{"id":604176,"date":"2024-06-02T20:00:00","date_gmt":"2024-06-03T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/mettl3-mediated-pre-mir-665-dlx3-m6a-methylation-facilitates-the-committed-differentiation-of-stem-cells-from-apical-papilla-experimental-molecular-medicine\/"},"modified":"2024-06-02T23:34:11","modified_gmt":"2024-06-03T03:34:11","slug":"mettl3-mediated-pre-mir-665-dlx3-m6a-methylation-facilitates-the-committed-differentiation-of-stem-cells-from-apical-papilla-experimental-molecular-medicine","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/mettl3-mediated-pre-mir-665-dlx3-m6a-methylation-facilitates-the-committed-differentiation-of-stem-cells-from-apical-papilla-experimental-molecular-medicine\/","title":{"rendered":"METTL3-mediated pre-miR-665\/DLX3 m6A methylation facilitates the committed differentiation of stem cells from apical papilla – Experimental & Molecular Medicine","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
SCAPs were isolated and exhibited a characteristic spindle-like morphology (Fig. 1a, b<\/a>). Flow cytometric analysis confirmed that the extracted SCAPs were undifferentiated stem cells derived from the mesenchyme, as these cells expressed CD29, CD73, CD90, and CD105 but did not express CD34 or CD45 (Supplementary Fig. 1<\/a>). Additionally, the ability of SCAPs to differentiate into three lineages, namely, chondrogenic, osteogenic, and adipogenic lineages, was confirmed through Alcian blue staining, Alizarin red staining, and Oil red O staining, respectively (Supplementary Fig. 2<\/a>). The results indicate that SCAPs have the ability to differentiate in multiple directions.<\/p>\n a<\/b>, b<\/b> SCAPs were isolated according to the literature. SCAPs displayed a typical spindle-like morphology in the original and third generations. Scale bar = 100\u2009\u00b5m. c<\/b>, d<\/b> Annotation of the m6<\/sup>A RNA methylation quantification kit and the expression of m6<\/sup>A at 0, 3, and 7 days of mineralization; n<\/i>\u2009=\u20096. e<\/b> m6<\/sup>A dot blot showing an increase in global m6<\/sup>A levels during osteogenic induction in SCAPs at 3 and 7 days. n<\/i>\u2009=\u20093. f<\/b>, g<\/b> Western blotting was performed to analyze the expression of METTL3, METTL14 and WTAP during SCAP odonto\/osteogenic differentiation at 0, 3 and 7 days. n<\/i>\u2009=\u20093. h<\/b> Immunofluorescence staining was conducted on CD1 mice at postnatal Day 7. Scale bar = 40\u2009\u00b5m. i<\/b> Representative images of immunohistochemistry assays demonstrating the enrichment of METTL3 in the papilla tissue of human apical teeth. PBS was used as a negative control. Scale bar = 1\u2009m; 100\u2009\u00b5m. j<\/b> Immunohistochemically stained samples of the METTL3-enriched zone in the papilla tissue of human apical teeth. PBS was used as a negative control. Scale bar = 500\u2009\u00b5m; 40\u2009\u00b5m. ns indicates no significance; **<\/i> indicates P<\/i>\u2009<\u20090.01; ***<\/i> indicates P<\/i>\u2009<\u20090.001.<\/p>\n<\/div>\n<\/div>\n To assess METTL3-mediated m6<\/sup>A modification during odontogenic induction, we cultured SCAPs in odontogenic induction medium for 0, 3, or 7 days. The results demonstrated a gradual increase in the overall level of m6<\/sup>A during SCAP mineralization (Fig. 1c\u2013e<\/a>). Additionally, western blotting analysis revealed a progressive increase in METTL3 expression with prolonged induction time, accompanied by increased expression of odonto\/osteoblast markers (DSPP\/RUNX2\/OSX). Conversely, the expression of the main \u201cwriter\u201d proteins WTAP and METTL14 did not significantly differ (Fig. 1f, g<\/a>). To observe the in vivo expression pattern of METTL3, we conducted immunofluorescence staining on postnatal Day 7 CD1 mouse samples. The findings demonstrated that METTL3 is primarily present in the apical papilla tissue of tooth roots (Fig. 1h<\/a>). Additionally, immunohistochemistry assays confirmed that enriched localization was observed in the human dental papilla (Fig. 1i<\/a>). METTL3 was also expressed in the apical tissue of third molars that displayed almost complete root development (Fig. 1j<\/a>). In general, these findings indicate that METTL3-induced m6<\/sup>A alterations might affect the differentiation of odonto\/osteoblasts in SCAPs.<\/p>\n To investigate the role of METTL3 in tooth root and bone development, we established a METTL3 knockdown mouse model using the CRISPR\/Cas9 system. As METTL3 homozygous knockout (METTL3-\/-<\/sup>) mice showed early embryonic lethality, we used METTL3 heterozygous knockdown (METTL3+\/\u2212<\/sup>) mice for subsequent experiments22<\/a><\/sup>. The genotype was initially confirmed through PCR and sequencing and further verified by western blotting (Fig. 2a\u2013d<\/a>, Supplementary Fig. 3a<\/a>). H&E staining revealed no discernible differences in the size or morphology of the lung, spleen, kidney, liver, or heart between METTL3+\/\u2212<\/sup> mice and wild-type (WT) mice (Supplementary Fig. 3b<\/a>). Subsequently, stereoscopic microscopy and micro-CT analyses of mandibular first molars revealed a reduction in molar root length and root dentin width in METTL3+\/\u2212<\/sup> mice compared to their WT littermates. Furthermore, the mandibular first molars of METTL3+\/\u2212<\/sup> mice exhibited a significant reduction in dentin width, as evidenced by H&E staining (Fig. 2e, f<\/a>). Additionally, the results obtained from micro-CT, H&E staining, and Goldner staining collectively indicated that METTL3 knockdown significantly decreased bone mass and bone formation (Fig. 2g-i<\/a>). Taken together, the above results suggest the important and notable role of METTL3 in root development and bone formation.<\/p>\n a<\/b>\u2013d<\/b> Schematic illustration of the generation of METTL3+\/\u2212<\/sup> knockout mice (C57BL\/6\u2009N) via a CRISPR\/Cas9-mediated genome engineering strategy. Six exons were identified. Exon 4 was selected as the target site. Cas9 and guide RNA (gRNA) were coinjected into fertilized eggs for mouse production, followed by genotype identification of METTL3+\/\u2212<\/sup> KO mice. e<\/b> A reduction in root length and dysplasia of the tooth root in METTL3+\/\u2212<\/sup> knockout mice compared to WT mice were observed via stereoscopic microscopy and micro-CT analyses. Scale bar = 1\u2009mm. f<\/b> Representative images of HE staining indicating that the width of the root dentin in the mandibular first molar was greater in METTL3\u2009+\/- mice than in WT mice. Scale bar = 200\u2009\u00b5m; 20\u2009\u00b5m. g<\/b> Representative micro-CT images of the femoral metaphysis and entire proximal femur. Scale bar = 10\u2009mm. h<\/b>, i<\/b> Representative images of HE staining and Golder staining showing that METTL3+\/\u2212<\/sup> mice exhibited reduced bone formation relative to that of their WT counterparts. Scale bar = 500\u2009\u00b5m; 40\u2009\u00b5m.<\/p>\n<\/div>\n<\/div>\n To assess the regulatory impact of METTL3 on the odontogenic\/osteogenic differentiation of SCAPs, we suppressed METTL3 via transfection of specific small interfering RNAs (siRNAs) (Fig. 3a<\/a>). As expected, m6<\/sup>A dot blot analysis revealed a decrease in the overall m6<\/sup>A level in SCAPs upon METTL3 knockdown (Fig. 3b<\/a>). Furthermore, the expression levels of genes and proteins associated with odonto\/osteogenic differentiation decreased, whereas METTL14 did not significantly change (Fig. 3c\u2013e<\/a>, Supplementary Fig. 3c<\/a>). Reduced ALP staining and activity, as well as decreased ARS staining, revealed a diminished degree of odonto\/osteogenesis and mineralization in SCAPs (Fig. 3f\u2013i<\/a>). Immunofluorescence staining analysis additionally demonstrated a significant reduction in the protein expression levels of RUNX2 and ALP in the SCAPs transfected with si-METTL3 compared to those in the si-NC group (Fig. 3j, k<\/a>).<\/p>\n a<\/b> The efficiency was measured by qRT\u2012PCR in SCAPs transfected with si-METTL3. b<\/b> m6<\/sup>A dot blot assay showing the negative effects of METTL3 inhibition on the m6A content. c<\/b> qRT\u2012PCR showed that the mRNA levels of DSPP, DMP1, ALP, RUNX2<\/i> and OSX<\/i> were decreased upon METTL3 silencing. d<\/b>, e<\/b> The protein expression of METTL3, DSPP, DMP1, ALP, RUNX2 and OSX decreased after METTL3 inhibition. f<\/b>\u2013i<\/b> Representative images of ALP staining and ARS staining and relative quantification showing the negative effects of METTL3 silencing compared with si-NC treatment at 7 and 14 days in the mineralized medium of SCAPs. Scale bar = 200\u2009\u00b5m. j<\/b>, k<\/b> Representative images of immunofluorescence staining for METTL3, ALP and RUNX2 in SCAPs after METTL3 silencing. Scale bar = 40\u2009\u00b5m. n<\/i>\u2009=\u20093, *<\/i> indicates P<\/i>\u2009<\u20090.05; **<\/i> indicates P<\/i>\u2009<\u20090.01; ***<\/i> indicates P<\/i>\u2009<\u20090.001.<\/p>\n<\/div>\n<\/div>\n In contrast, the impact of METTL3 overexpression on the differentiation of SCAPs was investigated using recombinant lentiviruses to introduce METTL3 (METTL3-OE) into SCAPs (Fig. 4a\u2013c<\/a>). Overexpression of METTL3 led to elevated levels of m6<\/sup>A, as shown by the m6<\/sup>A dot blot assay (Fig. 4d<\/a>). qRT\u2012PCR and western blotting revealed that the upregulation of METTL3 substantially increased the levels of odonto\/osteogenic biomarkers (Fig. 4e\u2012g<\/a>). ALP staining and ARS staining revealed a greater number of calcified nodules in the METTL3-OE group than in the NC-OE group, as further confirmed by increased ALP activity and CPC quantification analysis (Fig. 4h\u2013k<\/a>). To thoroughly examine the role of METTL3-mediated m6<\/sup>A modification in the process of bone regeneration, we generated a bilateral mandibular defect model in SD rats. As expected, compared with the control group, the METTL3-overexpressing SCAP group exhibited more bone-like structures, as shown by micro-CT and H&E staining (Fig. 4l, m<\/a>). Collectively, these findings suggest that METTL3-mediated m6<\/sup>A modification potentially exerts a beneficial effect on the odonto\/osteogenic differentiation of SCAPs.<\/p>\n a<\/b> Structural diagram of lentivirus-mediated infection of SCAPs with METTL3-OE or NC-OE. b, c<\/b> Immunofluorescence staining and qRT\u2012PCR were used to verify the efficiency of METTL3-OE and NC-OE infection at 72\u2009h in SCAPs. Scale bar = 100\u2009\u00b5m. d<\/b> m6<\/sup>A dot blot assay showing that METTL3 overexpression increased m6<\/sup>A levels in SCAPs. e<\/b> qRT\u2012PCR showed that METTL3 overexpression increased the mRNA expression of DSPP, DMP1, ALP, RUNX2<\/i> and OSX<\/i>. f<\/b>, g<\/b> Western blot analysis of METTL3, DSPP, DMP1, ALP, RUNX2 and OSX after METTL3 overexpression. h<\/b>\u2013k<\/b> Representative images and relevant quantification of ARS-stained ALP staining and mineralized nodule formation. Scale bar = 200\u2009\u00b5m. l<\/b>, m<\/b> Micro-CT and H&E staining were used to verify the role of METTL3 in regulating SCAP osteogenesis. S: scaffold; B: bone-like tissue; scale bar = 100\u2009\u00b5m. n<\/i>\u2009=\u20093, *<\/i> indicates P<\/i>\u2009<\u20090.05; **<\/i> indicates P<\/i>\u2009<\u20090.01; ***<\/i> indicates P<\/i>\u2009<\u20090.001.<\/p>\n<\/div>\n<\/div>\n After identifying the METTL3-mediated promotion of SCAP odonto\/osteogenic differentiation, we investigated the molecular mechanism through which METTL3 exerts its regulatory effects. Previous research has suggested that METTL3 can decrease the levels of mature miRNAs by modifying hairpin-shaped precursor miRNAs (pre-miRNAs) with m6<\/sup>A, thereby indirectly increasing the expression of miRNA target genes and ultimately participating in the regulation of cell differentiation23<\/a>,24<\/a><\/sup>. Hence, to determine the target pre-miRNAs of METTL3, we performed a m6<\/sup>A RNA immunoprecipitation (RIP) microarray analysis of METTL3-OE- and NC-OE-infected SCAPs (Fig. 5a<\/a>). The resulting list of the top 5 m6<\/sup>A-methylated pre-miRNAs in SCAPs was compiled. Among these, pre-miR-665 and pre-miR-933 exhibited the highest levels of m6<\/sup>A methylation (\u2009>\u20091.2-fold change) and displayed a high degree of conservation across different species (Fig. 5b, c<\/a>). Notably, miR-665 was found to promote the transition from acetylation to methylation of the promoters of the odontogenic differentiation markers DSPP and DMP125<\/a><\/sup>, suggesting the potential role of this molecule as a repressor during odontoblast induction. Consequently, we screened pre-miR-665 as a promising candidate for further investigation of METTL3 activity.<\/p>\n a<\/b> Flowchart of the m6<\/sup>A-RIP microarray analysis procedure used to identify the targets of METTL3. b<\/b> The top 5 pre-miRNAs with upregulated METTL3-mediated m6<\/sup>A methylation are listed. c<\/b> The combined sites of pre-miR-665 and m6<\/sup>A methylation are labeled. d<\/b> MeRIP-qPCR showed the positive effect of METTL3-OE on pre-miR-665 methylation by m6<\/sup>A compared with that of NC-OE. e<\/b> qRT\u2012PCR analysis of the inhibitory impact of pre-miR-665 and miR-665 on METTL3 overexpression. f<\/b> MeRIP-qPCR showed the negative effect of si-METTL3 on pre-miR-665 methylation by m6<\/sup>A compared with that of si-NC. g<\/b> qRT\u2012PCR analysis of the ability of pre-miR-665 and miR-665 to promote METTL3 inhibition. h<\/b> The mRNA expression of DSPP, DMP1, ALP, RUNX2<\/i> and OSX<\/i> in the SCAPs transfected with miR-665 mimics and inhibitors. i<\/b>, j<\/b> The protein expression of DSPP, DMP1, ALP, RUNX2 and OSX and quantitative analysis in the SCAPs transfected with miR-665 mimics and inhibitors. k<\/b>\u2013n<\/b> Representative images of ALP- and ARS-stained transfected SCAPs at 7 and 14 days in mineralized medium. Scale bar = 200\u2009\u00b5m. o<\/b> Immunofluorescence staining results of transfected SCAPs stained for ALP and RUNX2. Scale bar = 40\u2009\u00b5m. p<\/b> qRT\u2012PCR indicated pre-miR-665 was a target of YTHDF2. q<\/b> si-YTHDF2 increased the expression of pre-miR-665 after cotransfection of METTL3-OE. n<\/i>\u2009=\u20093, *<\/i> indicates P<\/i>\u2009<\u20090.05; **<\/i> indicates P<\/i>\u2009<\u20090.01; ***<\/i> indicates P<\/i>\u2009<\u20090.001.<\/p>\n<\/div>\n<\/div>\n Subsequently, we investigated the effects of amplifying or suppressing METTL3 on alterations in the m6<\/sup>A levels of pre-miR-665 utilizing MeRIP-qPCR. MeRIP-qPCR revealed that increased METTL3 expression led to an increase in the m6<\/sup>A methylation level of pre-miR-665, while METTL3 inhibition had the opposite effect (Fig. 5d, f<\/a>). Furthermore, the expression levels of both pre-miR-665 and miR-665 were notably decreased when METTL3 was overexpressed in SCAPs. In contrast, si-METTL3 increased the production of pre-miR-665 and miR-665, indicating that METTL3-induced pre-miR-665 m6<\/sup>A enrichment can suppress miR-665 expression (Fig. 5e, g<\/a>).<\/p>\n To verify whether METTL3 regulates the odonto\/osteogenic differentiation of SCAPs by mediating the m6<\/sup>A modification of pre-miR-665, we further examined the impact of miR-665 on the odonto\/osteogenic differentiation of SCAPs. Transient transfection of miR-665 mimics and inhibitors was conducted (Supplementary Fig. 4a, b<\/a>). qRT\u2012PCR and western blotting demonstrated that miR-665 mimics hindered the odonto\/osteoblast differentiation of SCAPs, while miR-665 inhibitors increased the expression of relevant proteins and genes (Fig. 5h\u2012j<\/a>). Furthermore, ALP and ARS staining, along with subsequent quantitative analyses, was performed, revealing a diminished degree of odonto\/osteogenesis and mineralization in the SCAPs transfected with miR-665 mimics, while miR-665 inhibitors resulted in increased formation of calcium nodules. The immunofluorescence staining results also demonstrated an identical pattern (Fig. 5k\u2013o<\/a>).<\/p>\n The involvement of YTH domain family proteins, specifically YTHDF2\/3 and YTHDC1\/2, in m6<\/sup>A-related RNA degradation has been demonstrated26<\/a>,27<\/a>,28<\/a>,29<\/a><\/sup>. Consequently, we investigated the presence of collaborative \u201creader\u201d proteins in the process of METTL3-mediated pre-miR-665\u2009m6<\/sup>A methylation in SCAPs. si-YTHDF2 increased the expression of pre-miR-665, indicating that pre-miR-665 may be a potential target of YTHDF2 (Fig. 5p<\/a>; Supplementary Fig. 4c<\/a>). Furthermore, the inhibitory effect of METTL3-OE on pre-miR-665 expression was counteracted by cotransfection of SCAPs with si-YTHDF2 (Fig. 5q<\/a>).<\/p>\n To explore the possible downstream target genes associated with the control of METTL3\/miR-665 axis-mediated differentiation, we conducted a computational prediction using the TargetScan, MiRDB, and miRWalk databases to identify candidate target genes. Our analysis revealed a total of 177 potential target genes that bind to miR-665 (Fig. 6a<\/a>). Moreover, Gene Ontology (GO) analysis indicated a significant correlation between these target genes and diverse cellular biological processes (Fig. 6b<\/a>). These three databases predicted that DLX3<\/i> can simultaneously bind to miR-665, and GO analysis indicated that DLX3<\/i> is involved in tissue development. Furthermore, previous research has demonstrated the contribution of DLX3<\/i> to the odonto\/osteoblastic differentiation of human DPSCs, and DLX3<\/i> mutations have been linked to abnormal tooth development30<\/a>,31<\/a><\/sup>. Additionally, a significant association between DLX3 and bone mass has been reported32<\/a><\/sup>. Interestingly, Heair et al. revealed that miR-665 decreased DLX3<\/i> mRNA expression in dental pulp cells25<\/a><\/sup>. Therefore, the selection of DLX3<\/i> as a target of miR-665 is justified in SCAPs.<\/p>\n<\/a><\/div>\n
METTL3 knockdown (METTL3+\/\u2212<\/sup>) mice exhibit tooth root and bone dysplasia<\/h3>\n
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METTL3 positively regulates the odonto\/osteogenic differentiation capacity of SCAPs<\/h3>\n
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Pre-miR-665\/miR-665 is a functional target of METTL3 in SCAPs<\/h3>\n
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\n DLX3<\/i> is directly targeted by miR-665 in SCAPs<\/h3>\n