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Objective:To analyze the change of differential genes and signaling pathways in high glucose induced BV2 cells, and to explore the mechanism of transgelin-2 (TAGLN2) regulating cellular inflammatory response and metabolic process.Methods:An experimental study. The cultured BV2 cells were divided into mannitol treatment (Man) group, glucose treatment (Glu) group, overexpression control Glu treatment (Con) group, overexpression TAGLN2 Glu treatment group, silence control Glu treatment (shCon Glu) group, and silence TAGLN2 Glu treatment (shTAGLN2 Glu) group. Cells in the Man group were cultured in modified Eagle high glucose medium (DMEM) containing 25 mmol/L mannitol and 25 mmol/L glucose, cells in other groups (Glu group, Con Glu group, TAGLN2 Glu group, shCon Glu group and shTAGLN2 Glu group) were cultured in DMEM medium containing 50 mmol/L glucose. After 24 hours of cells culture, transcriptome sequencing of cells in each group were performed using high-throughput sequencing technology, and significantly differentially expressed genes (DEG) were screened. |log 2 (fold change)|≥1 and P≤0.05 were adopted as criteria to screen for DEG. Gene Ontology (GO) function enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were performed. Real-time polymerase chain reaction (RT-PCR) was used to detect the relative expression level of DEG mRNA. The data between groups were compared by independent sample t-test. Results:When compared with Man group, a total of 517 differentially expressed genes were screened in Glu group, which including 277 up-regulated genes and 240 down-regulated genes. KEGG pathway enrichment analysis showed that the up-regulated genes were significantly enriched in immune system processes such as nuclear factor (NF)-κB signal pathway, Jak-signal transducers and activators of transcription (STAT) signal pathway, while down-regulated genes were significantly enriched in glycosaminoglycan degradation and glyceride metabolic pathway. Compared with Con Glu group, a total of 480 DEG were screened in TAGLN2 Glu group, among which 147 up-regulated and 333 down-regulated genes were detected. Up-regulated genes were significantly enriched in the metabolic processes of fatty acid, glyceride and pyruvate, while down-regulated genes were significantly enriched in immune system processes such as NF-κB signal pathway, Jak-STAT signal pathway and tumor necrosis factor (TNF) signal pathway. Compared with shCon Glu group, a total of 582 DEG were screened in shTAGLN2 Glu group, among which 423 up-regulated and 159 down-regulated genes were detected. Up-regulated DEG were significantly enriched in immune system processes such as TNF signal pathway and chemokine signal pathway, while down-regulated DEG were significantly enriched in pattern recognition receptor signal pathway. RT-PCR results showed that the relative expression levels of DEG mRNA Card11 ( t=13.530), Icos ( t=3.482), Chst3 ( t=6.949), Kynu ( t=5.399), interleukin (IL)-1β ( t=2.960), TNF-α ( t=5.800), IL-6 ( t=3.130), interferon-γ ( t=7.690) and IL-17 ( t=6.530) in the TAGLN2 Glu treatment group were decreased significantly compared with Con Glu group, and the difference was statistically significant. Conclusion:TAGLN2 can inhibit glucose induced microglia inflammation by NF-κB and Jak-STAT signaling pathways, Card11, Icos, Chst3 and Kynu play an important role in the anti-inflammatory process of TAGLN2.
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Objective@#To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison.@*Methods@#Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated.@*Results@#①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH.@*Conclusion@#The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.
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Objective@#To analyze the percentage of myeloperoxidase (MPO)-positive acute myeloid leukemia (AML) blast cells, and to explore the correlation of MPO expression with the clinical features, gene alterations, therapeutic response and prognosis of AML.@*Methods@#The expressions of MPO in BM blasts cells of 233 newly diagnosed AML were retrospectived analyzed, they were divided into two groups using the percentage of MPO-positive blast [low (≤70%) and high (>70%)], clinical features, gene alterations, chemotherapy efficacy and prognosis were compared between the two groups.@*Results@#①Of the 233 patients, 121(51.9%) were in the low MPO group, and the rest 112(48.1%) in the high MPO group. Favorable-risk group according NCCN guidelines of AML was always MPO-high (χ2=32.773, P<0.001), while MPO-low was closely related to poor-risk (χ2=7.078, P=0.008); ②DNMT3A mutation (χ2=6.905, P=0.009), spliceosome genes mutation (SF3B1/SRSF2/U2AF1) (χ2=5.246, P=0.022), RUNX1 mutation (χ2=4.577, P=0.032), ASXL1 mutation (χ2=7.951, P=0.005) and TP53 mutation (P=0.004) were more likely to be seen in the low MPO group, while C-KIT mutation (χ2=8.936, P=0.003) and CEBPA mutation (χ2=12.340, P<0.001) were more frequent in the high MPO group, especially CEBPA double mutation; ③The rates of first complete remission in the low MPO group were significantly lower than that in the high MPO group (38.8% vs 68.1%, χ2=15.197, P<0.001). Multivariate analysis showed that low MPO positivity significantly affected the CR1 unfavourably. ④The overall survival (OS) and the progression-free survival (PFS) were significantly worse in the low MPO group (18.0% vs 89.4% for OS, and 11.5% vs 56.7% for PFS, P<0.001). Multivariate analysis disclosed that the low number of MPO was significantly unfavourable prognostic factor. ⑤The low MPO group still showed a worse survival even when restricted to the patients with normal karyotype, the OS and the PFS were 31.1% and 18.8% respectively.@*Conclusions@#AML with different MPO expression percentage had a unique gene mutation spectrum. Low expression of MPO was an independent risk factor for CR1, OS and PFS in AML patients, which may be a simple and highly significant factor for AML patients when evaluating the therapeutic efficacy and prognosis.
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Objective To evaluate the influence of single nucleotide polymorphism of Wilms tumor 1(WT1)gene rs16754 on the chemosensitivity and clinical outcomes of elderly patients with acute myeloid leukemia(AML).Methods A total of 178 AML patients aged 60 years and over who received cytarabine-based chemotherapy were enrolled in this retrospective study.The peripheral blood was extracted from 178 AML patients receiving chemotherapy for DNA preparation and study.And bone marrow specimens were collected in 65 AML patients before chemotherapy.The Wilms' Tumor-1 (WT1) rs16754 polymorphism was detected by PCR-RFLP method.The association between genotypes and other variables were analyzed by using Logistic regression model.Variables were adjusted by Cox regression analysis.Results The locus of WT1 gene rs16754 is located in coding region of WT1 gene.The genotype frequency and distribution of the studied population were 55.62％ (99/178)in GG,37.64％(67/178)in GA,and 6.74％(12/178)in AA,with minimum allele frequencies of 0.26.The distributions of the three genotypes were in accordance with Hardy-Weinberg Equilibrium (P=0.884).There was no statistical difference in the data distribution of the genotypes on clinical indexes at baseline.Overall survival time(OS)was longer in patients with allele A and genotype GA plus AA[2.73 years(95 ％CI:1.03-5.11 years)]than in patients with GG genotype[1.64 years(95 ％ CI:0.71-4.34),(P=0.003)].The replase free survival(RFS) was longer in patients with allele A and genotype GA plus AA[2.06 years(95％CI:0.95-4.87)]than in patients with GG genotype[1.12 years(95％CI:0.56-4.11),P =0.032)].Adjusted by using multivariate Cox regression analysis,GA plus AA genotypes still showed a better effect on OS (HR =0.51,P =0.013)than did GG genotypes.In the 65 pretreatment bone marrow specimens,the expression level of WT1 mRNA in bone marrow cells was higher in patients with GG genotype than in patients with GA plus AA genotype(P ＜ 0.001).Conclusions Among elderly AML patients treated by cytarabine-based chemotherapy,the WT1 rs16754 may impact the clinical prognosis of AML patients by influencing the mRNA expression of WT1.