Identifying MiR-140-3p as a stable internal reference to normalize MicroRNA qRT-PCR levels of plasma small extracellular vesicles in lung cancer patients.

Exploration of a stably expressed gene as a reference is critical for the accurate evaluation of miRNAs isolated from small extracellular vesicles (sEVs). In this study, we analyzed small RNA sequencing on plasma sEV miRNAs in the training dataset (n = 104) and found that miR-140-3p was the most stably expressed candidate reference for sEV miRNAs. We further demonstrated that miR-140-3p expressed most stably in the validation cohort (n = 46) when compared to two other reference miRNAs, miR-451a and miR-1228-3p, and the commonly-used miRNA reference U6. Finally, we compared the capability of miR-140-3p and U6 as the internal reference for sEV miRNA expression by evaluating key miRNAs expression in lung cancer patients and found that miR-140-3p was more suitable as a sEV miRNA reference gene. Taken together, our data indicated miR-140-3p as a stable internal reference miRNA of plasma sEVs to evaluate miRNA expression profiles in lung cancer patients.

HMGB2 is a biomarker associated with poor prognosis promoting radioresistance in glioma by targeting base excision repair pathway.

BACKGROUND: High mobility group box 2 (HMGB2) is considered as a biomarker of poor prognosis in various cancers.This study aims to investigate the effect and mechanism of HMGB2 in gliomas. METHODS: With the glioma related on-line and our local hospital databases, the expression differences of HMGB2,Kaplan-Meier survival analysis and COX regression analysis were performed.The correlation analysis between the clinicopathological features and imaging parameters with the HMGB2 expression had been done. Then GSEA and PPI networks were carried out to find out the most significant pathway. The pathway inhibitor was applied to verify HMGB2's participation. CCK8,EDU assays,γ-H2AX immunofluorescence staining and colony formation assay were conducted to observe effects on glioma cells. RESULTS: Available datasets showed that HMGB2 was highly expressed in glioma and patients with high expression of HMGB2 had poorer prognosis and molecular characteristics. Protein level evidence of western blot and immunohistochemistry from our center supported the conclusions above. Analysis on imaging features suggested that HMGB2 expression level had an inverse association with ADCmean but positively with the thickness of enhancing margin. Results from GSEA and PPI network analysis exhibited that HMGB2 was involved in base excision repair (BER) signaling pathway. Experimental evidence demonstrated that the overexpression of HMGB2 promoted the proliferation of glioma cells and enhanced the radio-resistance. CONCLUSIONS: HMGB2 could promote glioma development and enhance the radioresistance of glioma cells, potentially related to the BER pathway, suggesting it may serve as an underlying biomarker for patients with glioma.

Proline-rich protein PRPL1 enhances Panax notoginseng defence against Fusarium solani by regulating reactive oxygen species balance and strengthening the cell wall barrier.

The root rot mainly caused by Fusarium solani is a bottleneck in the cultivation of Panax notoginseng. In this study, we reported a gene encoding a plant cell wall structural protein, P. notoginseng proline-rich protein (PnPRPL1), whose transcription was upregulated by F. solani and induced by some hormone signals. The PnPRPL1 recombinant protein significantly inhibited the growth and conidial germination of the root rot pathogens. Downregulation of PnPRPL1 by RNA interference (RNAi) in P. notoginseng leaves increased the susceptibility to F. solani, whereas overexpression of PnPRPL1 in tobacco (Nicotiana tabacum) enhanced the resistance to F. solani. Compared with wild-type tobacco, the PnPRPL1-overexpressing transgenic tobacco had higher reactive oxygen species (ROS)-scavenging enzyme activities, lower ROS levels, and more lignin and callose deposition. The opposite results were obtained for the P. notoginseng expressing PnPRPL1 RNAi fragments. Furthermore, the PnPRPL1 promoter transcription activity was induced by several plant hormones and multiple stress stimuli. In addition, the transcription factor PnWRKY27 activated the expression of PnPRPL1 by directly binding to the promoter region. Thus, PnPRPL1, which is positively regulated by a WRKY transcription factor, encodes an antimicrobial protein that also mediates ROS homoeostasis and callose/lignin deposition during the response to F. solani infection.

Panax notoginseng transcription factor WRKY15 modulates resistance to Fusarium solani by up-regulating osmotin-like protein expression and inducing JA/SA signaling pathways.

BACKGROUND: Panax notoginseng (Burk) F. H. Chen is a valuable traditional Chinese medicinal plant, but its commercial production is seriously affected by root rot caused by some pathogenic fungi, including Fusarium solani. Nevertheless, the genetic breeding for disease resistance of P. notoginseng remains limited. The WRKY transcription factors have been revealed to play important roles in plant defense responses, which might provide an inspiration for resistance improvement in P. notoginseng. RESULTS: In this study, the regulatory mechanism of transcription factor PnWRKY15 on P. notoginseng resistance to F. solani infection was revealed. The suppressed expression of PnWRKY15 via RNA interference increased the sensitivity of P. notoginseng to F. solani and decreased the expression levels of some defense-related genes, including PnOLP1, which encodes an osmotin-like protein that confers resistance to F. solani. Ectopic expression of PnWRKY15 in the model plant tobacco significantly enhanced the resistance to F. solani. Moreover, the transcriptome sequencing analysis discovered that some pathogenesis-related genes were expressed at higher levels in the PnWRKY15-overexpressing tobacco than that in the wild-type tobacco. In addition, the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were evidently induced by PnWRKY15-overexpression, that was evidenced by that the JA and SA contents were significantly higher in the PnWRKY15-overexpressing tobacco than that in the wild-type. Furthermore, PnWRKY15, which was localized in the nucleus, can trans-activate and up-regulate PnOLP1 expression according to the EMSA, yeast one-hybrid and co-expression assays. CONCLUSIONS: PnWRKY15 contributes to P. notoginseng resistance to F. solani by up-regulating the expression of resistance-related gene PnOLP1 and activating JA/SA signaling pathways. These findings will help to further elucidate the transcriptional regulatory mechanism associated with the P. notoginseng defense response to F. solani.

Deacetylation of IRF8 inhibits iNOS expression and inflammation via SIRT1 in macrophages.

AIMS: Dysregulated interferon regulatory factor 8 (IRF8) mediated inducible nitric oxide synthase (iNOS) transcription is crucial to the pathogenesis of several inflammatory disorders. However, the molecular mechanism that control the transcription activity of IRF8 in the regulation of iNOS is not fully elucidated. This study is undertaken to determine whether SIRT1 impacts IRF8 acetylation level in the macrophages. MAIN METHODS: The silver stain, mass spectrum, bone marrow-derived monocytes differentiation, lentiviral transduction, immunoprecipitation and chromatin immunoprecipitation assay were used to investigate the relationship between IRF8 and SIRT1. KEY FINDINGS: We demonstrate that deacetylation of IRF8 is induced by lipopolysaccharide (LPS) and suppresses iNOS expression. Macrophages expressing acetylation-defective iNOS are highly septic upon transfer to macrophages cleaned up mice. Mechanistically, deacetylation IRF8 facilitates the binding of silent information regulator 1 (SIRT1) to the iNOS promoter and restricts iNOS transcription. The expression of iNOS was enhanced in the macrophages from SIRT1 conditional knockout mice and the progression of sepsis is more serious. SIGNIFICANCE: The discovery of the IRF8-SIRT1 interaction that governs iNOS expression may exploit new therapeutic strategies for inflammatory disorders.

Panax notoginseng WRKY Transcription Factor 9 Is a Positive Regulator in Responding to Root Rot Pathogen Fusarium solani.

Panax notoginseng (Burk) F.H. Chen is a rare and valuable Chinese herb, but root rot mainly caused by Fusarium solani severely affects the yield and quality of P. notoginseng herbal materials. In this study, we isolated 30 P. notoginseng WRKY transcription factors (TFs), which were divided into three groups (I, II, and III) on the basis of a phylogenetic analysis. The expression levels of 10 WRKY genes, including PnWRKY9, in P. notoginseng roots increased in response to a methyl jasmonate (MeJA) treatment and the following F. solani infection. Additionally, PnWRKY9 was functionally characterized. The PnWRKY9 protein was localized to the nucleus. The overexpression of PnWRKY9 in tobacco (Nicotiana tabacum) considerably increased the resistance to F. solani, whereas an RNAi-mediated decrease in the PnWRKY9 expression level in P. notoginseng leaves increased the susceptibility to F. solani. The RNA sequencing and hormone content analyses of PnWRKY9-overexpression tobacco revealed that PnWRKY9 and the jasmonic acid (JA) signaling pathway synergistically enhance disease resistance. The PnWRKY9 recombinant protein was observed to bind specifically to the W-box sequence in the promoter of a JA-responsive and F. solani resistance-related defensin gene (PnDEFL1). A yeast one-hybrid assay indicated that PnWRKY9 can activate the transcription of PnDEFL1. Furthermore, a co-expression assay in tobacco using ß-glucuronidase (GUS) as a reporter further verified that PnWRKY9 positively regulates PnDEFL1 expression. Overall, in this study, we identified P. notoginseng WRKY TFs and demonstrated that PnWRKY9 positively affects plant defenses against the root rot pathogen. The data presented herein provide researchers with fundamental information regarding the regulatory mechanism mediating the coordinated activities of WRKY TFs and the JA signaling pathway in P. notoginseng responses to the root rot pathogen.

The silicon active sites driven by oxygen vacancies in iron silicate for activating peroxymonosulfate.

Most metal sites and some non-metallic sites such as carbon and nitrogen are usually considered to be traditional active sites during peroxymonosulfate (PMS) activation. However, as an important non-metallic element, the actual role of silicon (Si) in PMS activation still remains unclear. In this work, taking iron silicate (FeSi) as an example, the role of the Si region in PMS activation was clearly revealed. The experiments and density functional theory (DFT) calculation results showed that besides the traditional Fe sites, the Si also played a non-negligible role during PMS activation. In FeSi containing oxygen vacancies (Ovac), Fe-Si was the active site instead of Fe-Fe. The Bard charge results implied that the presence of Ovac tuned the electronic properties of FeSi, making the Si participate in PMS activation. This work deepened understanding of the role of Si in silicates for PMS activation and provided a theoretical basis for the development of excellent Si-based catalysts.

ANXA1: An Important Independent Prognostic Factor and Molecular Target in Glioma.

Objective: The expression, prognosis, and related mechanisms of ANXA1 are investigated in glioma, with the objective to find potential therapeutic molecular targets for glioma. Methods: We analyzed the gene expression of ANXA1 using glioma-related databases, including the Chinese Glioma Genome Atlas (CGGA) database, The Cancer Genome Atlas (TCGA) database, and the Gene Expression Omnibus (GEO) database. Moreover, we collected the sample tissues and corresponding paracancerous tissues of 23 glioma patients and then conducted a Western blot experiment to verify the expression and correlate survival of ANXA1. Moreover, we generated survival ROC curves, performing univariate and multivariate Cox analyses and the construction of the nomogram. Differential expression analysis was conducted by high and low grouping based on the median of the ANXA1 gene expression values. We conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and Gene Set Enrichment Analysis (GSEA) to explore possible mechanisms, and gene co-expression analysis was also performed. Results: The results showed that the ANXA1 expression level was higher in gliomas than in normal tissues, and a high expression level of ANXA1 in gliomas was associated with poorer prognosis. The independent prognosis analysis showed that the ANXA1 gene was an independent prognostic factor of glioma. In the analysis of KEGG and Gene Set Enrichment Analysis (GSEA), it is shown that ANXA1 may play an important role in glioma patients by affecting extracellular matrix (ECM)-receptor interaction and the focal adhesion signal pathway. The core genes, including COL1A1, COL1A2, FN1, ITGA1, and ITGB1, were screened for gene correlation and prognosis analysis. The expression level of the five genes was verified by qPCR in glioma. We concluded that these five core genes and ANXA1 could play a synergistic role in gliomas. Conclusion: The results indicated that a high expression level of ANXA1 leads to worse prognosis and ANXA1 is an independent prognostic factor and a potentially important target for the treatment of gliomas.

Lilium regale Wilson WRKY3 modulates an antimicrobial peptide gene, LrDef1, during response to Fusarium oxysporum.

BACKGROUND: WRKY transcription factors (TFs) play vital roles in plant growth and development, secondary metabolite synthesis, and response to biotic and abiotic stresses. In a previous transcriptome sequencing analysis of Lilium regale Wilson, we identified multiple WRKY TFs that respond to exogenous methyl jasmonate treatment and lily Fusarium wilt (Fusarium oxysporum). RESULTS: In the present study, the WRKY TF LrWRKY3 was further analyzed to reveal its function in defense response to F. oxysporum. The LrWRKY3 protein was localized in the plant cell nucleus, and LrWRKY3 transgenic tobacco lines showed higher resistance to F. oxysporum compared with wild-type (WT) tobacco. In addition, some genes related to jasmonic acid (JA) biosynthesis, salicylic acid (SA) signal transduction, and disease resistance had higher transcriptional levels in the LrWRKY3 transgenic tobacco lines than in the WT. On the contrary, L. regale scales transiently expressing LrWRKY3 RNA interference fragments showed higher sensitivity to F. oxysporum infection. Moreover, a F. oxysporum-induced defensin gene, Def1, was isolated from L. regale, and the recombinant protein LrDef1 isolated and purified from Escherichia coli possessed antifungal activity to several phytopathogens, including F. oxysporum. Furthermore, co-expression of LrWRKY3 and the LrDef1 promoter in tobacco enhanced the LrDef1 promoter-driven expression activity. CONCLUSIONS: These results clearly indicate that LrWRKY3 is an important positive regulator in response to F. oxysporum infection, and one of its targets is the antimicrobial peptide gene LrDef1.

An MYB Transcription Factor Modulates Panax notoginseng Resistance Against the Root Rot Pathogen Fusarium solani by Regulating the Jasmonate Acid Signaling Pathway and Photosynthesis.

Root rot of Panax notoginseng, a precious Chinese medicinal plant, seriously impacts its sustainable production. However, the molecular regulatory mechanisms employed by P. notoginseng against root rot pathogens, including Fusarium solani, are still unclear. In this study, the PnMYB2 gene was isolated, and its expression was affected by independent treatments with four signaling molecules (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) as assessed by quantitative real-time PCR. Moreover, the PnMYB2 expression level was induced by F. solani infection. The PnMYB2 protein localized to the nucleus and may function as a transcription factor. When overexpressed in transgenic tobacco, the PnMYB2 gene conferred resistance to F. solani. Jasmonic acid (JA) metabolism and disease resistance-related genes were induced in the transgenic tobacco, and the JA content significantly increased compared with in the wild type. Additionally, transcriptome sequencing, Kyoto Encyclopedia of Genes and Genomes annotation enrichment, and metabolic pathway analyses of the differentially expressed genes in the transgenic tobacco revealed that JA metabolic, photosynthetic, and defense response-related pathways were activated. In summary, PnMYB2 is an important transcription factor in the defense responses of P. notoginseng against root rot pathogens that acts by regulating JA signaling, photosynthesis, and disease-resistance genes.

Overexpression of miR-101 suppresses collagen synthesis by targeting EZH2 in hypertrophic scar fibroblasts.

BACKGROUND: MicroRNA-101 (miR-101) is a tumor suppressor microRNA (miRNA) and its loss is associated with the occurrence and progression of various diseases. However, the biological function and target of miR-101 in the pathogenesis of hypertrophic scars (HS) remains unknown. METHODS: We harvested HS and paired normal skin (NS) tissue samples from patients and cultured their fibroblasts (HSF and NSF, respectively). We used quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH), enzyme-linked immunosorbent assays (ELISA) and Western blot analyses to measure mRNA levels and protein expression of miR-101, enhancer of zeste homolog 2 (EZH2), collagen 1 and 3 (Col1 and Col3) and α-smooth muscle actin (α-SMA) in different in vitro conditions. We also used RNA sequencing to evaluate the relevant signaling pathways and bioinformatics analysis and dual-luciferase reporter assays to predict miR-101 targets. We utilized a bleomycin-induced fibrosis mouse model in which we injected miR-101 mimics to evaluate collagen deposition in vivo. RESULTS: We found low expression of miR-101 in HS and HSF compared to NS and NSF. Overexpressing miR-101 decreased Col1, Col3 and α-SMA expression in HSF. We detected high expression of EZH2 in HS and HSF. Knockdown of EZH2 decreased Col1, Col3 and α-SMA in HSF. Mechanistically, miR-101 targeted the 3'-untranslated region (3'UTR) of EZH2, as indicated by the decreased expression of EZH2. Overexpressing EZH2 rescued miR-101-induced collagen repression. MiR-101 mimics effectively suppressed collagen deposition in the bleomycin-induced fibrosis mouse model. CONCLUSIONS: Our data reveal that miR-101 targets EZH2 in HS collagen production, providing new insight into the pathological mechanisms underlying HS formation.

Identification of MCM4 as a Prognostic Marker of Hepatocellular Carcinoma.

METHODS: MCM4 expression difference in HCC were analyzed from TCGA and GEO data and verified by real-time PCR and western blot. ROC curve was used to analyze the diagnostic value of MCM4 and AFP. Additionally, the relationship between MCM4 and stage or nodal metastasis status or grade or age in TCGA cohort with HCC was observed from the UALCAN website. The univariate and multivariate Cox and functional analyses were done to explore the prognostic value of MCM4 in TCGA cohort. RESULTS: It was found that MCM4 was significantly highly expressed in HCC tissues from TCGA, GEO, and experimental data. Furthermore, ROC curve analysis showed that MCM4 was superior to be a diagnostic biomarker than AFP from TCGA (AUCMCM4 = 0.9461, AUCAFP = 0.7056) and GEO (GSE19665: AUCMCM4 = 0.8800, AUCAFP = 0.5100; GSE64041 AUCMCM4 = 0.8038, AUCAFP = 0.6304). AUC of MCM4 from real-time PCR result in 60 pairs of HCC and adjacent tissues was 0.7172, demonstrating the prediction value of MCM4. Besides, different expression tendencies of MCM4 among different stages or nodal metastasis status or grade or age were observed from the UALCAN website. In addition, multiROC analysis showed the advantage of MCM4 as a survival prediction at 1, 3, and 5 years with the higher AUC at 0.69 of 1 year, 0.65 of 3 years, and 0.61 of 5 years. It was shown that MCM4 was independently associated with OS in univariate and multivariate Cox analysis. And GSEA displayed that MCM4 was highly enriched in KEGG_CELL_CYCLE signaling pathway following higher correlation positively with CDC6, PLK1, CRC1, and BUB1B in HCC. CONCLUSION: MCM4 might be a potential biomarker in guiding the prognostic status of HCC patients.

Upregulated Expression of Cancer-Derived Immunoglobulin G Is Associated With Progression in Glioma.

OBJECTIVE: Gliomas are the most aggressive intracranial tumors accounting for the vast majority of brain tumors with very poor prognosis and overall survival (OS). Cancer-derived immunoglobulin G (cancer-IgG) has been found to be widely expressed in several malignancies such as breast cancer, colorectal cancer, and lung cancer. Cancer-IgG could promote tumorigenesis and progression. However, its role in glioma has not been revealed yet. METHODS: We mined open databases including the Chinese Glioma Genome Atlas (CGGA), The Cancer Genome Atlas (TCGA), and the Gene Expression Omnibus (GEO) to study the role of IGHG1, which encodes cancer-IgG in glioma. Examination of the differential expression of IGHG1 was carried out in the GEO and TCGA databases. Furthermore, its expression in different molecular subtypes was analyzed. Stratified analysis was performed with clinical features. Subsequently, immune infiltration analysis was conducted using single-sample gene set enrichment analysis (ssGSEA). GSEA was performed to reveal the mechanisms of IGHG1. Lastly, immunohistochemistry was processed to validate our findings. RESULTS: In this study, we found that the expression of IGHG1 was higher in glioma and molecular subtypes with poor prognosis. The overall survival of patients with a high expression of IGHG1 was worse in the stratified analysis. Immune infiltration analysis indicated that the expression level of IGHG1 was positively correlated with the stromal score, ESTIMATE score, and immune score and negatively correlated with tumor purity. Results from the GSEA and DAVID demonstrated that IGHG1 may function in phagosome, antigen processing and presentation, extracellular matrix structural constituent, antigen binding, and collagen-containing extracellular matrix. Finally, immunohistochemistry assay validated our findings that patients with a high expression of cancer-IgG had poor OS and disease-free survival (DFS). CONCLUSION: Cancer-IgG is a promising biomarker of diagnosis and treatment for patients with glioma.

Lilium regale Wilson WRKY2 Regulates Chitinase Gene Expression During the Response to the Root Rot Pathogen Fusarium oxysporum.

Root rot, mainly caused by Fusarium oxysporum, is the most destructive disease affecting lily (Lilium spp.) production. The WRKY transcription factors (TFs) have important roles during plant immune responses. To clarify the effects of WRKY TFs on plant defense responses to pathogens, a WRKY gene (LrWRKY2) was isolated from Lilium regale Wilson, which is a wild lily species highly resistant to F. oxysporum. The expression of LrWRKY2, which encodes a nuclear protein, is induced by various hormones (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide) and by F. oxysporum infection. In this study, LrWRKY2-overexpressing transgenic tobacco plants were more resistant to F. oxysporum than the wild-type plants. Moreover, the expression levels of jasmonic acid biosynthetic pathway-related genes (NtAOC, NtAOS, NtKAT, NtPACX, NtJMT, NtOPR, and NtLOX), pathogenesis-related genes (NtCHI, NtGlu2, and NtPR-1), and antioxidant stress-related superoxide dismutase genes (NtSOD, NtCu-ZnSOD, and MnSOD) were significantly up-regulated in LrWRKY2 transgenic tobacco lines. Additionally, the transient expression of a hairpin RNA targeting LrWRKY2 increased the susceptibility of L. regale scales to F. oxysporum. Furthermore, an F. oxysporum resistance gene (LrCHI2) encoding a chitinase was isolated from L. regale. An electrophoretic mobility shift assay demonstrated that LrWRKY2 can bind to the LrCHI2 promoter containing the W-box element. Yeast one-hybrid assay results suggested that LrWRKY2 can activate LrCHI2 transcription. An examination of transgenic tobacco transformed with LrWRKY2 and the LrCHI2 promoter revealed that LrWRKY2 activates the LrCHI2 promoter. Therefore, in L. regale, LrWRKY2 is an important positive regulator that contributes to plant defense responses to F. oxysporum by modulating LrCHI2 expression.

A Focal Adhesion-Related Gene Signature Predicts Prognosis in Glioma and Correlates With Radiation Response and Immune Microenvironment.

BACKGROUND: Glioma is the most frequent brain malignancy presenting very poor prognosis and high recurrence rate. Focal adhesion complexes play pivotal roles in cell migration and act as hubs of several signaling pathways. METHODS: We used bioinformatic databases (CGGA, TCGA, and GEO) and identified a focal adhesion-related differential gene expression (FADG) signature by uniCox and LASSO regression analysis. We calculated the risk score of every patient using the regression coefficient value and expression of each gene. Survival analysis, receiver operating characteristic curve (ROC), principal component analysis (PCA), and stratified analysis were used to validate the FADG signature. Then, we conducted GSEA to identify the signaling pathways related to the FADG signature. Correlation analysis of risk scores between the immune checkpoint was performed. In addition, the correlation of risk scores and genes related with DNA repair was performed. CIBERSORT and ssGSEA were used to explore the tumor microenvironment (TME). A nomogram that involved our FADG signature was also constructed. RESULTS: In total, 1,726 (528 patients diagnosed with WHO II, 591 WHO III, and 603 WHO IV) cases and 23 normal samples were included in our study. We identified 29 prognosis-related genes in the LASSO analysis and constructed an eight FADG signature. The results from the survival analysis, stratified analysis, ROC curve, and univariate and multivariate regression analysis revealed that the prognosis of the high-risk group was significantly worse than the low-risk group. Correlation analysis between risk score and genes that related with DNA repair showed that the risk score was positively related with BRCA1, BRCA2, RAD51, TGFB1, and TP53. Besides, we found that the signature could predict the prognosis of patients who received radiation therapy. SsGSEA indicated that the high-risk score was positively correlated with the ESTIMATE, immune, and stromal scores but negatively correlated with tumor purity. Notably, patients in the high-risk group had a high infiltration of immunocytes. The correlation analysis revealed that the risk score was positively correlated with B7-H3, CTLA4, LAG3, PD-L1, and TIM3 but inversely correlated with PD-1. CONCLUSION: The FADG signature we constructed could provide a sensitive prognostic model for patients with glioma and contribute to improve immunotherapy management guidelines.

DNMT family induces down-regulation of NDRG1 via DNA methylation and clinicopathological significance in gastric cancer.

BACKGROUND: Aberrant DNA methylation of tumor suppressor genes is a common event in the development and progression of gastric cancer (GC). Our previous study showed NDRG1, which could suppress cell invasion and migration, was frequently down-regulated by DNA methylation of its promoter in GC. PURPOSE AND METHODS: To analyze the relationship between the expression and DNA methylation of NDRG1 and DNA methyltransferase (DNMT) family. We performed a comprehensive comparison analysis using 407 patients including sequencing analysis data of GC from TCGA. RESULTS: NDRG1 was down-regulated in GC, and was negatively correlative to DNMT1 (r = -0.11, p = 0.03), DNMT3A (r = -0.10, p = 0.01), DNMT3B (r = -0.01, p = 0.88), respectively, whereas the DNA methylation of NDRG1 was positively correlative to DNMT family (DNMT1 r = 0.20, p < 0.01; DNMT3A r = 0.26, p < 0.001; DNMT3B r = 0.03, p = 0.57, respectively). NDRG1 expression was significantly inverse correlated with invasion depth (p = 0.023), but DNMT1 was significantly positive correlated with invasion depth (p = 0.049). DNMT3B was significantly correlated with the degree of tumor cell differentiation (p = 0.030). However, there was no association between the expression of DNMT3A and clinicopathological features. The KM plotter showed that NDRG1 (HR = 0.95, 95% CI [0.8-1.12], p = 0.53) and DNMT1 (HR = 1.04, 95% CI [0.88-1.23], p = 0.67) had no association with prognosis of GC patients, while, DNMT3A (p = 0.0064) and DNMT3B (p = 0.00025) displayed significantly association. But the overall survival of high expression of NDRG1 tended to be prolonged. CONCLUSION: These data suggest that down-regulation of NDRG1expression in GC may be due to its promoter DNA methylation via DNMT family. The demethylating agent maybe a potential target drug for GC patients.

KR­12­a6 promotes the osteogenic differentiation of human bone marrow mesenchymal stem cells via BMP/SMAD signaling.

Considering the increased resistance to antibiotics in the clinic and the ideal antibacterial properties of KR­12, the effects of KR­12­a6, an important analogue of KR­12, on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) were investigated. Osteogenic differentiation­associated experiments were conducted in hBMSCs, and KR­12­a6 was used as an additional stimulating factor during osteogenic induction. Quantitative analysis of alkaline phosphatase (ALP) and alizarin red staining, and reverse transcription­quantitative PCR analysis of the expression of osteogenesis­associated genes were performed to determine the effects of KR­12­a6 on the osteogenic differentiation of hBMSCs. LDN­212854 was selected to selectively suppress BMP/SMAD signaling. Western blotting was performed to investigate the underlying mechanisms. The intensity of ALP and alizarin red staining gradually increased with increasing KR­12­a6 concentrations. KR­12­a6 induced the strongest staining at 40 µg/ml, whereas 60 µg/ml and 80 µg/ml concentrations did not further increase the intensity of staining. The mRNA expression levels of RUNX2 and ALP increased in a dose­dependent manner as early as 3 days post­KR­12­a6 treatment. The mRNA expression of COL1A1, BSP and BMP2 exhibited significant upregulation from day 7 post­KR­12­a6 treatment. In contrast, the mRNA levels of OSX, OCN and OPN were enhanced dramatically at day 14 following KR­12­a6 stimulation. Additionally, KR­12­a6 significantly promoted the phosphorylation of Smad1/5. Furthermore, LDN­212854 suppressed the activation of Smad1/5 and inhibited the upregulation of several osteogenic differentiation­associated genes in KR­12­a6­treated hBMSCs. KR­12­a6 promoted the osteogenic differentiation of hBMSCs via BMP/SMAD signaling.

The Akt/FoxO/p27Kip1 axis contributes to the anti-proliferation of pentoxifylline in hypertrophic scars.

Hypertrophic scars (HS) are characterized by the excessive production and deposition of extracellular matrix (ECM) proteins. Pentoxifylline (PTX), a xanthine derived antioxidant, inhibits the proliferation, inflammation and ECM accumulation of HS. In this study, we aimed to explore the effect of PTX on HS and further clarify the mechanism of PTX-induced anti-proliferation. We found that PTX could significantly attenuate proliferation of HS fibroblasts and fibrosis in an animal HS model. PTX inhibited the proliferation of HSFs in a dose- and time-dependent manner, and this growth inhibition was mainly mediated by cell cycle arrest. Transcriptome sequencing showed that PTX affects HS formation through the PI3K/Akt/FoxO1 signalling pathway to activate p27Kip1 . PTX down-regulated p-Akt and up-regulated p-FoxO1 in TGF-ß1 stimulated fibroblasts at the protein level, and simultaneously, the expression of p27Kip1 was activated. In a mouse model of HS, PTX treatment resulted in the ordering of collagen fibres. The results revealed that PTX regulates TGFß1-induced fibroblast activation and inhibits excessive scar formation. Therefore, PTX is a promising agent for the treatment of HS formation.