Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by stimulating the release of neutrophil extracellular traps in the tumor immune microenvironment.

BACKGROUND: The aim of this study was to investigate the impact of Porphyromonas gingivalis (P. gingivalis) on the progression of oral squamous cell carcinoma (OSCC) through neutrophil extracellular traps (NETs) in the tumor immune microenvironment. METHODS: The expression of NETs-related markers was identified through immunohistochemistry, immunofluorescence, and Western blotting in different clinical stages of OSCC samples. The relationship between NETs-related markers and clinicopathological characteristics in 180 samples was analyzed using immunohistochemistry data. Furthermore, the ability to predict the prognosis of OSCC patients was determined by ROC curve analysis and survival analysis. The effect of P. gingivalis on the release of NETs was identified through immunofluorescence and immunohistochemistry, both in vitro and in vivo. CAL27 and SCC25 cell lines were subjected to NETs stimulation to elucidate the influence of NETs on various cellular processes, including cell proliferation, migration, invasion, and metastasis in vitro. Furthermore, the impact of NETs on the growth and metastatic potential of OSCC was assessed using in vivo models involving tumor-bearing mice and tumor metastasis mouse models. RESULTS: Immunochemistry analysis revealed a significant correlation between the NETs-related markers and clinical stage, living status as well as TN stage. P. gingivalis has demonstrated its ability to effectively induce the release of NETs both in vivo and in vitro. NETs have the potential to facilitate cell migration, invasion, and colony formation. Moreover, in vivo experiments have demonstrated that NETs play a pivotal role in promoting tumor metastasis. CONCLUSION: High expression of NETs-related markers demonstrates a strong correlation with the progression of OSCC. Inhibition of the NETs release process stimulated by P. gingivalis and targeted NETs could potentially open up a novel avenue in the field of immunotherapy for patients afflicted with OSCC.

A randomized controlled clinical trial of concentrated growth factor combined with sodium hyaluronate in the treatment of temporomandibular joint osteoarthritis.

OBJECTIVE: To investigate the effect of concentrated growth factor (CGF) combined with sodium hyaluronate (SH) on temporomandibular joint osteoarthritis (TMJOA). METHODS: Sixty patients with TMJOA who were diagnosed by cone-beam computed tomography (CBCT) between March 2020 and March 2023 at the Stomatological Hospital of Xi'an Jiaotong University were randomly divided into a control group (n = 30) and an experimental group (n = 30). The patients in the experimental group were treated with CGF + SH, and those in the control group were treated with SH only. The visual analogue scale (VAS) score indicating pain in the temporomandibular joint (TMJ) area; the Helkimo Clinical Dysfunction Index (Di); and changes in condylar CBCT at the first visit and 2 weeks, 3 months and 6 months after treatment were recorded. The CBCT data of the patients in the experimental and control groups were collected, and the three-dimensional CBCT image sequences were imported into Mimics Medical 19.0 software in DICOM format for condylar reconstruction. RESULTS: The VAS scores at 2 weeks, 3 months and 6 months after treatment were significantly lower in the experimental group than in the control group (P < 0.05), and the pain in the experimental group was significantly relieved. The Di was significantly lower in the experimental group than in the control group (P < 0.05), and the clinical function of the TMJ improved. After treatment, the CBCT score was significantly lower in the experimental group than in the control group (P < 0.05), and the condylar bone cortex was obviously repaired. Observation of the condylar bone cortex by three-dimensional reconstruction showed the same results as those obtained by CBCT. CONCLUSION: CGF combined with SH is effective in the treatment of TMJOA and can improve muscle pain, TMJ pain, Impaired TMJ function, Impaired range of movement, Pain on movement of the mandible and promote bone repair. THE REGISTRATION NUMBER (TRN): ChiCTR2400082712. THE DATE OF REGISTRATION: April 5, 2024.

Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by activating the neutrophil chemotaxis in the tumour microenvironment.

BACKGROUND: We aimed to determine the significance of Porphyromonas gingivalis (P. gingivalis) in promoting tumour progression in the tumour microenvironment (TME) of oral squamous cell carcinoma (OSCC). METHODS: The Gene Expression Omnibus (GEO) was used to screen out the differentially expressed genes from the two datasets of GEO138206 and GSE87539. Immunohistochemistry and immunofluorescence analysis of samples, cell biological behaviour experiments, and tumour-bearing animal experiments were used to verify the results in vivo and in vitro. The mechanism was revealed at the molecular level, and rescue experiments were carried out by using inhibitors and lentiviruses. RESULTS: CXCL2 was selected by bioinformatics analysis and was found to be related to a poor prognosis in OSCC patients. Samples with P. gingivalis infection in the TME of OSCC had the strongest cell invasion and proliferation and the largest tumour volume in tumour-bearing animal experiments and exhibited JAK1/STAT3 signalling pathway activation and epithelial-mesenchymal transition (EMT). The expression of P. gingivalis, CXCL2 and TANs were independent risk factors for poor prognosis in OSCC patients. A CXCL2/CXCR2 signalling axis inhibitor significantly decreased the invasion and proliferation ability of cells and the tumour volume in mice. When lentivirus was used to block the CXCL2/CXCR2 signalling axis, the activity of the JAK1/STAT3 signalling pathway was decreased, and the phenotype of EMT was reversed. CONCLUSION: Porphyromonas gingivalis promotes OSCC progression by recruiting TANs via activation of the CXCL2/CXCR2 axis in the TME of OSCC.

Mena as a key enhancer factor of EMT to promote metastasis of human tongue squamous cell carcinoma.

OBJECTIVE: The aim of this study was to investigate the effect of mammalian-enabled (Mena) on tongue squamous cell carcinoma (TSCC) metastasis and its mechanism. MATERIALS AND METHODS: Immunochemistry was performed to investigate the Mena and tumor-related markers expression, and its clinicopathological characteristics in 46 TSCC specimens. TSCC cell SCC9 and Cal27 untransfected or stable transfected with Mena overexpression and small interfering RNA were used to determine the role of Mena in cell proliferation, cell migration, invasion and metastasis, and EMT-related markers in vitro, and the effect of Mena on TSCC growth and metastasis through tumor-bearing and tumor metastasis immunodeficient mice models in vivo. RESULTS: Immunochemistry showed that the expression of Mena was significantly correlated with lymphatic metastasis and TNM stage, E-cadherin, Vimentin, and MMP2. Mena did not affect cell proliferation and colony formation in vitro, and tumor growth in vivo. However, it promoted cell migration and invasion in vitro, and TSCC metastasis in vivo. CONCLUSIONS: Mena expression is associated with lymphatic metastasis and tumor stage and promotes TSCC invasion and metastasis by inducing the EMT process. Thus, Mena may be a biomarker for prognosis and targeted therapy in TSCC patients.

An Item Response Theory Model for Incorporating Response Times in Forced-Choice Measures.

Forced-choice (FC) measures have been widely used in many personality or attitude tests as an alternative to rating scales, which employ comparative rather than absolute judgments. Several response biases, such as social desirability, response styles, and acquiescence bias, can be reduced effectively. Another type of data linked with comparative judgments is response time (RT), which contains potential information concerning respondents' decision-making process. It would be challenging but exciting to combine RT into FC measures better to reveal respondents' behaviors or preferences in personality measurement. Given this situation, this study aims to propose a new item response theory (IRT) model that incorporates RT into FC measures to improve personality assessment. Simulation studies show that the proposed model can effectively improve the estimation accuracy of personality traits with the ancillary information contained in RT. Also, an application on a real data set reveals that the proposed model estimates similar but different parameter values compared with the conventional Thurstonian IRT model. The RT information can explain these differences.

The AT-rich DNA-binding protein SATB2 promotes expression and physical association of human (G)γ- and (A)γ-globin genes.

Matrix attachment region (MAR)-binding protein (MARBP) has profound influence on gene transcriptional control by tethering genes to the nuclear scaffold. MARBP SATB2 is recently known as a versatile regulator functioning in the differentiation of multiple cell types including embryonic stem cells, osteoblasts and immunocytes. Roles of SATB2 in erythroid cells and its working mechanism in orchestrating target gene expression are largely unexplored. We show here that SATB2 is expressed in erythroid cells and activates γ-globin genes by binding to MARs in their promoters and recruiting histone acetylase PCAF. Further analysis in higher-order chromatin structure shows that SATB2 affects physical proximity of human (G)γ- and (A)γ-globin promoters via self-association. We also found that SATB2 interacts with SATB1, which specifically activates ε-globin gene expression. Our results establish SATB2 as a novel γ-globin gene regulator and provide a glimpse of the differential and cooperative roles of SATB family proteins in modulating clustered genes transcription and mediating higher-order chromatin structures.

Inflammatory response-related genes predict prognosis in patients with HNSCC.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the most common malignancy of the head and neck, and the inflammatory microenvironment can impact the prognosis of HNSCC. However, the contribution of inflammation to tumour progression has not been fully elucidated. METHODS: The mRNA expression profiles and corresponding clinical data of HNSCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The least absolute shrinkage and selection operator (LASSO) Cox analysis model was used to identify prognostic genes. The overall survival (OS) between high- and low-risk patients was compared by Kaplan‒Meier analysis. The independent predictors of OS were determined by univariate and multivariate Cox analyses. Single-sample gene set enrichment analysis (ssGSEA) was used to assess immune cell infiltration and immune-related pathway activity. GSEA was used to analyse Gene Ontology (GO) terms and Kyoto encyclopaedia of Genes and Genomes (KEGG) pathways. The Gene Expression Profiling Interactive Analysis (GEPIA) database was used to examine prognostic genes in HNSCC patients. Immunohistochemistry was used to verify the protein expression of prognostic genes in HNSCC samples. RESULTS: An inflammatory response-related gene signature was constructed by LASSO Cox regression analysis. HNSCC patients in the high-risk group showed significantly reduced OS compared with those in the low-risk group. The predictive capacity of the prognostic gene signature was confirmed by ROC curve analysis. Multivariate Cox analysis revealed that the risk score was an independent predictor for OS. Functional analysis indicated that the immune status was markedly different between the two risk groups. The risk score was significantly related to tumour stage and immune subtype. The expression levels of the prognostic genes were significantly related to the sensitivity of cancer cells to antitumour drugs. Furthermore, high expression of the prognostic genes significantly predicted poor prognosis of HNSCC patients. CONCLUSIONS: The novel signature containing 9 inflammatory response-related genes reflects the immune status of HNSCC and can be used for prognosis prediction. Furthermore, the genes may be potential targets for HNSCC treatment.

PLAU and LAMC2 can predict a poor prognosis in patients with HNSCC.

Objectives: Head and neck squamous cell carcinoma (HNSCC) is the most common malignancy of the head and neck. However, the molecular mechanisms governing the development of HNSCC have not been fully elucidated. Materials and Methods: Differentially expressed genes (DEGs) were screened out from The Cancer Genome Atlas (TCGA) and GSE23036 datasets. Weighted gene coexpression network analysis (WGCNA) was used to reveal the correlations among genes and to search for significantly correlated gene modules. The expression levels of genes in HNSCC and normal samples according to antibody-based detected methods was assessed by utilizing the Human Protein Atlas (HPA). The impact of the selected hub genes on the prognosis of HNSCC patients was assessed by analysing immunohistochemistry (IHC) and immunofluorescence (IF) expression levels and clinical data. Results: Twenty-four genes positively correlated with tumour status and 15 genes negatively correlated with tumour status were screened out by WGCNA. PLAU and LAMC2 were associated with a poor prognosis in patients with HNSCC and were finally screened out and verified by GEPIA and HPA database analysis. Immunohistochemistry of samples collected from 175 patients with HNSCC and subsequent statistical analysis also showed that PLAU and LAMC2 were associated with a poor prognosis in patients with HNSCC, and the levels of these two factors were positively correlated. The expression and co-localization of PLAU and LAMC2 in HNSCC tissues were confirmed by double immunofluorescence labeling. Conclusions: There was a positive correlation between PLAU and LAMC2 expression in HNSCC samples, and PLAU and LAMC2 might be independent prognostic biomarkers for HNSCC.

Overexpression of Mena is associated with tumor progression and poor prognosis in oral squamous cell carcinoma via EMT.

Background: Mena, a cytoskeletal regulatory protein, is involved in actin-based regulation of cell motility and adhesion, and contributes to tumor invasion and metastasis. However, the role of Mena in oral squamous cell carcinoma remains unclear. This is the first research focusing on the prognostic value of Mena in OSCC. In this study, we aimed to investigate the correlation between Mena expression and clinicopathological significance, as well as prognostic value in OSCC. Methods: Mena gene expression profiles of OSCC and normal tissues were collected from Oncomine, TCGA, and GEO databases. Biological function was analyzed through GO, KEGG and GSEA enrichment. Further, the expression level of Mena and tumor-related markers in 151 OSCC specimens was examined by IHC staining based on tissue microarray. Kaplan-Meier analysis was used to assess the prognostic performance of Mena in OSCC. Result: Mena was generally upregulation in various malignancies, especially OSCC. The functional analyses indicated that Mena was involved in the assembly and regulation of actin, cell movement, and EMT. IHC staining revealed that high expression of Mena in OSCC was correlated with Lymphatic metastasis, TNM stage, E-cadherin, Vimentin, and MMP-2, but insignificantly Ki67. Kaplan-Meier analysis demonstrated that elevated expression of Mena was significantly associated with poor overall survival and disease-free survival of OSCC patients. Conclusion: Mena could be a novel biomarker for predicting the prognosis of OSCC patients, which supports a theoretical basis for developing molecular target therapy.

Positive regulation of hepatic miR-122 expression by HNF4α.

BACKGROUND & AIMS: miR-122 is the most abundant microRNA in the liver and regulates metabolic pathways including cholesterol biosynthesis, fatty acid synthesis, and oxidation. However, little is known about mechanisms that regulate the expression of miR-122 in the liver. The aim of this study was to identify key transcriptional regulators for miR-122 expression through intensively studying its primary transcript and promoter region. METHODS: Bioinformatics analysis, Northern blotting, RT-PCR, and 5'/3' RACE were performed to analyze miR-122 primary transcript structure, its promoter region, and potential transacting factor binding sites. Reporter gene assays integrated with truncation and site-mutation in miR-122 promoter were performed to determine the trans-activation effect of HNF4α to miR-122-promoter in vitro. ChIP and EMSA assays were performed to determine HNF4α binding to miR-122 promoter. Finally, forced expression and RNAi were performed to verify the regulatory roles of HNF4 to miR-122 expression in vitro and in vivo. RESULTS: Here, we show that miR-122 is processed from a long spliced primary transcript directed by a distal upstream promoter region conserved across species. We dissected this promoter region and identified putative binding sites for liver-enriched transcriptional factors that contribute to the regulation of miR-122 expression, including a putative binding site for hepatocyte nuclear factor 4α (HNF4α). We demonstrate that HNF4α binds to the miR-122 promoter region through the conserved DR-I element. We observed the DR-1-element-dependent activation effect of HNF4α on the conserved miR-122 promoter and the activation could be further enhanced by the addition of PGC1α. Using overexpression and knockdown strategies, we show that HNF4α positively regulates miR122 expression in both Huh7 cells and the mouse liver. CONCLUSIONS: Our results suggest that HNF4α is a key regulator of miR-122 expression in the liver.

Bioinformatics and immunohistochemistry analyses of expression levels and clinical significance of CXCL2 and TANs in an oral squamous cell carcinoma tumor microenvironment of Prophyromonas gingivalis infection.

The present study aimed to detect the immunoexpression and clinical significance of Porphyromonas gingivalis (P. gingivalis) in the tumor microenvironment (TME) of oral squamous cell carcinoma (OSCC). The immunoexpression of P. gingivalis in OSCC tissues was detected via immunohistochemistry (IHC) after P. gingivalis was infected into the TME of OSCC. To identify the differentially expressed genes in the carcinogenesis and progression of OSCC with P. gingivalis infection, microarray datasets (GSE87539 and GSE138206) were downloaded from the Gene Expression Omnibus database. The immunoexpression levels of C-X-C motif chemokine ligand 2 (CXCL2) and tumor-associated neutrophils (TANs) were also evaluated via IHC, and the immunoexpression levels of all three clinical variables were analyzed using χ2 or Fisher's exact tests. The survival rates were calculated using the Kaplan-Meier method and the survival curves were compared using log-rank tests. Predominantly strong immunoexpression of P. gingivalis was identified in OSCC samples. CXCL2 was considered to be a differential gene in the two datasets. Immunoexpression of P. gingivalis was positively associated with CXCL2 and TANs expression. Furthermore, P. gingivalis was associated with survival status (P<0.001) and differentiation (P<0.001). CXCL2 was associated with age (P=0.038) and survival status (P=0.003), while TANs were associated with T stage (P=0.015) and clinical stage (P=0.002). These clinical variables were considered to be independent risk factors for the poor prognosis of patients with OSCC. Collectively, the results suggested that the immunoexpression of P. gingivalis may be positively associated with CXCL2 and TANs. In addition, the strong immunoexpression levels of P. gingivalis, CXCL2 and TANs may be associated with a poor prognosis in patients with OSCC.

Identification of long range regulatory elements of mouse alpha-globin gene cluster by quantitative associated chromatin trap (QACT).

Chromatin from different regions of the genome frequently forms steady associations that play important roles in regulating gene expression. The widely used chromatin conformation capture (3C) assay allows determination of the in vivo structural organization of an active endogenous locus. However, unpredicted chromatin associations within a given genomic locus can not be identified by 3C. Here, we describe a new strategy, quantitative associated chromatin trap (QACT), which incorporates a modified 3C method and a quantitative assay tool, to capture and quantitatively analyzes all possible associated chromatin partners (ACPs) of a given chromatin fragment. Using QACT, we have analyzed the chromatin conformation of the mouse alpha-globin gene cluster and proved the extensive interaction between HS26 and alpha-globin genes. In addition, we have identified a candidate alpha1-globin gene specific silencer 475A8 which shows the differentiation-stage specific DNase I hypersensitivity. Functional analysis suggests that 475A8 may regulate the alpha1-globin gene during terminal differentiation of committed erythroid progenitor cells. ChIP (chromatin immunoprecipitation) and cotransfection assays demonstrate that GATA-1, a hemopoietic specific transcriptional factor, may increase alpha1-globin gene expression by suppressing the function of 475A8 in terminally differentiated erythroid cells.

Improvement of SSO-mediated gene repair efficiency by nonspecific oligonucleotides.

Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only approximately 0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells.

[Clinical characteristics of oral and maxillofacial space infection in patients with different ages].

PURPOSE: To analyze the general information,clinical symptoms, etiology of infection, and complications of oral and maxillofacial space infection in patients with different ages, in order to provide references for prevention of complications. METHODS: Three hundred and forty-eight patients with oral and maxillofacial space infection treated in the Oncology Department of Oral and Maxillofacial Surgery of the First Affiliated Hospital of Xinjiang Medical University were retrospectively analyzed from March 2007 to Feburary 2017. Statistical analysis was performed with SPSS 20.0 software package. RESULTS: All patients were divided into 2 groups. 152(43.68%) patients were senior and 196(56.32% ) patients were younger. In the two groups, male patients accounted for 59.69% in the younger group, 59.87% in the senior group. There was no significant difference. We also have found that label test and interval times of symptoms appeared to visit. There was no significant difference. 51.53% of the younger patients had negative bacterial culture results, which was significantly more than those of the senior groups. CONCLUSIONS: In patients with oral and maxillofacial space infection, senior patients had many similar clinical characteristics to younger patients, but senior patients suffered from more and severe complications.

Evaluation of optimal expression cassette in retrovirus vector for beta-thalassemia gene therapy.

Trials of retroviral vector-mediated human beta-globin gene transfer were hampered by low titers, unstable vector transmission, and low-level expression of transferred gene. With the goal of optimizing the retrovirally encoded human beta-globin gene expression cassette for gene therapy of beta-thalassemia, we generated 3 series of vector constructs (a total of 12 constructs) and investigated the effects of the proximal promoter, 3' - enhancer, and derivatives from the beta-locus control region or alpha-major regulatory element on virus titer, vector transmission stability, and gene expression. The virus titers for 9 of the 12 vector constructs ranged between 2.8 x 10(4) cfu/mL and 1.0 x 10(6) cfu/mL. We found that proviral DNA was intact in most G418- resistant murine erythroleukemia (MEL) cell clones for 5 vector constructs, while obvious genetic instability was observed for 4 other vector constructs. MEL cells harboring the intact provirus were induced to differentiate, and human beta-globin gene expression was analyzed with RNase protection assay. The percentage of human beta-globin transcript relative to endogenous murine alpha-globin transcript were 101.8 +/- 64.3% (n = 10), 40.1 +/- 28.7% (n = 4), 31.1 +/- 31.9% (n = 12), 52.4 +/- 11.2% (n = 12), and 53.6 +/- 8.6% (n = 12) for the 5 constructs, respectively, demonstrating the development of optimized retroviral vectors for beta-globin gene therapy with murine erythroid cell lines as a model. Unexpectedly, we also documented that the point mutation 8700(C-->T) in DNase I hypersensitive site 2 (HS2) core fragment might contribute to low-level expression of the human beta-globin gene, based on a comparison of results from transfected and transduced MEL cells and sequence analysis of proviral DNA.

[The possible mechanisms underlying low expression of human beta-globin gene cloned in a retroviral vector].

Murine erythroleukemia (MEL) cell line was used as a model to evaluate the potential value of retroviral construct containing human beta-globin express io n cassette in gene therapy of beta-thalassemia and to explore possible mechanisms underlying low expression of retrovirally cloned human beta-globin gene. A recombinant retroviral vector was constructed, which harbored 2.0 kb beta-globin gene w it h a 374 bp deletion in intervening sequence II coupled with a mini locus control region (miniLCR) composed of DNaseI hypersensitive sites (HS) 2 and 3 from human beta-LCR. The recombinant retroviruses were generated from an established psi-2 producer cell line, and by transient transfection of amphotropic packaging cell l ine psi-A, respectively. The integrity of provirus in transduced MEL cells was determined using Southern blot, and the expression of transferred human beta-globin gene was detected using RNase protection assay. The structure of provirus was further analyzed by sequence analysis of PCR products from genomic DNA of MEL individual clone as template. The results demonstrated that the average expression of human beta-globin gene was (52.4-/+11.2)% (n=12) and (73.8-/+14.3)% (n=12, without copy-number determination), compared with that of endogenous murine alpha-globin ge ne, in MEL cells transduced with the recombinant retrovirus from transient transfection of psi-A and MEL cells transfected with the construct, respectively. In M EL cells transduced with virus from psi-2 producer cell line, however, the average expression was less than 3%. A point mutation was detected in HS2 of provirus i n MEL cell clone with low expression of human beta-globin gene. The possible mechanisms involved in low expression, including position effect, DNA methylation a nd RNA interference are discussed in addition to the point mutation.

Endothelium-specific SIRT1 overexpression inhibits hyperglycemia-induced upregulation of vascular cell senescence.

The rapidly increasing prevalence of diabetes mellitus worldwide is one of the most serious and challenging health problems in the 21st century. Mammalian sirtuin 1 (SIRT1) has been shown to decrease high-glucose-induced endothelial cell senescence in vitro and prevent hyperglycemia-induced vascular dysfunction. However, a role for SIRT1 in prevention of hyperglycemia-induced vascular cell senescence in vivo remains unclear. We used endothelium-specific SIRT1 transgenic (SIRT1-Tg) mice and wild-type (WT) mice to construct a 40-week streptozotocin (STZ)-induced diabetic mouse model. In this mode, 42.9% of wild-type (WT) mice and 38.5% of SIRT1-Tg mice were successfully established as diabetic. Forty weeks of hyperglycemia induced significant vascular cell senescence in aortas of mice, as indicated by upregulation of expression of senescence-associated markers including p53, p21 and plasminogen activator inhibitor-1 (PAI-1). However, SIRT1-Tg diabetic mice displayed dramatically decreased expression of p53, p21 and PAI-1 compared with diabetic WT mice. Moreover, manganese superoxide dismutase expression (MnSOD) was significantly downregulated in the aortas of diabetic WT mice, but was preserved in diabetic SIRT1-Tg mice. Furthermore, expression of the oxidative stress adaptor p66Shc was significantly decreased in aortas of SIRT1-Tg diabetic mice compared with WT diabetic mice. Overall, these findings suggest that SIRT1-mediated inhibition of hyperglycemia-induced vascular cell senescence is mediated at least partly through the reduction of oxidative stress.

Inter-MAR association contributes to transcriptionally active looping events in human beta-globin gene cluster.

Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the beta-globin gene cluster, it is unclear that how these MAR elements are involved in regulating beta-globin genes expression. Here, we report the identification of a new MAR element at the LCR (locus control region) of human beta-globin gene cluster and the detection of the inter-MAR association within the beta-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in beta-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the alpha-like globin genes and beta-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure.

Site-specific transfer of an intact beta-globin gene cluster through a new targeting vector.

The ideal gene-therapy vector for treating genetic disorders should deliver intact therapeutic genes and their essential regulatory elements into the specific "safe genomic site" and realize long-term, self-regulatory expression. For beta-thalassemia gene therapy, viral vectors have been broadly used, but the accompanying insertional mutation and immunogenicity remain problematic. Hence, we aimed to develop new non-viral vectors that are efficient and safe in treating diseases. As previous studies have demonstrated that physiological expression of beta-globin genes requires both a 5' locus control region and 3' specific elements, we constructed a new human chromosome-derived targeting vector to transfer the intact beta-globin gene cluster into K562 cells. The whole beta-globin gene cluster was precisely integrated into the target site and expressed in a self-regulatory pattern. The results proved that the human chromosome-derived vector was specifically targeted to the human genome and this could provide a novel platform for further gene therapy research.