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. 2021 Jan 28;11(2):99. doi: 10.1007/s13205-021-02650-w

MiR-192-5p regulates the proliferation and apoptosis of cholangiocarcinoma cells by activating MEK/ERK pathway

Chaofeng Tang 1,#, Peng Yuan 1,#, Jian Wang 2, Yubo Zhang 1, Xiaowei Chang 3, Dong Jin 1, Peng Lei 1, Zhenhui Lu 1, Bendong Chen 1,✉
PMCID: PMC7843823  PMID: 33552829

Abstract

Objective

Cholangiocarcinoma (CCA) is the second most common liver cancer, characterized by late diagnosis and fatal outcome. Although miR-192-5p has been shown to have a vital role in various cancers, its role in CCA is unknown. Here, we investigated the role of miR-192-5p in CCA cell proliferation and apoptosis, and elucidated its potential mechanism of action.

Methods

The miR-192-5p expression in CCA tissues and cell lines was detected by real-time quantitative reverse transcription-polymerase chain reaction. Cell proliferation was analyzed using the cell counting Kit-8 and 5-bromodeoxyuridine staining assays, while apoptosis was examined by flow cytometry and the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay. Western blot analysis was used to measure the expression of cell proliferation and apoptosis-related proteins, as well as MEK/ERK signaling pathway-related proteins.

Results

MiR-192-5p was highly expressed in CCA tissues and cell lines. Overexpression of miR-192-5p significantly promoted CCA proliferation, and inhibited apoptosis. The MEK inhibitor, PD98059, reversed these miR-192-5p-induced effects on MEK/ERK signaling-associated protein expression, proliferation promotion, and apoptosis inhibition in TFK-1 cells.

Conclusion

MiR-192-5p promotes proliferation and suppressed apoptosis of CCA cells via the MEK/ERK pathway, which may be a potential therapeutic strategy for CCA treatment.

Keywords: MiR-192-5p, MEK/ERK, Cholangiocarcinoma, Proliferation, Apoptosis

Highlights

1. MiR-192-5p is highly expressed in CCA tissues and cell lines.
2. MiR-192-5p promotes proliferation and inhibits apoptosis of CCA cells.
3. MiR-192-5p regulates CCA process via MEK/ERK signaling pathway.
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Introduction

Cholangiocarcinoma (CCA) is a hepatocellular carcinoma that arises from bile duct epithelial cells (Klöppel et al. 2013; Rizvi and Gores 2013). The three subtypes of CCA, intrahepatic, perihilar, and distal, are based on the location of the tumor (Wu et al. 2019). CCA is the second most common primary liver cancer after hepatocellular carcinoma (Rizvi et al. 2018; Siegel et al. 2019) and its worldwide incidence is increasing (Khan et al. 2005). While surgical resection is still the most effective treatment for patients with CCA, the absence of an identifiable clinical manifestation of the disease and tumor-specific biomarkers means that most patients present with advanced disease that provides little opportunity for surgery, and often results in recurrence after curative resection (Jung et al. 2017; Liao et al. 2019; Sirica et al. 2019). The first-line treatment option for patients with advanced CCA is chemotherapy (gemcitabine in combination with cisplatin); however, the median overall survival of patients is approximately 1 year (Razumilava and Gores 2014). Thus, elucidation of the molecular mechanisms and pathways associated with this disease is essential to improve the survival of CCA patients.

MicroRNAs (miRNAs) are endogenous, small (~ 21–25 bases), non-coding, highly conserved RNAs (Chan and Tay 2018; Jin et al. 2015; Weidle et al. 2019) that are involved in various cellular processes. Dysregulated miRNA expression has been associated with the development of malignant tumors, including CCA (Alcantara and Garcia 2019; Hu et al. 2016; Singh and Adams 2017; Wan et al. 2018; Wang et al. 2018). Of the previously characterized miRNAs, miR-192-5p is of particular interest due to its diverse roles in tumorigenesis. For example, in hepatocellular carcinoma, overexpression of miR-192-5p facilitates cell proliferation and metastasis by targeting SEMA3A (Yan-Chun et al. 2017), while in squamous cell lung carcinoma, miR-192-5p enhances the chemoresistance and invasiveness of cancer cells (Filipska et al. 2018). In osteosarcoma, miR-192-5p promotes tumor development and progression by targeting USP1 (Yang et al. 2015). However, the fundamental mechanisms underlying the miR-192-5p-dependent effects on proliferation and apoptosis in CCA are still poorly understood.

A recent study has demonstrated that the 3′-UTR region of epiregulin (EREG) is predicted to contain a binding site for miR-192-5p. (Morimoto et al. 2017). EREG, a member of the epidermal growth factor (EGF) family, activates the EGF receptor (EGFR) in the cell membrane, which leads to the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, and subsequent cell proliferation and differentiation (Cui et al. 2019). EREG promotes stem cell proliferation via activation of the MEK/ERK signaling pathway (Cao et al. 2013). Based on these studies, we hypothesized that miR-192-5p regulated CCA progression by targeting EREG, thereby modulating MEK/ERK activity.

Here, we examined the role of miR-192-5p on the proliferation and apoptosis of CCA cells, and found that its actions were mediated via the MEK/ERK pathway. Based on our findings, we propose that miR-192-5p may be a potential therapeutic target in pharmacological strategies for CCA.

Materials and methods

Tissue samples

A total of 21 paired samples of CCA and their adjacent normal tissues were obtained from patients who underwent surgical procedures at the local University. All of the patients provided written consent and approval was obtained from the Ethics Committee of the local University.

Cell culture and drug treatment

Human intrahepatic biliary epithelial cells (HIBECs) and human CCA cell lines (TFK-1, CCLP-1, HCCC 9810, RBE, HuCCT-1) were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Life technologies, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere of 5% CO2. PD98059 (MCE, Monmouth Junction, NJ, USA) was fully dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), then stored at −20 °C in the dark. Stock solutions were diluted according to the required concentration. HuCCT-1 cells were pretreated with 100 nM PD98059 for 48 h at 37 °C.

Cell transfection

A miR-192-5p mimic, miR-192-5p inhibitor, and their corresponding control oligonucleotides were acquired from Tsingke Biotech Co., Ltd. (Beijing, China). 50 nmol/L miR-192-5p mimic and 50 nmol/L NC mimic were transfected into HuCCT-1 cells; 200 nmol/L miR-192-5p inhibitor and 200 nmol/L NC inhibitor were transfected into CCLP-1 cells. Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for transient transfection according to the manufacturer’s instructions. The transfected cells were incubated for 48 h or 72 h before use in subsequent experiments.

Cell counting Kit-8 (CCK-8) assay

HuCCT-1 cells were transfected with 50 nmol/L miR-192-5p mimic and 50 nmol/L NC mimic; CCLP-1 cells were transfected with 200 nmol/L miR-192-5p inhibitor and 200 nmol/L NC inhibitor. After 48 h transfection, cells collected at the logarithmic growth phase were digested with 0.25% trypsin. The cell suspension was seeded into 96-well plates at 104 cells/well (100 μL). The wells on the margin area were filled with sterile PBS, and the cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 for 0 h, 24 h, 48 h and 72 h. 10 μL CCK-8 solution was added to each well and cells were cultured at 37 °C for 4 h in a humidified atmosphere of 5% CO2. The optical density (OD) value was determined with a microplate reader at a wavelength of 450 nm.

The 5-bromodeoxyuridine (BrdU) staining assay

HuCCT-1 cells were transfected with 50 nmol/L miR-192-5p mimic and 50 nmol/L NC mimic; CCLP-1 cells were transfected with 200 nmol/L miR-192-5p inhibitor and 200 nmol/L NC inhibitor. After 48 h transfection, cell were seeded in 96-well plates for 5 days. Cell proliferation was determined using a BrdU staining ELISA Kit (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.

Flow cytometry assay

CCLP-1 cells and HuCCT-1 cells were seeded in 6-well plates (1 × 105 cells/well). HuCCT-1 cells were transfected with 50 nmol/L miR-192-5p mimic and 50 nmol/L NC mimic; CCLP-1 cells were transfected with 200 nmol/L miR-192-5p inhibitor and 200 nmol/L NC inhibitor. After 48 h transfection, annexin V-FITC and/or propidium iodide (PI) were added to collected cells and the cells were incubated in the dark at room temperature for 15 min. Next, the binding buffer (0.1 M HEPES, pH 7.4, 1.4 M NaCl, 25 mM CaCl2, dilute to 10% before use) was added, and apoptotic cells were detected using a BD FACSC anto II flow cytometer (BD Biosciences, San Jose, CA). FITC-positive and PI-negative cells were considered to be apoptotic.

Terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay

Apoptotic cells were visualized using the Dead-End Colorimetric TUNEL system (Promega). Briefly, cells were fixed with 4% paraformaldehyde (PFA), then permeabilized using 0.1% Triton X-100 (Sigma-Aldrich). Apoptotic cells were stained using the TUNEL assay and visualized under a fluorescence microscope according to the manufacturer’s instructions.

Real-time quantitative reverse transcription-polymerase chain reaction (q-RT-PCR)

Total RNA was extracted from samples using TRIzol. The cDNA was synthesized using the PrimeScript RT reagent kit (TaKaRa Bio, Dalian, China) according to the manufacturer’s instructions. MiR-32-5p expression was detected using the SYBR Green kit (TaKaRa, Otsu, Shiga, Japan). The designed primers were as follows: miR-192-5p, forward: 5ʹ-GGACTTTCTTCATTCACACCG, reverse: 5ʹ-GACCACTGAGGTTAGAGCCA-3′; Ki-67, forward: 5′-GAGGGCAAGTACGAGTGGCA-3′, reverse: 5′-GCAGGTCGCTTCCTTATTCC-3′; PCNA, forward: 5ʹ-GCGTGTGCCTGTGACAGTTA-3ʹ, reverse: 5ʹ-CCTAGCGTTTTTGCTTCCCTT-3ʹ; Bax, forward: 5′-CCTTTTGCTTCAGGGTTTCAT-3′, reverse: 5′-CTCCATGTTACTGTCCAGTTCGT-3′; and GAPDH, forward: 5ʹ-GGAAAGCTGTGGCGTGAT-3ʹ, reverse: 5ʹ-AAGGTGGAAGAATGGGAGTT-3ʹ. Results obtained were calculated by the 2−ΔΔCt method.

Western blot

Protein concentrations in cell lysates were quantified using the BCA kit (Beyotime, Shanghai, China). A total of 20 μg of protein from each sample was used for western blot analysis. The following primary antibodies were used: MEK1/2 (1:1,000, #4694, Cell Signaling Technology), phospho-MEK1/2 (1:2,000, #2338, Cell Signaling Technology), Erk1/2 (1:1,000,#4695,Cell Signaling Technology), phospho-Erk1/2 (1:1,000,#9101,Cell Signaling Technology), c-myc (1:1,000,#9402,Cell Signaling Technology), Ki-67 (1:5,000,ab92742,Abcam), PCNA (1:1,000, ab13110, Abcam), Bcl-2 (1:1,000, ab15071, Abcam), Bax (1:5,000,ab32503,Abcam), Caspase-3 (1:2,000, ab184787, Abcam), and GAPDH (1:500, ab8245, Abcam). The membranes were incubated with primary antibodies overnight at 4℃. The membranes were washed thrice with PBS and then incubated with HRP Goat Anti-Rabbit (IgG) secondary antibody (1:2,000, ab6721, Abcam) for 1 h at room temperature. The membrane was washed thrice with PBS for 5 min, then visualized using enhanced chemiluminescence (ECL, Thermo Fisher Scientific). GAPDH was used as the loading control. All data analyses were repeated three times independently.

Statistical analysis

All experiments were performed in triplicate. The mean ± SD was calculated to evaluate the data. Where appropriate, we used the Student's t test and the Mann–Whitney U test for two-group comparisons, while multiple groups of data were analyzed using One-way ANOVA. p < 0.05 was considered to be statistically significant. Data were analyzed using SPSS software (version19).

Results

MiR-192-5p expression is significantly upregulated in CCA tissues and cells.

To confirm whether the expression of miR-192-5p was different between CCA and paracancerous tissues, we examined miR-192-5p expression by q-RT-PCR. As shown in Fig. 1a, miR-192-5p expression was markedly upregulated in CCA tissues compared with adjacent non-cancerous tissues (p < 0.001). Similarly, miR-192-5p expression was increased in the CCA cell lines, TFK-1, CCLP-1, HCCC9810, RBE, and HuCCT-1 compared to the control HIBECs (p < 0.001, Fig. 1b). These data suggested that miR-192-5p may have a role in CCA progression.

Fig. 1.

Fig. 1

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Relative expression of miR-192-5p in CCA tissues and cells. a Relative expression of miR-192-5p in CCA tissues and adjacent normal tissues was examined by q-RT-PCR. b Relative expression of miR-192-5p in CCA cell lines and human intrahepatic bile ducts endothelial cells (HIBEC) was examined by q-RT-PCR. CCA cholangiocarcinoma; ***p < 0.001

MiR-192-5p promotes CCA cell proliferation.

To investigate the role of miR-192-5p on CCA cell proliferation, cells were transfected with either a miR-192-5p mimic or miR-192-5p inhibitor. Increased cell viability and number of BrdU-positive cells were observed by CCK-8 and BrdU staining assays in CCA cells treated with the miR-192-5p mimic compared to the NC mimic group. In contrast, decreased viability and number of BrdU-positive cells were observed in the miR-192-5p inhibitor group (p < 0.01, Fig. 2a, b). The mRNA and protein expression levels of the cell proliferation-related proteins, Ki-67 and PCNA, were significantly increased in miR-192-5p mimic-transfected CCA cells compared to the NC mimic group. In contrast, Ki-67 and PCNA mRNA and protein levels were significantly decreased in the miR-192-5p inhibitor group (p < 0.05, Fig. 2c, d). Taken together, our results demonstrated that miR-192-5p promotes CCA cell proliferation.

Fig. 2.

Fig. 2

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Effects of miR-192-5p on the proliferation of CCA cells. HuCCT-1 cells were transfected with miR-192-5p mimic and its negative controls; CCLP-1 cells were transfected with miR-192-5p inhibitor and its negative controls. a CCK-8 assay was used to detect cell proliferation. b BrdU staining was used to detect cell proliferation; c The mRNA expression of Ki-67 and PCNA was detected by q-RT-PCR. d Western blot was used to detect the protein expression of Ki-67 and PCNA. CCA cholangiocarcinoma; *p < 0.05, **p < 0.01

MiR-192-5p suppresses apoptosis of CCA cells.

Both flow cytometry and TUNEL staining revealed significantly lower rates of apoptosis in miR-192-5p mimic-treated cells than NC mimic-treated cells. In contrast, increased levels of apoptosis were observed after treatment with the miR-192-5p inhibitor (p < 0.05, Fig. 3a, b). The mRNA and protein expression levels of Bcl-2, a pro-survival protein, and Bax and Caspase-3, pro-apoptotic proteins were detected by q-RT-PCR and western blot analysis. A significant increase in Bcl-2 mRNA and protein expression, with a concomitant significant decrease in Bax and Caspase-3, was observed in miR-192-5p mimic-treated CCA cells compared to the NC mimic-treated group. In contrast, a decrease in Bcl-2 and an increase in Bax and Caspase-3, was observed after treatment with the miR-192-5p inhibitor (p < 0.05, Fig. 3c, d). Taken together, our results showed that miR-192-5p can inhibit CCA cell apoptosis.

Fig. 3.

Fig. 3

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Effects of miR-192-5p on the apoptosis of CCA cells. HuCCT-1 cells were transfected with miR-192-5p mimic and its negative controls; CCLP-1 cells were transfected with miR-192-5p inhibitor and its negative controls. a Apoptosis was detected by flow cytometry assay. b Apoptosis was detected by TUNEL staining; c The mRNA expressions of Bcl-2, Bax and caspase-3 were detected by q-RT-PCR. d The protein expressions of Bcl-2, Bax and caspase-3 were detected by western blot. CCA cholangiocarcinoma; *p < 0.05,

MiR-192-5p activates the MEK/ERK signaling pathway

Because our data suggested that miR-192-5p had a role in the regulation of proliferation and apoptosis of CCA cells, we next examined the role of miR-192-5p in the activation of the MEK/ERK signaling pathway in CCA. p-MEK1/2, p-ERK1/2, and c-myc protein expression levels were increased in miR-192-5p mimic-treated TFK-1 cells compared with the control NC mimic group (p < 0.05, Fig. 4a, b). In contrast, treatment with the miR-192-5p inhibitor led to a decrease on mRNA and protein expression of p-MEK1/2, p-ERK1/2, and c-myc (p < 0.05, Fig. 4a, b). We then treated miR-192-5p mimic-transfected HuCCT-1cells with the MEK inhibitor, PD98059, or DMSO for the control group. We found that p-MEK1/2, p-ERK1/2 and c-myc expression levels were significantly reduced in the PD98059-treated group, compared with the DMSO-treated group (p < 0.05, Fig. 4c, d). Taken together, these findings indicated that miR-192-5p can activate the MEK/ERK signaling pathway in CAA cells.

Fig. 4.

Fig. 4

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Mir-192-5p activates MEK/ERK signaling pathway. HuCCT-1 cells were transfected with miR-192-5p mimic and its negative controls; CCLP-1 cells were transfected with miR-192-5p inhibitor and its negative controls. HuCCT-1 cells were pretreated with 100 nM PD98059 and then were transfected with miR-192-5p mimic. a, b Western blot was used to detect the effect of miR-192-5p interference on the protein expression of MEK/ERK signaling pathway-related proteins. c, d Western blot was used to detect the protein expression of MEK / ERK signaling pathway-related proteins in HuCCT-1 cells treated with PD98059. CCA cholangiocarcinoma; *p < 0.05

MiR-192-5p regulates proliferation and apoptosis of CCA cells via the MEK/ERK signaling pathway

Based on these findings, we speculated that MEK/ERK signaling may be involved in the miR-192-5p-dependent regulation of proliferation and apoptosis of CCA cells. Addition of PD98059 to miR-192-5p-treated HuCCT-1 cells reduced the proliferation induced by the miR-192-5p mimic while increasing apoptosis (p < 0.05, p < 0.01, Fig. 5a, b, c). A reduction in proliferation-related protein (Ki-67, PCNA) and pro-survival protein (Bcl-2) protein expression levels were observed after treatment of miR-192-5p-treated HuCCT-1 cells with PD98059, along with a concomitant increase in pro-apoptotic protein (Bax, Caspase-3) protein expression levels (Fig. 5d). Our results indicated that miR-192-5p regulates CCA proliferation and apoptosis via the MEK/ERK signaling pathway.

Fig. 5.

Fig. 5

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MiR-192-5p regulates proliferation and apoptosis of CCA cells via MEK/ERK signaling pathway. HuCCT-1 cells were pretreated with 100 nM PD98059 and then were transfected with miR-192-5p mimic. a CCK-8 assay was used to detect cell proliferation. b Apoptosis was detected by flow cytometry assay. c BrdU staining was used to detect cell proliferation and TUNEL was used to detect cell apoptosis. d Western blot was used to detect the protein expression of cell proliferation-and-apoptosis-related factors. CCA cholangiocarcinoma; *p < 0.05, **p < 0.01

Discussion

CCA is one of the most malignant diseases in the world, but the development of effective treatment regimes remains challenging due to uncontrolled tumor growth and distant metastasis (Liao et al. 2019). Surgical resection and conventional chemotherapy are not effective in prolonging long-term survival due to the late diagnosis and early metastasis of the disease (Blechacz and Gores 2008). Hence, the development of new strategies for the diagnosis and treatment of CCA is urgently required. In this study, we explored the molecular mechanisms underlying the tumorigenesis and progression of CCA to identify novel therapeutic targets for CCA.

The miRNAs typically function as post-transcriptional regulators of gene expression. The miRNAs interact with the 3′UTR of target mRNAs to reduce their stability or translational efficiency (Chiaretti et al. 2014; Li et al. 2013). The miRNAs are essential for multiple biological processes including cell proliferation, apoptosis, migration, and autophagy (Lai 2002; Stark et al. 2005). Previous studies have demonstrated that a variety of miRNAs are dysregulated in many human cancers. A number of these miRNAs, including miR-383, miR-92b, miR-378, miR-19b, miR-124, and miR-424, have been associated with CCA (Ma et al. 2018; Wan et al. 2018; Wu et al. 2019; Zhou et al. 2018; Zhou and Ma 2019). Recently, miR-192–5p has been found to be markedly upregulated in prostate cancer tissues (Chen et al. 2019). In addition, elevated miR-192 expression has been reported in CCA serum, indicating that miR-192 could be a potential diagnostic marker for CCA (Loosen et al. 2019). However, a role for miR-192-5p in CCA tumorigenesis and progression has yet to be shown. In the present study, we examined the effects of miR-192-5p on CCA cell proliferation and apoptosis. We found that the expression of miR-192-5p was significantly upregulated in CCA tissues and cell lines compared to adjacent normal tissues and HIBECs, suggesting that miR-192-5p may promote tumorigenesis and development in human CCA.

Uncontrolled cell proliferation or dysregulated apoptosis can lead to tumorigenesis (Wang et al. 2013). Recently, miR-192-5p was shown to be involved in regulating the effects of intestinal microflora metabolites on colon cancer cell proliferation (Zhao et al. 2020), while Chen et al. demonstrated that overexpression of miR-192–5p promoted proliferation of prostate cancer cells (Chen et al. 2019). Consistent with these studies, we found that miR-192-5p enhanced proliferation in CCA cells.

Apoptosis plays a vital role in cell death and survival, and its dysregulation is regarded as one of the main causes of cancer development (Yang et al. 2016). Here, we found that miR-192-5p over-expression significantly suppressed apoptosis in HuCCT-1 cells. This suppression was accompanied by a significant increase in expression of the pro-survival marker, Bcl-2, and a significant decrease in the expression of the pro-apoptotic markers, Bax, and Caspase-3. These findings suggested that miR-192-5p suppressed apoptosis in CCA cells.

The MEK/ERK signaling pathway is involved in mediating diverse cellular functions including proliferation, differentiation, migration, and apoptosis (Bai et al. 2014; Reddy et al. 2003). Abnormal expression of the MEK/ERK pathway associated with dysregulated miRNAs has been reported in a number of cancers. For example, Qin et al. demonstrated that miR-96-5p promotes the migration of breast cancer cells by activation of the MEK/ERK pathway (Qin et al. 2020) whereas Yang et al. found that miRNA-133a suppresses the MEK/ERK signaling pathway in esophageal cancer (Yang et al. 2017). Here, we demonstrated that miR-192-5p activated the MEK/ERK signaling pathway in CCA cells, indicating that MEK/ERK signaling may have a role in the regulation of proliferation and apoptosis in CCA cells.

To determine whether the MEK/ERK pathway was responsible for the tumor-promoting effects of miR-192-5p, we performed rescue experiments. We found that the MEK inhibitor, PD98059, reversed the proliferation-promoting and apoptosis-inhibiting effects of the miR-192-5p mimic on HuCCT-1 cells. These findings suggested that miR-192-5p promoted proliferation and inhibited apoptosis through the MEK/ERK signaling pathway.

Multiple studies have suggested that miRNAs may promote tumorigenesis through the MEK/ERK signaling pathway in a diverse range of cancers including hepatocellular carcinoma (Zhang et al. 2020), esophageal cancer (Yang et al. 2017), and Ewing's sarcoma (Ye et al. 2018). Here, we showed that miR-192-5p mediates CCA cancer progression via the MEK/ERK pathway.

Conclusion

Our present study showed that miR-192-5p enhanced proliferation and repressed apoptosis of CCA cells through upregulation of the MEK/ERK signaling pathway. To our knowledge, this is the first time that a role for the miR-192-5p/MEK/ERK axis in the modulation of CCA cell proliferation and apoptosis has been described. Our data provided insight into the potential mechanisms underlying CCA tumorigenesis. Furthermore, our finding indicated that the miR-192-5p/MEK/ERK axis could be a potential therapeutic target for patients with CCA.

Author contributions

CT and BC conceived and designed the project, CT, PY, JW and Yubo Zhang acquired the data, Xiaowei Chang, Dong Jin and Peng Lei analysed and interpreted the data, Zhenhui Lu and Bendong Chen wrote the paper.

Availability of data and materials

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

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Ethical approval and consent to participate

This study was approved by the hospital ethics committee. Informed consent was obtained from all participating subjects.

Footnotes

Chaofeng Tang and Peng Yuan contributed equally to this work and should be regarded as co-first authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

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

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