Quercetin, a Natural Dietary Flavonoid, Emerges a Novel USP7 Inhibitor with Anti-colorectal Cancer Effects

Abstract

The human deubiquitinating enzyme ubiquitin-specific peptidase 7 (USP7) has emerged as a promising anti-tumor target, particularly in colorectal cancer, due to its regulation of the MDM2/p53 axis. Through a combination of ubiquitin C-terminal 7-amido-4-methylcoumarin hydrolysis assay and ubiquitin-propargylamide protease profiling, we identified the natural dietary flavonoid quercetin as a potent USP7 inhibitor in both cell-free and cellular contexts. Cellular thermal shift and surface plasmon resonance analyses demonstrated that quercetin is directly bound to USP7. Consistent with USP7 target engagement in cells, quercetin decreased MDM2 levels and subsequently increased the levels of p53. Moreover, quercetin also suppressed colorectal cancer cell proliferation by inducing G2/M cell cycle arrest and inhibiting cell migration. Collectively, our study identified quercetin as a novel and effective USP7 inhibitor with potent anti-colorectal cancer activity, highlighting its therapeutic potential for targeting the USP7–MDM2–p53 axis and warranting further exploration as a promising therapeutic agent in colorectal cancer treatment.

INTRODUCTION

Colorectal cancer (CRC) ranks among the top 3 cancers in terms of both incidence and mortality worldwide.¹⁾ Although treatment methods are available, many challenges still exist, such as toxicity and severe complications.²⁾ Therefore, developing safe and effective drugs against CRC is still one of the important research focuses. Ubiquitination plays an important role in protein degradation and is crucial in basic cellular processes, including cell survival and tumor suppression.³⁾ Deubiquitinating enzymes (DUBs), which remove ubiquitin (Ub) from ubiquitinated proteins, have gained great attention as drug targets in recent years.⁴⁾ There are approximately 100 DUBs, which are subdivided into 6 families, with Ub-specific proteases (USPs) being the largest and most diverse subfamily.⁵⁾ USPs can remove Ub from their substrates, thereby protecting proteins from degradation.⁶⁾ Among all USPs, USP7 is the most widely studied DUB because of its involvement in the MDM2/p53 pathway.⁷⁾ USP7 contains a tumor necrosis factor receptor-associated factor (TRAF)-like domain, a middle catalytic domain, and five carboxy-terminal Ub-like domains.⁸⁾ USP7 is overexpressed in CRC cells and tissues, and its expression is correlated with advancing tumor stage and poor prognosis.^9,10) USP7 inhibition promotes MDM2 degradation and activates p53 signaling.⁷⁾ Thus, USP7 has been considered a promising therapeutic target in cancer treatment. To date, several USP7 inhibitors have been reported, including P5091, P22077, HBX19818, XL188, and GNE6776.¹¹⁾ Natural sources harbor structural diversities and are important for drug discovery and development.¹²⁾ However, to date, USP7 inhibitors derived from natural compounds have not been extensively explored.¹³⁾ We previously reported that the USP7 inhibitor parthenolide inhibited CRC proliferation by regulating Wnt/β-catenin signaling pathway.¹⁴⁾ Flavonoids are natural products that are abundant in plant-derived foods, such as tea, fruits, and vegetables.¹⁵⁾ Flavonoids have been reported to have benefits against inflammatory diseases, cardiovascular diseases, and cancers.^16,17) As an important flavonoid, quercetin has been shown to exert anti-tumor properties through the phosphatidylinositol 3-kinase (PI3K)/AKT, nuclear factor-kappaB (NF-𝜅B), and Wnt/β-catenin signaling pathways.¹⁸⁾ However, the biological effects of quercetin on USP7 remain unexplored.

In this study, we first demonstrated that quercetin is a novel and effective USP7 inhibitor that suppresses USP7 enzymatic activity via direct interaction. Moreover, quercetin-mediated USP7 suppression resulted in MDM2 destabilization, p53 accumulation, and inhibition of CRC cell growth.

MATERIALS AND METHODS

Cell Lines and Culture Conditions

HCT116 cells were obtained from ATCC, and HEK293T and RKO cells were obtained from the Cell Bank of the Chinese Academy of Sciences. For cell growth, all cells were incubated at 37°C in a cell culture incubator (Thermo Fisher Scientific, Waltham, MA, U.S.A.) with 5% CO₂. Cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS). DMEM and FBS were purchased from Biological Industries (Kibbutz Beit Haemek, Israel).

Ub-AMC Assay

USP7 was incubated in reaction buffer containing 50 mM Tris–HCl (pH 7.6), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 20 mM NaCl, and 5 mM MgCl₂ with different concentrations of quercetin. Following incubation at room temperature for 30 min, the Ub C-terminal 7-amido-4-methylcoumarin (Ub-AMC) substrate (Boston Biochem, Cambridge, MA, U.S.A.) was added. Fluorescence was measured immediately.

Ub-PA Assay

For the cell-free system, USP7 protein was preincubated with quercetin (0–320 nM) in buffer containing 50 mM Tris (pH 7.6), 5 mM MgCl₂, 250 mM sucrose, 2 mM dithiothreitol (DTT), and 0.5 mM EDTA, followed by incubation with the Ub-propargylamide (Ub-PA) probe (Boston Biochem) in a cell culture incubator for another 20 min. For living cells, HEK293T cells in the logarithmic growth phase were lysed with TE buffer containing 50 mM Tris–HCl (pH 7.4), 5 mM MgCl₂, 150 mM NaCl, 0.5 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, and 2 mM DTT). After centrifugation, the supernatant was obtained and incubated with different doses of quercetin. To check the inhibitory effects of quercetin on USP7 in intact HCT116 cells, the cells were pre-treated with quercetin for a period of 12 h, harvested, and lysed with TE buffer; the supernatant was obtained after centrifugation. The reaction was initiated by adding the Ub-PA probe. All samples were boiled and then processed for Western blotting.

Cellular Thermal Shift Assay

The cellular thermal shift assay (CETSA) has been used to study ligand–protein engagement within both cellular lysates and intact cells. For cellular lysates, cells were lysed with phosphate-buffered saline (PBS) lysis buffer (PBS containing 1 mM phenylmethanesulfonyl fluoride fluoride; Beyotime, Shanghai, China). Samples were broken by liquid nitrogen and collected by centrifugation. The cell lysate was divided into 2 equal parts. One was incubated with quercetin (100 μM), and the other was incubated with an equivalent amount of dimethyl sulfoxide (DMSO). After 2 h of incubation, each sample was sub-packaged into 4 smaller parts (50 μL) and heated at the indicated temperatures (45, 50, 55, and 60°C) for 3 min using a PCR instrument, and then cooled for another 3 min. For intact cells, HCT116 cells were seeded into 10-cm plates and incubated with quercetin (100 μM) or vehicle (DMSO) for 6 h. After that, the cells were lysed with 250 μL PBS lysis buffer and centrifuged. The suspension was divided into 4 equal parts (50 μL) and heated at 45, 50, 55, and 60°C for 3 min. All heated samples were centrifuged at 20000 × g to separate soluble proteins from cell lysates. The soluble proteins were collected and prepared for Western blot assay.

Cycloheximide (CHX) Chase Assay

HCT116 and RKO cells were seeded in 6-well plates and allowed to attach overnight. The next day, fresh medium containing 100 μM quercetin was added, and the cells were incubated for 24 h. CHX was then added to a final concentration of 100 μg/mL to block new protein synthesis. Cells were harvested at 0, 1, 2, 4, and 8 h after CHX addition. At each time point, the medium was aspirated, the cells were washed twice with ice-cold PBS, and lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors. Lysates were cleared by centrifugation (12000 rpm, 10 min, 4°C). Supernatants were mixed with 5× loading buffer, heated at 100°C for 5 min, and subjected to Western blot analysis.

Co-immunoprecipitation (Co-IP)

Quercetin-treated HCT116 and RKO cells were lysed after 24 h of treatment, and the lysates were quantified. A 40 μL aliquot was reserved as input. The remaining lysate was incubated with an anti-p53 antibody (Santa Cruz Biotechnology, Dallas, TX, U.S.A.) for 5–6 h, followed by the addition of Protein A/G beads (Santa Cruz Biotechnology) and incubation overnight at 4°C. The beads were washed 3 times, and 2× loading buffer was added. The samples were boiled at 100°C for 10 min to elute the proteins from the beads. The eluted proteins were then analyzed using antibodies against p53 and Ub (Santa Cruz Biotechnology).

Western Blotting Assay

After pre-treatment, cells were harvested and lysed with cold RIPA buffer (Beyotime) supplemented with the protease inhibitor phenylmethanesulfonyl fluoride before use. Cells were lysed on ice and centrifuged at 12600 rpm for 12 min to remove debris. Total protein concentrations were quantified using a BCA protein assay kit (Proteintech, Rosemont, IL, U.S.A.). Samples were then heated and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Blots were carried out using antibodies against USP7 (Bethyl Laboratories, Montgomery, TX, U.S.A.), p53, MDM2, GAPDH, and actin (Proteintech). After primary antibody incubation, the polyvinylidene fluoride membranes were washed with TBST (0.1% Tween-20 in Tris-buffered saline), followed by incubation with horseradish peroxidase-conjugated anti-goat, anti-rabbit, and anti-mouse secondary antibodies (Proteintech) for 1–2 h. Finally, the membranes were incubated with Pierce ECL substrate (Proteintech), and then developed using the ChemiDoc™ MP imaging system (Bio-Rad, Hercules, CA, U.S.A.).

Cell Proliferation Assay and Colony Formation Assay

Cell viability of quercetin on CRC cells was detected by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Cells were counted, prepared in a tube, and seeded into 96-well plates, followed by quercetin treatment for 24, 48, and 72 h. After incubation, the old culture medium was discarded, and 100 μL of MTS working solution (fresh culture medium: MTS [Promega, Madison, WI, U.S.A.] = 50 : 1) was added to each well. Cells were incubated at 37°C for 20–60 min, and the optical density values were measured at 492 nm. The IC₅₀ values were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, U.S.A.).

For the colony formation assay, single cells were treated with various doses of quercetin for 7 d. Then, the colonies were fixed with 4% paraformaldehyde for 15 min, followed by staining with 1% crystal violet (Solarbio, Beijing, China) for another 20 min. Finally, cellular colonies were washed with distilled water and imaged with a scanner.

Wound Healing and Transwell Assay

HCT116 and RKO cells were incubated in 6-well plates and allowed to adhere. The adherent cells were wounded with a 200 μL micropipette tip, the old culture medium was discarded, the cells were gently washed with PBS, and 2 mL of FBS-free medium with 0–50 μM quercetin was added. The width of the wounds was recorded at 0, 24, and 48 h using an inverted microscope. Finally, ImageJ software was used to measure the migration distance.

For the transwell assay, cells in 6-well plates were treated with quercetin (0, 25, and 50 μM) for 24 h. Then, 1 × 10⁵ cells in 200 μL of FBS-free medium were added to the upper chamber (8 μm pore size; Corning, Transwell, Corning^®, NY, U.S.A.), with 600 μL of medium containing 20% FBS in the lower chamber. Following 48 h of incubation at 37°C with 5% CO₂, migrated cells on the lower surface were fixed with ice-cold methanol for 20 min, stained with 1% crystal violet for 15 min, and washed. Non-migrated cells in the upper chamber were removed with a cotton swab. Images were then observed under a ×10 objective lens.

Quantitative Real-Time PCR Analysis

Cells were treated with quercetin for 24 h and harvested. Total cellular RNA was isolated from HCT116 and RKO cells using the Eastep™ Total RNA Extraction Kit (Promega) and quantified with a NanoDrop spectrophotometer. Subsequently, 1 μg of RNA was used to generate cDNA by reverse transcriptase (Promega). The prepared cDNA was analyzed using a real-time PCR system with the Easep^® qPCR Master Mix (2×) (Promega). The relative mRNA expression levels of puma, p21, and noxa genes were determined and normalized to the housekeeping gene GAPDH. The PCR primers were listed as follows:

Surface Plasmon Resonance (SPR) Analysis

Studies of the binding effects between USP7 and quercetin were performed on a Biacore S200 (GE Healthcare, Chicago, IL, U.S.A.). Briefly, USP7 protein was immobilized on a CM5 sensor chip according to a standard amine-coupling procedure. Different doses of quercetin were injected in immobilization buffer supplemented with 5% (v/v) DMSO at a flow rate of 30 μL/min, a 120 s contact time, and a 180 s dissociation time. Data were collected and analyzed using the Biacore Evaluation Software S200 with an appropriate binding model to obtain the dissociation equilibrium constant (K_D).

Cell Cycle Analysis

The cell cycle analysis of quercetin on HCT116 cells was performed using flow cytometry. Briefly, cells (2 × 10⁵) were plated in 6-well plates and different doses of quercetin were added. After 24 h, cells were harvested, washed twice with cold PBS, and fixed in 70% ethanol at −20°C. Cells were resuspended in PBS containing 50 μg/mL RNase A (Sigma-Aldrich, St. Louis, MO, U.S.A.) to remove interference from RNA. Finally, samples were stained with 50 μg/mL propidium iodide (PI) in the dark at room temperature. Samples were filtered and analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, U.S.A.). Cell cycle distribution in each phase was analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, U.S.A.).

Data Analysis

Data were given as mean ± standard deviation (S.D.). Student’s t-test was used to analyze differences in mRNA expression, cell cycle distribution, and migration activity. If p < 0.05, it was considered to be statistically significant. *p < 0.05, **p < 0.01, and ***p < 0.001.

RESULTS

Quercetin Is Identified as a Novel and Effective USP7 Inhibitor

To obtain USP7 inhibitors, a screening based on the hydrolysis of Ub-AMC by USP7 was conducted, leading to the discovery of quercetin as a USP7 inhibitor. The results showed that quercetin markedly reduced the USP7 activity in the Ub-AMC assay (Fig. 1A), with an IC₅₀ of 75 nM. N-ethylmaleimide, a non-selective protease inhibitor, served as a positive control.¹⁹⁾ Next, the Ub-PA probe was employed to confirm the USP7 inhibitory activity of quercetin. As shown in Fig. 1B, incubation of purified USP7 protein with the Ub-PA probe resulted in the formation of Ub-USP7 conjugates. Quercetin effectively competed with the Ub-PA probe in a concentration-dependent manner (Fig. 1B). These data suggest that quercetin markedly inhibited USP7 activity in cell-free systems. Next, we also utilized the Ub-PA assay to examine whether quercetin could inhibit USP7 activity in cellular contexts. HEK293T crude cell extracts were incubated with increasing concentrations of quercetin for 1 h, followed by labeling with the Ub-PA probe. As expected, USP7 labeling by the Ub-PA probe was inhibited by quercetin (Fig. 1C, left). Similarly, living HCT116 cells were pre-treated with quercetin, followed by labeling with the Ub-PA probe at 37°C for 20 min. As indicated in Fig. 1C (right), USP7 labeling by the Ub-PA probe was also blocked by quercetin in intact HCT116 cells. Together, these data demonstrate that quercetin inhibits USP7 activity in both cell-free systems and cellular environments.

Fig. 1. Quercetin Is Identified as a Novel and Effective USP7 Inhibitor

(A) Quercetin inhibited enzymatic activity of USP7 in a dose- and time-dependent manner. NEM was used as a positive control. (B) Quercetin inhibited USP7 labeling by the Ub-PA probe in a concentration-dependent manner. Purified USP7 protein was treated with quercetin (0, 5, 10, 20, 40, and 80 μM) for 20 min and incubated with the indicated Ub-PA probe. (C) HEK293T crude cell extracts (left) or living HCT116 cells (right) preincubated with quercetin were labeled with the Ub-PA probe for 20 min. USP7 was identified using an anti-USP7 antibody.

Quercetin Directly Binds to USP7

CETSA and SPR are 2 powerful label-free methods for studying ligand–protein interactions.^20,21) In this study, CETSA was used to identify whether quercetin directly binds to USP7 in both cell lysates and intact cells. For cell lysate experiments, HCT116 cells in the logarithmic growth phase were lysed and incubated with quercetin for 2 h. As shown in Fig. 2A, compared to the DMSO-treated control, quercetin treatment enhanced the thermal stability of USP7 in HCT116 cell lysates. Next, CETSA was also performed in intact HCT116 cells to validate this interaction. Cells were pre-treated with quercetin or DMSO, lysed, and subjected to heating at graded temperatures. As expected, quercetin treatment markedly increased the heat stability of USP7 protein in the intact HCT116 cells (Fig. 2B). SPR analysis further confirmed the direct binding between quercetin and USP7, with a K_D value of 18.76 μM (Fig. 2C). Collectively, CETSA and SPR revealed that quercetin directly interacts with USP7.

Fig. 2. Quercetin Directly Binds to USP7

(A) Quercetin (100 μM) significantly increased the heat stability of USP7 in HCT116 cell lysates, as assessed by CETSA at 45–60°C. (B) As shown by CETSA, quercetin enhances the heat stability of USP7 in intact HCT116 cells. (C) SPR analysis demonstrates direct binding between quercetin and USP7 protein.

Quercetin Modulates the p53/MDM2 Pathway in CRC Cells

The MDM2/p53 pathway plays a pivotal role in regulating cancer cell growth, apoptosis, and senescence. USP7 inhibition decreases MDM2 protein, activates the p53 signaling pathway, and causes cell cycle arrest and apoptosis.⁷⁾ To confirm the inhibitory effect of quercetin on USP7 activity in cells, we monitored the levels of p53 and MDM2 in HCT116 and RKO cells, both of which express wild-type p53. Quercetin treatment decreased the level of MDM2, which was accompanied by a strong induction of p53 (Figs. 3A and 3B). Furthermore, quercetin dramatically increased p53 levels in a dose- and time-dependent manner (Figs. 3C and 3D). Moreover, RT-qPCR results showed that quercetin upregulated the mRNA expression of p53 target genes, including p21, PUMA and NOXA (Figs. 3E and 3F).

Fig. 3. Quercetin Modulates the MDM2/p53 Pathway in Colorectal Cancer Cells

(A, B) Quercetin regulates p53 and MDM2 protein levels in HCT116 (A) and RKO (B) cells. (C, D) Analysis of endogenous p53 levels in HCT116 and RKO cells following quercetin (0, 20, and 80 μM) treatment for different times. (E, F) Quercetin regulates the mRNA expression of p21, PUMA, and NOXA in HCT116 (E) and RKO (F) cells. (G, H) Quercetin decreases p53 ubiquitination levels in HCT116 (G) and RKO (H) cells. (I, J) Quercetin increases the half-life of p53 in HCT116 (I) and RKO (J) cells. Cells were treated with 100 μM quercetin for 24 h, and then cycloheximide (CHX) was added for 0–8 h. Values are mean ± S.D.; *p < 0.05, **p < 0.01, and ***p < 0.001.

It has been reported that USP7 mediates the ubiquitination of p53 in cancer.⁷⁾ Next, using Co-IP, we investigated whether quercetin modulates p53 ubiquitination levels in CRC cells. HCT116 and RKO cells were treated with DMSO or quercetin, and p53 ubiquitination was analyzed. As shown in Figs. 3G and 3H, quercetin reduced the ubiquitination of p53. Moreover, quercetin markedly extended the half-life of p53 in these cells (Figs. 3I and 3J). Taken together, these results demonstrated that quercetin-mediated USP7 inhibition induced MDM2 degradation and led to the subsequent functional activation of p53 in CRC cells.

Quercetin Suppresses CRC Cell Growth by Inducing G2/M Cell Cycle Arrest and Inhibiting Cell Migration

p53 is a critical regulator of the cell cycle and cell growth.²²⁾ As a USP7 inhibitor, quercetin’s effects on cell growth inhibition and the underlying mechanisms were further investigated. First, we employed the MTS assay to study the inhibitory effects of quercetin on HCT116 and RKO cells. As shown in Fig. 4A, quercetin decreased the viability of HCT116 and RKO cells in a dose- and time-dependent manner, and the IC₅₀ values are shown in Fig. 4B. Additionally, the colony formation assay was performed to further confirm the inhibitory effects of quercetin. As shown in the results, quercetin also significantly suppressed the colony-forming ability of HCT116 and RKO cells (Fig. 4C). We further analyzed the cell cycle distribution in quercetin-treated CRC cells. As shown in Fig. 5A, quercetin treatment effectively induced G2/M cell cycle arrest in HCT116 cells. In addition, USP7 inhibition has been shown to reduce the migration ability of CRCs.¹⁰⁾ We next investigated whether the USP7 inhibitor quercetin could affect CRC cell migration. Wound healing and transwell assays showed that quercetin treatment reduced the migratory capability of HCT116 and RKO cells in a dose-dependent manner (Figs. 5B–5D). Taken together, our data suggest that quercetin-induced USP7 inhibition suppresses CRC cell proliferation by inducing G2/M phase arrest and inhibiting cell migration.

Fig. 4. Quercetin Suppresses Colorectal Cancer Cell Growth

(A, B) MTS assay showed that quercetin inhibited cell viability of HCT116 and RKO cells. The IC₅₀ values were computed. (C) Quercetin inhibited the colony formation abilities of the colorectal cancer cell lines.

Fig. 5. Quercetin Induces G2/M Cell Cycle Arrest and Inhibits Cell Migration

(A) Flow cytometry experiments showed that quercetin induced cell cycle arrest at the G2/M phase in HCT116 cells. (B, D) Quercetin attenuated the migratory capabilities of colorectal cancer cells in scratch (B, C) and transwell assays (D). *p < 0.05, **p < 0.01, and ***p < 0.001.

DISCUSSION

After years of research, USP7 has come to be a recognized as a critical protein in cellular physiology.²³⁾ To date, USP7 has become a new and important molecular target for cancer therapy.²⁴⁾ While many USP7 inhibitors have been discovered, fewer natural products with USP7 inhibitory activity have been reported. Quercetin, the most widely distributed flavonoid, is extensively used in botanical medicine and traditional Chinese medicine.²⁵⁾ It exhibits diverse biological activities by affecting various signaling pathways, such as p53, NF-κB, PI3K/AKT, and Wnt/β-catenin pathways.^26,27) Our study demonstrated that quercetin is a novel and effective USP7 inhibitor. Ub-AMC and Ub-PA probe assays demonstrated that quercetin effectively inhibits USP7 enzymatic activity in both cell-free and cellular systems. CETSA and SPR assays found that quercetin directly interacted with USP7. At the cellular level, quercetin dramatically induced MDM2 degradation, subsequently leading to the stabilization of p53 protein. Moreover, quercetin also suppressed the proliferation of CRC cells by inducing G2/M cell arrest and inhibiting cell migration.

Quercetin has remarkable anticancer effects in vitro and in vivo. Research indicated that quercetin inhibits tumor formation and progression through mechanisms such as p53 activation, cell growth inhibition, apoptosis induction, and metastasis suppression.²⁸⁾ p53, the most important USP7-interacting protein, plays a crucial role in tumorigenesis and growth.²⁹⁾ Our study also found that the USP7 inhibitor quercetin could activate the p53 signaling in CRC cells. Based on these findings, we considered that quercetin might stabilize p53 by inhibiting USP7 activity. Our future work aims to determine whether quercetin stabilizes p53 through USP7 inhibition.

Flavonoids are important natural compounds with diverse molecular structures. While previous studies have reported on the inhibitory effects of flavonoids on USP7,^30,31) our study provides a comprehensive exploration of quercetin’s binding mechanism and biological effects on USP7. Specifically, we demonstrate that quercetin directly binds to USP7, leading to p53 activation and suppression of cancer cell proliferation. In summary, our results not only provide new insights into the anticancer properties of quercetin but also provide a new structural scaffold for the development of USP7 inhibitors in the future.

Funding

This study was funded by the Yunnan Provincial Department of Science and Technology—Kunming Medical University Joint Special Project on Applied Basic Research (202201AY070001-246); the Science and Technology Project of Yunnan Province (202201AU070016); and the Opening Foundation of the First People’s Hospital of Yunnan Province (2023YJZX-YX08).

Author Contributions

Conceptualization: L.K. and X.L. Methodology: X.L., H.Z., and Q.Y. Validation: Q.L. Data curation: X.L., Y.H., and L.K. Writing—original draft preparation: X.L. Writing—review and editing: L.K. Supervision: L.K. All authors have read and agreed to the published version of the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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