Introduction
Hepatocellular carcinoma (HCC) is a common primary liver neoplasm with high mortality, and it imposes a significant health and economic burden worldwide.1 HCC risk factors include hepatitis virus B and/or C infection and alcohol consumption leading to the dysregulation of cell signaling transduction pathways, such as MAPK, AKT and ERK, imbalance between the activities of proto-oncogenes and tumor suppressor genes, and immortal proliferation of liver cancer stem cells.2–4 High HCC mortality and poor prognosis are mainly due to tumor metastasis, and the underlying molecular mechanism of HCC metastasis has been extensively studied. Many genes participate in the process of HCC metastasis, including EGFR, TP53, APP, VEGFA, MAPK1, PI3KCA, and MMP9.5 However, further investigation of the genes and gene pathways in HCC metastasis could help us control HCC more effectively in the future.
Dermcidin (DCD) was originally identified in eccrine sweat glands. With a molecular weight of 11.2 kDa, as a precursor protein, DCD is composed of 110 amino acid residues.6,7 After the removal of the first 19-amino acid signal peptide, the precursor matures to a secreted protein with a 9.5 kDa molecular weight. In eccrine sweat, DCD is further proteolytically processed into many active peptides with different antimicrobial activities.6–9 Moreover, DCD is putatively produced and processed by cancer cells, including those of melanoma,10 pancreatic cancer,11,12 breast cancer,11,13,14 gastroesophageal tumors,15 leukemia,16,17 and HCC.18 DCD participates in oncogenesis19 and induces cancer cachexia and cancer cell growth and survival20 but reduces serum dependency19 for tumor invasion21 and migration.22 Thus, DCD expression may contribute to cancer progression and a poor cancer prognosis.13
In HCC, DCD has been reported as a factor affecting survival.12 DCD levels were found to be significantly elevated in HCC tissues and the sera of patients, and serum DCD levels were associated with tumor metastasis and thus could potentially be a biomarker for HCC diagnosis.23 Previous studies also revealed that noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), a Src homology (SH) 2 and SH3 domain-bearing protein, can bind to Wiskott-Aldrich syndrome protein (WASP) and modulate reorganization of the actin cytoskeleton and cell mobility.24 The phosphotyrosine residue at position 20 of the DCD molecule was found to be crucial to its interaction with Nck1.18 Moreover, actin cytoskeleton reorganization caused by different Rho GTPases (e.g., Ras-related C3 botulinum toxin substrate 1 [Rac1] and cell division control protein 42 homologue [Cdc42]) binds to WASP and activates the WASP/ actin-related protein 2/3 (Arp2/3) complex25 for cell growth, migration, and adhesion.26–29 In this context, fibronectin, a glycoprotein of the extracellular matrix,30 also influences cell growth, adhesion, migration, and/or differentiation.30 Dysregulated fibronectin expression was found to cause cancer and liver fibrosis.31,32 Other publications showed that DCD can bind to the Nck1 SH2 domain and activate Rac1, Cdc42,18 and ρ-GTPases18 to promote HCC cell migration;18 although, the signaling mechanisms that facilitate cell adhesion, growth and migration in HCC have not been thoroughly elucidated.
In this study, we assessed the effects of DCD overexpression and knockdown on the regulation of HCC cell phenotypes and the expression of Nck1, Rac1, Cdc42, WASP, and Arp2/3 in vitro and in nude mice.
Methods
Patients
The study protocol was approved by the Institutional Human Research Committee of Guangzhou Hospital of Traditional Chinese Medicine (Guangzhou, China), with approval number 2015NK001, and conducted following the standards set by the Declaration of Helsinki. Each patient provided written informed consent. The study cohort included 105 patients with HCC and 42 healthy controls (non-cirrhotic and non-HCC individuals).
The patients were treated at Guangzhou Hospital of Traditional Chinese Medicine between October 2016 and May 2017. There were 83 men and 22 women, with a median age of 56.37 years. The control individuals visited our hospital for an annual health check, and had no abnormal findings. The patients and healthy controls were demographically matched. The inclusion criteria were HCC diagnosed histologically according to the Standardization of Diagnosis and Treatment for Hepatocellular Carcinoma (2017 edition) without any pretreatment or other malignancies and aged 18 years or older.
Enzyme-linked immunosorbent assay (ELISA)
Blood samples were requisitioned from both the patients and controls for ELISA analysis of the serum DCD levels using a human DCD ELISA kit (Cat. #KT-13259; Kaniya Biomedical, Fullerton, CA, USA). According to the manufacturer, this ELISA was a double-antibody sandwich type with a high sensitivity and excellent specificity for DCD detection, with no significant cross-reactivity or interference between DCD and its analogue using the DCD antibody that recognizes Homo sapiens antigen. The procedures were conducted in accordance with the manufacturer’s protocol. In brief, 100-µL serum samples were added to ELISA plates in triplicate and incubated at 37°C for 2 h. The solution was then rinsed out, and Buffer A from the kit was added for incubation at 37°C for 1 h. The wells were washed 3 times with 350 µL of the washing solution for 2 m, after which 100 µL of Buffer B was added and incubated at 37°C for 30 m. Thereafter, the wells were washed 5 times with the washing solution and 90 µL of the color solution was added; after incubation at 37°C for up to 25 m in the dark, stop solution was added and mixed well. Then, the solution was measured at 450 nm using a spectrophotometer (Fenghua, Guangzhou, China). The measurement was repeated at least once.
Cell line, culture, and transfection
HCC SK-HEP-1 cells were purchased from the cell bank of the Chinese Academy of Medical Sciences (Shanghai, China) and grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Clayton, VIC, Australia) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified incubator containing 5% CO2 at 37°C.
The DCD cDNA was PCR-amplified and subcloned into pReciever M06 (FulenGen, Guangzhou, China), while the GST-tagged SH2 domain of Nck cDNA was constructed using PCR amplification with a human Nck cDNA template and ligated into pGEX-4T-3 (GE Healthcare, Fairfield, CT, USA). After confirming the DNA sequencing, these vectors were transfected into SK-HEP-1 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. DCD small interfering (si)RNA and negative control siRNA (NC) were obtained from FulenGen and transfected into the SK-HEP-1 cells using Lipofectamine 2000. The DCD siRNA targeting sequence was 5′-AGACGTCCTTGACTCAGTA-3′, while the NC sequences were not disclosed by the manufacturer.
Immunofluorescent detection of NCK and DCD in SK-HEP-1 cells
DCD cDNA-, siRNA-, or NC-transfected SK-HEP-1 cells were subjected to immunofluorescence staining of DCD and NCK proteins using anti-DCD and NCK antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), respectively. The procedures were conducted in accordance with the manufacturer’s recommended protocol. Images were then captured with a fluorescence microscope (Olympus, Tokyo, Japan).
Western blot analysis
Western blotting was utilized to assess changed protein levels according to a previous study.33 Primary antibodies against DCD, Nck1, and GAPDH were purchased from Abcam (Cambridge, MA, USA), Santa Cruz Biotechnology, and Sigma-Aldrich (St. Louis, MO, USA), respectively.
RT-quantitative (q)PCR
Total cellular RNA was isolated using TRIzol reagent (Invitrogen) in accordance with the manufacturer’s instructions. cDNA was synthesized via reverse transcription with 1-µg RNA samples of each using a Geneseed II First Strand cDNA Synthesis Kit (Geneseed Biotech, San Diego, CA, USA).
qPCR was then used to amplify different genes using Geneseed qPCR SYBR Green Master Mix in an ABI Prism 700 machine in accordance with the manufacturer’s protocol. The primer sequences are listed in Supplementary Table 1. The PCR conditions were set to a hot start at 95°C for 5 m, followed by 40 cycles of 95°C for 10 s and 60°C for 34 s, and a melting program of 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The relative level of mRNA was normalized to GAPDH mRNA and expressed as 2−ΔΔCt.
Transwell assay
SK-HEP-1 cells (0.5–1.0×105 cells/well) were cultured in the upper chambers of 24-well Transwell plates with 8.0-µm pore filters (BD Biosciences, San Jose, CA, USA). The bottom chambers were filled with complete cell growth medium. The cells were treated in duplicate with vehicle alone, doxorubicin (Adriamycin; 5 µg/mL), or different concentrations (10–30%) of Mahong and cultured for 8 h. The difference between the migration and invasion assays was the filter used in the Transwells, i.e. precoated with 50 µL of Matrigel (BD Biosciences) for the invasion assay or not subjected to any pretreatment for the migration assay.
The cells on the surface of the upper chamber membrane were carefully removed using cotton swabs, and the migrated or invaded cells at the bottom surface of the upper chamber membrane were fixed and stained with 0.5% crystal violet in 70% ethanol and photographed under a light microscope (YS100; Nikon, Tokyo, Japan). The numbers of migrated or invaded cells in five fields of each upper chamber were counted in a blinded manner. For the invasion assay, the Transwell filters were precoated with Matrigel (BD Biosciences) for 24 h.
Rac1 and Cdc42 activation assay
The intracellular activity of the Rac1 and Cdc42 GTPases was measured with Rac1 and Cdc42 activation assay kits (Upstate Biotechnology, Lake Placid, NY, USA), respectively, and conducted in accordance with the manufacturer’s instructions. In particular, cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with Mg2+ lysis/wash buffer. The samples were clarified using glutathione agarose and quantified, and equal aliquots of protein were incubated with the Rac/Cdc42 assay reagent (PAK-1PBD, agarose) at 4°C for 1 h. GTP-S-pretreated lysate was used as a positive control. GTP-bound Rac1 and Cdc42 were precipitated and eluted in Laemmli reducing sample buffer, resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted with monoclonal anti-Rac1 or Cdc42 antibody. The cell lysate (5%) was also subjected to Western blotting using these two monoclonal antibodies to assay the total amount of Rac1 and Cdc42.
SK-HEP2 cell orthotopic mouse model
The animal study protocol was approved by the ethics committee of Guangzhou Hospital of Traditional Chinese Medicine. This study was conducted in accordance with the Guidelines of the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research. Specifically, 20 male Balb/c nude mice, 6–7 weeks-old, were obtained from the Animal Experimental Center of Guangzhou Yuanshen Biomedical Technology (Guangzhou, China), quarantined in a specific pathogen-free (SPF) barrier facility, and housed under controlled temperature and humidity with alternating 12-h light and dark cycles. The mice received SPF mouse chow and were allowed to drink sterile water ad libitum for 7 days before the experiments.
For our experiments, SK-HEP2-luc and HEP2-luc-DCD cells (1×106 cells/injection), obtained by the stable transfection of DCD cDNA into SK-HEP-1 cells using lentivirus carrying pCDH-DCD-copGFP or pCDH-DCD-LUC-copGFP, were injected into mouse liver. The mice were anesthetized using 3% isoflurane and the abdomen opened to expose the liver for a direct injection of the tumor cells (2×107 cells/mL in 50 µL PBS) into the liver. The injection site was sealed with biogum, and the abdomen was then sutured. These mice had free access to food and water; water bottles were fitted with extended 3.5-inch spouts. Mice that were unable to walk were hand-watered. The mice were left to recover. The mice were monitored daily, and the data were recorded every 3 days. On day 26 after tumor cell transplantation, metastatic lesions within the liver and abdomen were examined under luciferin light (Abcam). At the end of the experiments, the mice were euthanized by intraperitoneal injection of 160 mg/kg sodium pentobarbital.
Statistical analysis
The data were expressed as the mean±standard deviation of three independent experiments. Statistical analyses of normally distributed continuous data were performed using the unpaired t-test. Comparisons of multiple groups of data with confidence intervals among and between groups were analyzed using one-way analysis of variance and then Bonferroni correction or Dunnett’s tests, respectively. The diagnostic value of the serum DCD level in patients was assessed using a receiver operating characteristic (ROC) curve and logistic regression analyses by calculation of the area under the ROC curve (AUC). All statistical analyses were performed using Statistica 19.0 (Palo Alto, CA, USA). A p value <0.05 was considered statistically significant.
Results
Demographic and clinicopathological characteristics of patients
Our data showed no significant difference in the demographic data between the patient and control groups (Supplementary Table 2). However, the serum DCD levels were significantly higher in the patients than in the healthy controls. The ROC analysis showed that DCD could be used as a diagnostic marker of HCC (AUC=0.856, 95% confidence interval [CI]: 0.789–0.908). The optimum DCD cutoff value was 18.87 ng/mL, with sensitivity of 74.29% (95% CI: 64.8–82.3%) and specificity of 92.86% (95% CI: 80.5–98.5%); the positive predictive value and negative predictive value at this cutoff level were 96.3% (95% CI: 89.6–99.2%) and 59.1% (95% CI: 46.3–71.0%), respectively (Table 1, Fig. 1 and Supplementary Table 2).
Table 1ROC analysis of the DCD value in HCC diagnosis
| Value | 95% CI |
|---|
| AUC | 0.856 | 0.789–0.908 |
| DCD cutoff | 18.87 | – |
| Se | 74.29 | 64.8–82.3 |
| Sp | 92.86 | 80.5–98.5 |
| +LR | 10.4 | 3.5–31.1 |
| –LR | 0.28 | 0.2–0.4 |
| +PV | 96.3 | 89.6–99.2 |
| –PV | 59.1 | 46.3–71.0 |
Upregulated expression of DCD and Nck1 in HCC cells
DCD protein was able to bind to the Nck1-SH2 domain in HCC tissues. Their coexpression in vitro was verified using immunofluorescence microscopy. DCD overexpression plasmids or DCD-siRNA were transfected for 48 h into SK-HEP-1 cells, and the DCD and NCK levels were detected by immunofluorescence. Images were captured under an immunofluorescence microscope. The data showed that both DCD and NCK were localized in the cytoplasm and nuclei in the control cells and increased after DCD cDNA transfection. However, DCD-siRNA transfection led to the expression of both DCD and NCK in the nuclei (although their levels were lower; Fig. 2).
Effects of DCD on the expression of Rac1, Cdc42, fibronectin, WASP, and Arp2/3 in SK-HEP-1 cells
DCD cDNA-transfected SK-HEP-1 cells showed a significant increase in the mRNA levels of DCD, Arp2/3, WASP and fibronectin, whereas transfection of DCD-siRNA significantly decreased the mRNA levels of these genes (Fig. 3A, E), while transfection of DCD-siRNA significantly increased the level of Arp2/3 (mRNA; Fig. 3C). Moreover, transfection of DCD cDNA also significantly induced the protein levels of DCD, active Rac1, active Cdc42, fibronectin, WASP, and Arp2/3, whereas transfection of DCD-siRNA significantly decreased the protein levels of DCD, active Rac1, active Cdc42, fibronectin, WASP, and Arp2/3 in HCC cells (Fig. 3F, G).
Effects of DCD manipulation on SK-HEP-1 cell migration and invasion
Transfection of DCD cDNA significantly increased tumor cell migration and invasion, whereas transfection of DCD-siRNA significantly decreased these cell numbers in SK-HEP-1 cells (Fig. 4A, B).
Rac1 attenuation of DCD-siRNA-mediated inhibition of SK-HEP-1 cell migration and invasion
Transfection of Rac1 cDNA significantly increased HCC cell migration and invasion, and transfection of Rac1 cDNA into HCC cells after DCD-siRNA transfection also attenuated the inhibitory effects of DCD-siRNA on SK-HEP-1 cell migration and invasion (Fig. 4C, D).
Rac1 rescued the expression of active Rac1/total Rac1, WASP, Arp2/3, and fibronectin in DCD-siRNA-transfected SK-HEP-1 cells
The levels of different proteins were assessed after transfection of Rac1 into SK-HEP-1 cells with previous DCD-siRNA transfection. The data showed a significant decrease in the levels of DCD, active Rac1/total Rac1, WASP, Arp2/3, and fibronectin proteins in DCD-siRNA-transfected SK-HEP-1 cells (Fig. 5). Transfection of Rac1 cDNA in SK-HEP-1 cells significantly (p<0.01) increased the levels of active Rac1/total Rac1, WASP, Arp2/3 and fibronectin protein. However, cotransfection of DCD-siRNA and Rac1 cDNA led to the levels of active Rac1/total Rac1, WASP, Arp2/3 and fibronectin proteins reaching those of the control in SK-HEP-1 cells.
Effect of DCD on the regulation of SK-HEP-1 cell metastasis in mice
SK-HEP2-luc and HEP2-luc-DCD cells were injected into mouse liver, and on day 26 after transplantation, fluorescence revealed that two-thirds of the mice implanted with HEP2-luc-DCD cells showed metastatic lesions in the liver and abdomen. SK-HEP2-luc cells only showed metastatic lesions within the liver but without abdominal lesions in the mice (Fig. 6).
Discussion
In the current study, we found that the DCD level was higher in the sera of patients with HCC and in SK-HEP-1 cells. The detection of serum DCD levels could be used as a diagnostic marker for HCC. Furthermore, both DCD overexpression and knockdown influenced the expression of Nck1, Rac1, Cdc42, fibronectin, WASP, and Arp2/3, the ability of HCC cells to migrate and invade in vitro and tumor cell metastasis in nude mice. These results showed that DCD-promoted HCC cell migration, invasion, and metastasis were affected through regulation of NCK and its downstream signaling pathway (Fig. 7).
Indeed, our current data on an increase in serum DCD levels are consistent with our previous study23 and support the possibility of serum DCD levels being used as a biomarker for HCC diagnosis.23 However, there was a different serum DCD cutoff value used between the current study and our previous study,23 i.e. 18.87 ng/mL and 25.75 ng/mL, respectively. This may be due to the differences in sizes of the patient and control populations of the two studies. A future study with a larger sample size from multiple institutions is needed to verify the true cutoff for serum DCD in HCC diagnosis. Moreover, DCD overexpression led to increases in HCC cell migration and invasion, while knockdown of DCD expression reduced the capacity of SK-HEP-1 cell migration and invasion in culture, and DCD overexpression induced HCC cell abdominal metastasis in vivo. Similar data have been observed in other cancers19–22 with further confirmation that DCD expression is associated with cancer progression and unfavorable prognosis.13 Taken together, DCD has the potential to be used as a diagnostic and prognostic biomarker for HCC.
The colocalization of DCD and Nck1 proteins in SK-HEP-1 cell cytoplasm and nuclei was consistent with the data showing that DCD binds to the Nck1-SH2 domain in HCC after coimmunoprecipitation-Western blot analysis.18 Nck1, as a SH2 and SH3 domain-bearing protein, can bind to WASP and modulate actin cytoskeleton reorganization through the WASP/Arp2/3 complex and promote cell directional migration via pseudopodia.24 Indeed, the phosphorylated tyrosine residue at position 20 of the DCD molecule was crucial to its interaction with Nck1.18 However, tyrosine residue 20 is not a part of the signaling peptides, the survival promoting peptide (Y-P30) or proteolysis inducing factor. It may not be possible to attribute the DCD function to interaction with Nck1; thus, further investigation using specific antibodies and genetic approaches is needed to better understand the underlying molecular mechanisms. Moreover, our observations showed that overexpression of DCD increased the expression of Nck1 and cell migration and invasion, whereas knockdown of DCD expression decreased the expression of Nck1. Cell migration and invasion in cultured SK-HEP-1 cells revealed that Nck1 was involved in DCD-mediated HCC progression.
Reorganization of the actin cytoskeleton could be caused by different Rho GTPases, such as Rac1 and Cdc42, after binding to WASP and activation of the WASP/Arp2/3 complex.25 Rac1, a Rho-like GTPase, modulates the cytoskeleton and influences cell growth, migration, and adhesion.26–29 Rac1 is activated to induce the establishment of actin-rich lamellipodia protrusions at the leading edge of migrating cells and drives the cell membrane to extend for cell movement,34,35 which leads to cell epithelial mesenchymal transition.36,37
Cdc42, a GTPase of the Rho family, modifies signaling pathways for diverse cellular functions, such as cell migration.38 Activated Cdc42 binds to WASP, stimulates actin cytoskeleton remodeling, promotes the formation of filopodia39–41 and pseudopodia,42 and aids cell migration, invasion and metastasis.42
The WASP family comprises five members, including WASP and N-WASP, with a similar protein structure.43 The Arp2/3 complex comprises seven-subunit proteins and plays a key role in regulation of the actin cytoskeleton.44 WASP is activated after binding to Cdc42, PIP2,43 and GTP-bound Rac1.45,46 After activation, WASP can bind to the Arp2/3 complex.47 The complex has affinity for the existing filaments and triggers elongation of a new filament as actin nucleation cores.44 The formation of filopodia is required for cell motility48 and promotes cell migration.45,46
In the current study, we also found that DCD overexpression increased the expression of WASP and Arp2/3, whereas knockdown of DCD expression decreased the expression of WASP and Arp2/3 in cultured SK-HEP-1 cells. Overexpression of DCD also increased the levels of active Rac1 and Cdc42 proteins, whereas transfection of DCD-siRNA decreased the levels of active Rac1 and Cdc42. All of these results indicate that DCD also regulates the activation of Rac1 and Cdc42 as well as the expression of WASP and Arp2/3, influences actin cytoskeleton modelling, and promotes cell migration, invasion, and metastasis in HCC (Fig. 7).18,45,46 This was further confirmed by our observation that transfection of Rac1 cDNA into DCD-siRNA-transfected SK-HEP-1 cells attenuated DCD-siRNA-induced suppression of HCC cell mobility and invasion and rescued the expression of active Rac1/total Rac1, WASP, and Arp2/3 proteins. Thus, overall, DCD protein binds to Nck through the Nck-SH2 domain to, in turn, activate WASP, leading to Rac1, Cdc42, and Arp2/3 activation and therefore to enhanced cell mobility. We also observed that DCD reduced the level of Arp2/3 mRNA but increased the level of Arp2/3 protein, indicating that DCD could regulate Arp2/3 at the transcriptional level but stabilized Arp2/3 protein; however, further study is needed to confirm and clarify this.
Fibronectin is a glycoprotein of the extracellular matrix30 that influences cell growth, adhesion, mobility, and differentiation.30 Deregulated fibronectin expression causes cancer and fibrosis.31,32 Overexpression of DCD increased the expression of fibronectin, while knockdown of DCD decreased the expression of fibronectin in cultured SK-HEP-1 cells. This suggests that DCD also promotes cell migration and invasion via fibronectin-mediated cell adhesion in HCC,49 which was further demonstrated by Rac1 rescue in DCD-siRNA-transfected SK-HEP-1 cells.
However, our current study does have some limitations. For example, the study is a proof-of-principle, and DCD’s effects on HCC progression in vivo need further confirmation. Moreover, our nude mouse data only showed the effects of DCD cDNA in vivo, and there are no data on the inverse effects of DCD knockout in vivo. In conclusion, DCD protein levels were higher in the sera of patients with HCC and in SK-HEP-1 cells. DCD expression induced HCC cell migration, invasion, and metastasis in vitro and in nude mice by modulating Nck1, WASP, Arp2/3, and fibronectin expression and activating Rac1/Cdc42.
Supporting information
Supplementary Table 1
PCR primer sequences.
(DOCX)
Supplementary Table 2
Baseline characteristics of 105 patients with HCC.
(DOCX)
Abbreviations
- Arp2/3:
actin-related protein 2/3
- AUC:
area under the ROC curve
- Cdc42:
cell division control protein 42 homologue
- CI:
confidence interval
- DCD:
dermcidin
- ELISA:
enzyme-linked immunosorbent assay
- HCC:
hepatocellular carcinoma
- NC:
negative control
- Nck1:
noncatalytic region of tyrosine kinase adaptor protein 1
- qPCR:
quantitative PCR
- PBS:
phosphate-buffered saline
- Rac1:
Ras-related C3 botulinum toxin substrate 1
- ROC:
receiver operating characteristic
- SH:
Src homology
- si:
small interfering
- SPF:
specific pathogen-free
- WASP:
Wiskott-Aldrich syndrome family protein
Declarations
Data sharing statement
The data from the current study are available from the corresponding author upon reasonable request.
Funding
This study was supported in part by grants from Natural Science Foundation of Guangdong Province (#2021A1515010668), the National Natural Science Foundation of China (#81503424), the Guangzhou Science and Technology Innovation Commission (#201704020171), the Traditional Chinese Medicine Bureau of Guangdong Province (#20201263), and the Guangzhou Municipal Health and Health Committee (#20191A011014, #20202A011006, #20192A010005).
Conflict of interest
The authors have no conflict of interests related to this publication.
Authors’ contributions
Study design (XH), Western blot experiments (FQ), in vitro studies (HL, LZ), biostatistical analyses (JL, ZY), and drafting of the manuscript (FQ, XH). All authors have read and approved the final version of this manuscript.