YTHDF2 promotes anaplastic thyroid cancer progression via activating the DDIT4/AKT/mTOR signaling

doi:10.21203/rs.3.rs-5241567/v1

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Research Article

YTHDF2 promotes anaplastic thyroid cancer progression via activating the DDIT4/AKT/mTOR signaling

https://doi.org/10.21203/rs.3.rs-5241567/v1

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Background

RNA methylation, an important reversible post-transcriptional modification in eukaryotes, has emerged as a prevalent epigenetic alteration. However, the role of the m6A reader YTH domain family 2 (YTHDF2) has not been reported in anaplastic thyroid cancer (ATC) and its biological mechanism is unclear.

Methods

The relationship between YTHDF2 expression and ATC was determined through the utilization of data sets and tissue samples. A range of analytical techniques were employed to investigate the regulatory mechanism of YTHDF2 in ATC, including bioinformatics analysis, M6A dot-blot analysis, methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA immunoprecipitation (RIP) assays, RNA sequencing, RNA stability assays and dual luciferase reporter gene assays. In vitro and in vivo assays were also conducted to determine the contribution of YTHDF2 to ATC development.

Results

In this study, we found that YTHDF2 expression was significantly increased in ATC. Our comprehensive in vitro and in vivo experiments demonstrated that YTHDF2 knockdown significantly attenuated ATC proliferation, invasion, migration, and promotion of apoptosis, whereas the opposite trend was obtained with YTHDF2 overexpression. Mechanically, RNA-seq, MeRIP-seq and RIP-seq analysis, and molecular biology experiments demonstrated that YTHDF2 accelerated the degradation of DNA damage and Development 1 (DDIT4, or REDD1) mRNA in an m6A-dependent manner, which in turn activated the AKT/mTOR signaling pathway and induced activation of epithelial-mesenchymal transition (EMT), thereby promoting ATC tumor progression.

Conclusions

This study is the first to demonstrate that elevated YTHDF2 expression levels suppress DDIT4 expression in an m6A-dependent manner and activate the AKT/mTOR signaling pathway, thereby promoting ATC progression. YTHDF2 plays a pivotal role in ATC progression, and it may serve as a promising therapeutic target in the future.

Anaplastic thyroid cancer

m6A

YTHDF2

AKT

mTOR

Thyroid cancer is the most prevalent malignant endocrine tumor, with an increasing incidence rate over recent years. It ranks as the ninth most common cancer worldwide and the third most common cancer in China (1, 2). ATC is a rare but highly malignant form of thyroid cancer, accounting for 1–2% of all thyroid cancers and the majority of thyroid cancer-related deaths (3). According to the American Joint Committee on Cancer tumor-lymph node-metastasis classification system, ATC patients are generally classified as stage IV (4). High invasiveness and lethality are the distinguishing features of ATC, the median overall survival of ATC from the date of diagnosis was 4 months, and the disease-specific mortality was nearly 100% (5). More than 40% of ATC patients present with distant metastases, which often result in death from local tumor expansion or asphyxiation due to distant metastasis (6). Currently, there is no effective treatment to extend the overall survival time of patients (3, 7). However, understanding the ATC molecular landscape, in particular the characterization of such drivers of carcinogenesis in malignancies, has identified potential opportunities for targeted therapies (8).

m6A is the most common internal modification of eukaryotic mRNA, which is dynamically reversible and widely involved in mRNA splicing, nuclear output, stability and translation (9). This modification is regulated by m6A methylases, demethylases, and recognition proteins (10, 11). Alterations in the expression of these proteins can affect tumor transcripts and oncoprotein expression, cancer cell proliferation, survival, tumorigenesis, progression, and metastasis (12). YTHDF2, the earliest binding recognition protein, is capable of regulating the stability of m6A-modified mRNAs by recognizing and binding to m6A-modified sites to degrade target genes(13, 14), This process has been shown to promote the progression of different tumors, as evidenced by studies in which YTHDF2 was found to be upregulated in tumor cells and to promote tumor growth and metastasis(15–18). Moreover, the absence of YTHDF2 in tumor-associated macrophages (TAMs) impedes tumor growth by reprogramming TAMs to an antitumor phenotype and enhancing their capacity to cross-present antigens. These findings suggest that the inhibition of YTHDF2 may enhance the efficacy of cancer immunotherapy(19). Nevertheless, the role and molecular mechanism of YTHDF2 in ATC remain incompletely understood.

In the process of metastasis, cells undergo epithelial-to-mesenchymal transition (EMT), which involves the loss of epithelial cell surface markers and the acquisition of characteristics associated with mesenchymal cells. These cells play a role in the distant metastasis of tumors, as evidenced by studies (20, 21). Recent studies have indicated that EMT and its transcription factors are regulated by m6A modification (22, 23). Furthermore, intervention with m6A modification has been shown to have potential for cancer therapy(24). Previous studies have indicated that the AKT/mTOR pathway and EMT are closely related (25, 26).

A series of in vitro and in vivo experiments was conducted with the objective of delineating the biological functions of YTHDF2 and its potential molecular mechanisms in the pathogenesis of human ATC. Our findings indicate that YTHDF2 activates the AKT/mTOR signaling pathway by degrading DDIT4, thereby promoting the proliferation, migration, and invasion of ATC.

Specimens from patients

From June 2014 to December 2023, human thyroid cancer and its adjacent tissues were collected from the Guangdong General Hospital for formalin fixation and paraffin embedding. The thyroid cancer were diagnosed by histopathology. These clinical materials were used for research purposes with the prior consent of the patients and the approval of the institutional research ethics committee.

Cell lines and cell culture

The thyroid cancer cell lines (BHT101, KHM-5M, FTC133) were purchased from Wuhan Pricella Biotechnology Co., Ltd. The thyroid cancer cell lines (ARO, CAL-62) and the normal thyroid cell line (Nthy-ori 3 − 1) were purchased from the Beijing Bina Biological BNCC cell bank, and all were confirmed by short tandem repeat analysis. The KHM-5M and Nthy-ori 3 − 1 cells were cultured in RPMI1640(Gibco, USA) medium containing 10% fetal bovine serum (FBS, Procell, Wuhan, China). The Cal-62, BHT101, ARO and FTC133 cell lines were cultured in 10% DMEM (Gibco, USA) medium at 37°C and 5% CO₂ in the cell incubator.

Immunohistochemistry (IHC) staining Hematoxylin and Eosin (H&E) staining

The paraffin-embedded tissues were sectioned at a thickness of 5 µm. The deparaffinization process involved the use of xylene, followed by rehydration with a gradient alcohol series. The sections were then incubated with 3% hydrogen peroxide for 30 minutes at room temperature. Antigen retrieval was achieved through microwave heating in sodium citrate buffer. Finally, the sections are blocked with 5% bovine serum albumin (BSA). The sections were then incubated with anti-YTHDF2 (Proteintech, China) overnight at 4℃. The sections were then treated with peroxidase-labeled streptavidin for 30 minutes at room temperature after addition of the polymer enhancer. The antibody reaction was observed with a fresh substrate solution containing 3,3'-diaminobenzidine tetrahydrochloride (DAB). Paraffin sections were deparaffinized and hematoxylin stained for three minutes. The sections should then be rinsed with running water and stained with eosin for 30 s -1 min. The stained sections were then examined under a microscope.

Immunofluorescence staining

The appropriate cells were added to confocal dishes at 37°C overnight. The cells were then fixed with 4% paraformaldehyde for 20 minutes and permeated with 0.3% triton X-100 for 10 minutes. The cells were then blocked with 5% BSA for 30 minutes and incubated overnight with anti-YTHDF2 (Proteintech, China) and anti-m6A (Proteintech, China) antibodies at 4°C. Subsequently, the cells were incubated with a secondary antibody for one hour at room temperature. A cytoskeleton fluorescence assay was conducted using a cytoskeleton red fluorescence probe (KeyGEN Biotech, China) on cells that had been blocked at room temperature for 20 minutes. The nuclei were then stained with DAPI for a period of five minutes. Subsequently, fluorescent images were captured using a confocal fluorescence microscope.

CCK-8 assay

Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8, Beyotime, China). 2000/well cells (6 double wells) were seeded on 96-well plates at 37°C and adhered to the wall overnight. At the designated time point, the culture medium in six replicate wells was replaced with 100 µl of fresh medium containing 10% CCK-8 reagent.

EdU assay

A 5-ethynyl-20-deoxyuridine (EdU) assay kit (Ribobio, Guangzhou, China) was utilized to quantify cell proliferation. The cells were seeded into six-well plates at a density of 10 × 10^5 cells per well and incubated overnight. The following day, the cells were incubated with 50 µM EdU buffer at 37°C for 2 h, fixed with 4% paraformaldehyde for 0.5 h, and permeabilized with 0.5% Triton X-100 for 10 min in flow tubes. Each tube was stained with 1× Apollo and incubated at room temperature for 10 minutes in the dark. The supernatant was then discarded, and the cells were resuspended in 500 µL of PBS. Flow cytometry was then employed to detect the cells.

Trans-well invasion assay

The matrix gel was diluted in accordance with the instructions and applied to an 8-µm pore size trans-well filter insert in a 24-well plate for invasive determination. 5 × 10^4 cells were placed in the upper chamber without serum medium, while 10% FBS medium was added to the lower chamber. Following a 36-hour incubation period at 37°C, the cells situated beneath the membrane were fixed and stained with crystal violet (Beyotime, China). The penetrated cells were counted in five random fields under the microscope.

Wound healing assay

Cells were seeded in six-well plates at a density of 5 × 10^5 cells per well at 37°C overnight. The cells were then replaced with serum-free medium and a 200 µL pipette tip was used to create a scratch. The migration rate of cells was quantified by microscopy at 0 hours and 36 hours post-scratch. All tests were conducted in triplicate.

RNA extraction and quantitative real-time PCR (qPCR)

Total RNA was extracted from cells using Esciece RNA Rapid Extraction Kit(YiShan Biotech, Shanghai, China) according to the manufacturer's instructions, and quantified with NanoDrop 2000. The complementary DNA was generated from 1µg RNA and real-time qPCR was performed to determine determine the levels of the target RNA. The data were analyzed according to the ΔCt method. The primers utilized in this study are presented in Supplementary Table S2.

Western blotting(WB)

Cells and animal tissue were lysed in RIPA lysis buffer (New Cell and Molecular Biotech, Suzhou, China) supplemented with protease and phosphatase inhibitors to extract protein. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.45 µm polyvinylidene difluoride membranes (Millipore, USA). The membranes were then incubated in Tris-buffered saline with Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 1 hour at room temperature. Subsequently, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies were then detected with secondary antibodies conjugated with horseradish peroxidase. The immunoblots were imaged using an imaging system (Bio-Rad, USA) and the ECL western blotting kit (Millipore, USA). The antibodies utilized were detailed in Supplementary Table S3.

m6A dot-blot analysis

Total RNA was extracted using the RN001 RNA Quick Purification kit(ESScience, Shanghai, China), and quantified with a NanoDrop 2000. Then 2 µL of the RNA sample was applied to the positively-charged nylon transfer membrane (BersinBio, Guangzhou, China) and subjected to UV crosslinking twice to the nylon transfer membrane. Subsequently, the membrane was blocked with 5% BSA, followed by an overnight incubation with the anti-m6A antibody at 4°C. After incubating with the corresponding second antibody for 1h, the membrane was developed and the blots were visualized using an ECL western blotting kit (Millipore).

RNA immunoprecipitation (RIP) assays

The RIP assays were performed according to the manufacturer’s instructions (Beyotime, Shanghai, China). The cell lysate was divided into three samples: anti-YTHDF2, anti-IgG, and input. Immunoprecipitation was performed for overnight at 4°C using 5 µg specific antibody or IgG as the negative control. qPCR was performed to quantify the levels of the target RNA.

Transient transfection with pDDIT4

The lipofectamine™ 3000 transfection reagent (Invitrogen, USA) was used to transfect the overexpression plasmids pDDIT4 and the control pNull (empty vector) (Miaoling Biology, Wuhan, China) according to the manufacturer’s protocol.

Lentivirus vector infection

Lentiviral constructs expressing YTHDF2 and GFP, along with the negative control lentivirus, were purchased from Hanbio (Shanghai, China). The sequences of the lentiviruses that overexpress YTHDF2 (YTHDF2), YTHDF2-shRNA (shYTHDF2-1, shYTHDF2-2), the empty vector (Vec), and the scrambled shRNA (shNC) are listed in Supplementary Table S1. CAL-62 cells were used to establish YTHDF2 knockdown cell line. BHT101 cells were selected to establish stable YTHDF2 overexpression cell lines. Transfection was conducted in accordance with the manufacturer’s instructions. Briefly, 5 × 10^4 cells were plated into a 6-well plate and transfected with the indicated lentivirus. Infected cells were selected using 2µg/ml puromycin (MCE, USA) for ≥ 1 week, and the transfection efficiency was determined by qPCR and Western blotting analysis.

Dual-luciferase reporter assay

Wild-type and mutant segments were synthesized using m6A motifs in DDIT4-5’UTR(A-T). The indicated cells were transfected with DDIT4 wild-type or mutant dual-luciferase reporter plasmid. After 48 h post-transfection, the luciferase activities were detected using the Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China).

RNA decay assays

After treated with 5 µg/ml actinomycin D for 0, 1, 2 and 3 h, total RNA was extracted from CAL-62 or BHT101 cells using the RNA Quick Purification Kit (ESScience, Shanghai, China). The expression levels were then quantified by qPCR analysis.

Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-qPCR

Total RNA was isolated and purified using TRIzol reagent (Invitrogen, USA) following the manufacturer's instructions. Poly (A) RNA was purified from 50µg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, USA) by two rounds of purification. Subsequently, the poly(A) RNA was fragmented into small pieces using the Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA) under 86℃ for 7 minutes. The cleaved RNA fragments were incubated with m6A-specific antibody for immunoprecipitated. Both the input RNA samples and the immunoprecipitated RNA samples were prepared for subsequent sequencing procedures or qPCR analyses.

Animal experiments

All animal experiments were approved by the Ethics Review Committee of Guangdong Provincial People's Hospital (acceptance No. S2023-357-02.20160006). For the purpose of establishing tumor xenograft models, 5×10^6 CAL-62 shYTHDF2-1 or BHT101 OEYTHDF2 cells, along with empty vector cells, suspended in 200 µl of PBS were separately implanted into the right flank of 4-week-old nude mice which were randomly divided into 4 groups (4 mice per group). The volumes of the tumors (V = width^2 × length × 0.52) were recorded at 4-day intervals using a caliper. The mice were euthanized at 28 days post-implantation, and the tumors were photographed and weighed. 1× 10^6 transfected cells suspended in 100 µl of PBS were injected into the tail vein of BALB/c nude mice (4 weeks old), which were randomly divided into 4 groups (5 mice per group). One month following injection, the average radiation efficiency (p/s/cm^2/sr) was quantified by an in vivo imaging system (IVIS), after which the mice were sacrificed. The GFP-positive tissues were excised and subjected to IHC and H&E Staining.

Statistical analysis

The data were expressed as the mean ± standard deviation(SD). To assess differences between groups, a student’s t-test, a non-parametric test (Mann-Whitney test), or a one-way ANOVA test with Bonferroni’s correction was employed. All analyses were conducted using GraphPad Prism 9.0, and a two-tailed value of *P < 0.05, **P < 0.01, and ***P < 0.001 was considered statistically significant.

The expression of YTHDF2 and m6A in ATC

To investigate the expression pattern of YTHDF2 in thyroid cancer, UCSC XENA (https://xenabrowser.net/datapages/) was utilized. This revealed that YTHDF2 is up-regulated between thyroid carcinoma tissues and normal tissues (Fig. 1A). Furthermore, the level of YTHDF2 protein was found to be increased in ATC paraffin-embedded specimens compared to FTC and normal specimens using an IHC assay (Fig. 1B). A comparable pattern was observed in different subtypes of thyroid cancer cell lines, including thyroid cancer cell lines (CAL-62, KMH-2, KHM-5M, BHT101, FTC133), as well as a human normal thyroid cell line (Nthy-ori 3 − 1) (Fig. 1C). The results of the immunofluorescence assay indicated that YTHDF2 was distributed in both the cytoplasm and the nucleus (Fig. 1D). To identify the biological function of YTHDF2 in ATC, the endogenous expression of YTHDF2 was effectively knocked down in the CAL-62 cell line, and a stable YTHDF2-overexpressing transfection was established in the BHT101 cell line (Fig. 1E, F). Previous studies have demonstrated that YTHDF2 upregulates m6A levels by inducing targeted mRNA decay(14, 27). To understand the alterations of m6A levels, an RNA m6A dot blot assay was used, and the results revealed an upregulation of total m6A levels in YTHDF2 knock-down cell lines when RNA concentrations varied (200 ng and 400 ng) (Fig. 1G). This was also consistently corroborated by cell immunofluorescence of m6A (Fig. 1H).

YTHDF2 promoted the proliferation, invasion and migration of ATC cells in vitro

To investigate the role of YTHDF2 in ATC tumorigenesis, we conducted a colony formation assay. Our findings revealed that YTHDF2 knockdown in CAL-62 cells resulted in a significant reduction in colony formation rates, whereas YTHDF2 overexpression in BHT101 cells led to an increase in colony formation rates (Fig. 2A). The results of the EdU assay indicated that YTHDF2 knockdown suppressed the ability of DNA replication in CAL-62 cells. This was evidenced by the decreased percentage of EdU-positive cells in the YTHDF2 knockdown group compared with the control group. Conversely, overexpression of YTHDF2 had the opposite effect (Fig. 2B). The results of the CCK-8 assay were also consistent with those of the EdU assay (Fig. 2C). Actin–microtubule crosstalk plays a pivotal role in regulating cell shape and polarity during cell migration and division (28, 29). Therefore, we adopted fluorescent staining of F-actin to analyze the cytoskeleton. In comparison to the control cells, the YTHDF2 knock-down group exhibited a cobblestone-like shape and shrinkable F-actin fibers, which impeded cell migration, elongated cellular morphology, and stretched F-actin fibers. Conversely, cells overexpressing the protein exhibited elongated cellular morphology and stretched F-actin fibers, which are well-suited for cellular migration (Fig. 2D and Supplementary Fig. S1). The invasion rate of CAL-62 cells in the YTHDF2-deficient group was significantly reduced compared to that of the control group after 36 hours of incubation in serum-free medium, as determined by trans-well assay. In contrast, the opposite effect was observed in BHT101 cells overexpressing YTHDF2 (Fig. 2E). The cell migration rate was also evidently impeded by the knockdown of YTHDF2, as evidenced by the results of the wound-healing assay (Fig. 2F). A further flow cytometry assay of two cell lines indicated that YTHDF2 knock-down had an inhibitory effect on cell apoptosis. However, YTHDF2 overexpression appeared to attenuate the apoptosis rate (Fig. 2G). In conclusion, these results collectively indicated that YTHDF2 promoted the proliferation, migration, and invasion of ATC cells.

YTHDF2 induced AKT-mTOR activation and EMT via suppression of DDIT4 expression

To investigate the potential and direct m6A-modified targets of YTHDF2, we conducted a MeRIP-seq in YTHDF2 stably knocked down CAL-62 cell line (CAL-62 shYTHDF2) and its control (CAL-62 shNC). The volcano plot indicated that the m6A levels of 1736 genes were upregulated and 1718 genes were downregulated after knocking down YTHDF2 (Fig. 3A). In order to study the biological function and molecular mechanism of YTHDF2 in ATC, KEGG analysis and GO-BP enrichment analysis were performed on the differential genes (|log₂FC| > 0.5, P < 0.05). The TNF signaling pathway, NF-kappa B signaling pathway and PI3K-AKT pathway were significantly enriched and were closely related to biological functions such as proliferation, invasion and metastasis (Fig. 3B, C). GSEA enrichment analysis revealed that YTHDF2-related highly expressed genes were significantly enriched in the EMT pathway (Fig. 3D).

To identify YTHDF2 target genes, RNA-seq, MeRIP-seq, and RIP-seq were combined, focusing on genes with high m6A levels and significant changes in mRNA expression. This approach yielded 12 genes (Fig. 3E).

Given that the most typical role of YTHDF2 is to accelerate the degradation of mRNAs by recognizing m6A modifications(13, 14), we hypothesized that YTHDF2 might activate the EMT-associated pathway by degrading negative regulatory genes. Through a literature review, we found that has been identified as a negative regulator of mTORC1 and mTORC2, acting as a central signal transduction hub involved in cellular metabolism, energy homeostasis, and cell growth networks(30–33). To assess the impact of YTHDF2 knockdown on DDIT4 mRNA binding, YTHDF2 RIP-qPCR was conducted. The results showed that the enrichment of YTHDF2 to DDIT4 mRNA was diminished in cells overexpressing YTHDF2 (Fig. 3F). YTHDF2 exhibited a significant negative regulatory effect on DDIT4 mRNA levels in CAL-62 and BHT101 cells, as evidenced by qPCR and Western blot analysis (Fig. 3G-I). The knockdown of YTHDF2 resulted in a downregulation of EMT-related proteins and an inhibition of AKT phosphorylation, while overexpression of YTHDF2 exhibited the opposite trend (Fig. 3I).

YTHDF2 mediated the mRNA degradation of DDIT4 in m6A-dependent way

To further validate the existence of m6A modification on DDIT4 mRNA in ATC, Integrative genomics viewer (IGV) plots demonstrated the m6A modification sites, the YTHDF2 binding sites and the change in MeRIP-seq peak of DDIT4 mRNA upon YTHDF2 knockdown in CAL-62 cell line. The results demonstrated the presence of peaks mapping to DDIT4 mRNA in m6A antibody-precipitated RNA, and reads of peaks mapping to DDIT4 mRNA were also increased in YTHDF2-knockdown cells (Fig. 4A). Two significantly upregulated m6A sites in 5’UTR of DDIT4 at single site resolution were identified in the YTHDF2 knock-down CAL-62 cell line from MeRIP-seq data analysis (Fig. 4B). The motifs of the m6A peaks were consistent with the consensus sequence (RRACH (R = G/A, H = A/C/U)) (Fig. 4C). Furthermore, a dual luciferase activity assay demonstrated that the knockdown of YTHDF2 significantly elevated luciferase activity in the wild-type group (GGACU/GGACA, m6A sites included in 5’UTR), but not in the mutated group (mutated m6A sites from A to T), and vice versa (overexpression of YTHDF2) (Fig. 4D, E).

To ascertain whether this regulation was m6A-dependent, we employed the global methylation inhibitor, 3-deazaadenosine (DAA), to treat the CAL-62 and BHT101 cell lines. Our findings revealed that the total m6A levels, as determined by RNA m6A dot blot assay, exhibited a notable reduction compared to the DMSO control group at 400 ng RNA concentration (Fig. 4F). Consequently, the mRNA expression of DDIT4 was also upregulated with the same concentration of DAA treatment (Fig. 4G). Moreover, MeRIP-qPCR demonstrated a considerable elevation in DDIT4 mRNA levels in the m6A antibody-precipitated RNA relative to that of IgG. Additionally, DDIT4 mRNA precipitated by the m6A antibody exhibited a notable decline in Mettl3-knockdown CAL-62 cells (Fig. 4H). Subsequently, the impact of YTHDF2 knockdown and overexpression on the stability of DDIT4 mRNA was studied. The stability of DDIT4 mRNA was increased in CAL-62 cells following YTHDF2 knockdown and decreased in BHT101 cells overexpressing YTHDF2 (Fig. 4I).

Collectively, YTHDF2 facilitated the mRNA degradation of DDIT4 by recognizing m6A-modified sites, and this regulation was m6A-dependent.

YTHDF2 accelerated tumor growth and metastasis in vivo

To investigate the effect of YTHDF2 on the malignant phenotype in vivo, we also constructed stable YTHDF2 knockdown and overexpressing cells, expressing YTHDF2 and GFP constructed by lentivirus. The size of the tumor was observed and measured every four days until it reached an appropriate size. The results demonstrated that knocking down YTHDF2 resulted in a dramatic retardation of tumor growth and weight (Fig. 5A). In contrast, xenografts overexpressing YTHDF2 exhibited accelerated tumor progression (Fig. 5B). IHC staining of subcutaneous tumor tissues revealed that YTHDF2 expression levels were significantly lower in the shYTHDF2 group and significantly higher in the YTHDF2 overexpression group (Fig. 5C). Protein levels were verified by extracting subcutaneous tumor proteins, and it was found that the expression levels of EMT-related proteins N-cadherin, TWIST1 were decreased and E-cadherin were increased in the shYTHDF2 group, while the opposite trend was shown in the YTHDF2 overexpression group (Fig. 5D). A tail vein injection metastatic model was utilized to assess the extent of metastasis. The results indicated that YTHDF2 knockdown significantly inhibited the metastasis of CAL-62 and BHT101 cells at the 4th week. This was further corroborated by the reduced radiance value measured by the IVIS after 28 days (Fig. 5E). The metastatic organs were then anatomized and imaged using the in vivo imaging system, which facilitated the precise localization and identification of the metastases. The above metastatic tissues were prepared as slides and stained with H&E to identify the metastatic sites (Fig. 5F). In conclusion, the results demonstrated that YTHDF2 significantly accelerated tumor growth and metastasis in vivo.

DDIT4 overexpression reversed the influence of YTHDF2 on the malignant phenotype of tumors and AKT-mTOR activation

To further validate the role of DDIT4 in YTHDF2 promotion of ATC progression and AKT-mTOR activation, we overexpressed DDIT4 in cells overexpressing YTHDF2. Western blotting also showed that DDIT4 overexpression suppressed AKT-mTOR activation and EMT-associated proteins induced by YTHDF2 overexpression (Fig. 6A). The CCK-8 and Edu assays indicated that DDIT4 overexpression significantly attenuated the proliferation of BHT101 cells overexpressing YTHDF2 (Fig. 6B, C). Meanwhile, wound-healing assay and trans-well migration analysis showed the DDIT4 overexpression reduced the increased migration of BHT101 cells caused by YTHDF2 overexpression (Fig. 6D, E). Flow cytometry showed that DDIT4 attenuated the effect of YTHDF overexpression on apoptosis (Fig. 6F). These findings indicated that DDIT4 overexpression reversed the influence of YTHDF2 on the malignant phenotype of tumors and AKT-mTOR activation. Thus, in this study, we found that YTHDF2 exerted its degradation function by targeting the tumor suppressor DDIT4, consequently activating the DDIT4/AKT/mTOR signaling pathway and inducing EMT in ATC. All these results were summarized in a schematic diagram (Fig. 6G).

RNA methylation, an important reversible post-transcriptional modification in eukaryotes, has emerged as a broad regulatory mechanism with implications in almost all significant biological processes, including the initiation and progression of cancer (9, 34, 35). Previous studies have found that the m6A reader YTHDF2 mediates mRNA degradation of tumor suppressor genes LHPP and NKX3-1 in an m6A-dependent manner, thereby regulating AKT phosphorylation-induced tumor progression in prostate cancer (15). YTHDF2 recognized the m6A modification in the 5’-untranslational(5’UTR) region of ETS variant transcription factor 5 mRNA and recruited eukaryotic translation initiation factor 3 subunit B to facilitate its translation, thereby promoting liver cancer immune evasion and angiogenesis (36). Deletion of YTHDF2 in myeloid cells after ionizing radiation has been shown to enhance anti-tumor immunity and overcome tumor radiation resistance (37). Similarly, in this study, YTHDF2 was found to promote ATC progression by recognizing the binding 5'UTR m6A modification site and accelerating the degradation of the target gene DDIT4.

In previous studies, the AKT-mTOR signaling pathway was found to be closely related to tumor proliferation (38), and it also participated in the EMT process of tumors (39, 40). ATC is characterized by the rapid proliferation of local lesions, high rate of invasion and metastasis, and poor prognosis (5). ATC displays a more active epithelial-mesenchymal transition than PTC, which enhances its invasion and metastasis outside the thyroid (41). A review of the literature, we found that DDIT4 has been identified as a potent inhibitor of the mTORC1 pathway, regulating various cellular processes, including protein synthesis, autophagy, and cell growth (42, 43). Previous studies have found that up-regulation of DDIT4 in human myeloma cells inhibits AKT and mTOR signaling and exerts anti-tumor progression effects(44). Furthermore, it has been proposed that DDIT4 facilitates the progression of urothelial carcinoma of the bladder and antagonizes REDD1, thereby sensitizing BUC cells to paclitaxel.(45) In this study, overexpression of DDIT4 significantly reduced the phosphorylation of Akt and mTOR. Further experiments have confirmed that DDIT4 is the direct target of YTHDF2. To confirm the dependence of this regulation on m6A, demethylation of ATC cells by DAA demonstrated a significant decrease in m6A levels and an increase in DDIT4 RNA expression. Moreover, the knockdown of METTL3 levels in CAL-62 cell lines yielded consistent results. This further substantiates the conclusion that the aforementioned regulation is m6A-dependent. Consequently, we hypothesized that YTHDF2 induces the degradation of DDIT4 by binding to m6A modification sites, which was confirmed by dual luciferase experiments on GGACU/GGACA in the 5'UTR, respectively.

Previous studies suggested that DDIT4 promoted protein phosphatase 2A-dependent dephosphorylation of AKT on Thr308 but not on Ser473(30), as well as the finding that phosphorylation of AKT on Thr308 was required for TSC2 phosphorylation, but not Akt phosphorylation on Ser473(46).we observed that the AKT-mTOR pathway was activated following YTHDF2-mediated degradation of the DDIT4 target gene. Concurrently, the expression level of EMT-related proteins was significantly elevated. Western blot experiments revealed that following the decrease in the expression of DDIT4, the phosphorylation levels of the main phosphorylation sites of AKT, Thr308 and Ser473, were increased. Of these, the change in the phosphorylation level of Thr308 was more correlated to the change in DDIT4. It is possible that DDIT4 more significantly affects the phosphorylation site of AKT, Thr308. and This hypothesis is consistent with the findings of the aforementioned study.

Previous studies have found that the TWIST1 transcription factor can promote tumor metastasis and induce EMT (47, 48). Previous studies have demonstrated that TWIST1 upregulation of SOX2 was associated with the promotion of cancer stem cell-like properties in prostate cancer cells and also with the progression of triple-negative breast cancer(49, 50). The findings of this study indicate that the protein expression level of TWIST1 is consistent with that of EMT-related proteins and YTHDF2, but opposite to that of DDIT4. This indicates that YTHDF2 may additionally influence the progression of EMT by regulating TWIST1, this requires further research to discover the underlying mechanisms.

RNA methylation is widely involved in a multitude of biological processes, yet little has been reported in ATC. In this study, YTDHF2 was found to play an important role in ATC for the first time. However, due to the small number of ATC cases and the extremely poor prognosis, there is a lack of valid clinical data to verify the clinical significance of this study. Subsequent studies will focus on the key targets of YTHDF2 involved in the progression of ATC, thereby providing theoretical support for further targeted drug research.

In conclusion, our study demonstrates for the first time that elevated YTHDF2 expression levels can inhibit DDIT4 expression levels in an m6A-dependent manner and activate the AKT/mTOR signaling pathway to promote ATC proliferation, metastasis, and inhibit apoptosis. These findings indicate that YTHDF2 plays a pivotal role in ATC progression and may serve as a promising therapeutic target.

Anaplastic Thyroid Cancer (ATC)

YTH domain family 2 (YTHDF2)

RNA immunoprecipitation (RIP)

N6-methyladenosine (m6A)

epithelial-mesenchymal transition (EMT)

the American Joint Committee on Cancer (AJCC)

tumor-lymph node-metastasis (TNM)

Tumor-associated macrophage (TAM)

DNA damage and Development 1 (DDIT4, or REDD1)

fetal bovine serum (FBS)

Immunohistochemistry (IHC)

Hematoxylin and Eosin (H&E)

bovine serum albumin (BSA)

3,3′-diaminobenzidine tetrahydrochloride (DAB)

the Cell Counting Kit-8 (CCK-8)

5-ethynyl-20-deoxyuridine (EdU)

3-deazaadenosine (DAA)

Author information

Authors and Affiliations

Department of Thyroid and Hernia Surgery, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, 510080, China

Bao Dai, Lei Xu, Ziteng Lan, Weijian Chen, Yongchen Liu, Linhe Wang, Jian Chen, Zeyu Wu

Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences

Lei Xu

Department of Thyroid Surgery, Shenzhen People’s Hospital (The Second Clinical Medical College, Jinan University, The First Affiliated Hospital, Southern University of Science and Technology), Shenzhen, Guangdong, China

Shikuo Rong

Department of anesthesiology, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, Guangzhou, Guangdong, 510080, China

Muye Song

Department of Laboratory, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, 106 Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China

Jinghua Li

Contributions

Bao Dai was involved in protocol design, in vitro and in vivo experiments, data analysis, and drafting the manuscript; Lei Xu was involved in in vitro experiments and data analysis. Shikuo Rong was involved in protocol design, data analysis, drafting the manuscript. Muye Song was involved in in vitro experiments and data analysis. Ziteng Lan, Weijian Chen, Yongchen Liu, and Linhe Wang were involved in data analysis. Jinghua Li, Jian Chen, Zeyu Wu were involved in grant support, protocol design, and manuscript review. All authors read and approved the final manuscript.

Corresponding authors

Correspondence and requests for materials should be addressed to Jinghua Li, Jian Chen, Zeyu Wu.

Ethics approval and consent to participate

The study was approved by the Ethical Review Committee of the Guangdong Provincial People's Hospital and was conducted in accordance with the Declaration of Helsinki (Project No. KY2023-306). All animals were housed and handled according to the guidelines of the Ethical Review Committee of Guangdong Provincial People's Hospital (acceptance No. S2023-357-02.20160006).

Consent for publication

Not applicable.

Competing interests

The authors declare no potential conflicts of interest.

Additional information

Not applicable.

Funding

This study was supported by the Natural Science Foundation of Guangdong Province (No. 2020A1515010127, No. 2023A1515010175) and Guangdong Provincial People’s Hospital Scientific Foundation for Distinguished Young Scholars of Guangdong Province (No. KJ012019441). Guangzhou Science and Technology Scheme (2024A04J4974). Young and Middle-Aged Thyroid Physician Research Program (a9Z04JKM2022E036a0).

Author Contribution

Bao Dai was involved in protocol design, in vitro and in vivo experiments, data analysis, and drafting the manuscript.Lei Xu was involved in in vitro experiments and data analysis. Shikuo Rong was involved in protocol design, data analysis, drafting the manuscript. Muye Song was involved in in vitro experiments and data analysis. Ziteng Lan, Weijian Chen, Yongchen Liu, and Linhe Wang were involved in data analysis. Jinghua Li, Jian Chen, Zeyu Wu were involved in grant support, protocol design, and manuscript review. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Availability of data and materials

Upon reasonable request, the data supporting the conclusions of this paper will be made available by the corresponding author.

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No competing interests reported.

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YTHDF2 promotes anaplastic thyroid cancer progression via activating the DDIT4/AKT/mTOR signaling

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Abstract

Figures

Introduction

Materials and Methods

Specimens from patients

Cell lines and cell culture

Immunohistochemistry (IHC) staining Hematoxylin and Eosin (H&E) staining

Immunofluorescence staining

CCK-8 assay

EdU assay

Trans-well invasion assay

Wound healing assay

RNA extraction and quantitative real-time PCR (qPCR)

Western blotting(WB)

m6A dot-blot analysis

RNA immunoprecipitation (RIP) assays

Transient transfection with pDDIT4

Lentivirus vector infection

Dual-luciferase reporter assay

RNA decay assays

Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-qPCR

Animal experiments

Statistical analysis

Results

The expression of YTHDF2 and m6A in ATC

YTHDF2 promoted the proliferation, invasion and migration of ATC cells in vitro

YTHDF2 induced AKT-mTOR activation and EMT via suppression of DDIT4 expression

YTHDF2 mediated the mRNA degradation of DDIT4 in m6A-dependent way

YTHDF2 accelerated tumor growth and metastasis in vivo

Discussion

Conclusions

Abbreviations

Declarations

Author information

Additional information

Funding

Author Contribution

Acknowledgements

Availability of data and materials

References

Additional Declarations

Supplementary Files

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Version 1