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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2012 Apr 6;138(8):1321–1328. doi: 10.1007/s00432-012-1201-7

The FGF2-binding peptide P7 inhibits melanoma growth in vitro and in vivo

Yonglin Yu 1, Susu Gao 1, Quchou Li 1, Cong Wang 2, Xinqiang Lai 1, Xilei Chen 1, Ruixue Wang 1, Jingfang Di 1, Tao Li 1, Wenhui Wang 1, Xiaoping Wu 1,2,✉
PMCID: PMC11824751  PMID: 22481251

Abstract

Purpose

Melanoma is a malignant tumor and causes majority of deaths related to skin cancer. Fibroblast growth factor 2 (FGF2) greatly contributes to melanoma growth and progress. In this paper, we attempt to evaluate the therapeutic potential of FGF2-binding peptide (named P7) using as a potent FGF2 antagonist via exploration of its antitumor effect on melanoma in vitro and in vivo.

Methods

Cell viability was measured by WST-1. Cell cycle progression was determined by propidium iodide staining and flow cytometry. Western blotting was carried out to detect the activation of Erk1/2, P38, Akt, and MEK, and the expression of apoptosis-associated proteins. The influence of P7 on FGF2 internalization was assessed by separation of nuclear and cytoplasmic protein fractions followed by Western blotting. Female C57BL/6 mice bearing xenograft melanoma were established and used to evaluate the antitumor effect of P7 in vivo.

Results

In this study, we first proved that P7 peptides significantly inhibited proliferation of FGF2-induced melanoma cell line B16-F10. Further investigations revealed that the mechanisms of P7 peptides inhibiting cell proliferation of melanoma cells stimulated with FGF2 in vitro involved cell cycle arrest at the G0/G1 phase, blockade of the activation of Erk1/2, P38, and Akt cascades, and inhibition of FGF2 internalization. Finally, treatment of P7 peptides in a murine melanoma model resulted in significant inhibition of tumor growth and angiogenesis in vivo, which was associated with blockade of mitogen-activated protein kinase signal activation, and suppression of the expressions of anti-apoptotic Bcl-2 protein and angiogenic factor in the melanoma tumors.

Conclusions

The FGF2-binding peptide with potent antiproliferation and anti-angiogenic activity may have therapeutic potential in melanoma.

Keywords: FGF2-binding peptide, Proliferation, Angiogenesis, Melanoma

Introduction

Fibroblast growth factor 2 (FGF2) belongs to the family of fibroblast growth factors (FGFs) and is a potent inducer involved in the processes of proliferation and differentiation of a wide variety of cells derived from mesoderm and neuroectoderm (Beenken and Mohammadi 2009; Dailey et al. 2005). The biological activities of FGF2 are mediated by binding to and activating the transmembrane FGFR family of tyrosine kinase receptors (Dailey et al. 2005). It also translocated to the cytosol and the nucleus, which is necessary for FGF2 inducing full mitogenic response in target cells (Bossard et al. 2003; Sorensen et al. 2006). Overexpression of FGF2 and/or its receptors is commonly observed in malignant tumors (Cronauer et al. 2003; Korc and Friesel 2009). Therefore, FGF2 has been considered as a potential target for tumor therapy.

Melanoma predominantly occurred in skin is a malignant tumor of melanocytes. Although it is less common than other skin cancers, melanoma is much more dangerous and causes up to 75 % of deaths related to skin cancer (Shoo and Kashani-Sabet 2009). Being as a strong mitogenic and angiogenic factor, FGF2 is up-regulated in melanoma patients and contributes to melanoma growth and progress (Ribatti et al. 2003; Meier et al. 2003; Tsunoda et al. 2007). In the previous study, we have used phage display technology to isolate a high-affinity FGF2-binding peptide (named P7) with strong inhibitory activity against FGF2-induced cell proliferation and angiogenesis (Wu et al. 2010, 2011; Wang et al. 2010). The results suggest that P7 peptides may bind to FGF2 and block the biological activities of FGF2 by interrupting its interactions with its receptors. Herein, we attempt to evaluate the therapeutic potential of P7 using as a potent FGF2 antagonist via exploration of its antitumor effect on melanoma in vitro and in vivo.

Materials and methods

Materials

Recombinant human FGF2 was obtained from PeproTech Inc. (Rocky Hill, NJ, USA). P7 peptides (PLLQATLGGGS) and the scrambled peptides (QLGPGLASLGT, named as H7) with purity higher than 98 % were synthesized at SBS Genetech (Beijing, China). Dynabeads® M-280 Streptavidin, Dynamag-2 magnet, Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA); the cell proliferation reagent WST-1 was from Roche (Mannheim, Germany); propidium iodide (PI) was from Sigma (USA); and anti-phospho-MEK, anti-phospho-Erk1/2, anti-Erk1/2, anti-phospho-P38, anti-P38, anti-phospho-Akt, anti-Akt, anti-Bcl-2, anti-Bcl-xL, and anti-GAPDH antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-FGF2 antibody was the product of Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-CD34 antibody was obtained from Boster (Wuhan, China). Biotin (long arm) maleimide was purchased from Vector Laboratories (USA). Nuclear and Cytoplasmic Protein Extraction Kit was the product of KeyGEN (Nanjing, China). Ultrasensitive TM S–P (Rabbit) kit, 3,3′-diaminobenzidine (DAB), and hematoxylin were obtained from Maxin-Bio (Fuzhou, China). Polyvinylidenedifluoride (PVDF) membrane was from Millipore (Billerica, MA, USA). The enhanced chemiluminescence (ECL) detection kit was the product of Pierce (Rockford, IL, USA). Female C57BL/6 mice (5–6 weeks old) were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China).

Cell culture

B16-F10 cells kept by our laboratory were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and maintained in a humidified atmosphere containing 5 % CO2 at 37 °C.

Cell viability assay

Cells were seeded in a volume of 200 μl at a density of 2,000 cells/well in 96-well plates and allowed to attach overnight. After starved cultivation in DMEM with 0.4 % FBS for 24 h, cells were treated with serially diluted peptides, 30 ng/ml FGF2 alone, or 30 ng/ml FGF2 plus serially diluted peptides for 48 h. The number of viable cells was finally determined by WST-1. Briefly, 20 μl of WST-1 was added to each well (1:10 final dilution) and incubated with cells for 2 h. The absorbance was immediately measured at 450 nm to determine the number of viable cells.

Cell cycle analysis

B16-F10 cells were seeded in 12-well plates with 4 × 104 cells per well, starved for 24 h, and treated with 30 ng/ml FGF2 alone or 30 ng/ml FGF2 plus serially diluted peptides for 48 h. Cells were harvested and fixed in 70 % ice-cold ethanol for 12 h at 4 °C. After washed with PBS for three times, cells were stained for DNA content by use of 300 μl PBS containing 50 μg/ml propidium iodide and 50 μg/ml preboiled RNase A. The suspension was incubated in dark at room temperature for 30 min and then subjected to FACSCalibur flow cytometer analysis with excitation at 488 nm and emission at 560–640 nm (FL2 mode). Data were analyzed with the ModFit DNA analysis program.

MAP kinase and AKT activation assay

Starved B16-F10 cells were pretreated with serially diluted peptides for 5 min before stimulation with FGF2 (30 ng/ml) for 20 min. After washed with cold PBS, cells were lysed in 1 × SDS–PAGE loading buffer. The lysate was syringed, boiled for 5 min, and then centrifuged at 12,000×g for 10 min at 4 °C to remove the insoluble components. The supernatant was run on 10 % SDS–PAGE gel followed by being transferred to PVDF membrane. The membrane was incubated with TBST (25 mM Tris, pH 7.4, 150 mM NaCl, and 0.1 % Tween-20) containing 5 % nonfat dry milk at room temperature for 1 h. After washed with TBST for 3 times, the membrane was probed with the primary antibody (an anti-phospho-Erk1/2 rabbit mAb or an anti-phospho-P38 rabbit mAb or an anti-phospho-Akt rabbit mAb) at 4 °C overnight followed by incubation with goat antirabbit IgG, HRP-linked antibody for 1 h at room temperature. The blots were detected with an ECL detection kit according to the manufacturer’s procedure. Nonphosphorylated Erk1/2, P38, or Akt was used as the reference control. The results were analyzed by Quantity One software to determine the relative ratio.

FGF2 internalization assay

FGF2 was biotinylated on cysteine residues as described by the manufacturer’s instructions. Modification of FGF2 at cysteine residues has no effect on the biological activity of the growth factor (Bonnet et al. 1996). B16-F10 cells were seeded and allowed to grow to 70 % confluence. After starved cultivation for 24 h, cells were pretreated with peptides (4 μM) for 5 min before stimulation with biot-FGF2 (30 ng/ml) for 4 h. Approximately 1 × 107 cells were harvested in 5 ml of DMEM-10 % FBS, centrifuged for 5 min at 500×g at 4 °C, and washed twice in cold PBS. Nuclear and cytoplasmic proteins were prepared with the Nuclear and Cytoplasmic Protein Extraction Kit according to the manufacturer’s procedure. The protein concentrations were determined using the Bradford method (Bradford 1976). An equal amount of protein samples were incubated in the presence of streptavidin beads for 30 min at room temperature using gentle rotation. After extensive washing, bound proteins were directly resuspended in 0.1 % SDS by boiling beads for 5 min and analyzed by Western blotting using anti-FGF2 antibodies.

In vivo antitumor studies

Mice were inoculated subcutaneously into the right flank with B16-F10 cells (2 × 105 cells). When tumors were visible on all mice on day 9, Mice were randomly assigned to 3 groups (n = 6 per group) and received intraperitoneal injections of the synthetic peptides on alternate days for a total of 6 injections at a dosage of 50 and 250 mg/kg, whereas control mice were injected with PBS.

Body weights were monitored, and tumors were measured using vernier calipers before each injection. The tumor volumes were determined by measuring length (l) and width (w) and calculating volume (V = lw 2/2) as described (Sun et al. 1999). On the next day after the last injection, mice were killed and the tumors were extracted for further Western blot and immunohistochemical analysis, respectively.

For Western blotting, tumors were minced, ground, and lysed in RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 1 % NP-40, 0.1 % SDS, and 1 % PMSF). The lysates were centrifuged at 12,000×g for 10 min at 4 °C to remove the insoluble components. The supernatants whose protein concentrations were determined by BCA Protein Assay Kit (Pierce, USA) were separated by SDS–PAGE and transferred to PVDF membranes. After blocked with 5 % nonfat dry milk in TBST for 1 h, the membranes were incubated with primary antibodies against phospho-MEK, Bcl-2, Bcl-xL, FGF2, and GAPDH overnight at 4 °C followed by the corresponding HRP-conjugated IgG antibodies. The signals were detected with an ECL detection kit according to the manufacturer’s procedure. The intensity of the signals was determined by Quantity One.

For immunohistochemical analysis, the tumors were extracted and fixed in 10 % neutral buffered formalin, dehydrated, and embedded in paraffin. Four-micrometer sections of formalin-fixed and paraffin-embedded tissue specimens were cut and stained by established method as described previously (Umemoto et al. 2001). Briefly, tissue sections were deparaffinized and processed to microwave antigen retrieval before being incubated with anti-CD34 rabbit polyclonal antibody (1:200 dilution) overnight at 4 °C followed by secondary biotinylated antibodies and a streptavidin-conjugated horseradish peroxidase. Tissue sections incubated with PBS instead of primary antibodies were used as the negative control.

For assessment of microvascular density, CD34-stained slides were examined at a low-power view (4×) for areas of increased numbers of vessels. After five areas of highest neovascularization were identified, individual microvessels were counted on a 100 × (10 × objective lens and 10 × ocular lens) and a 250 × (25 × objective lens and 10 × ocular lens) field. Any brown-staining endothelial cell or endothelial cell cluster, clearly separated from adjacent microvessels, was considered as a single, countable microvessel (Maeda et al. 1995).

Statistical analysis

Data were presented as mean ± standard deviations (SD) from at least three independent experiments. Statistical differences between the groups were determined with one-way ANOVA (GraphPad Prism 5.0), followed by Tukey’s multiple comparison test. A p value of <0.05 was considered statistically significant.

Results

P7 inhibits FGF2-stimulated proliferation of B16-F10 cells

The effects of synthetic peptides on the proliferation of B16-F10 cells were determined by WST-1 assay. Starved cells were treated with FGF2 alone, FGF2 plus various concentrations of P7, or P7 peptides alone for 48 h. Scrambled peptide H7 was used as normal control to evaluate the selective effects of P7 on FGF2. As shown in Fig. 1, synthetic P7 peptides had a dose-dependent inhibitory effect on the proliferation of B16-F10 cells stimulated with 30 ng/ml of FGF2, whereas administration of FGF2 plus H7, or P7 peptides alone had no inhibitory effect on the growth of B16-F10 cells. A 50 % inhibition (IC50) of cell proliferation by P7 was observed at about 1 μM (Fig. 1b).

Fig. 1.

Fig. 1

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Effects of synthetic P7 peptides on FGF2-stimulated proliferation of B16-F10 cells. Starved cells were treated with 30 ng/ml FGF2 alone, 30 ng/ml FGF2 plus P7 (a and b) or H7 (d), or P7 peptides alone (c) at the indicated concentrations, and cell proliferation was measured 48 h later using WST-1 assays. Data are presented as the mean ± SD of three independent experiments performed in triplicate. *p < 0.01 versus control group; **p < 0.01 versus FGF2 group

P7 arrests FGF2-induced cells at the G0/G1 phase

Propidium iodide staining combined with flow cytometry analysis was performed to investigate the effect of the synthetic peptides on cell cycle progression of B16-F10 cells induced by FGF2. The results shown in Fig. 2 indicated that FGF2-treated cells presented an increased S-phase population and a decreased G0/G1-phase population compared with the control, whereas addition of P7 peptides increased the percentage of FGF2-induced cell in G0/G1 phase and decreased the S-phase percentage. Administration of the scrambled H7 peptides had no effect on the cell cycle distribution stimulated with FGF2, suggesting that the specific sequence of P7 plays essential role in counteracting the regulation effect of FGF2 on the cell cycle progression.

Fig. 2.

Fig. 2

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Flow cytometric analysis of cell cycle using propidium iodide. A B16-F10 cells were starved for 24 h and then treated with 30 ng/ml FGF2 (b) or 30 ng/ml FGF2 plus various concentrations of P7 or H7 (c–f: 0.25, 1, 4, and 16 μM) for 48 h. (a) Control cells without treatment of bFGF, P7, or H7. The representative pictures shown are from one of three independent experiments. B Cell cycle distribution of the control and treated cells. Data are presented as the mean ± SD of three independent experiments. *p < 0.01 versus control group; #p < 0.05 versus FGF2 group; **p < 0.01 versus FGF2 group

P7 blocks FGF2-induced phosphorylation of Akt and MAP kinases

The effects of P7 on FGF2-triggered signal transduction were determined by detecting its capacity to inhibit FGF2-induced Akt and MAP kinases activation. As shown in Fig. 3, intense phosphorylation of Erk1/2, P38, and Akt was observed in cells treated with FGF2 for 20 min (Fig. 3, lane 2). Pretreatment with P7 peptides (0.25–16 μM) for 5 min before stimulation with FGF2 resulted in significant blockage of Erk1/2, P38, and Akt activation (Fig. 3, lanes 3–6). P7 at the concentration of 1 μM suppressed the level of the detected signal molecule activation to the control.

Fig. 3.

Fig. 3

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Synthetic P7 peptides inhibit FGF2-induced MAP kinase and Akt activation. B16-F10 cells were pretreated with serially diluted peptides for 5 min before stimulation with 30 ng/ml FGF2 for 20 min. The phosphorylated and total levels of Erk1/2, P38, and Akt were determined by Western blot analysis. GAPDH served as the loading control. Density ratios of phosphorylated proteins to total proteins were presented as the mean ± SD of three independent experiments. *p < 0.01 versus control group; **p < 0.01 versus FGF2 group

P7 suppresses FGF2 internalization

FGF2 binding with its receptors not only activates the signal transduction cascades, but also internalizes into cells via endocytosis. Internalization of FGF2 is required for exerting its full mitogenic activity. Separation of nuclear and cytoplasmic proteins followed by Western blotting analysis of the exogenous biotinylated FGF2 enriched with streptavidin-coated Dynabeads was carried out to analyze the effect of P7 peptides on FGF2 internalization. The results showed that a strong signal was observed in cytoplasmic protein extract of FGF2-stimulated cells (Fig. 4a, lane 3). However, pretreatment of the cells with P7 (4 μM) for 5 min before stimulation with FGF2 suppressed FGF2 internalization, presenting a weak signal in cytoplasmic protein extract (Fig. 4a, lane 5). No signal was detected in nuclear protein extract from cells treated with or without P7 peptides (Fig. 4a, lanes 4 and 6).

Fig. 4.

Fig. 4

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P7 peptides suppress FGF2 internalization. Starved B16-F10 cells were pretreated with 4 μM peptides for 5 min before stimulation with 30 ng/ml biot-FGF2 for 4 h. And 107 cells were harvested and fractionated in cytoplasm (Cyt) and nuclei (Nuc). An equal amount of protein samples were incubated with streptavidin beads prior to Western blotting analysis with anti-FGF2 antibody. Data are representative of three independent experiments

P7 inhibits tumor growth and angiogenesis in vivo

The antitumor efficacy of P7 peptides in vivo was investigated in a mouse model of melanoma. Treatment with P7 peptides at a dose of 250 mg/kg resulted in a significant reduction in tumor volume compared to that observed in PBS group (p < 0.05 versus PBS group). No significant differences were observed in total body weight between P7-treated groups and PBS group, and no visible signs of toxicity were evident even at the maximal dose of 250 mg/kg (Fig. 5a). Western blot analysis showed that P7 peptides suppressed MEK activation and reduced the expressions of FGF2 and Bcl-2 in tumors. But the expression levels of Bcl-xL in P7-treated tumors were similar to that in tumors received PBS (Fig. 5b). Tumor angiogenesis was demonstrated by microvessel density (MVD) in CD34-stained sections (Fig. 5c). The MVD counts were 18.00 ± 8.88/8.80 ± 2.28 and 13.40 ± 5.98/4.60 ± 2.08 in tumors treated with 50 and 250 mg/kg P7, which were significantly lower than 40.40 ± 10.88/18.00 ± 4.58 in tumors treated with PBS (p < 0.01), demonstrating that P7 strongly inhibited angiogenesis in melanoma (Table 1). The data revealed that the mechanisms of P7 peptides inhibiting tumor growth involve regulations of the MAPK signal transduction and the expressions of proteins related to the mitogenesis, anti-apoptosis, and angiogenesis.

Fig. 5.

Fig. 5

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P7 peptides inhibit tumor growth and angiogenesis in xenograft mouse model. a B16-F10 cells were subcutaneously injected into the right flank of 7-week-old female C57BL/6 mice. When tumors were visible on all mice on day 9, mice were received intraperitoneal injections of P7 peptides on alternate days at a dosage of 50 and 250 mg/kg, whereas control mice were injected with PBS. Body weights were monitored, and tumors were measured before each injection. *p < 0.05 versus PBS group. b On the next day after last injection, tumors were extracted, lysed, and probed with antibodies against phospho-MEK, Bcl-2, Bcl-xL, FGF2, and GAPDH. *p < 0.05 versus PBS group; **p < 0.01 versus PBS group. c Tumors were extracted, fixed, and embedded in paraffin. Tissue sections were immunostained with CD34 as described in the “Materials and methods.” Negative control was incubated with PBS instead of anti-CD34 antibody. Original magnification, 100× (upper), 250× (lower). Data are representative of three independent experiments

Table 1.

P7 inhibits tumor angiogenesis

Tumor Group Microvessel count (mean ± SD)
Magnifications: 100× 250×
B16-F10 Control 40.40 ± 10.88 18.00 ± 4.58
P7 (50 mg/kg) 18.00 ± 8.88* 8.80 ± 2.28*
P7 (250 mg/kg) 13.40 ± 5.98* 4.60 ± 2.07**
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* p < 0.01 versus control group; ** p < 0.001 versus control group

Discussion

Our previous studies showed that the FGF2-binding peptide P7 with strong inhibitory activity against FGF2-induced cell proliferation and angiogenesis may have potential in cancer therapy. In order to determine the effects of P7 peptides on melanoma tumor growth and progression, we first investigated the effects of P7 on FGF2-induced proliferation of melanoma cell line B16-F10. The results confirmed that P7 rather than its scrambled peptide H7 significantly inhibited the growth of FGF2-stimulated B16-F10 cells in a dose-dependent manner, suggesting that the inhibitory effects of P7 targeting FGF2 are sequence specific.

It was reported that the mitogenic activity of FGF2 is closely related to its regulations of cell cycle progress. FGF2 promotes the expressions of cell cycle regulatory proteins involved in cell cycle entry and progression through the G1 and S phases (Neary et al. 2005). The results of cell cycle analysis showed that P7 arrested FGF2-stimulated cells at the G0/G1 phase, whereas H7 peptides have little effect on cell cycle distribution. The specific sequence of P7 may exert inhibitory effects on FGF2-induced proliferation in B16-F10 cells by suppressing the ability of FGF2 to stimulate cell progress through the cell cycle phase from G0/G1 to S phase.

The mitogen-activated protein kinases (MAPK) signaling begins with Ras and consists of several parallel pathways controlling cellular processes particularly related to cell proliferation. In order to determine which MAPK pathway involved in mediating the inhibitory effect of P7 on FGF2-induced proliferation, phosphorylation levels of Erk1/2, P38, and JNK, chosen as markers presenting the activation of three parallel MAPK pathways, respectively, were detected by Western blotting. The results showed that P7 suppressed FGF2-induced Erk1/2 and P38 phosphorylation in a dose-dependent manner, whereas administration of FGF2 had no effect on the phosphorylation of JNK in B16-F10 cells (data not shown), suggesting that the Erk and P38 cascades rather than JNK cascade involved in mediating the inhibitory effects of P7 on FGF2-induced proliferation. We also investigated the influences of P7 peptides on the activation of other proliferation-related pathway, PI3K/Akt. The results revealed that P7 peptides also inhibited cell growth via PI3K/Akt pathway. Because PI3K/Akt pathway often controls cell survival and apoptosis (Zhou et al. 2000), and FGF2 promotes cell survival by regulating anti-apoptotic PI3K/Akt signaling (Park et al. 2009), it seems reasonable to speculate that P7 peptides may regulate the effects of FGF2 on cell survival and apoptosis, which is currently being addressed in our laboratory.

FGF2 forms dimer, interacts with its receptors, and triggers signal transduction and internalization. Both activation of the signal transduction pathways and internalization of FGF2 are essential for exerting its full mitogenic activity. Facchiano et al. (2003) reported that FREG, a short peptide derived from the predicted interface of the FGF2 dimer, strongly inhibited FGF2 internalization likely via inhibition of FGF2 self-interaction. Our studies have showed that P7 peptides may affect interaction between FGF2 and its receptors by binding with FGF2. Therefore, the observed P7-induced suppression of FGF2 internalization may be due to reduced FGF2 interaction with its receptors. However, the possibility of P7 inhibiting FGF2 internalization via affecting FGF2 dimerization cannot be ruled out.

In vivo experiments performed in a murine melanoma model demonstrated that systemic administration of P7 peptides can significantly reduce tumor volume and the expressions of anti-apoptotic Bcl-2 protein and angiogenic factor FGF2 and suppress MEK activation in treated melanoma versus control, suggesting that P7 peptides may exert antimelanoma activity in vivo. Additionally, upon P7 peptide treatment, mice present good general conditions. No significant differences were observed in total body weight versus controls, and no visible signs of toxicity were evident even at the maximal dose of 250 mg/kg. Therefore, the observed antimelanoma effect of P7 peptides is unlikely related to toxic effects.

In summary, the results demonstrate that the previously isolated FGF2-binding peptide specifically inhibiting FGF2 activity has antimelanoma effects in vitro and in vivo. Further studies will be aimed at characterizing its potential therapeutic role in melanoma.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (30973671, 81071800), the Natural Science Foundation of Zhejiang Province of China (Y2090292), the Natural Science Foundation of Guangdong Province of China (9151064001000031), the Science and Technology Planning Project of Wenzhou (Y20090244), the Fundamental Research Funds for the Central Universities (X. P. Wu), Guangdong Provincial “Thousand-Hundred-Ten Talent Project” (X. P. Wu), and Guangdong Provincial Key Discipline in Biochemistry and Molecular biology.

Conflict of interest

All authors declare that there are no potential conflicts of interest.

Footnotes

Yonglin Yu and Susu Gao have contributed equally to this work.

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