Prognostic Analysis of Lactic Acid Metabolism Genes in Oral Squamous Cell Carcinoma.

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the oral and maxillofacial region. Lactic acid accumulation in the tumour microenvironment (TME) has gained attention for its dual role as an energy source for cancer cells and an activator of signalling pathways crucial to tumour progression. This study aims to reveal the impact of lactate-related genes (LRGs) on the prognosis, TME, and immune characteristics of OSCC, with the ultimate goal of developing a novel prognostic model. METHODS: Unsupervised clustering analysis of LRGs in OSCC patients from The Cancer Genome Atlas database was conducted to evaluate and compare TME, immune features, and clinical characteristics across various lactate subtypes. A refined prognostic model was developed through the application of Cox and Least absolute shrinkage and selection operator (LASSO) regression techniques. External validation sets were then utilised to improve model accuracy, along with a detailed correlation analysis of drug sensitivity. RESULTS: The Cancer Genome Atlas-OSCC patients were categorised into 4 distinct lactate subtypes based on LRGs. Notably, patients in subtype 1 and subtype 2 exhibited the least and most favourable prognoses, respectively. Subtype 1 patients showed elevated expression levels of immune checkpoint genes. Further analysis identified 1086 genes with significant expression differences between cancer and noncancer tissues, as well as between subtype 1 and subtype 2 patients. Selected genes for the prognostic model included ZNF662, CGNL1, VWCE, and ZFP42. The high-risk group defined by this model had a significantly poorer prognosis (P < .0001) and functioned as an independent prognostic factor (P < .001), accurately predicting 1-, 3-, and 5-year survival rates. Additionally, individuals in the high-risk category exhibited heightened sensitivity to chemotherapy drugs such as AZ6102 and Venetoclax. CONCLUSIONS: The predictive model based on the genes ZNF662, CGNL1, VWCE, and ZFP42 can serve as a reliable biomarker, providing accurate prognostic predictions for OSCC patients and potential opportunities for pharmaceutical interventions.

Liproxstatin-1 alleviates cartilage degradation by inhibiting chondrocyte ferroptosis in the temporomandibular joint.

BGROUND INFORMATION: Ferroptosis contributes to temporomandibular joint osteoarthritis (TMJOA) lesion development and is still poorly understood. RESULTS: In this study, we used different TMJOA animal models to examine whether ferroptosis was related to disease onset in TMJOA induced by monosodium iodoacetate (MIA), IL-1ß, occlusion disorder (OD), and unilateral anterior crossbite (UAC). Immunohistochemical staining and Western blot analysis were used to detect ferroptosis- and cartilage degradation-related protein expression. Our results revealed reduced levels of the ferroptosis-related protein GPX4 in the cartilage layer, but the levels of ACSL4 and P53 were increased in the condyle. Injection of the ferroptosis inhibitor liproxstatin-1 (Lip-1) effectively decreased ACSL4, P53 and TRF expression. In vitro, IL-1ß reduced cartilage extracellular matrix expression in mandibular condylar chondrocytes (MCCs). Lip-1 maintained the morphology and function of mitochondria and ameliorated the exacerbation of lipid peroxidation and reactive oxygen species (ROS) production induced by IL-1ß. CONCLUSION: These results suggest that chondrocyte ferroptosis plays an important role in the development and progression of TMJOA. SIGNIFICANCE: Inhibiting condylar chondrocyte ferroptosis could be a promising therapeutic strategy for TMJOA.

PRMT1 promotes extracellular matrix degradation and apoptosis of chondrocytes in temporomandibular joint osteoarthritis via the AKT/FOXO1 signaling pathway.

Temporomandibular joint osteoarthritis (TMJOA) is a chronic degenerative joint disease characterized by extracellular matrix (ECM) degradation and chondrocyte apoptosis. The aim of this study was to investigate the role of PRMT1 in TMJOA pathogenesis and its underlying molecular mechanism. Compared to the control group, PRMT1 was highly expressed in IL-1ß-treated chondrocytes and articular cartilage following MIA injection into rat TMJs. Furthermore, knocking down PRMT1 considerably inhibited ECM degradation and apoptosis induced by IL-1ß. Mechanistic analyses further revealed that PRMT1 knockdown activated the PI3K/AKT signaling pathway and prevented FOXO1 from translocating to the nucleus. Moreover, an inhibitor of AKT (LY294002) rescued the effect of PRMT1 knockdown on IL-1ß-induced ECM degradation and apoptosis, and AMI-1, a selective inhibitor of PRMT1, inhibited PRMT1 expression and reversed the pathological progress of TMJOA. Thus, our findings suggest that PRMT1 plays an essential role in ECM degradation and chondrocyte apoptosis in TMJOA via the AKT/FOXO1 signaling pathway.

BMP2-dependent gene regulatory network analysis reveals Klf4 as a novel transcription factor of osteoblast differentiation.

Transcription factors (TFs) regulate the expression of target genes, inducing changes in cell morphology or activities needed for cell fate determination and differentiation. The BMP signaling pathway is widely regarded as one of the most important pathways in vertebrate skeletal biology, of which BMP2 is a potent inducer, governing the osteoblast differentiation of bone marrow stromal cells (BMSCs). However, the mechanism by which BMP2 initiates its downstream transcription factor cascade and determines the direction of differentiation remains largely unknown. In this study, we used RNA-seq, ATAC-seq, and animal models to characterize the BMP2-dependent gene regulatory network governing osteoblast lineage commitment. Sp7-Cre; Bmp2fx/fx mice (BMP2-cKO) were generated and exhibited decreased bone density and lower osteoblast number (n > 6). In vitro experiments showed that BMP2-cKO mouse bone marrow stromal cells (mBMSCs) had an impact on osteoblast differentiation and deficient cell proliferation. Osteogenic medium induced mBMSCs from BMP2-cKO mice and control were subjected to RNA-seq and ATAC-seq analysis to reveal differentially expressed TFs, along with their target open chromatin regions. Combined with H3K27Ac CUT&Tag during osteoblast differentiation, we identified 2338 BMP2-dependent osteoblast-specific active enhancers. Motif enrichment assay revealed that over 80% of these elements were directly targeted by RUNX2, DLX5, MEF2C, OASIS, and KLF4. We deactivated Klf4 in the Sp7 + lineage to validate the role of KLF4 in osteoblast differentiation of mBMSCs. Compared to the wild-type, Sp7-Cre; Klf4fx/+ mice (KLF4-Het) were smaller in size and had abnormal incisors resembling BMP2-cKO mice. Additionally, KLF4-Het mice had fewer osteoblasts and decreased osteogenic ability. RNA-seq and ATAC-seq revealed that KLF4 mainly "co-bound" with RUNX2 to regulate downstream genes. Given the significant overlap between KLF4- and BMP2-dependent NFRs and enriched motifs, our findings outline a comprehensive BMP2-dependent gene regulatory network specifically governing osteoblast differentiation of the Sp7 + lineage, in which Klf4 is a novel transcription factor.

Klf4 Promotes Dentinogenesis and Odontoblastic Differentiation via Modulation of TGF-ß Signaling Pathway and Interaction With Histone Acetylation.

Transcription factors bind to cell-specific cis-regulatory elements, such as enhancers and promoters, to initiate much of the gene expression program of different biological process. Odontoblast differentiation is a necessary step for tooth formation and is also governed by a complex gene regulatory network. Our previous in vitro experiments showed that Krüppel-like factor 4 (KLF4) can promote odontoblastic differentiation of both mouse dental papillary cells (mDPCs) and human dental pulp cells; however, its mechanism remains unclear. We first used Wnt1-Cre; KLF4fx/fx (Klf4 cKO) mice to examine the role of KLF4 during odontoblast differentiation in vivo and demonstrated significantly impaired dentin mineralization and enlarged pulp/root canals. Additionally, combinatory analysis using RNA-seq and ATAC-seq revealed genomewide direct regulatory targets of KLF4 in mouse odontoblasts. We found that KLF4 can directly activate the TGF-ß signaling pathway at the beginning of odontoblast differentiation with Runx2 as a cofactor. Furthermore, we found that KLF4 can directly upregulate the expression levels of Dmp1 and Sp7, which are markers of odontoblastic differentiation, through binding to their promoters. Interestingly, as a transcription factor, KLF4 can also recruit histone acetylase as a regulatory companion to the downstream target genes to positively or negatively regulate transcription. To further investigate other regulatory companions of KLF4, we chose histone acetylase HDAC3 and P300. Immunoprecipitation demonstrated that KLF4 interacted with P300 and HDAC3. Next, ChIP analysis detected P300 and HDAC3 enrichment on the promoter region of KLF4 target genes Dmp1 and Sp7. HDAC3 mainly interacted with KLF4 on day 0 of odontoblastic induction, whereas P300 interacted on day 7 of induction. These temporal-specific interactions regulated Dmp1 and Sp7 transcription, thus regulating dentinogenesis. Taken together, these results demonstrated that KLF4 regulates Dmp1 and Sp7 transcription via the modulation of histone acetylation and is vital to dentinogenesis. © 2019 American Society for Bone and Mineral Research.

Dental pulp tissue engineering with bFGF-incorporated silk fibroin scaffolds.

The clinical translation of regenerative endodontics demands further development of suitable scaffolds. Here, we assessed the possibility of using silk fibroin scaffold for pulp regeneration with dental pulp stem cells (DPSCs) and basic fibroblast growth factor (bFGF) in ectopic root canal transplantation model. Porous silk fibroin scaffolds were fabricated using freeze-drying technique (with or without bFGF incorporation), and characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy. DPSCs were isolated, characterized, seeded onto scaffolds, and inserted into the tooth root fragments. Cell viability and morphology were tested in the 3D model in vitro using CCK8 assay and SEM. Furthermore, the ectopic transplantation model was used to verify the generation of pulp-like tissue in DPSCs seeded silk fibroin scaffold with bFGF, as examined by histological analysis. DPSCs seeded in silk fibroin scaffold survived, exhibited cytoplasmic elongation in scaffolds at least 4 weeks in culture. bFGF promoted DPSCs viability in tooth fragments/scaffolds (TSS) between 7 and 28 days. Pulp-like tissue was generated in the bFGF-incorporated TSS with DPSCs. Histologically, the generated tissue was shown to be with well vascularity, have new matrix deposition and dentin-like tissue formation, and consist of both the transplanted and host-derived cells. Collectively, these data support the use of bFGF-incorporated silk fibroin scaffold as a highly promising scaffold candidate for future treatment concepts in regenerative endodontics to save teeth.

Autophagy in SDF-1α-mediated DPSC migration and pulp regeneration.

Critical morphological requirements for pulp regeneration are tissues replete with vascularisation, neuron formation, and dentin deposition. Autophagy was recently shown to be related to angiogenesis, neural differentiation, and osteogenesis. The present study aimed to investigate the involvement of autophagy in stromal cell-derived factor-1α (SDF-1α)-mediated dental pulp stem cell (DPSC) migration and pulp regeneration, and identify its presence during pulp revascularisation of pulpectomised dog teeth with complete apical closure. In vitro studies showed that SDF-1α enhanced DPSCs migration and optimised focal adhesion formation and stress fibre assembly, which were accompanied by autophagy. Moreover, autophagy inhibitors significantly suppressed, whereas autophagy activator substantially augmented SDF-1α-stimulated DPSCs migration. Furthermore, after ectopic transplantation of tooth fragment/silk fibroin scaffold with DPSCs into nude mice, pulp-like tissues with vascularity, well-organised fibrous matrix formation, and new dentin deposition along the dentinal wall were generated in SDF-1α-loaded samples accompanied by autophagy. More importantly, in a pulp revascularisation model in situ, SDF-1α-loaded silk fibroin scaffolds improved the de novo ingrowth of pulp-like tissues in pulpectomised mature dog teeth, which correlated with the punctuated LC3 and Atg5 expressions, indicating autophagy. Our findings provide novel insights into the pulp regeneration mechanism, and SDF-1α shows promise for future clinical application in pulp revascularisation.