). In addition, RT-qPCR demonstrated that the expression of FLVCR1-AS1 was higher in CCA cell lines (RBE, HCCC-9810, HuCCT1 and CCLP1) compared with the HIBEC noncancerous cholangiocyte cell line (P<0.01; Fig. 1B). These results indicated that FLVCR1-AS1 may act as an oncogene in CCA. HuCCT1 and CCLP1 cells exhibited the highest expression of FLVCR1-AS1 and were used for subsequent experiments.

Figure 1. Expression of FLVCR1-AS1 in CCA tissues samples cell lines. (A) The expression level of FLVCR1-AS1 in CCA tissue samples was measured by RT-qPCR (n=22). ***P<0.001. (B) RT-qPCR analysis was performed to determine the expression of FLVCR1-AS1 in the noncancerous cholangiocyte cell line HIBEC and the CCA cell lines RBE, CCLP1, HuCCT1 and HCCC-9810. Data are presented as the mean ± standard deviation. **P<0.01, ***P<0.001 vs. HIBEC. CCA, cholangiocarcinoma; FLVCR1-AS1, FLVCR1 antisense RNA 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; lncRNA, long non-coding RNA.

Downregulation of FLVCR1-AS1 inhibits cell proliferation in vitro and in vivo

To investigate whether FLVCR1-AS1 influences cell viability of HuCCT1 and CCLP1 cells, FLVCR1-AS1 was silenced by transfection with shFLVCR1-AS1. The transfection efficiency in HuCCT1 and CCLP1 cells was determined by measuring the expression level of FLVCR1-AS1 using RT-qPCR (Fig. 2A). MTT and colony formation assays were performed to detect cell proliferation and the results revealed that silencing of FLVCR1-AS1 significantly suppressed cell viability and proliferation of HuCCT1 and CCLP1 cells compared with the respective shNC groups (P<0.01; Fig. 2B and C).

Figure 2. Downregulation of FLVCR1-AS1 inhibits cell proliferation in vitro and in vivo. (A) Relative expression level of FLVCR1-AS1 was assessed by reverse transcription-quantitative polymerase chain reaction following transfection with shFLVCR1-AS1 or shNC plasmids. (B) Cell proliferation was evaluated by MTT assay following transfection with shFLVCR1-AS1 or shNC. (C) Cell cloning capabilities of HuCCT1 and CCLP1 cells transfected with shFLVCR1-AS1 or shNC were evaluated by colony formation assay, magnification, ×100. (D) Tumor growth was identified by measuring tumor volume every 7 days for 28 days after injection (n=4/group). Tumor weights isolated from nude mice in each treatment group were determined on day 28 after injection (n=4/group). Data are presented as the mean ± standard deviation. **P<0.01, ***P<0.001 vs. shNC. FLVCR1-AS1, FLVCR1 antisense RNA 1; sh, short hairpin; NC, negative control; OD, optical density.

The biological role of FLVCR1-AS1 on CCA tumor growth was evaluated in a xenograft mouse model. HuCCT1 cells, transfected with shNC or shFLVCR1-AS1, were implanted subcutaneously into mice. Subsequently, tumor growth was measured every 7 days for a period of 28 days. FLVCR1-AS1-knockdown significantly delayed tumor growth in vivo (Fig. 2D). At 4 weeks post-implantation, mice were sacrificed and tumors were harvested and weighed. Silencing of FLVCR1-AS1 significantly decreased the tumor weight (Fig. 2D).

FLVCR1-AS1 suppresses cell migration and invasion

To identify the effect of FLVCR1-AS1 on cell migration and invasion, wound-healing and Transwell assays were performed. As presented in Fig. 3A, the migration ability of cells transfected with shFLVCR1-AS1 was significantly suppressed compared with the shNC groups (P<0.01). The invasion of HuCCT1 and CCLP1 cells transfected with shFLVCR1-AS1 was also significantly reduced compared with the shNC groups (P<0.01; Fig. 3B). Expression levels of migration and invasion-associated proteins, including Twist, MMP-2 and MMP-9, were determined by western blot analysis. The expression levels of Twist, MMP-2 and MMP-9 in the shFLVCR1-AS1 groups were significantly decreased compared with the shNC groups (P<0.01; Fig. 3C).

Figure 3. Silencing of FLVCR1-AS1 inhibits cell migration and invasion. (A) Cell migration was assessed by wound-healing assay and (B) invasion was evaluated by Transwell assay, magnification, ×100. (C) Protein expression levels of Twist, MMP-2 and MMP-9 were determined by western blot analysis and quantified. Data are presented as the mean ± standard deviation. **P<0.01, ***P<0.001 vs. shNC. FLVCR1-AS1, FLVCR1 antisense RNA 1; sh, short hairpin; NC, negative control; MMP, matrix metalloproteinase.

FLVCR1-AS1 interacts with miR-485-5p in HuCCT1 and CCLP1 cells

To investigate the molecular mechanism by which FLVCR1-AS1 promotes CCA cell proliferation, migration and invasion, target miRNAs of FLVCR1-AS1 were predicted with miRanda. miR-485-5p, which is commonly reported to be involved in many types of human tumor (21,22), was selected as a candidate target of FLVCR1-AS1 (Fig. 4A). Furthermore, luciferase reporter vectors carrying either the predicted miR-485-5p binding site (wild-type FLVCR1-AS1) or its corresponding mutant fragment (mutant FLVCR1-AS1) were constructed. As presented in Fig. 4B, co-transfection of wild-type FLVCR1-AS1 and miR-485-5p mimic significantly reduced the luciferase activity, while co-transfection of mutant FLVCR1-AS1 and miR-485-5p mimic did not affect luciferase activity. The present study further evaluated the expression of miR-485-5p in HuCCT1 and CCLP1 cells transfected with shFLVCR1-AS1 or shNC. As presented in Fig. 4C, the expression of miR-485-5p was significantly increased in the shFLVCR1-AS1 groups compared with the shNC groups (P<0.001). Furthermore, the expression of miR-485-5p in CCA samples and normal tissues was determined by RT-qPCR. A significant decrease was observed in CCA tumor samples compared with the normal tissue samples (P<0.001; Fig. 4D). Pearson's correlation analysis revealed that FLVCR1-AS1 expression was negatively correlated with miR-485-5p expression in CCA tissues (P<0.001; Fig. 4E). These data indicated that FLVCR1-AS1 may interact with miR-485-5p in CCA cells.

Figure 4. FLVCR1-AS1 sponges miR-485-5p in CCA cells. (A) A putative binding site of miR-485-5p in FLVCR1-AS1 was predicted using miRanda. (B) Luciferase activity was measured following co-transfection with miR-485-5p mimic and wt-FLVCR1-AS1 or mut-FLVCR1-AS1. ***P<0.001 vs. NC mimic. (C) miR-485-5p expression in HuCCT1 and CCLP1 cells transfected with shFLVCR1-AS1 or shNC was detected by RT-qPCR. ***P<0.001 vs. shNC. (D) miR-485-5p expression level in CCA and normal tissues was analyzed by RT-qPCR. **P<0.01. (E) Correlation between FLVCR1-AS1 and miR-485-5p expression in CCA tissues was determined by Pearson's correlation analysis. Data are presented as the mean ± standard deviation. FLVCR1-AS1, FLVCR1 antisense RNA 1; sh, short hairpin; NC, negative control; miR-485-5p, microRNA-485-5p; wt, wild-type; mut, mutant; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCA, cholangiocarcinoma.

Discussion

A number of studies have reported that lncRNAs serve a crucial role in the occurrence, development and prognosis of malignant neoplasms, including CCA (12,23). The present study determined the expression of FLVCR1-AS1 in CCA tissues and cell lines, and examined the effect of FLVCR1-AS1 on CCA cell growth, migration and invasion. In addition, the mechanism by which FLVCR1-AS1 functions as an oncogene in CCA was evaluated.

FLVCR1-AS1 is understood to exhibit tumor-promoting activity in hepatocellular carcinoma (24). Initially, the present study identified that the expression of FLVCR1-AS1 was significantly increased in CCA tissues compared with the adjacent normal tissues. The expression of FLVCR1-AS1 was further upregulated in CCA cell lines. These results indicate that FLVCR1-AS1 may serve a role in the development and progression of CCA. CCA cell lines (HuCCT1 and CCLP1) stably transfected with shFLVCR1-AS1 or shNC were established, and cell proliferation and colony formation assays were performed. Silencing of FLVCR1-AS1 significantly inhibited cell proliferation and colony formation. Furthermore, FLVCR1-AS1-knockdown significantly inhibited tumor growth in a xenograft model, which supports the in vitro data. Therefore, silencing FLVCR1-AS1 was demonstrated to inhibit tumor growth in vitro and in vivo. Immoderate tumor cell migration and invasion promote cancer metastasis (25). The effects of FLVCR1-AS1-knockdown on cell migration and invasion were evaluated by wound-healing and Transwell assays, respectively. Knockdown of FLVCR1-AS1 significantly weakened the migration and invasion abilities of CCA cells. Additionally, increased expression levels of Twist, MMP-2 and MMP-9 serve important roles in the development of malignant tumors (26,27), and the expression levels of these proteins were identified to be significantly reduced following silencing of FLVCR1-AS1.

A previous study indicated that FLVCR1-AS1 promotes cancer cell proliferation, migration and invasion by sponging miR-513c in hepatocellular carcinoma (24). To elucidate the underlying molecular mechanisms by which FLVCR1-AS1 serves a tumor-promoting role in CCA, bioinformatics analysis and a luciferase reporter assay were performed. The analysis and reporter assay revealed that FLVCR1-AS1 competitively bound to miR-485-5p in CCA cells. miR-485-5p has been reported to act as a negative regulator in the progression of gastric and breast cancer (21,22). Previous studies have indicated that miR-485-5p exerts its antitumor effect by targeting nucleoside diphosphate linked moiety X, peroxisome proliferator-activated receptor-g coactivator-1α, flotillin-1 and survivin (28–30). In the present study, the expression of miR-485-5p in CCA tissues was significantly decreased compared with the normal tissues. Furthermore, FLVCR1-AS1 expression was identified to be inversely correlated with miR-485-5p expression in the CCA tissues.

In summary, lncRNA FLVCR1-AS1 was revealed to be significantly upregulated in CCA tissues and cell lines, and was demonstrated to function as an oncogene in CCA by sponging miR-485-5p. The present study provided novel insights that may promote understanding of the molecular mechanism of lncRNA FLVCR1-AS1 in CCA tumorigenesis and metastasis. Investigating other downstream targets of lncRNA FLVCR1-AS1 may be the focus of future studies. lncRNA FLVCR1-AS1 may serve as a potential therapeutic target and a novel diagnostic marker for CCA.

Acknowledgments

Not applicable.

Funding

This study was supported by the Shanghai Scientific Research Project Fund (grant no. 16411967000).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Authors' contributions

All authors (WB, FC, SN, HL, JY, ZS and BZ) were involved in the conception and design of this study. SN and HL performed the literature search and data extraction. SN, HL, FC, JY and ZS analyzed the data. BZ drafted the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The use of human tissues was approved by the Ethics Committee of Shanghai Seventh People's Hospital affiliated to Shanghai University of Traditional Chinese Medicine (Shanghai, China) and written informed consent was obtained from all patients. All animal experiments were performed in accordance with institutional and international animal regulations. The animal experimental protocol was approved by the Animal Care and Use Committee of Shanghai Seventh People's Hospital affiliated to Shanghai University of Traditional Chinese Medicine.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References