PRMT6/LMNA/CXCL12 signaling pathway regulated the osteo/odontogenic differentiation ability in dental stem cells isolated from apical papilla.

Tooth loss and maxillofacial bone defect are common diseases, which seriously affect people's health. Effective tooth and maxillofacial bone tissue regeneration is a key problem that need to be solved. In the present study, we investigate the role of PRMT6 in osteo/odontogenic differentiation and migration capacity by using SCAPs. Our results showed that knockdown of PRMT6 promoted the osteo/odontogenic differentiation compared with the control group, as detected by alkaline phosphatase activity, alizarin red staining, and the indicators of osteo/odontogenic differentiation measured by Western blot. In addition, overexpression of PRMT6 inhibited the osteo/odontogenic differentiation potentials of SCAPs. Then, knockdown of PRMT6 promoted the migration ability and overexpression of PRMT6 inhibited the migration ability in SCAPs. Mechanically, we discovered that the depletion of PRMT6 promoted the expression of CXCL12 by decreasing H3R2 methylation in the promoter region of CXCL12. In addition, PRMT6 formed a protein complex with LMNA, a nuclear structural protein. Depletion of LMNA inhibited the osteo/odontogenic differentiation and CXCL12 expression and increased the intranucleus PRMT6 in SCAPs. To sum up, PRMT6 might inhibit the osteo/odontogenic differentiation and migration ability of SCAPs via inhibiting CXCL12. And LMNA might be a negative regulator of PRMT6. It is suggested that PRMT6 may be a key target for SCAP-mediated bone and tooth tissue regeneration.

Epiregulin promotes osteogenic differentiation and inhibits neurogenic trans-differentiation of adipose-derived mesenchymal stem cells via MAPKs pathway.

Mesenchymal stem cells (MSCs) exists low efficiency to trans-differentiate into other germinal layer cell types. One key issue is to discover the effect of important factor on MSCs differentiation abiltiy. In this study, we investigated the role and mechanism of epiregulin (EREG) on the osteogenic differentiation and neurogenic trans-differentiation in adipose-derived stem cells (ADSCs). We discovered that the depletion of EREG inhibited the osteogenic differentiation in vitro. And 25 ng/mL recombinant human epiregulin protein (rhEREG) effectively improved the osteogenic differentiation of EREG-depleted-ADSCs. Depletion of EREG promoted the formation of neural spheres, and increased the expressions of nestin, ßIII-tubulin, NeuroD, NCAM, TH, and NEF in ADSCs. Then, 25 ng/mL rhEREG significantly inhibited these neurogenic differentiation indicators. Inhibition of p38 MAPK, JNK, or Erk1/2 signaling pathway separately, blocked the rhEREG-enhanced osteogenic differentiation ability and the rhEREG-inhibited neurogenic trans-differentiation ability of ADSCs. In conclusions, EREG promoted the osteogenic differentiation and inhibited the neurogenic trans-differentiation potentials of ADSCs via MAPK signaling pathways.

FAM96B inhibits the senescence of dental pulp stem cells.

Dental pulp stem cells (DPSCs) are considered a remarkable source for the regeneration of dental pulp tissues, but their therapeutic effectiveness remains limited, especially in elderly people. Previous studies found that senescence has a negative effect on the proliferation and differentiation potential of DPSCs. Moreover, numerous long non-coding RNA (lncRNA) and messenger RNA were significantly differentially regulated in DPSCs from young and elderly donors. However, the changes in DPSCs protein during senescence have not been addressed. In this study, differences in DPSC protein expression profiles and coexpression of protein and lncRNA were analyzed using proteomics and bioinformatics. The results showed 75 upregulated proteins and 69 downregulated proteins in DPSCs from elderly donors. Vasopressin-regulated water reabsorption, Parkinson's disease, Alzheimer's disease, and protein export were the top four functional pathways associated with DPSCs. High mobility group N1 (HMGN1), HMGN2, UCHL1, and the family with sequence similarity 96 member B homeobox gene (FAM96B) were associated with DPSCs senescence. Then, we investigated FAM96B function in DPSCs. After FAM96B depletion, telomerase reverse transcriptase (TERT) activity decreased, but the number of senescence-associated ß-galactosidase (SA-ß-gal) positive cells and the protein levels of p16, p53 were significantly increased. Gain-of-function assays suggested that FAM96B overexpression was positively correlated with TERT activity, but negatively correlated with the number of SA-ß-gal positive cells and the protein levels of P16 and P53. Moreover, after FAM96B overexpression, the results showed a significant increase in alkaline phosphatase activity and an enhanced mineralization ability of DPSCs. The reverse-transcription polymerase chain reaction results also showed that dentin sialophosphoprotein and osteocalcin were expressed at greater levels. The carboxyfluorescein succinimidyl ester (CFSE) results displayed that FAM96B increased the proliferation potential of DPSCs. Our study revealed candidate proteins that might be related to DPSCs senescence and provided information to elucidate the mechanism of the biological changes in DPSCs' aging. Moreover, FAM96B was demonstrated to play an important role in suppressing DPSCs senescence and promoting osteogenic differentiation and proliferation.

The miR-3940-5p inhibits cell proliferation of gingival mesenchymal stem cells.

OBJECTIVES: Drug-induced gingival overgrowth (DIGO) is a well-recognized side effect of nifedipine (NIF). However, the molecular mechanisms of DIGO are still unknown. Here, we explored the possible role of miR-3940-5p in DIGO using NIF-treated gingival mesenchymal stem cells (GMSCs). MATERIAL AND METHODS: CFSE and cell cycle assays were used to examine cell proliferation. The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, quantitative calcium analysis, and osteogenesis-related gene expression were used to examine osteo/dentinogenic differentiation. RESULTS: The CFSE assay showed that NIF enhanced cell proliferation, and the over-expression of miR-3940-5p inhibited the proliferation of GMSCs with or without NIF stimulation. Cell cycle assays revealed that the cell cycle was arrested at the G0/G1 phase. Furthermore, it was found that the over-expression of miR-3940-5p upregulated p15INK4b , p18INK4c , p19INK4d , and Cyclin A and downregulated Cyclin E in GMSCs with or without NIF treatment. In addition, the over-expression of miR-3940-5p enhanced ALP activity and mineralization in vitro and increased the expression of the osteo/dentinogenic differentiation markers DSPP and DMP1 and the key transcription factor DLX5 in GMSCs. CONCLUSIONS: miR-3940-5p inhibited cell proliferation, enhanced the osteo/dentinogenic differentiation of GMSCs, and might play a role in DIGO as a potent agent in the treatment of nifedipine-induced gingival overgrowth.

SFRP2 enhances the osteogenic differentiation of apical papilla stem cells by antagonizing the canonical WNT pathway.

BACKGROUND: Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms. METHODS: SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of ß-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs. RESULTS: SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear ß-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential. CONCLUSIONS: The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

METTL3 Promotes Osteo/Odontogenic Differentiation of Stem Cells by Inhibiting miR-196b-5p Maturation.

Mesenchymal stem cells (MSCs) have been considered a potential method for the regeneration of tooth and maxillofacial bone defects based on the multidirectional differentiation characteristics of MSCs. miRNAs have been found to play a key role in the differentiation of MSCs. However, its effectiveness still needs to be improved, and its internal mechanism is still unclear. In the present study, our data discovered that the knockdown of miR-196b-5p promoted alkaline phosphatase (ALP) activity assay, mineralization in vitro, and expressions of osteo/odontogenic differentiation markers DSPP and OCN and enhanced in vivo osteo/odontogenic differentiation of stem cells of the apical papilla (SCAPs). Mechanistically, the results indicated that METTL3-dependent N6-methyladenosine (m6A) methylation inhibited miR-196b-5p maturation by the microprocessor protein DGCR8. Moreover, miR-196b-5p indirectly negatively regulates METTL3 in SCAPs. Then, METTL3 was found to strengthen the ALP activity assay, mineralization, and expressions of osteo/dentinogenic differentiation markers. Taken together, our findings highlight the critical roles of the METTL3-miR-196b-5p signaling axis in an m6A-dependent manner in osteo/odontogenic differentiation of SCAPs, identifying some potential targets for tooth and maxillofacial bone defects.

The cannabinoid receptor I (CB1) enhanced the osteogenic differentiation of BMSCs by rescue impaired mitochondrial metabolism function under inflammatory condition.

BACKGROUND: Periodontitis is a chronic infectious disease leading to bone resorption and periodontal tissue disruption under inflammatory stimulation. The osteogenic differentiation ability of mesenchymal stem cells (MSCs) is impaired under the inflammatory environment, which limits the effect of treatment. The cannabinoid receptor I (CB1) is the main effector of the endogenous cannabinoid system (ECS), and our previous study verified that CB1 could enhance the osteo/dentinogenic differentiation of dental MSCs, which might be a target for alveolar bone regeneration. However, the effect of CB1 on the osteogenic differentiation of MSCs derived from bone remains unknown. In present study, we investigated the role and mechanism of CB1 on mitochondrial function and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) under inflammatory environment. METHODS: Alkaline phosphatase (ALP) activity, alizarin red staining, quantitative calcium analysis, and osteogenic markers were used to detect the osteogenic differentiation ability of BMSCs. Real-time RT-PCR and Western blot were used to detect the gene expression. Seahorse Cell Mito Stress Test was used to detect the oxygen consumption rate (OCR). JC-10 assay was used to determine the mitochondrial membrane potential (MMP). RESULTS: CB1 increased osteogenic differentiation potential and mitochondrial energy metabolism, including the OCR, MMP, and enhanced the expressions of Nrf1 and Nrf2 in hBMSCs without or with TNF-α or INF-γ stimulation. Then, the inhibitor of mitochondrial electron transport chain (ETC), rotenone (ROT), inhibited the osteogenic differentiation in hBMSCs, and CB1 could rescue ROT impaired osteogenic differentiation potentials of hBMSCs without or with TNF-α or INF-γ stimulation. Activation of ETC by Coenzyme Q10 (CoQ10) could restore the impaired osteogenic differentiation of hBMSCs by depletion of CB1 without or with TNF-α or INF-γ stimulation. Mechanismly, CB1 could activate the JNK signaling pathway, p38 MAPK signaling pathway, and inhibit the Erk1/2 signaling pathway. CONCLUSIONS: The activating of CB1 enhanced the osteogenic differentiation by rescuing the mitochondrial metabolism function in hBMSCs under the inflammatory environment, suggesting that CB1 is a potential target for enhancing bone regeneration under the inflammatory environment.

A Comprehensive Analysis of SE-lncRNA/mRNA Differential Expression Profiles During Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Objective: Articular cartilage injury is common and difficult to treat clinically because of the characteristics of the cartilage. Bone marrow-derived mesenchymal stem cell (BMSC)-mediated cartilage regeneration is a promising therapy for treating articular cartilage injury. BMSC differentiation is controlled by numerous molecules and signaling pathways in the microenvironment at both the transcriptional and post-transcriptional levels. However, the possible function of super enhancer long non-coding RNAs (SE-lncRNAs) in the chondrogenic differentiation of BMSCs is still unclear. Our intention was to explore the expression profile of SE-lncRNAs and potential target genes regulated by SE-lncRNAs during chondrogenic differentiation in BMSCs. Materials and Methods: In this study, we conducted a human Super-Enhancer LncRNA Microarray to investigate the differential expression profile of SE-lncRNAs and mRNAs during chondrogenic differentiation of BMSCs. Subsequent bioinformatic analysis was performed to clarify the important signaling pathways, SE-lncRNAs, and mRNAs associated with SE-lncRNAs regulating the chondrogenic differentiation of BMSCs. Results: A total of 77 SE-lncRNAs were identified, of which 47 were upregulated and 30 were downregulated during chondrogenic differentiation. A total of 308 mRNAs were identified, of which 245 were upregulated and 63 were downregulated. Some pathways, such as focal adhesion, extracellular matrix (ECM)-receptor interaction, transforming growth factor-ß (TGF-ß) signaling pathway, and PI3K-Akt signaling pathway, were identified as the key pathways that may be implicated in the chondrogenic differentiation of BMSCs. Moreover, five potentially core regulatory mRNAs (PMEPA1, ENC1, TES, CDK6, and ADIRF) and 37 SE-lncRNAs in chondrogenic differentiation were identified by bioinformatic analysis. Conclusion: We assessed the differential expression levels of SE-lncRNAs and mRNAs, along with the chondrogenic differentiation of BMSCs. By analyzing the interactions and co-expression, we identified the core SE-lncRNAs and mRNAs acting as regulators of the chondrogenic differentiation potential of BMSCs. Our study also provided novel insights into the mechanism of BMSC chondrogenic and cartilage regeneration.

miR-140-3p enhanced the osteo/odontogenic differentiation of DPSCs via inhibiting KMT5B under hypoxia condition.

Human dental pulp stem cells (DPSCs) have emerged as an important source of stem cells in the tissue engineering, and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration. Nevertheless, little is known about the complicated molecular mechanisms. In this study, we aimed to investigate the influence and mechanism of miR-140-3p on DPSCs under hypoxia condition. Hypoxia was induced in DPSCs by Cobalt chloride (CoCl2) treatment. The osteo/dentinogenic differentiation capacity of DPSCs was assessed by alkaline phosphatase (ALP) activity, Alizarin Red S staining and main osteo/dentinogenic markers. A luciferase reporter gene assay was performed to verify the downstream target gene of miR-140-3p. This research exhibited that miR-140-3p promoted osteo/dentinogenic differentiation of DPSCs under normoxia environment. Furthermore, miR-140-3p rescued the CoCl2-induced decreased osteo/odontogenic differentiation potentials in DPSCs. Besides, we investigated that miR-140-3p directly targeted lysine methyltransferase 5B (KMT5B). Surprisingly, we found inhibition of KMT5B obviously enhanced osteo/dentinogenic differentiation of DPSCs both under normoxia and hypoxia conditions. In conclusion, our study revealed the role and mechanism of miR-140-3p for regulating osteo/dentinogenic differentiation of DPSCs under hypoxia, and discovered that miR-140-3p and KMT5B might be important targets for DPSC-mediated tooth or bone tissue regeneration.

Peroxiredoxin1 Knockdown Inhibits Oral Carcinogenesis via Inducing Cell Senescence Dependent on Mitophagy.

PURPOSE: Cellular senescence is a physiological phenomenon by which cells irreversibly lose their proliferative potential. It is not clear whether senescent cells are related to malignant transformation in oral precancerous lesions. The role of peroxiredoxin1 (Prx1)-induced cell senescence in OLK malignant transformation has not been reported. The aim of this study is to investigate the role and mechanism of cell senescence in oral carcinogenesis. METHODS: In this study, 4-nitro-quinoline-1-oxide (4NQO) induced tongue carcinogenesis model in Prx1+/+ and Prx1+/- mice and dysplastic oral keratinocyte (DOK) were used. Prx1 knockdown DOK cells were harvested with shRNA injection, and cell senescence was detected via the senescence-associated ß-galactosidase (SA ß-gal) assay. The senescence and mitophagy-related proteins were observed by immunohistochemistry (IHC), Western blot and qRT-PCR. The binding of Prx1 with prohibitin 2 (PHB2) and light chain 3 (LC3) was predicted via ZDOCK and measured in mice by Duolink analysis. RESULTS: Histologically, 4NQO treatment induced epithelial hyperplasia, dysplasia (mild, moderate and severe), carcinomas in situ and oral squamous cell carcinoma (OSCC) in mouse tongue mucosa. The malignant transformation rate in Prx1+/- mice (37.5%) was significantly lower compared with Prx1+/+ mice (57.1%). In Prx1+/+ mice, a higher number of senescent cells and greater expression of p53 and p21 were observed in hyperplastic and dysplastic tongue tissues when compared with those in OSCC tissues. Prx1 knockdown induced a greater number of senescent cells in hyperplastic tissues, and DOK cells accompanied cell cycle arrest at the G1 phase and PHB2/LC3II downregulation. Prx1 was predicted to dock with PHB2 and LC3 via ZDOCK, and the interactions were confirmed by in situ Duolink analysis. CONCLUSION: Prx1 silencing inhibits the oral carcinogenesis by inducing cell senescence dependent on mitophagy.

Clinicopathological Significance of FOXO4 Expression and Correlation with Prx1 in Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: Forkhead box O 4 (FOXO4), a key albumen in the forkhead box O (FOXOs) family, plays crucial roles as a tumor suppressor in the cancer development. In our previous study, Peroxiredoxin1 (Prx1) promoted the development of oral cancer and was predicted to bind to FOXO4. The aim of this study was to investigate the clinicopathological significance of FOXO4 expression and its potential mechanism in head and neck squamous cell carcinomas (HNSCC). METHODS: The function of FOXO4 correlation with HNSCC prognosis was analyzed via ONCOMINE, UALCAN, Human Protein Atlas, and cBioPortal. The expression of FOXO4 was detected in Prx1 silenced CaL27 and SCC9 cell lines by Western blot. FOXO4 protein expression was observed via immunohistochemistry (IHC) and the binding of Prx1 to FOXO4 measured by Duolink analysis in a 4-nitro-quinoline-1-oxide- (4NQO-) induced tongue carcinogenesis model in Prx1+/+ and Prx1+/- mice. RESULTS: By the analysis of Bioinformation Databases, there was a significant interaction of FOXO4 down expression to clinical tumor stages and pathological grades in the patients with HNSCC. Reduced mRNA and protein expression of FOXO4 were found to be significantly correlated with the poor overall survival (OS) of HNSCC patients. FOXO4 expression is negatively related to Prx1 significantly in HNSCC tissues. By employing a 4NQO-induced oral carcinogenesis mouse model, we confirmed that FOXO4 expression was reduced in 4NQO-induced squamous cell carcinoma (SCC) tongue tissues compared with those in normal tissues. Prx1 knockdown resulted in the upregulation of FOXO4 expression in the SCC tissues and CaL27 and SCC9 cell lines. Furthermore, the interaction of Prx1 with FOXO4 was observed in mouse tongue tissues by Duolink analysis. CONCLUSION: FOXO4 plays an important role in the development of HNSCC. The lower expression of FOXO4 is significantly correlated with the shorter OS in patients with HNSCC. FOXO4 is negatively regulated via interaction with Prx1. FOXO4 could be a potential molecular target for the treatment and prognosis of HNSCC.

Azoxystrobin Reduces Oral Carcinogenesis by Suppressing Mitochondrial Complex III Activity and Inducing Apoptosis.

PURPOSE: The five-year survival rate of patients with oral cancer is approximately 50%; thus, alternative drugs with higher efficacy are urgently required. Azoxystrobin (AZOX), a natural, novel methoxyacrylate fungicide isolated from mushrooms, has a broad-spectrum, with highly efficient bactericidal effect. However, studies on AZOX have focused on antifungal effects. Here, we explore the potential cancer-preventive effects of AZOX and the underlying mechanisms. MATERIALS AND METHODS: The effects of AZOX on oral carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO) were investigated in C57BL/6 mice. Cell proliferation and apoptosis were examined by Ki67 immunohistochemistry and TUNEL staining, respectively. The main organ coefficients of each group were calculated to evaluate the biosafety of AZOX. CCK8 and flow cytometry were used to detect the effects of AZOX on cell viability and apoptosis in oral cancer cell line CAL27 and SCC15 cells in vitro. Cell cycle, mitochondrial complex III activity, intercellular reactive oxygen species (ROS) level, mitochondrial ROS level, and mitochondrial membrane potential (MMP) were detected by flow cytometry in AZOX-treated CAL27 cells. RESULTS: AZOX significantly inhibited the occurrence of 4NQO-induced tongue cancer and delayed the progression of tongue precancerous lesions in mice. High-dose AZOX obviously inhibited cell viability and induced apoptosis in epithelial dysplastic and oral squamous cell carcinoma (OSCC) lesions in mouse tongue mucosa. AZOX was confirmed to have high biosafety. Similarly, in vitro cell viability was suppressed, and apoptosis was induced in AZOX-treated CAL27 and SCC15 cells. AZOX induced cell cycle arrest at the S phase. AZOX inhibited mitochondrial complex III activity, increased intracellular and mitochondrial ROS levels, and decreased MMP in CAL27 cells. CONCLUSION: AZOX inhibited the development of oral cancer through specific inhibition of the activity of mitochondrial complex III, which led to ROS accumulation, and MMP decrease, ultimately inducing apoptosis. AZOX may be a novel agent for the prevention and treatment of OSCC.

DLX5 and HOXC8 enhance the chondrogenic differentiation potential of stem cells from apical papilla via LINC01013.

BACKGROUND: Mesenchymal stem cell (MSC)-based cartilage tissue regeneration is a treatment with great potential. How to enhance the MSC chondrogenic differentiation is a key issue involved in cartilage formation. In the present study, we seek to expound the phenotypes and mechanisms of DLX5 in chondrogenic differentiation function in MSCs. METHODS: Stem cells from apical papilla (SCAPs) were used. The Alcian Blue staining, pellet culture system, and cell transplantation in rabbit knee cartilage defect were used to evaluate the chondrogenic differentiation function of MSCs. Western blot, real-time RT-PCR, and ChIP assays were used to evaluate the molecular mechanisms. RESULTS: DLX5 and HOXC8 expressions were upregulated during chondrogenic differentiation. In vitro results showed that DLX5 and HOXC8 enhanced the expression of chondrogenic markers including collagen II (COL2), collagen V (COL5), and sex-determining region Y box protein 9 (SOX9) and promoted the chondrogenic differentiation and the formation of cartilage clumps in the pellet culture system. Mechanically, DLX5 and HOXC8 formed protein complexes and negatively regulated the LncRNA, LINC01013, via directly binding its promoter. In vivo transplantation experiment showed that DLX5 and HOXC8 could restore the cartilage defect in the rabbit knee model. In addition, knock-down of LINC01013 enhanced the chondrogenic differentiation of SCAPs. CONCLUSIONS: In conclusion, DLX5 and HOXC8 enhance the chondrogenic differentiation abilities of SCAPs by negatively regulating LINC01013 in SCAPs, and provided the potential target for promoting cartilage tissue regeneration.

Proteome profiling to identify peroxiredoxin 1 interacting protein partners in nicotine-associated oral leukoplakia.

OBJECTIVE: Tobacco smoking is one of the main risk factors for oral squamous cell carcinoma (OSCC) and can induce generation of reactive oxygen species (ROS). In our previous studies, we demonstrated that nicotine, the major ingredient in tobacco, can upregulate an important antioxidant enzyme Peroxiredoxin 1 (Prx1), in oral leukoplakia (OLK), an oral precancerous lesion. The underlying regulatory mechanisms, however, remain unclear. This study aims to identify regulatory mechanisms of nicotine and identify Prx1 interacting proteins in nicotine-associated OLK. DESIGN: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with bioinformatics analysis was conducted to profile Prx1 binding proteins in human dysplastic oral keratinocyte (DOK) cells. Candidate interaction proteins were further verified using Co-immunoprecipitation (Co-IP), Western blot or Duolink assay in 4-nitro-quinoline-1-oxide (4NQO)-induced OLK in mice and human OLK tissues. RESULTS: We identified Thioredoxin (Trx), Nucleolar GTP-binding protein 1 (GTPBP4), GTP-binding protein Di-Ras2 (DIRAS2) and apoptosis signal-regulating kinase 1 (ASK1) as key Prx1 interacting proteins regulated by nicotine. Our data showed that nicotine upregulated Trx, GTPBP4, DIRAS2, and downregulated ASK1 in 4NQO-induced OLK in mice, at least in part dependent on Prx1. The modulations of Trx, GTPBP4, DIRAS2 and ASK1 by nicotine were also found in OLK smokers compared to OLK non-smokers. The in-situ interaction of Trx, GTPBP4, DIRAS2 and ASK1 with Prx1 were validated in human OLK tissues. CONCLUSION: Nicotine may promote OLK development via regulating Prx1 binding proteins Trx, GTPBP4, DIRAS2 and ASK1. The results of this study will help to develop therapeutic approaches for OLK in humans targeting Prx1 interacting protein network.

Nicotine promotes cervical metastasis of oral cancer by regulating peroxiredoxin 1 and epithelial-mesenchymal transition in mice.

BACKGROUND: Tobacco is a major risk factor for oral squamous cell carcinoma (OSCC). However, the role of nicotine in OSCC is not completely understood. MATERIALS AND METHODS: To analyze the mechanisms of nicotine-induced cervical metastasis, we investigated whether nicotine induced invasion, migration, and epithelial-mesenchymal transition (EMT) via regulating peroxiredoxin 1 (Prx1) in CAL 27 cells. In addition, we established a mouse model to confirm the roles of nicotine in regulating Ets1/Prx1/EMT signaling in OSCC metastasis. RESULTS: We showed that nicotine induced CAL 27 cell invasion, migration, EMT, and Prx1 and Ets1 expression. Prx1 knockdown inhibited cell invasion, migration, and EMT. Ets1 silencing downregulated Prx1 expression and EMT. Prx1 and Ets1 were shown to interact in CAL 27 cells treated with nicotine, and nicotine could significantly upregulate the binding of the transcription factor Ets1 to the Prx1 gene promoter region. Additionally, an in vivo study showed that nicotine induced tumor metastasis and EMT. Prx1 knockdown inhibited cervical metastasis rates and EMT progression. No significant differences in metastasis rates and EMT-related marker expression levels were observed between vehicle- and nicotine-treated mice. CONCLUSION: The results indicate that nicotine promotes cervical lymph node metastasis through regulating Ets1/Prx1/EMT signaling during OSCC pathogenesis; consequently, Prx1 may represent a potential target for the prevention and treatment of OSCC.

Anti-tumor Effect of Rhaponticum uniflorum Ethyl Acetate Extract by Regulation of Peroxiredoxin1 and Epithelial-to-Mesenchymal Transition in Oral Cancer.

Objective: To explore whether Rhaponticum uniflorum (R. uniflorum) had anti-tumor effects in oral cancer and investigate the molecular mechanisms involved in these anti-tumor effects. Methods: Chemical compositions of R. uniflorum ethyl acetate (RUEA) extracts were detected by ultra-performance liquid chromatography-Q/time-of-flight mass spectrometry (UPLC-Q/TOF-MS), followed by pharmacology-based network prediction analysis. The effects of RUEA extracts on proliferation, apoptosis, migration, and invasion ability of human oral squamous cell carcinoma (OSCC) cell line SCC15 were evaluated by CCK8 assay, Annexin V- fluorescein isothiocyanate/propidium iodide staining, wound healing assay, and Matrigel invasion assay, respectively. The mRNA and protein expression of peroxiredoxin1 (Prx1), the epithelial-to-mesenchymal transition (EMT) marker E-cadherin, vimentin, and Snail were determined by quantitative real-time reverse transcription polymerase chain reaction and western blotting. A mouse xenograft model of SCC15 cells was established to further evaluate the effect of RUEA extracts in vivo. Immunohistochemical assessment of Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining of apoptotic cells were performed on the tumor tissues to assess the effects of RUEA extracts on proliferation and apoptosis. Results: Fourteen compounds were identified from RUEA extracts by UPLC-Q/TOF-MS. The pharmacology-based network prediction analysis showed that Prx1 could be a potential binder of RUEA extracts. In SCC15 cells, RUEA extracts inhibited cell viability, induced apoptosis, and suppressed cell invasion and migration in a concentration-dependent manner. After treatment with RUEA extracts, the mRNA and protein expression of E-cadherin increased, whereas those of Prx1, vimentin, and Snail decreased. RUEA extracts also affected the EMT program and suppressed cell invasion and migration in Prx1 knockdown SCC15 cells. In an OSCC mouse xenograft model, RUEA extracts (25 and 250 mg/kg) significantly inhibited the growth of tumors. Compared with the control group, Ki67 expression was reduced and apoptosis rates were elevated in the transplanted tumors treated with RUEA extracts. RUEA extracts increased the expression of E-cadherin and decreased the expression of Prx1, vimentin, and Snail in vivo. Conclusion: RUEA extracts inhibited tumor growth and invasion by reducing Prx1 expression and suppressing the EMT process in OSCC. RUEA extracts may be a potential candidate for OSCC treatment.

Nicotine suppresses apoptosis by regulating α7nAChR/Prx1 axis in oral precancerous lesions.

Nicotine, a tumor promoter in tobacco, can increase Peroxiredoxin (Prx1) and nicotinic acetylcholine receptors (nAChRs) in oral squamous cell carcinoma (OSCC). In the present study, we investigate the effects of nicotine in oral precancerous lesions focusing on apoptosis and nAChR/Prx1 signaling. We detected expression of Prx1, α3nAChR, α7nAChR, phosphorylation of mitogen-activated protein kinases (MAPK) and apoptosis in dysplastic oral keratinocyte (DOK) cells as well as in 4-nitroquinoline 1-oxide (4NQO) or 4NQO + nicotine - induced oral precancerous lesions in Prx1 wild-type (Prx1+/+) and Prx1 knockdown (Prx1+/-) mice. In DOK cells, Prx1 knockdown and blocking α7nAChR activated apoptosis, and nicotine increased the expression of Prx1, α3nAChR and α7nAChR, and inhibited MAPK activation. Moreover, nicotine suppressed apoptosis depending on Prx1 and α7nAChR in DOK cells. In animal bioassay, nicotine and Prx1 promoted growth of 4NQO-induced precancerous lesions in mouse tongue. 4NQO plus nicotine suppressed MAPK activation in Prx1 wild-type mice but not in Prx1 knockdown mice. Our data demonstrate that nicotine inhibits cell apoptosis and promotes the growth of oral precancerous lesions via regulating α7nAChR/Prx1 during carcinogenesis of OSCC.

Peroxiredoxin 1 has an anti-apoptotic role via apoptosis signal-regulating kinase 1 and p38 activation in mouse models with oral precancerous lesions.

Peroxiredoxin 1 (Prx1) is important in the protection of cells from oxidative damage and the regulation of cell proliferation and apoptosis. Prx1 is overexpressed in oral precancerous lesions of oral leukoplakia (OLK) and oral cancer; however, the association between Prx1 expression and OLK pathogenesis remains unknown. The present study investigated the role of Prx1 and its molecular mechanisms in oxidative stress-induced apoptosis during the pathogenesis of OLK. Wild-type and Prx1 knockout mice were treated with 50 µg/ml 4-nitroquinoline-1-oxide (4NQO) or 4NQO + H2O2 for 16 weeks to establish mouse models with tongue precancerous lesions. Apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The expression of Prx1, apoptosis signal-regulating kinase 1 (ASK1), phosphor-ASK1, p38 and phosphor-p38 was analyzed using immunohistochemical staining, and their mRNA expression levels were evaluated by reverse transcription quantitative polymerase chain reaction. The present results demonstrated that 4NQO or 4NQO + H2O2 induced the development of tongue precancerous lesions in Prx1 knockout and wild-type mice. Prx1 was overexpressed in tongue precancerous lesions compared with normal tongue mucosa. There was a significant decrease in the degree of moderate or severe epithelial dysplasia, and mild epithelial dysplasia was clearly elevated, in Prx1 knockout mice treated with 4NQO + H2O2 compared with wild-type mice treated with 4NQO + H2O2. Prx1 suppressed apoptosis and upregulated phosphor-ASK1 and phosphor-p38 expression in tongue precancerous lesions. The present results suggest that Prx1 suppresses oxidative stress-induced apoptosis via the ASK1/p38 signalling pathway in mouse tongue precancerous lesions. In conclusion, Prx1 and H2O2 have a coordination role in promoting the progression of tongue precancerous mucosa lesions. The present findings provide novel insight into Prx1 function and the mechanisms of Prx1 in OLK pathogenesis.

Involvement of the Nonneuronal Cholinergic System in Bone Remodeling in Rat Midpalatal Suture after Rapid Maxillary Expansion.

Few studies sought to analyze the expression and function of the nonneuronal acetylcholine system in bone remodeling in vivo due to the lack of suitable models. We established a rat maxilla expansion model in which the midline palatine suture of the rat was rapidly expanded under mechanical force application, inducing tissue remodeling and new bone formation, which could be a suitable model to investigate the role of the nonneuronal acetylcholine system in bone remodeling in vivo. During the expansion, the expression pattern changes of the nonneuronal cholinergic system components and the mRNA levels of OPG/RANKL were detected by immunohistochemistry or real-time PCR. The value of the RANKL/OPG ratio significantly increased after 1 day of expansion, indicating dominant bone resorption induced by the mechanical stimulation; however after 3 days of expansion, the value of the RANKL/OPG ratio significantly decreased, suggesting a dominant role of the subsequent bone formation process. Increasing expression of Ach was detected after 3 days of expansion which indicated that ACh might play a role in bone formation. The mRNA expression levels of other components also showed observable changes during the expansion which confirmed the involvement of the nonneuronal cholinergic system in the process of bone remodeling in vivo. Further researches are still needed to figure out the detailed functions of the nonneuronal cholinergic system and its components.

Peroxiredoxin 1 promotes invasion and migration by regulating epithelial-to-mesenchymal transition during oral carcinogenesis.

Tobacco smoking is the major risk factor for oral squamous cell carcinoma (OSCC). Previously, we found that nicotine up-regulates peroxiredoxin 1 (Prx1), an important antioxidant enzyme, and nuclear factor kappa B (NFκB) in OSCC cells. However, the molecular mechanism of Prx1 in oral carcinogenesis remains obscure. To improve our understanding of the functional role of Prx1 during the cascade of tobacco-associated oral carcinogenesis, we characterized Prx1, NFκB, and epithelial-to-mesenchymal transition (EMT) markers including E-cadherin, vimentin and Snail in 30 primary oral tumors (15 from smokers with OSCC and 15 from non-smokers with OSCC) and 10 normal oral mucosa specimens from healthy individuals. The expression levels of Prx1, nuclear NFκB, vimentin and Snail were higher in the tumors from smokers with OSCC than in those from non-smokers with OSCC or the healthy controls. The expression levels of E-cadherin showed an opposite trend. Prx1 silencing suppressed the nicotine-induced EMT, cell invasion and migration in SCC15 cells in vitro. Furthermore, Prx1 activated the NFκB pathway in SCC15 cells. Prx1 might therefore play an oncogenic role in tobacco-related OSCC and thus serve as a target for chemopreventive and therapeutic interventions.