BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

OBJECTIVES: To reprogram human placental fibroblasts (HPFs) into chemically induced epithelioid-like cells (ciEP-Ls) using a combination of small-molecule compounds. METHODS: HPFs cultured under normoxic conditions were identified using immunofluorescence assay, PCR and chromosomal karyotyping. Under hypoxic conditions (37 ℃, 5% O₂), HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ (CHIR99021, 616452, TTNPB, SAG, EPZ5676, DZNep, Ruxolitinib, VTP50469, Afuresertib, JNK-IN-8, and EZM0414), and the cell morphology was observed daily. The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence, Western blotting and PCR. Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated. RESULTS: Before induction, HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers. PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype. After one day of induction, the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape, which was morphologically characteristic of ciEP-Ls. On day 4 of induction, the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A. RT-qPCR results also showed that the cells expressed the epithelial markers Smad3, GLi3, PAX8, WT1, KRT19, and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype. The reprogramming efficiency of HPFs into ciEP-Ls ranged from (64.53±2.8)% to (68.10±3.6)%. CONCLUSIONS The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.
Humans ; Fibroblasts/drug effects* ; Pregnancy ; Female ; Cell Differentiation/drug effects* ; Pyrimidines/pharmacology* ; Placenta/cytology* ; Cells, Cultured ; Pyridines/pharmacology* ; Pyrazoles/pharmacology* ; Epithelial Cells/cytology*

OBJECTIVES: To investigate the regulatory effect of LINC00837/miR-671-5p/SERPINE2 functional axis on pathological processes of fibroblast-like synovial cells (FLS) in rheumatoid arthritis (RA). METHODS: RA-FLS were transfected with a LINC00837 overexpression plasmid (pcDNA3.1-LINC00837), a LINC00837 interference plasmid (siRNA-LINC00837), or their respective negative control plasmids (pcDNA3.1-NC and siRNA-NC). Dual luciferase was used to verify the targeting relationship between LINC00837 and miR-671-5p and between miR-671-5p and SERPINE2. RT-qPCR was used to detect the expression levels of LINC00837, miR-671-5p and SERPINE2 in normal FLS or the transfected cells, whose proliferation and migration abilities were assessed using Edu assay and scratch healing assay and by detecting the expression levels of Ki-67, PCNA, E-cadherin and N-cadherin with Western blotting. The changes in cellular secretion of the inflammatory cytokines (TNF‑α, IL-17, IL-4 and IL-10) were examined using ELISA. RESULTS: Dual luciferase reporter gene assay showed that LINC00837 was capable of binding to the 3'-UTR of miR-671-5p, and the latter bound to the 3-UTR of SERPINE2 at specific binding sites between them. Compared with normal FLS, RA-FLS showed significantly increased expressions of LINC00837 and SERPINE2, lowered miR-671-5p expression and enhanced proliferation and migration abilities with increased expressions of pro-inflammatory cytokines and reduced expressions of anti-inflammatory cytokines. Transfection of RA-FLS with pcDNA-LINC00837 further enhanced cell proliferation and migration and the changes in the inflammatory cytokines, while transfection with si-LINC00837 produced the opposite changes. CONCLUSIONS RA-FLS have a LINC00837/miR-671-5p/SERPINE2 functional axis, which regulates cell proliferation, migration and secretion of inflammatory factors, and interventions targeting LINC00837 may provide a potential strategy to regulate the pathological processes in RA-FLS.
Arthritis, Rheumatoid/metabolism* ; MicroRNAs/metabolism* ; Humans ; Cell Proliferation ; Cell Movement ; Synovial Membrane/pathology* ; RNA, Long Noncoding/genetics* ; Fibroblasts/metabolism* ; Synoviocytes/metabolism* ; Cells, Cultured ; Transfection

OBJECTIVES: To investigate the effect of Haematococcus pluvialis (HP) on bleomycin (BLM)-induced pulmonary fibrosis in mice and on TGF-β1-induced human fetal lung fibroblasts (HFL1). METHODS: Thirty male C57BL/6 mice were randomly divided into control group, BLM-induced pulmonary fibrosis model group, low- and high-dose HP treatment groups (3 and 21 mg/kg, respectively), and 300 mg/kg pirfenidone (positive control) group. The effects of drug treatment for 21 days were assessed by examining respiratory function, lung histopathology, and expression of fibrosis markers in the lung tissues of the mouse models. In TGF-β1-induced HFL1 cell cultures, the effects of treatment with 120, 180 and 240 μg/mL HP or 1.85 μg/mL pirfenidone for 48 h on expression levels of fibrosis markers were evaluated. Transcriptome analysis was carried out using the control cells and cells treated with TGF-β1 and 240 μg/mL HP. RESULTS: HP obviously alleviated BLM-induced lung function damage and fibrotic changes in mice, evidenced by improved respiratory function, lung tissue morphology and structure, inflammatory infiltration, and collagen deposition and reduced expressions of fibrotic proteins. HP at the high dose produced similar effect to PFD. In TGF-β1-induced HFL1 cells, treatment with 240 μg/mL HP significantly reduced the mRNA and protein expression levels of α-SMA and FN. Transcriptome analysis revealed that multiple key genes and pathways mediated the protective effect of HP against pulmonary fibrosis. CONCLUSIONS HP alleviates pulmonary fibrosis in both the mouse model and cell model, possibly as the result of the synergistic effects of its multiple active components.
Animals ; Pulmonary Fibrosis/chemically induced* ; Bleomycin/adverse effects* ; Mice, Inbred C57BL ; Male ; Mice ; Fibroblasts/drug effects* ; Lung/pathology* ; Transforming Growth Factor beta1/pharmacology* ; Myofibroblasts/drug effects* ; Humans ; Pyridones

Perineural invasion (PNI) by tumor cells is a key phenotype of highly-invasive oral squamous cell carcinoma (OSCC). Since Schwann cells (SCs) and fibroblasts maintain the physiological homeostasis of the peripheral nervous system, and we have focused on cancer-associated fibroblasts (CAFs) for decades, it's imperative to elucidate the impact of CAFs on SCs in PNI⁺ OSCCs. We describe a disease progression-driven shift of PNI^- towards PNI⁺ during the progression of early-stage OSCC (31%, n = 125) to late-stage OSCC (53%, n = 97), characterized by abundant CAFs and nerve demyelination. CAFs inhibited SC proliferation/migration and reduced neurotrophic factors and myelin in vitro, and this involved up-regulated ER stress and decreased MAPK signals. Moreover, CAFs also aggravated the paralysis of the hind limb and PNI in vivo. Unexpectedly, leukemia inhibitory factor (LIF) was exclusively expressed on CAFs and up-regulated in metastatic OSCC. The LIF inhibitor EC330 restored CAF-induced SC inactivation. Thus, OSCC-derived CAFs inactivate SCs to aggravate nerve injury and PNI development.
Schwann Cells/metabolism* ; Mouth Neoplasms/metabolism* ; Humans ; Cancer-Associated Fibroblasts/metabolism* ; Animals ; Carcinoma, Squamous Cell/metabolism* ; Neoplasm Invasiveness/pathology* ; Male ; Female ; Mice ; Cell Movement/physiology* ; Cell Proliferation/physiology* ; Cell Line, Tumor ; Leukemia Inhibitory Factor/metabolism* ; Middle Aged

Abnormal accumulation of collagen fibrils is a hallmark feature of oral submucous fibrosis (OSF). However, the precise characteristics and underlying mechanisms remain unclear, impeding the advancement of potential therapeutic approaches. Here, we observed that collagen I, the main component of the extracellular matrix, first accumulated in the lamina propria and subsequently in the submucosa of OSF specimens as the disease progressed. Using RNA-seq and Immunofluorescence in OSF specimens, we screened the cartilage oligomeric matrix protein (COMP) responsible for the abnormal collagen accumulation. Genetic COMP deficiency reduced arecoline-stimulated collagen I deposition significantly in vivo. In comparison, both COMP and collagen I were upregulated under arecoline stimulation in wild-type mice. Human oral buccal mucosal fibroblasts (hBMFs) also exhibited increased secretion of COMP and collagen I after stimulation in vitro. COMP knockdown in hBMFs downregulates arecoline-stimulated collagen I secretion. We further demonstrated that hBMFs present heterogeneous responses to arecoline stimulation, of which COMP-positive fibroblasts secrete more collagen I. Since COMP is a molecular bridge with Fibril-associated collagens with Interrupted Triple helices (FACIT) in the collagen network, we further screened and identified collagen XIV, a FACIT member, co-localizing with both COMP and collagen I. Collagen XIV expression increased under arecoline stimulation in wild-type mice, whereas it was hardly expressed in the Comp^-/- mice, even with under stimulation. In summary, we found that COMP may mediates abnormal collagen I deposition by functions with collagen XIV during the progression of OSF, suggesting its potential to be targeted in treating OSF.
Oral Submucous Fibrosis/pathology* ; Cartilage Oligomeric Matrix Protein/genetics* ; Animals ; Mice ; Humans ; Fibroblasts/metabolism* ; Collagen Type I/metabolism* ; Arecoline/pharmacology* ; Mouth Mucosa/metabolism* ; Cells, Cultured ; Fluorescent Antibody Technique

Complement C3 plays a critical role in periodontitis. However, its source, role and underlying mechanisms remain unclear. In our study, by analyzing single-cell sequencing data from mouse model of periodontitis, we identified that C3 is primarily derived from periodontal fibroblasts. Subsequently, we demonstrated that C3a has a detrimental effect in ligature-induced periodontitis. C3ar^-/- mice exhibited significantly less destruction of periodontal support tissues compared to wild-type mice, characterized by mild gingival tissue damage and reduced alveolar bone loss. This reduction was associated with decreased production of pro-inflammatory mediators and reduced osteoclast infiltration in the periodontal tissues. Mechanistic studies suggested that C3a could promote macrophage polarization and osteoclast differentiation. Finally, by analyzing single-cell sequencing data from the periodontal tissues of patients with periodontitis, we found that the results observed in mice were consistent with human data. Therefore, our findings clearly demonstrate the destructive role of fibroblast-derived C3 in ligature-induced periodontitis, driven by macrophage M1 polarization and osteoclast differentiation. These data strongly support the feasibility of C3a-targeted interventions for the treatment of human periodontitis.
Animals ; Osteoclasts/cytology* ; Periodontitis/metabolism* ; Cell Differentiation ; Mice ; Fibroblasts/metabolism* ; Macrophages ; Disease Models, Animal ; Complement C3/metabolism* ; Humans ; Disease Progression ; Mice, Inbred C57BL ; Male ; Mice, Knockout

Smoking is a well-established risk factor for periodontitis, yet the precise mechanisms by which smoking contributes to periodontal disease remain poorly understood. Recent advances in spatial transcriptomics have enabled a deeper exploration of the periodontal tissue microenvironment at single-cell resolution, offering new opportunities to investigate these mechanisms. In this study, we utilized Visium HD single-cell spatial transcriptomics to profile gingival tissues from 12 individuals, including those with periodontitis, those with smoking-associated periodontitis, and healthy controls. Our analysis revealed that smoking disrupts the epithelial barrier integrity, induces fibroblast alterations, and dysregulates fibroblast-epithelial cell communication, thereby exacerbating periodontitis. The spatial analysis showed that endothelial cells and macrophages are in close proximity and interact, which further promotes the progression of smoking-induced periodontal disease. Importantly, we found that targeting the endothelial CXCL12 signalling pathway in smoking-associated periodontitis reduced the proinflammatory macrophage phenotype, alleviated epithelial inflammation, and reduced alveolar bone resorption. These findings provide novel insights into the pathogenesis of smoking-associated periodontitis and highlight the potential of targeting the endothelial-macrophage interaction as a therapeutic strategy. Furthermore, this study establishes an essential information resource for investigating the effects of smoking on periodontitis, providing a foundation for future research and therapeutic development for this prevalent and debilitating disease.
Humans ; Gingiva/cytology* ; Smoking/adverse effects* ; Male ; Periodontitis/pathology* ; Single-Cell Analysis ; Female ; Adult ; Middle Aged ; Macrophages ; Fibroblasts ; Endothelial Cells ; Case-Control Studies ; Chemokine CXCL12/metabolism*

Frostbite is the most common cold injury and is caused by both immediate cold-induced cell death and the gradual development of localized inflammation and tissue ischemia. Delayed healing of frostbite often leads to scar formation, which not only causes psychological distress but also tends to result in the development of secondary malignant tumors. Therefore, a rapid healing method for frostbite wounds is urgently needed. Herein, we used a mouse skin model of frostbite injury to evaluate the recovery process after frostbite. Moreover, single-cell transcriptomics was used to determine the patterns of changes in monocytes, macrophages, epidermal cells, and fibroblasts during frostbite. Most importantly, human-induced pluripotent stem cell (hiPSC)-derived skin organoids combined with gelatin-hydrogel were constructed for the treatment of frostbite. The results showed that skin organoid treatment significantly accelerated wound healing by reducing early inflammation after frostbite and increasing the proportions of epidermal stem cells. Moreover, in the later stage of wound healing, skin organoids reduced the overall proportions of fibroblasts, significantly reduced fibroblast-to-myofibroblast transition by regulating the integrin α5β1-FAK pathway, and remodeled the extracellular matrix (ECM) through degradation and reassembly mechanisms, facilitating the restoration of physiological ECM and reducing the abundance of ECM associated with abnormal scar formation. These results highlight the potential application of organoids for promoting the reversal of frostbite-related injury and the recovery of skin functions. This study provides a new therapeutic alternative for patients suffering from disfigurement and skin dysfunction caused by frostbite.
Animals ; Organoids/metabolism* ; Mice ; Humans ; Wound Healing ; Frostbite/metabolism* ; Skin/pathology* ; Induced Pluripotent Stem Cells/cytology* ; Cicatrix/pathology* ; Fibroblasts/metabolism* ; Disease Models, Animal ; Mice, Inbred C57BL ; Extracellular Matrix/metabolism* ; Male