Introduction
Mesenchymal stem cells (MSCs) residing in various tissues can still undergo self-renewal and differentiate into specific cell types to maintain tissue homeostasis and fulfill regenerative needs [
1]. Multiple adult stem cell types with diverse biological properties have been reported in many tissues and organs. Beyond their similarity in cellular and molecular functions, MSCs exhibit distinct features related to their original phenotypes. Dental pulp stem cells (DPSCs) have superior pluripotency capacity and high mineralization potential, which are essential for hard tissue formation [
2]. DPSCs can differentiate into odontoblasts and secrete mineralized matrix known as tertiary dentin bridge formation to seal the vital pulp chamber and prevent pulpal infection from potential insult [
3]. The differentiation of DPSCs is critical for tertiary dentin formation and dental repair in vital pulp therapy [
3,
4]. Meanwhile, accumulating evidence also supports that DPSCs are capable of differentiating into osteoblasts and forming lamellar bone, which is a promising stem cell source in bone engineering [
5]. The initiate signaling reprograms cellular differentiation and extracellular matrix secretion will greatly benefit therapeutic approaches in engineering DPSC-based vital pulp procedures. Epigenetic modulations are capable of temporally controlling the transcription programs in a heritable manner and subsequently guiding quiescent stem cells to undergo preferential trajectories toward differentiation [
6]. The key epigenetic regulators could reversibly modulate endogenous stem cell activities and promote functional mineralized tissue formation, which present therapeutic opportunities in regenerative strategies.
N
6-methyladenosine (m
6A) is the most prevalent posttranscriptional modification of messenger RNA (mRNA) and regulates almost every step of RNA metabolism in mammals [
7]. The methyltransferase complex formed by methyltransferase-like 3 (METTL3), METTL14, and Wilms tumor 1-associated protein (WTAP) is responsible for the m
6A modification of mRNA, which catalyzes adenosines with methyl groups from metabolite substrates [
8]. Large-scale transcripts are dynamically and timely tagged by m
6A marks to orchestrate the different stages of stem cells. RNA m
6A modification has emerged as a critical epitranscriptomic mechanism that regulates embryonic development, cell reprogramming and differentiation [
9,
10]. The disruption of m
6A modification in the stem cell program displays diverse effects across cell types and specific fate stages. Reduction of m
6A deposition improved pluripotency and blocked regeneration of embryonic stem cells [
11], while it limited self-renewal and triggered cell differentiation in epiblast stem cells and induced pluripotent stem cells [
12,
13]. It is critical to identify the regulatory mechanism of dynamic m
6A marks in the DPSC fate transition. Our previous study characterized the m
6A-tagged landscape in immature DPSCs, which is related to cell senescence and apoptosis [
14]. Meanwhile, how m
6A methylation participates in DPSC differentiation remains unclear. Clarifying the RNA epigenetic mechanism during DPSC differentiation and manipulating the key modulators in therapeutic applications would advance vital pulp therapy.
In this study, we revealed a dynamic and unique m6A-mRNA landscape with m6A RNA immunoprecipitation-sequencing (m6A RIP-seq), which provides an entry point to uncover the potential function of m6A methylation in DPSC differentiation. METTL3 was identified as a key molecule that mediates m6A modification in DPSC mineralization. Remarkably, dynamic m6A methylation of noggin (NOG) confers its stabilization by shortening the poly(A) tail in a stage-specific manner. Our results provide evidence for the critical role of m6A modification in DPSC differentiation and shed light on the epitranscriptomic mechanism in the temporal control of cell fate transition.
Materials and methods
DPSC isolation and culture
Dental pulp tissues were collected according to the appropriate guidelines after written informed consent was obtained by a protocol approved by the Human Research Committee of Stomatological Hospital, Southern Medical University (ethical code 2019(16)). Primary DPSCs were harvested and cultured as previously described [
15]. Briefly, dental pulp tissues were removed from extracted third molars and then digested with 3 mg/mL collagenase I (Gibco-Invitrogen, Carlsbad, CA, USA). DPSCs were collected and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin (all from Gibco-Invitrogen) at 37 °C with 5% CO
2. The medium was changed every 2 days, and DPSCs at passages 3–5 were used for subsequent experiments.
DPSCs (at a density of 2 × 105 cells/mL) were seeded in 6-well or 24-well plates (Corning Life Sciences, NY, US) and cultured until the cells reached 80–90% confluence. The culture medium was changed to osteo/odontogenic medium (OM) containing 10 mmol/L β-glycerophosphate, 50 μg/mL ascorbic acid and 0.1 μmol/L dexamethasone (all from Sigma‒Aldrich, St. Louis, MO, USA). The induction medium was changed every other day from day 0 to day 21 (the day of induction was defined as day 0). After 7 days of induction, the cells were fixed and stained for alkaline phosphatase (ALP) (Beyotime Biotechnology, Shanghai, China) (n = 5). Alizarin red staining (ARS) (Beyotime) was used to stain the accumulated mineralized matrix on day 14 (n = 5). For adipogenic differentiation, DPSCs were induced by the Adipogenesis Differentiation Kit (HUXXC-90031, Cyagen Biosciences, Guangzhou, China). Cells were cultured with adipogenic inducing solution A for 3 days, and the medium was replaced with solution B for 1 day and then replaced with solution A. This cycle was repeated four times and then subjected to oil red O staining (Cyagen Biosciences) (n = 5).
DPSCs were subjected to osteo/odontogenic induction for 0, 7, and 14 days, and RNA was extracted using TRIzol solution (Takara Biotechnology, Shiga, Japan). Total m6A was measured with an m6A RNA methylation quantification kit (P-9019-96, Epigentek, Farmingdale, NY, US) according to the manufacturer’s protocol.
Liquid chromatography with tandem mass spectrometry (LC‒MS/MS) analysis was utilized to assess the metabolite compounds related to m6A methylation, including S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Cell samples were subjected to methanol and homogenized before analysis with an ultrahigh-performance liquid chromatography (UHPLC) column (1290 Infinity LC, Agilent Technologies). Next, during MS/MS acquisition, a m/z range of 25–1000 Da was used, and the ion accumulation time was screened. Metabolite compounds were identified with a database of available standards and subjected to multivariate data analysis. Metabolites with a variable importance in projection (VIP) value > 1 were further subjected to statistical analysis.
m6A RIP-seq and m6A RIP-qPCR
m
6A RIP-seq was used to characterize m
6A modification during DPSC mineralization (osteo/odontogenic medium-induced DPSCs, OM-DPSCs). Total RNA was isolated from DPSCs induced for 0, 7, and 14 days with TRIzol solution. The extracted RNA enriched and purified with oligo(dT)-attached magnetic beads and an m
6A RIP kit (17-10499, Millipore, Burlington, MA, US) according to the kit’s protocol. The purified m
6A-RIP RNA fragments were then fragmented into small pieces with fragmentation buffer for sequencing. The RNA fragments were incubated with magnetic beads conjugated with an m
6A-specific antibody in buffer. The IP RNA and input RNA were reverse transcribed into cDNA and subjected to deep sequencing on an Illumina NovaSeq™ 6000 platform for m
6A RIP-seq [
16]. m
6A peak calling, distribution, motif mapping and enrichment analysis were performed by LC-BIO Technologies Co., Ltd. (Hangzhou, China). To identify specific genes targeted by METTL3, the enrichment of m
6A-modified
Noggin (NOG) mRNA in the immunoprecipitate (IP) and input RNA was quantified by qPCR analysis as described previously for m
6A RIP-qPCR [
14].
RNA sequencing and profile analysis
Total RNA was extracted from DPSCs, and poly(A) mRNA was purified with poly(T)-conjugated magnetic beads. Then, the mRNA was fragmented into small pieces and converted into double-stranded cDNA. Paired‐end runs with a read length of approximately 300 base pairs (bp) were used for RNA‐sequencing with the Illumina deep sequence platform by LC-BIO Technologies Co., Ltd. Significantly differentially expressed genes (DEGs) were identified in this study as genes with a fold change in expression ≥ 2.0 and corresponding p value < 0.05. The DEGs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses with the R packages GOseq and DAVID.
Lentiviral vector construction and infection
Two independent METTL3 shRNA sequences (shMETTL3-1 and shMETTL3-2) within lentiviral vectors were used. shMETTL3-1 lentivirus encoding METTL3-shRNA and shCTR-1 lentivirus encoding scrambled control (shCTR) sequences were constructed by GeneChem Company (Shanghai, China); shMETTL3-2 and shCTR-2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, US). cDNA encoding the full-length METTL3 gene was amplified and used to construct a lentiviral vector for METTL3 overexpression (LV-METTL3) by GeneChem Company. For lentivirus transfection, DPSCs were transfected with lentivirus at a multiplicity of infection (MOI) of 50 and cultured for 72 h before subsequent experiments. DPSCs with METTL3 knockdown were treated with 2 µg/mL human Noggin peptide (ab16380, Abcam, Cambridge, UK) to neutralize noggin protein during DPSC differentiation.
Quantitative polymerase chain reaction (qPCR)
The cells were digested and total RNA was extracted from DPSCs with a Total RNA Isolation Kit (Foregene Biotechnology, Chengdu, China). The RNA was then reverse transcribed with RT Master Mix (Takara) according to the manufacturer’s instructions to obtain complementary DNA (cDNA). Real-time qPCR was performed with TB Green qPCR Mix (Takara) according to the manufacturer’s protocol. Relative target gene expression was analyzed with a standard curve and normalized to Glyceraldehyde-3-Phosphate Dehydrogenase (
GAPDH) expression. The primer sequences used in qPCR are summarized in Additional file
1: Table S1.
Western blot analysis
DPSCs were lysed and assayed with a BCA protein assay kit (Beyotime). Samples containing 15–30 μg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins were then transferred to polyvinylidene fluoride membranes (Millipore). The membranes containing the transferred proteins were blocked with 5% bovine serum albumin and reacted with the primary antibody overnight at 4℃. The membranes were then labeled with corresponding secondary antibody of horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG for 1 h at room temperature before visualization with SuperSignal enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, US). Primary and secondary antibodies against the following proteins were used in this study: METTL3 (96391, 1:1000); METTL14 (51104, 1:1000); WTAP (56501, 1:1000); p-Smad1/5 (9516, 1:1000) were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). NOG (sc-293439, 1:1000); RUNX Family Transcription Factor 2 (RUNX2, sc-390351, 1:1000); Dentin Sialophosphoprotein (DSPP, sc-73632, 1:1000); Smad1/2/3 (sc-7960, 1:1000); p-Smad3 (sc-517575, 1:1000) were obtained from Santa Cruz Biotechnology. GAPDH (60004-1-Ig, 1:3000); goat anti-mouse IgG (SA00001-1, 1:3000) and goat anti-rabbit (SA00001-2, 1:3000) were purchased from ProteinTech Group (ProteinTech, Wuhan, China).
Animal model construction
The animal experiments were conducted in compliance with ARRIVE guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures followed protocols approved by the Ethics Committees of Stomatological Hospital, Southern Medical University (ethical code 2019(16)). For ectopic transplantation studies, porous beta-tricalcium phosphate/hydroxyapatite (β-TCP/HA) discs (diameter: 4 mm, thickness: 2 mm) were obtained from Biological Materials Manufacturing Core, Sichuan University. Approximately 1 × 10
6 transfected DPSCs were seeded on β-TCP/HA discs and cultured in a 24-well plate with odontogenic medium for 24 h [
17,
18]. The composites of DPSCs and the β-TCP/HA scaffold were transplanted into the subcutaneous dorsal pockets of 6-week-old BALB/c immunodeficient nude mice (n = 10) [
18,
19]. Two subcutaneous pockets were made on the right and left side of the dorsal surface, each allowing for one composite. The shCTR and shMETTL3 groups were carefully transplanted into the left and right subcutaneous regions, respectively (n = 5), as were the LV-METTL3 and LV-CTR groups (n = 5). After 4 weeks, the harvested implants were fixed with paraformaldehyde, followed by decalcification with Ethylenediaminetetraacetic acid (EDTA) for 2 weeks. The formation of new mineralized tissue was evaluated by Masson-trichrome staining.
Immunofluorescence staining
The composites of DPSCs and the β-TCP/HA scaffold were fixed, dehydrated, embedded in paraffin and then cut at 5 µm. The tissue slides were subjected to immunofluorescence with a standard protocol. The slides were incubated with primary antibody overnight at 4℃. After washing, samples were interacted with the corresponding secondary HRP-conjugated antibody (1:1000, ProteinTech) for 1 h and then labeled with Cy3 Tyramine (11065, AAT Bioquest, Inc. Sunnyvale CA, US) or AF 488 Tyramide reagent (11070 AAT Bioquest). Primary antibodies against the following proteins were used in this study: METTL3 (15073-1-AP, 1:200, ProteinTech); NOG (sc-293439, 1:200, Santa Cruz); p-Smad3 (sc-517575, 1:100, Santa Cruz) and Smad1/2/3 (sc-7960, 1:100, Santa Cruz).
Immunofluorescence staining of the induced DPSCs was performed as described in a previous study [
15]. Fixed cells were blocked and incubated with the primary antibodies anti-METTL3 and anti-NOG overnight and then the corresponding fluorescent secondary antibody of Cy3–conjugated anti-rabbit IgG (SA00009-2, 1:500, ProteinTech) or fluorescein (FITC)–conjugated anti-mouse (SA00003-1, 1:500, ProteinTech). Fluorescent images showing subcellular expression were obtained with a confocal microscope (LSM 900; Zeiss, Oberkohen, Germany).
m6A site-specific mutant plasmid construction
To investigate the biological effect of the m
6A methylated sites in the 3′ untranslated region (UTR) of NOG, full-length NOG cDNA was constructed and cloned into the NheI- and BamHI-digested Flag-PCDNA3.1(+) vector by Genecreate Biotechnology Co., Ltd. (Wuhan, China). The vector used to express NOG with the wild-type m
6A motif was referred to as NOG-WT. Two different mutants (NOG-MUT1 and NOG-MUT2) were generated by introducing single-nucleotide mutations (A to T) in the four m
6A motifs of the 3′ UTR after mapping the conserved m
6A motif sequence in m
6A RIP-seq. To examine mRNA expression, 293T cells were transfected with NOG-WT vector or a mutant NOG vector and subjected to qPCR [
20].
RNA and protein stability analysis
RNA transcription in DPSCs was inhibited by treatment with 5 µg/mL actinomycin D (ActD, 7240-37-1, Sigma‒Aldrich) as described in a previous study to analyze mRNA decay rates [
21]. mRNA was isolated after 0, 4, and 8 h and subjected to qPCR. The half-life of NOG mRNA has been reported.
Protein translation in DPSCs was inhibited by treatment with 100 µg/mL cycloheximide (CHX, A8244, APExBIO Technology, Houston, TX, US) to analyze protein stability. Total protein was isolated after 0, 4, and 8 h and subjected to western blot analysis. The protein expression level of NOG was used to analyze its stability.
3′-Rapid amplification of cDNA ends (3′-RACE)
3′-RACE to obtain the 3′ UTR sequences of NOG from shMETTL3 or shCTR DPSCs after osteogenic induction was performed with a 3′-RACE kit (6106, Takara) according to the protocol. The 3′-RACE products after two rounds of amplification were purified and subcloned into a vector, and the amplified fragments were further identified by sequencing [
22]. The
NOG-specific primers used for 3′-RACE analysis in this study were as follows: 5′-CATGGTGTGCAAGCCGTCCAAGTC-3′, 5′-TCACGGTGCTGCGGTGGCGCTGTC-3′.
Poly(A) tail assay
A poly(A) tail assay was performed with a Poly(A) Tail-Length Assay Kit (764551KT, Thermo Fisher) according to the protocol. Briefly, poly(A) polymerase was used to add G and I to the 3′ ends of the RNA, and the newly tailed RNA was converted to cDNA by reverse transcription. Then,
NOG-specific forward and reverse primers and universal reverse primers were used to generate a product consisting of
NOG with a poly(A) tail. The PCR products from METTL3 knockdown DPSCs in differentiated/undifferentiated stages were separated on agarose gels [
23]. The specific primers for
NOG were F: 5′-TAACCTGCTATTTATATTCCAGTGCCCTTC-3′ and R: 5′-TGAACTCTATAGCTTCTTCGAGGTCCAA-3′.
Quantification and statistical analysis
The experiments in this study were carried out biologically repeated at least three times, and the data are presented as the mean ± standard deviation. Statistical differences were evaluated by one‑way analysis of variance (ANOVA) and corresponding post hoc tests for multiple comparisons. Unpaired two-tailed Student’s t test was used to compare two groups. A p value < 0.05 was considered to be statistically significant and was analyzed by GraphPad Prism 7.0 (La Jolla, CA, US).
Discussion
Emerging evidence has proven that m
6A RNA methylation is a critical epitranscriptomic mechanism that permits additional specificity and plasticity to the transcriptome [
25]. Here, we revealed the dynamic and unique m
6A mRNA landscape in DPSC mineralization, and elevated m
6A marks in the 3′ UTR of certain transcripts are required for transcriptional prepatterning. METTL3 was identified as the essential m
6A modulator in regulating DPSC differentiation. Furthermore, increasing m
6A hallmarks in the 3′ UTR restricted the gene expression of NOG during DPSC mineralization. METTL3 mediated the m
6A modification of NOG and promoted its degradation via poly(A) tail shortening in a stage-specific manner. The present study addressed a critical role of m
6A modification in the temporal control of DPSC differentiation and provided new insight into the transcriptional coordination of stem cell regulation.
RNA m
6A deposition is redundant in the consensus motif RRm
6ACH ([G/A/U][G > A]m
6AC[U > A > C]), which is enriched in the CDS and 3′ UTR of RNA transcripts [
8,
26]. During embryonic cortical neurogenesis, m
6A-methylated transcripts are enriched in biological processes, such as neural stem cells, the cell cycle, and differentiation, which are essential to control the transcriptome composition of different stages [
25]. We characterized the dynamic and unique m
6A landscape in DPSC mineralization, and the m
6A-mRNA profile was mainly related to transcriptional regulation and cell differentiation. Moreover, the increasing total m
6A content and m
6A distribution in the 3′ UTR might result from a pronounced elevation of METTL3 expression. METTL3 was reported to participate in tooth root development by modulating translational efficiency [
27]. The inhibition of DPSC proliferation and osteogenesis by METTL3 knockdown was associated with an impaired glycolytic pathway [
28]. METTL3 is also involved in bone mesenchymal stem cell (BMSC) differentiation and function [
16,
29]. METTL3 depletion in BMSCs impaired osteogenic differentiation, while METTL3 overexpression partly abrogated the induction of osteoporosis in mice [
16]. Consistent with the current literature, METTL3 inhibition comprised DPSC differentiation, and METTL3 overexpression facilitated DPSC mineralization, indicating therapeutic potential. There are several in vivo models available to verify the regulatory mechanism of DPSC differentiation, such as dentin–pulp complex regeneration in situ and ectopic transplantation of DPSCs [
30,
31]. Ectopic mineralization models were used in this study and subcutaneous transplantation in immunocompromised mice supported that METTL3 is a positive regulator of DPSC differentiation and mineralized tissue formation. More evidences from orthotopic models are needed to support the therapeutic application in vital pulp procedures and DPSC-based therapy.
Various transcripts and signals are tagged in a timely manner by m
6A modification, which in turn controls proper development and differentiation. METTL3-mediated m
6A modification regulates the expression of some osteogenic markers and other related genes involved in bone metabolism [
32]. Parathyroid hormone (PTH)/Pth1r, TGF-β/SMAD, WNT and other signaling pathways are modulated by m
6A marks, which are essential in the cellular differentiation and cancer development [
16,
33,
34]. Here, NOG and downstream Smad pathway were identified as the target of METTL3-mediated m
6A modification during DPSC differentiation. NOG is a key player in ectoderm development, and its disruption can lead to organogenesis defects such as craniofacial defects and hypoplastic teeth [
35,
36]. Noggin is capable of binding and inactivating members of the TGF-β superfamily proteins as BMPs, subsequently blocking BMP-induced Smad pathway activation [
37]. BMSC osteogenesis and DPSC odontogenesis are regulated by NOG via the downstream Smad1/5 signaling pathway [
38,
39]. We found that the m
6A peaks in the 3′ UTR of
NOG mRNA increased during DPSC mineralization, which restricted its gene expression. METTL3 inhibited m
6A-tagged NOG expression and promoted its degradation in differentiated DPSCs. Consistent with our data, m
6A modification modulates RNA degradation and gene expression in neural stem cells, which is a critical epigenetic mechanism in the temporal control of neurogenesis [
25]. m
6A signaling clustered in the 3′ UTR is mainly responsible for cytoplasmic events related to RNA stability and translation [
40‐
42], and METTL3 can independently read and modulate m
6A marks in the 3′ UTR of certain transcripts [
40]. Taken together, these findings suggest that m
6A modification dynamically modulates the stability of specific transcripts, which is required for the transcriptional prepatterning of DPSC mineralization.
Poly(A) tails are 150–250 adenosine nucleotides acquired by the end of the 3′ UTR in the nucleus that subsequently undergo deadenylation in the cytoplasm. The length of a poly(A) tail changes throughout the lifetime of mRNA and has essential effects on its stability, degradation and translation [
43]. In the global transcriptome, transcripts with a longer poly(A) tail possess a longer average mRNA half-life [
23]. The deadenylation of shorter poly(A) tails can cause RNA decay or translational defects [
44]. A recent study also noted the correlation between m
6A marks and poly(A) tail regulation. The transcriptional dynamics of certain genes are related to differences in poly(A) tail length via m
6A modification and deadenylase complexes [
45]. m
6A signaling is capable of controlling RNA structural switching and RNA‒protein interactions [
46]. METTL3 and WATP can modulate RNA stabilization in an m
6A-HuR-dependent manner [
47,
48]. The m
6A reader YTH N
6-methyladenosine RNA binding protein (YTHDF) 2 is reported to directly interact with the CCR4-NOT complex. YTHDF3 can recruit the poly(A) specific ribonuclease subunit (PAN) 2-PAN3 complex, contributing to its deadenylation and degradation [
49,
50]. Nonadenosine residues, such as G modifications, are also related to high quality and delayed degradation of the poly(A) tail [
51,
52]. In our study, the temporal control of NOG stabilization by METTL3 relied on poly(A) tail shortening in the differentiation stage. Further studies are needed to identify the specific mechanism of how m
6A marks lead to shortened poly(A) tails in DPSC differentiation.
The osteo/odontogenic differentiation of DPSCs and tertiary dentin formation are of particular interest in relation to dental repair. Identifying the key signaling in DPSC differentiation and mineralized matrix formation, and recapitulating these processes in clinical strategies could preserve pulp vitality. In the present study, we demonstrated that dynamic m6A RNA methylation is essential for heightened transcriptional coordination during DPSC differentiation. METTL3-mediated m6A marks tag the 3′ UTR of NOG and inhibit its stabilization via poly(A) tail regulation in a stage-specific manner. The present study identifies a critical role of METTL3-mediated m6A methylation in the temporal control of cell fate transition and sheds light on the epitranscriptomic machinery of m6A-dependent poly(A) tail regulation in transcriptional dynamics.
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