Gremlin 1+ fibroblastic niche maintains dendritic cell homeostasis in lymphoid tissues.

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

Homeostatic functions of monocytes and interstitial lung macrophages are regulated via collagen domain-binding receptor LAIR1.

Myeloid cells encounter stromal cells and their matrix determinants on a continual basis during their residence in any given organ. Here, we examined the impact of the collagen receptor LAIR1 on myeloid cell homeostasis and function. LAIR1 was highly expressed in the myeloid lineage and enriched in non-classical monocytes. Proteomic definition of the LAIR1 interactome identified stromal factor Colec12 as a high-affinity LAIR1 ligand. Proteomic profiling of LAIR1 signaling triggered by Collagen1 and Colec12 highlighted pathways associated with survival, proliferation, and differentiation. Lair1-/- mice had reduced frequencies of Ly6C- monocytes, which were associated with altered proliferation and apoptosis of non-classical monocytes from bone marrow and altered heterogeneity of interstitial macrophages in lung. Myeloid-specific LAIR1 deficiency promoted metastatic growth in a melanoma model and LAIR1 expression associated with improved clinical outcomes in human metastatic melanoma. Thus, monocytes and macrophages rely on LAIR1 sensing of stromal determinants for fitness and function, with relevance in homeostasis and disease.

LRRC15+ myofibroblasts dictate the stromal setpoint to suppress tumour immunity.

Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.

Cross-tissue organization of the fibroblast lineage.

Fibroblasts are non-haematopoietic structural cells that define the architecture of organs, support the homeostasis of tissue-resident cells and have key roles in fibrosis, cancer, autoimmunity and wound healing1. Recent studies have described fibroblast heterogeneity within individual tissues1. However, the field lacks a characterization of fibroblasts at single-cell resolution across tissues in healthy and diseased organs. Here we constructed fibroblast atlases by integrating single-cell transcriptomic data from about 230,000 fibroblasts across 17 tissues, 50 datasets, 11 disease states and 2 species. Mouse fibroblast atlases and a DptIRESCreERT2 knock-in mouse identified two universal fibroblast transcriptional subtypes across tissues. Our analysis suggests that these cells can serve as a reservoir that can yield specialized fibroblasts across a broad range of steady-state tissues and activated fibroblasts in disease. Comparison to an atlas of human fibroblasts from perturbed states showed that fibroblast transcriptional states are conserved between mice and humans, including universal fibroblasts and activated phenotypes associated with pathogenicity in human cancer, fibrosis, arthritis and inflammation. In summary, a cross-species and pan-tissue approach to transcriptomics at single-cell resolution has identified key organizing principles of the fibroblast lineage in health and disease.

Vertucci's root canal configuration of 11,376 mandibular anteriors and its relationship with distolingual roots in mandibular first molars in a Cantonese population: a cone-beam computed tomography study.

BACKGROUND: Cone-beam computed tomography (CBCT) was used to study the root canal system of mandibular anteriors (MAs) in a Cantonese population and to evaluate the correlation between the complicated root canal configurations of mandibular lateral incisors (MLIs) and the presence of distolingual roots (DLRs) in mandibular first molars (MFMs). METHODS: A total of 11,376 mandibular anterior teeth were scanned by CBCT. Those whose images met the inclusion criteria were first analysed according to Vertucci's root canal configuration and then grouped based on gender, age, and side, and their effects on root canal morphology were analysed. Finally, statistical analysis was used to evaluate the correlation between the complicated root canal configurations of MLIs and the existence of DLRs in MFMs. All statistical analyses were performed by using SPSS 25.0 software. Quantitative data are presented as the mean ± standard deviation. Student's t tests were used to calculate statistical significance. P < 0.05 was considered statistically significant. RESULTS: In MAs in the Cantonese population, all mandibular central incisors (MCIs) and MLIs had one root, and 0.37% of mandibular canines (MCs) had two roots. The most common Vertucci's root canal configuration was Vertucci I followed by Vertucci III. A total of 30.91% of MLIs and approximately 8% of MCIs and MCs have complicated root canal configurations. There were no significant differences in the prevalence of DLRs in MFMs and the incidence of complicated root canal configurations of MLIs between males and females or between the right and left teeth. However, a significant difference was found in different age groups of root canal configurations in MLIs. Moreover, significant ipsilateral and contralateral correlations between MFMs with DLRs and MLIs with complicated root canal configurations were observed on both sides. CONCLUSION: In Cantonese population, the possibility of complicated root canal configuration in MLIs was higher, when DLR appeared in MFMs.

Diagnostic biomarker candidates for pulpitis revealed by bioinformatics analysis of merged microarray gene expression datasets.

BACKGROUND: Pulpitis is an inflammatory disease, the grade of which is classified according to the level of inflammation. Traditional methods of evaluating the status of dental pulp tissue in clinical practice have limitations. The rapid and accurate diagnosis of pulpitis is essential for determining the appropriate treatment. By integrating different datasets from the Gene Expression Omnibus (GEO) database, we analysed a merged expression matrix of pulpitis, aiming to identify biological pathways and diagnostic biomarkers of pulpitis. METHODS: By integrating two datasets (GSE77459 and GSE92681) in the GEO database using the sva and limma packages of R, differentially expressed genes (DEGs) of pulpitis were identified. Then, the DEGs were analysed to identify biological pathways of dental pulp inflammation with Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and Gene Set Enrichment Analysis (GSEA). Protein-protein interaction (PPI) networks and modules were constructed to identify hub genes with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape. RESULTS: A total of 470 DEGs comprising 394 upregulated and 76 downregulated genes were found in pulpitis tissue. GO analysis revealed that the DEGs were enriched in biological processes related to inflammation, and the enriched pathways in the KEGG pathway analysis were cytokine-cytokine receptor interaction, chemokine signalling pathway and NF-κB signalling pathway. The GSEA results provided further functional annotations, including complement system, IL6/JAK/STAT3 signalling pathway and inflammatory response pathways. According to the degrees of nodes in the PPI network, 10 hub genes were identified, and 8 diagnostic biomarker candidates were screened: PTPRC, CD86, CCL2, IL6, TLR8, MMP9, CXCL8 and ICAM1. CONCLUSIONS: With bioinformatics analysis of merged datasets, biomarker candidates of pulpitis were screened and the findings may be as reference to develop a new method of pulpitis diagnosis.

Classification and morphology of middle mesial canals of mandibular first molars in a southern Chinese subpopulation: a cone-beam computed tomographic study.

BACKGROUND: Cone-beam computed tomography (CBCT) was employed to study the morphology and curvature of middle mesial canals (MMCs) of mandibular first molars (MFMs). METHODS: CBCT scanning was performed on MFMs of 1100 patients. Patients' images that met the inclusion criteria were divided into group A (< 40 years old) and group B (≥ 40 years old) for further study. The images were used to study the incidence of MMCs at different ages, measure the curvature of MMCs in the mesiodistal and buccolingual directions using the Schneider method, and observe the anatomical morphology of the mesial root canal system. All statistical analyses were performed by using SPSS 21.0 software. Quantitative data were presented as mean ± standard deviation. Student's t-test was used to calculate the statistical significance. P < 0.05 was considered statistically significant. RESULTS: In 875 patients, 1750 MFM images met the inclusion criteria. Among these cases, 158 MFMs contained an MMC, yielding an incidence rate of 9.03%. The incidence rate of MMCs was 11.22% in group A and 6.61% in group B, and this difference was statistically significant (P < 0.05). The curvature in the mesiodistal direction was 29.39 ± 8.53° in group A and 26.06 ± 8.50° in group B, and this difference was also significant (P < 0.05). The curved regions in groups A and B were often located in the middle 1/3 of canal. No significant difference in the distance between MMC orifices and mesiobuccal canal orifices or mesiolingual canal orifices was noted (P > 0.05). The most common mesial root canal morphological type was type II (3-2) (53.80%). CONCLUSION: The incidence of MMCs in MFMs declined as age increased. The canal systems of MMCs were varied and complex, mainly exhibiting an obvious mesiodistal curve. CBCT is an outstanding method to help guide root canal therapy. *Yeqing Yang and Buling Wu have contribute equally to this article.

FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function.

Forkhead box A1 (FOXA1) is an FKHD family protein that plays pioneering roles in lineage-specific enhancer activation and gene transcription. Through genome-wide location analyses, here we show that FOXA1 expression and occupancy are, in turn, required for the maintenance of these epigenetic signatures, namely DNA hypomethylation and histone 3 lysine 4 methylation. Mechanistically, this involves TET1, a 5-methylcytosine dioxygenase. We found that FOXA1 induces TET1 expression via direct binding to its cis-regulatory elements. Further, FOXA1 physically interacts with the TET1 protein through its CXXC domain. TET1 thus co-occupies FOXA1-dependent enhancers and mediates local DNA demethylation and concomitant histone 3 lysine 4 methylation, further potentiating FOXA1 recruitment. Consequently, FOXA1 binding events are markedly reduced following TET1 depletion. Together, our results suggest that FOXA1 is not only able to recognize but also remodel the epigenetic signatures at lineage-specific enhancers, which is mediated, at least in part, by a feed-forward regulatory loop between FOXA1 and TET1.

Evaluation of Pharmacokinetics and Safety with Bioequivalence of Ibuprofen Sustained-Release Capsules of Two Formulations, in Chinese Healthy Volunteers: Bioequivalence Study.

Purpose: Ibuprofen is the first of the nonsteroidal anti-inflammatory drug (NSAID) to be used in the clinic. Our aim was to explore the pharmacokinetics (PK), bioequivalence, food effect, and safety of oral ibuprofen sustained-release capsules by two sponsors in healthy volunteers (HVs). Methods: Two separate randomized, open-label, single-dose, crossover-design studies were conducted: a fasting study (n = 24) and a fed study (n = 24). In each study, HVs were 1:1 divided into two groups (T-R and R-T) and received 0.3-g/capsule ibuprofen with a 3-day washout. The plasma was collected for up to 24 hours at the time point after dosing on Day 1/Day 4. The plasma concentrations of ibuprofen were measured using an HPLC-MS/MS method, and PK parameters were determined by noncompartmental methods. Results: Forty-eight healthy volunteers were enrolled. In fasting subjects, the maximum plasma concentration (Cmax, mean ± SD) was 14.86±3.19 µg/mL at 5.0 (4.0, 7.0) hours (median [min, max]) for sponsor T, and 13.88±2.60 µg/mL at 4.5 (3.0, 8.0) hours for sponsor R. In fed subjects, Cmax was 21.31±4.08 µg/mL at 5.6 (4.3, 10.0) hours for sponsor T, and 19.77±3.36 µg/mL at 6.0 (2.0, 8.0) hours for sponsor R. All 90% confidence intervals (CIs) for Cmax, AUC0-t, and AUC0-∞ were within the bioequivalence bounds (80-125%) both fasting and fed studies. Conclusion: Ibuprofen is well tolerated and has a favorable safety profile. In both fasting and fed study, there were no serious AEs, or AEs leading to withdrawal. Bioequivalence is achieved under fasting and fed conditions, supporting the demonstration of biosimilarity.

CCL5 might be a prognostic biomarker and associated with immuno-therapeutic efficacy in cancers: A pan-cancer analysis.

Purpose: Chemokine ligand 5 (CCL5), a vital member of the CC chemokine family, plays diverse roles in tumorigenesis, metastasis, and prognosis in various human tumors. However, no pan-cancer analysis has been conducted to illustrate its distinctive effects on clinical prognosis via underlying mechanisms and biological characteristics. Methods: Herein, we exploited the existed public bioinformatics database, primarily TCGA database and GTEx data, to comprehensively analyze the value of CCL5 involved in patient prognosis. Results: This study found that CCL5 was excessively expressed in most tumors and significantly associated with clinical prognosis in 10 out of 33 types of tumors. Notably, CCL5 might be an independent predictive biomarker of clinical outcome in SKCM patients, confirmed by univariate and multivariate Cox regression analysis. Furthermore, we acquired the genetic alteration status of CCL5 in multiple types of tumor tissues from TCGA cohorts. We revealed a potential correlation between the expression level of CCL5 and tumor mutational burden in 33 types of tumors. In addition, data showed that DNA methylation was associated with CCL5 gene expression in THCA, PRAD, LUSC, and BRCA cancers. Immune infiltration and immune checkpoints are fine indexes for evaluating immunotherapy. We uncovered that CCL5 was negatively correlated with the immune infiltration of CD8+ T cell, CD4+ T cell, macrophages, and gamma delta T cells in BRCA-basal and CESC tumors, while a significant positive correlation was observed in BLCA, COAD and other 7 types of tumors. Besides, CCL5 was closely associated with the immune checkpoint molecules in 8 types of tumors. The TIDE score was less in the CCL5 high-expressed group than in the CCL5 low-expressed group in SKCM patients, which indicated that CCL5 might be a fine monitor of immune response for immunotherapy. GO enrichment analysis data uncovered that cytokine-cytokine receptor interaction and chemokine signaling might be involved in the role of CCL5 in regulating tumor pathogenesis and prognosis. Conclusion: In conclusion, CCL5 was preliminarly identified as a biomarker of immune response and prognosis for tumors patients via our first comprehensive pan-cancer analysis.

Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

A novel hypoxic lncRNA, HRL-SC, promotes the proliferation and migration of human dental pulp stem cells through the PI3K/AKT signaling pathway.

BACKGROUND: Human dental pulp stem cells (hDPSCs) are critical for pulp generation. hDPSCs proliferate faster under hypoxia, but the mechanism by which long noncoding RNA (lncRNA) regulates this process is not fully understood. METHODS: Novel lncRNAs were obtained by reanalysis of transcriptome datasets from RNA-Seq under hypoxia compared with normoxia, and a differential expression analysis of target genes was performed. Bioinformatics analyses, including gene ontology analysis, Kyoto Encyclopedia of Genes and Genomes pathway analysis and gene set enrichment analysis, were used to understand the function of key novel lncRNAs. hDPSCs were isolated from dental pulp tissue. EdU and scratch wound healing assays were used to detect the proliferation and migration of hDPSCs. qRT-PCR was used to detect changes in the RNA expression of selected genes. RNA fluorescence in situ hybridization, small interfering RNA, qRT-PCR and Western blot analysis were used to explore the function of key novel lncRNAs. RESULTS: We identified 496 novel lncRNAs in hDPSCs under hypoxia, including 45 differentially expressed novel lncRNAs. Of these, we focused on a key novel lncRNA, which we designated HRL-SC (hypoxia-responsive lncRNA in stem cells). Functional annotation revealed that HRL-SC was associated with hypoxic conditions and the PI3K/AKT signaling pathway. HRL-SC was mainly located in the cytoplasm of hDPSCs and had stable high expression under hypoxia. Knockdown of HRL-SC inhibited the proliferation and migration of hDPSCs and the expression levels of PI3K/AKT-related marker proteins. Furthermore, the AKT activator SC79 partially offset the inhibitory effect caused by the knockdown, indicating that HRL-SC promoted hDPSCs through the PI3K/AKT signaling pathway. CONCLUSIONS: Hypoxia-responsive lncRNA HRL-SC promotes the proliferation and migration of hDPSCs through the PI3K/AKT signaling pathway, and this understanding may facilitate the regenerative application of hDPSCs.

METTL3-Mediated lncSNHG7 m6A Modification in the Osteogenic/Odontogenic Differentiation of Human Dental Stem Cells.

Background: Human dental pulp stem cells (hDPSCs) play an important role in endodontic regeneration. N6-methyladenosine (m6A) is the most common RNA modification, and noncoding RNAs have also been demonstrated to have regulatory roles in the expression of m6A regulatory proteins. However, the study on m6A modification in hDPSCs has not yet been conducted. Methods: Single base site PCR (MazF) was used to detect the m6A modification site of lncSNHG7 before and after mineralization of hDPSCs to screen the target m6A modification protein, and bioinformatics analysis was used to analyze the related pathways rich in lncSNHG7. After knockdown and overexpression of lncSNHG7 and METTL3, the osteogenic/odontogenic ability was detected. After METTL3 knockdown, the m6A modification level and its expression of lncSNHG7 were detected by MazF, and their binding was confirmed. Finally, the effects of lncSNHG7 and METTL3 on the Wnt/ß-catenin pathway were detected. Results: MazF experiments revealed that lncSNHG7 had a m6A modification before and after mineralization of hDPSCs, and the occurrence site was 2081. METTL3 was most significantly upregulated after mineralization of hDPSCs. Knockdown/ overexpression of lncSNHG7 and METTL3 inhibited/promoted the osteogenic/odontogenic differentiation of hDPSCs. The m6A modification and expression of lncSNHG7 were both regulated by METTL3. Subsequently, lncSNHG7 and METTL3 were found to regulate the Wnt/ß-catenin signaling pathway. Conclusion: These results revealed that METTL3 can activate the Wnt/ß-catenin signaling pathway by regulating the m6A modification and expression of lncSNHG7 in hDPSCs to enhance the osteogenic/odontogenic differentiation of hDPSCs. Our study provides new insight into stem cell-based tissue engineering.

Pulp revascularization on an adult mandibular right second premolar: A case report.

BACKGROUND: Pulp revascularization has become a new method for the treatment of periapical diseases in young permanent teeth in recent years. Through root canal flushing and disinfection, avoiding mechanical preparation, guiding apical stem cells into the root canal and promoting the continuous development of tooth roots, it has achieved good clinical curative effects. But in adult patients with chronic periapical periodontitis with immature roots and open apices, apical barrier technology is often used to treat these teeth. CASE SUMMARY: Pulp revascularization of a 26-year-old patient's tooth was performed using cefaclor instead of minocycline and iRoot BP instead of mineral trioxide aggregate as intracanal medication. The case was followed up for 36 mo. Observations showed evidence of regression of clinical signs and symptoms, resolution of apical periodontitis and no discolouration of affected teeth. CONCLUSION: For adult patients with chronic periapical periodontitis with immature roots and open apices, pulp revascularisation showed favourable results in treating these teeth.

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-tune Virtual Microdissection.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 µm and 300 µm diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Identifying transcriptional programs underlying cancer drug response with TraCe-seq.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.

METTL3-mediated m6A modification regulates cell cycle progression of dental pulp stem cells.

BACKGROUND: Dental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity. N6-methyladenosine (m6A) is the most prevalent, reversible internal modification in RNAs associated with stem cell fate determination. In this study, we aim to explore the biological effect of m6A methylation in DPSCs. METHODS: m6A immunoprecipitation with deep sequencing (m6A RIP-seq) demonstrated the features of m6A modifications in DPSC transcriptome. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence, and apoptosis were analyzed by ß-galactosidase, TUNEL staining, and flow cytometry. Bioinformatic analysis combing m6A RIP and shMETTL3 RNA-seq functionally enriched overlapped genes and screened target of METTL3. Cell cycle distributions were assayed by flow cytometry, and m6A RIP-qPCR was used to confirm METTL3-mediated m6A methylation. RESULTS: Here, m6A peak distribution, binding area, and motif in DPSCs were first revealed by m6A RIP-seq. We also found a relatively high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. A conjoint analysis of m6A RIP and RNA sequencing showed METTL3 depletion associated with cell cycle, mitosis, and alteration of METTL3 resulted in cell cycle arrest. Furthermore, the protein interaction network of differentially expressed genes identified Polo-like kinase 1 (PLK1), a critical cycle modulator, as the target of METTL3-mediated m6A methylation in DPSCs. CONCLUSIONS: These results revealed m6A methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and served new insight into stem cell-based tissue engineering.

circRNA Expression Profile in Dental Pulp Stem Cells during Odontogenic Differentiation.

INTRODUCTION: Odontogenic differentiation of human dental pulp stem cells (hDPSCs) is a key step of pulp regeneration. Recent studies showed that circular RNAs (circRNAs) have many biological functions and that competing endogenous RNA (ceRNA) is their most common mechanism of action. However, the role of circRNAs in hDPSCs during odontogenesis is still unclear. METHODS: Isolated hDPSCs were cultured in essential and odontogenic medium. Total RNA was extracted after 14 days of culture, and then, microarray analysis was performed to measure the differential expressions of circRNAs. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was then performed to validate the microarray results. Based on microarray data from this study and available in the database, a ceRNA network was constructed to investigate the potential function of circRNAs during odontogenesis. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to investigate the potential correlation between signaling pathways and circRNAs. In addition, qRT-PCR and Western blot analysis were used to explore the function of hsa_circRNA_104101. RESULTS: We found 43 upregulated circRNAs and 144 downregulated circRNAs during the odontogenic differentiation process (fold change > 1.5 and <-1.5, respectively; P < 0.05). qRT-PCR results were in agreement with the microarray results. Bioinformatic analysis revealed that the Wnt signaling pathway and the TGF-ß signaling pathway, as well as the other pathways associated with odontogenic differentiation, were correlated to the differentially expressed circRNAs. hsa_circRNA_104101 was proved to promote the odontogenic differentiation of hDPSCs. CONCLUSION: This study reported 187 circRNAs that were differentially expressed in hDPSCs during odontogenic differentiation. Bioinformatic analysis of the expression data suggested that circRNA-miRNA-mRNA networks might act as a crucial mechanism for hDPSC odontogenic differentiation, providing a theoretical foundation for the study of pulp regeneration regulation by circRNAs.

Genome-wide identification of long noncoding RNAs and their competing endogenous RNA networks involved in the odontogenic differentiation of human dental pulp stem cells.

BACKGROUND: Long noncoding RNAs (lncRNAs) play an important role in the multiple differentiations of mesenchymal stem cells (MSCs). However, few studies have focused on the regulatory mechanism of lncRNAs in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). METHODS: hDPSCs were induced to differentiate into odontoblasts in vitro, and the expression profiles of lncRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) in differentiated and undifferentiated cells were obtained by microarray. Bioinformatics analyses including Gene Ontology (GO) analysis, pathway analysis, and binding site prediction were performed for functional annotation of lncRNA. miRNA/odontogenesis-related gene networks and lncRNA-associated ceRNA networks were constructed. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to verify the expression of selected genes. RNA fluorescence in situ hybridization (FISH), qRT-PCR, and western blot analysis were used to explore the location and function of lncRNA-G043225. Dual-luciferase reporter assay was performed to confirm the binding sites of miR-588 with G043225 and Fibrillin 1 (FBN1). RESULTS: We identified 132 lncRNAs, 114 miRNAs, and 172 mRNAs were differentially expressed. GO analysis demonstrated that regulation of the neurogenic locus notch homolog (Notch), Wnt, and epidermal growth factor receptor (ERBB) signaling pathways and activation of mitogen-activated protein kinase (MAPK) activity were related to odontogenic differentiation. Pathway analysis indicated that the most significant pathway was the forkhead box O (FoxO) signaling pathway, which is related to odontogenic differentiation. Two odontogenesis-related gene-centered lncRNA-associated ceRNA networks were successfully constructed. The qRT-PCR validation results were consistent with the microarray analysis. G043225 mainly locating in cytoplasm was proved to promote the odontogenic differentiation of hDPSCs via miR-588 and FBN1. CONCLUSION: This is the first study revealing lncRNA-associated ceRNA network during odontogenic differentiation of hDPSCs using microarray, and it could provide clues to explore the mechanism of action at the RNA-RNA level as well as novel treatments for dentin regeneration based on stem cells.

Targeting FOXA1-mediated repression of TGF-ß signaling suppresses castration-resistant prostate cancer progression.

Prostate cancer (PC) progressed to castration resistance (CRPC) is a fatal disease. CRPC tumors develop resistance to new-generation antiandrogen enzalutamide through lineage plasticity, characterized by epithelial-mesenchymal transition (EMT) and a basal-like phenotype. FOXA1 is a transcription factor essential for epithelial lineage differentiation. Here, we demonstrate that FOXA1 loss leads to remarkable upregulation of transforming growth factor beta 3 (TGFB3), which encodes a ligand of the TGF-ß pathway. Mechanistically, this is due to genomic occupancy of FOXA1 on an upstream enhancer of the TGFB3 gene to directly inhibit its transcription. Functionally, FOXA1 downregulation induces TGF-ß signaling, EMT, and cell motility, which is effectively blocked by the TGF-ß receptor I inhibitor galunisertib (LY2157299). Tissue microarray analysis confirmed reduced levels of FOXA1 protein and a concordant increase in TGF-ß signaling, indicated by SMAD2 phosphorylation, in CRPC as compared with primary tumors. Importantly, combinatorial LY2157299 treatment sensitized PC cells to enzalutamide, leading to synergistic effects in inhibiting cell invasion in vitro and xenograft CRPC tumor growth and metastasis in vivo. Therefore, our study establishes FOXA1 as an important regulator of lineage plasticity mediated in part by TGF-ß signaling, and supports a novel therapeutic strategy to control lineage switching and potentially extend clinical response to antiandrogen therapies.