Clinical applications of abnormal DNA methylation in chronic myeloid leukemia. / 异常DNAç”²åŸºåŒ–åœ¨æ ¢æ€§é«“ç³»ç™½è¡€ç— ä¸­çš„ä¸´åºŠåº”ç”¨.

DNA methylation, a crucial biochemical process within the human body, fundamentally alters gene expression without modifying the DNA sequence, resulting in stable changes. The changes in DNA methylation are closely related to numerous biological processes including cellular proliferation and differentiation, embryonic development, and the occurrence of immune diseases and tumor. Specifically, abnormal DNA methylation plays a crucial role in the formation, progression, and prognosis of chronic myeloid leukemia (CML). Moreover, DNA methylation offers substantial potential for diagnosing and treating CML. Accordingly, understanding the precise mechanism of DNA methylation, particularly abnormal changes in the methylation of specific genes in CML, can potentially promote the development of novel targeted therapeutic strategies. Such strategies could transform into clinical practice, effectively aiding diagnosis and treatment of CML patients.

[Research Progress of Pyroptosis in Leukemia: from Mechanism to Treatment --Review].

Pyroptosis is a programmed death mediated by activated caspase and Gasdermin family proteins, characterized by cell swelling, cytosolysis and release of inflammatory factors. Leukemia is a malignant disease characterized by abnormal differentiation and proliferation of hematopoietic stem cells, thus seriously threating human health. In recent years, it has been found that the transformation, proliferation, metastasis and treatment response of leukemia cells are closely related to pyrodeath. Pyroptosis provides a new perspective for the study of leukemia. This paper reviews the types and molecular mechanisms of pyroptosis, the role of pyroptosis in the occurrence and development of leukemia and the treatment of leukemia, so as to provide some references for further study of the relationship between pyroptosis and leukemia, in order to provide a new strategy for the treatment of leukemia.

[Effect of Silencing UVRAG on Mitophagy in Leukemia Cells K562].

OBJECTIVE: To explore the effect of UVRAG on mitophagy in leukemia cells K562. METHODS: K562 cells were induced with different concentrations of mitophagy inducer carbonylcyanide-m-chlorophenylhydrazone (CCCP) for 6, 12 and 24 hours, and the cell viability was detected by the CCK-8 assay. K562 cells were divided into NC, UVRAG-siRNA, UVRAG-siRNA+CCCP, and CCCP group, while Western blot was used to detect the expression of UVRAG protein. Flow cytometry was used to detect the changes in reactive oxygen species (ROS) and mitochondrial structural integrity. The expressions of autophagy related proteins P62 and LC3-Ⅱ/LC3-Ⅰ were detected by Western blot. RESULTS: Compared with NC group, the expression of UVRAG protein in UVRAG -siRNA group significantly decreased (P<0.01). Compared with CCCP group, in UVRAG -siRNA+CCCP group ROS, mitochondrial structure damage, and the expression of LC3-Ⅱ/LC3-Ⅰ decreased significantly (P<0.05, P<0.05, P<0.01), while the expression of P62 protein increased (P<0.05). Compared with NC group, the differences in the expressions of P62 and LC3-Ⅱ/LC3-Ⅰ protein, ROS, and mitochondrial structural integrity in UVRAG -siRNA group were not obvious (P>0.05). CONCLUSION: Under the treatment of CCCP, silencing UVRAG can inhibit mitophagy in K562 cells.

Spatiotemporal analysis of pertussis in Hunan Province, China, 2009-2019.

OBJECTIVES: This study aims to explore the spatial and spatiotemporal distribution of pertussis in Hunan Province, and provide a scientific basis for targeting preventive measures in areas with a high incidence of pertussis. DESIGN: In this retrospective spatial and spatiotemporal (ecological) study, the surveillance and population data of Hunan Province from 2009 to 2019 were analysed. The ArcGIS V.10.3 software was used for spatial autocorrelation analysis and visual display, and SaTScan V.9.6 software was used for statistical analysis of spatiotemporal scan data. SETTINGS: Confirmed and suspected pertussis cases with current addresses in Hunan Province and onset dates between 1 January 2009 and 31 December 2019 were included in the study. PARTICIPANTS: The study used aggregated data, including 6796 confirmed and suspected pertussis cases. RESULTS: The seasonal peak occurred between March and September, and scattered children were at high risk. The global Moran's I was between 0.107 and 0.341 (p<0.05), which indicated that the incidence of pertussis in Hunan had a positive spatial autocorrelation. The results of local indicators of spatial autocorrelation analysis showed that the hot spots were mainly distributed in the northeast region of Hunan Province. Moreover, both purely space and spatiotemporal scans showed that the central and northeastern parts were the most likely cluster areas with an epidemic period between March and October in 2018 and 2019. CONCLUSION: The distribution of the pertussis epidemic in Hunan Province from 2009 to 2019 shows spatiotemporal clustering. The clustering areas of the pertussis epidemic were concentrated in the central and northeastern parts of Hunan Province between March and October 2018 and 2019. In areas with low pertussis incidence, the strengthening of the monitoring system may reduce under-reporting. In areas with high pertussis incidence where we could study whether the genes of endemic pertussis strains are mutated and differ from vaccine strains.

Expression of serum miR-27b and miR-451 in patients with congenital heart disease associated pulmonary artery hypertension and risk factor analysis.

This study investigated expression of serum miR-27b and miR-451 in patients with congenital heart disease associated pulmonary arterial hypertension (CHD-PAH), and analyzed the risk factors of CHD-PAH. A total of 114 patients with CHD admitted to the First Affiliated Hospital of the University of South China were recruited and allocated into a study group (61 patients with PAH) and a control group (53 patients without PAH). Reverse transcription-polymerase chain reaction (RT-PCR) was employed for the qualification of serum miR-27b and miR-451, and an automatic biochemical analyzer was used for the measurement of biochemical indexes in peripheral blood, and enzyme-linked immunosorbent assay (ELISA) for the detection of serum brain natriuretic peptide (BNP) and asymmetric dimethylarginine (ADMA). The patients with CHD-PAH showed higher serum miR-27b, BNP and ADMA but lower miR-451 than the controls. Serum miR-27b was positively correlated with mean pulmonary artery pressure (mPAP), BNP and ADMA, whereas serum miR-451 was negatively correlated with them. The combined detection of miR-27b and miR-451 was more valuable than a single detection in the diagnosis of CHD-PAH. Logistic regression analysis showed that ADMA, miR-27b, miR-451 and ventricular septal defect (VSD) were independent risk factors for CHD-PAH. In conclusion, miR-27b is highly expressed and miR-451 and the expression is low in patients with CHD-PAH. miR-27b and miR-451 are significantly correlated with BNP, ADMA, and the severity of the disease. The combination of miR-27b and miR-451 has high diagnostic value and can be used as a biomarker for the diagnosis and assessment of CHD-PAH. CHD-PAH is common in children with CHD, which poses a serious threat to the life and safety. At present, there are no effective methods for its early diagnosis and treatment. MicroRNAs (miRNAs, miRs) have been found to be closely related to the pathogenesis of CHD-PAH. In this study, miR-27b and miR-451 with differential expression in CHD-PAH were evaluated, and it was found that they were of great significance in the diagnosis and assessment of CHD-PAH.

[Adenanthin Induces Differentiation of Acute Promyelocytic Leukemia Cells by Targeting Peroxiredoxin III].

OBJECTIVE: To investigate the differentiation of acute promyelocytic leukemia (APL) cells induced by adenosine targeting Prx III. METHODS: HL-60 cells were divided into four groups: control group, all-trans retinoic acid (ATRA) group, adenanthin group and ATRA+adenanthin group. Cell morphologic changes were observed under optical microscope. The influence of adenanthin on the differentiation of HL-60 was observed by nitro blue tetrazolium chloride (NBT) test. Cell surface differentiation antigens CD11b expression was measured by flow cytometry. The protein expression of Prx III was detected by immunohistochemical assay. RESULTS: Adenanthin could induce the differentiation of HL-60 cells; the NBT reduction positive rate in ATRA+adenanthin group was significantly higher than that in ATRA group and adenanthin group (P＜0.05). The percentage of CD11b positive cells in ATRA+adenanthin group (43.62％±1.38％) was higher than that in adenanthin group (28.15％±1.78％), ATRA group (36.72％±1.33％) and control group (7.99％±1.78％) (P＜0. 05). The content of Prx Ⅲ protein in adenanthin group was significantly higher than that in control group and ATRA group (P＜0.05). CONCLUSION: Adenanthin and ATRA have a synergistic effect on the differentiation and maturation of HL-60 cells, and its mechanism may be related with regulation of Prx III expression.

[Value of S100A8 in evaluating the prognosis of children with acute lymphoblastic leukemia].

OBJECTIVE: To study the association between S100A8 expression and prognosis in children with acute lymphoblastic leukemia (ALL). METHODS: The clinical data of 377 children with ALL who were treated with the CCLG-2008-ALL regimen were retrospectively reviewed. ELISA and PCR were used to measure serum protein levels and mRNA expression of S100A8. The Kaplan-Meier method was used for survival analysis and a Cox regression analysis was also performed. RESULTS: The children were followed up for 56 months, and the overall survival rate of the 377 children was 89.1%. The prednisone good response group had significantly lower S100A8 protein and mRNA levels than the prednisone poor response group (P<0.01). In the children with standard or median risk, both S100A8 protein and mRNA levels were associated with event-free survival rate (P<0.05). There were significant differences in S100A8 protein and mRNA levels between the children with different risk stratifications (P<0.01). The children who experienced events had significantly higher S100A8 protein and mRNA levels than those who did not (P<0.01). The Kaplan-Meier survival analysis and the Cox regression model suggested that S100A8 overexpression was an independent risk factor for the prognosis of children with ALL. CONCLUSIONS: High S100A8 expression may be associated with the poor prognosis of children with ALL and is promising as a new marker for individualized precise treatment of children with ALL.

[The role of the PI3K/AKT signaling pathway in DADS-induced apoptosis of K562 cells].

OBJECTIVE: To study the role of the PI3K/AKT signaling pathway in the diallyl disulfide (DADS)-induced apoptosis of K562 cells. METHODS: K562 cells in the logarithmic growth phase were treated with 10, 20, 40, or 80 mg/L DADS for 48 hours, then fixed and stained with acridine orange/ethidium bromide (AO/EB), and examined for cellular morphological changes under an inverted microscope. Annexin V-FITC/PI staining was used for determining the apoptotic rates, and Western blot for measuring the expression of AKT, p-AKT, and Caspase-3. Two control groups, blank and solvent, were used as references. RESULTS: K562 cells treated with DADS for 48 hours exhibited the characteristic morphological features of apoptosis including cell shrinkage, irregular cell shape, and membrane blebbing. AO/EB staining results demonstrated that the number of apoptotic cells with cell shrinkage, pyknotic or bead-like nuclei, chromatin condensation, and orange staining increased with the increasing DADS concentration, and 40 mg/L DADS had the most significant effect. The apoptotic rates of cells treated with 10, 20, 40, and 80 mg/L DADS were all significantly higher than those in the control groups (P<0.05). There were no significant differences in AKT protein expression between the K562 cells treated with different concentrations of DADS; the p-AKT protein expression decreased with the increasing DADS concentration, while the Caspases-3 protein expression increased with the increasing DADS concentration (P<0.05). CONCLUSIONS: DADS induces the apoptosis of K562 cells, probably through inhibiting the protein expression in the PI3K/AKT signaling pathway.

[Mechanism of Fas/FasL signal transduction pathway in K562 cell apoptosis induced by diallyl disulfide].

The aim of this study was to investigate the effect of diallyl disulfide (DADS) on the apoptosis of K562 cells and to explore the mechanism of K562 apoptosis induced by DADS. The K562 cells were treated with different concentrations of DADS for 24, 48 and 72 hours. The concentrations of DADS were as follows: 0 (control group), 10, 20, 40 and 80 mg/L. The morphologic changes of leukemia K562 cells treated with DADS were observed by Hoechst33 258 staining. The apoptosis of K562 cells treated with different concentrations of DADS for 24, 48 and 72 hours was analyzed by flow cytometry. The mRNA expression changes of Fas and FasL were detected by reverse transcription-polymerase chain reaction (RT-PCR) after K562 cells were treated with different concentrations of DADS for 48 hours. The results indicated that the characteristics of apoptosis in K562 cells induced by DADS were as follows: reduction of nucleus, chromatin condensation and nuclear membrane rupture. The flow cytometry with PI straining showed that after 24 hours of DADS treatment the apoptosis rate of K562 cells increased from 11.60 ± 0.83% at the concentration of 10 mg/L to 37.94 ± 0.87% at the concentration of 40 mg/L. The apoptosis rate of K562 cells increased from 37.94 ± 0.87% (24 hours) to 47.02 ± 0.66% (72 hours) after treatment with DADS of 10 mg/L increasing to 40 mg/L DADS. The Fas mRNA expression levels of the related apoptotic genes increased after K562 cells were treated with different concentrations of DADS for 48 hours, while FasL mRNA expression decreased significantly after DADS treatment for 48 hours, compared with those in the control group (p < 0.05). It is concluded that DADS can induce the apoptosis of human leukemia K562 cells in a time-and concentration-dependent manners. The activation of Fas/FasL pathway may play an important role in the K562 cell apoptosis induced by DADS, which is associated with increasing Fas gene expression and decreasing FasL gene expression.

A critical role for UVRAG in apoptosis.

Autophagy and apoptosis are tightly regulated biological processes that are crucial for cell growth, development and tissue homeostasis. UVRAG (UV radiation resistance-associated gene), a mammalian homolog of yeast Vps38, activates the Beclin 1/PtdIns3KC3 (class III phosphatidylinositol-3-kinase) complex, which promotes autophagosome formation. Moreover, UVRAG promotes autophagosome maturation by recruiting class C Vps complexes (HOPS complexes) and Rab7 of the late endosome. We found that UVRAG has anti-apoptotic activity during tumor therapy through interactions with Bax. UVRAG inhibits Bax translocation from the cytosol to mitochondria during chemotherapy- or UV irradiation-induced apoptosis of human tumor cells. Moreover, deletion of the UVRAG C2 domain abolishes Bax binding and anti-apoptotic activity. These results suggest that, in addition to its previously recognized pro-autophagy activity in response to starvation, UVRAG has cytoprotective functions in the cytosol that control the localization of Bax in tumor cells exposed to apoptotic stimuli.

UV irradiation resistance-associated gene suppresses apoptosis by interfering with BAX activation.

Ultraviolet irradiation resistance-associated gene (UVRAG) is a well-known regulator of autophagy by promoting autophagosome formation and maturation. However, little is known about the non-autophagic functions of UVRAG. Here, we present evidence that UVRAG functions as an unusual BCL2-associated X protein (Bax) suppressor to regulate apoptosis. Chemotherapy and radiation induces UVRAG expression and subsequently upregulates autophagy and apoptosis in tumour cells. Depletion of UVRAG expression by RNA interference renders tumour cells more sensitive to chemotherapy- and radiation-induced apoptosis in vitro and in vivo. Moreover, UVRAG interacts with Bax, which inhibits apoptotic stimuli-induced mitochondrial translocation of Bax, reduction of mitochondrial membrane potential, cytochrome c release and activation of caspase-9 and -3. Our findings show that UVRAG has an essential role in the intrinsic mitochondrial pathway of apoptosis by regulating the localization of Bax. This pathway represents a target for clinical intervention against tumours.

WAVE1 regulates P-glycoprotein expression via Ezrin in leukemia cells.

For children with acute myeloblastic leukemia (AML), multidrug resistance (MDR) reduces treatment effectiveness, and often leads to poor patient survival. While a number of factors have been described that affect MDR, the mechanisms underlying this effect remain unclear. In this study, the role of WAVE1 in MDR was investigated. Among 62 children with AML, high levels of WAVE1 were associated with poor patient outcomes. Proteomic techniques were used to identify novel WAVE1-interacting proteins from leukemia cells, one of which was the cytoskeleton regulator Ezrin. In leukemia cells, WAVE1 co-localized with both Ezrin and P-glycoprotein (P-gp), a critical regulator of the MDR phenotype. Overexpression of WAVE1 in K562 leukemia cells up-regulated P-gp and Ezrin, and reduced K562 cells' sensitivity to the chemotherapy drug adriamycin. The opposite effect was seen when WAVE1 expression was reduced via RNA interference. Critically, overexpression of WAVE1 in the absence of Ezrin did not affect P-gp levels or MDR. These data suggest that WAVE1 affects P-gp and MDR of leukemia cells through Ezrin.

[Effects of diallyl disulfide on apoptosis of human leukemia K562 cells and expression of Fas, FasL and caspase-8].

OBJECTIVE: To study the effects of diallyl disulfide (DADS) on apoptosis of human leukemia K562 cells and possible mechanisms. METHODS: The morphologic changes of leukemia K562 cells after DADS treatment were observed by Hoechst 33258 staining. Cell apoptosis rates after different concentrations and different durations of DADS treatment were determined by flow cytometry. Fas, FasL and caspase-8 mRNA expression was estimated by reverse transcription-polymerase chain reaction (RT-PCR) 48 hrs after DADS treatment. RESULTS: The characteristics of apoptosis in K562 cells induced by DADS were observed. After 24 hrs of DADS treatment, the apoptosis rate of K562 cells increased from (11.60 ± 0.83)% at the concentration of 10 mg/L to (37.94 ± 0.87)% at the concentration of 40 mg/L. The apoptosis rate of K562 cells increased after 40 mg/L DADS with the increasing time from (37.94 ± 0.87)% (24 hrs) to (47.02 ± 0.66)% (72 hrs). Expression of Fas and caspase-8 mRNA increased, while FasL mRNA expression decreased significantly 48 hrs after DADS treatment compared with the control group (P<0.05). CONCLUSIONS: DADS can induce apoptosis of human leukemia K562 cells in a time- and concentration-dependent manner, possibly through increasing Fas and caspase-8 expression and decreasing FasL expression.

DAMP-mediated autophagy contributes to drug resistance.

Damage-associated molecular pattern molecules (DAMPs) are cellularly derived molecules that can initiate and perpetuate immune responses following trauma, ischemia and other types of tissue damage in the absence of pathogenic infection. High mobility group box 1 (HMGB1) is a prototypical DAMP and is associated with the hallmarks of cancer. Recently we found that HMGB1 release after chemotherapy treatment is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia. Overexpression of HMGB1 by gene transfection rendered leukemia cells resistant to cell death; whereas depletion or inhibition of HMGB1 and autophagy by RNA interference or pharmacological inhibitors increased the sensitivity of leukemia cells to chemotherapeutic drugs. HMGB1 release sustains autophagy as assessed by microtubule-associated protein 1 light chain 3 (LC3) lipidation, redistribution of LC3 into cytoplasmic puncta, degradation of p62 and accumulation of autophagosomes and autolysosomes. Moreover, these data suggest a role for HMGB1 in the regulation of autophagy through the PI3KC3-MEKERK: pathway, supporting the notion that HMGB1-induced autophagy promotes tumor resistance to chemotherapy.

Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARα oncoprotein.

PML-RARα oncoprotein is a fusion protein of promyelocytic leukemia (PML) and the retinoic acid receptor-α (RARα) and causes acute promyelocytic leukemias (APL). A hallmark of all-trans retinoic acid (ATRA) responses in APL is PML-RARα degradation which promotes cell differentiation. Here, we demonstrated that autophagy is a crucial regulator of PML-RARα degradation. Inhibition of autophagy by short hairpin (sh) RNA that target essential autophagy genes such as Atg1, Atg5 and PI3KC3 and by autophagy inhibitors (e.g. 3-methyladenine), blocked PML-RARα degradation and subsequently granulocytic differentiation of human myeloid leukemic cells. In contrast, rapamycin, the mTOR kinase inhibitor, enhanced autophagy and promoted ATRA-induced PML-RARα degradation and myeloid cell differentiation. Moreover, PML-RARα co-immunoprecipitated with ubiquitin-binding adaptor protein p62/SQSTM1, which is degraded through autophagy. Furthermore, knockdown of p62/SQSTM1 inhibited ATRA-induced PML-RARα degradation and myeloid cell differentiation. The identification of PML-RARα as a target of autophagy provides new insight into the mechanism of action of ATRA and its specificity for APL.

[Effects of CXCR4 silence induced by RNA interference on cell cycle distribution and apoptosis of Jurkat cells].

This study was aimed to investigate the effect of down-regulating the CXCR4 expression on cell cycle and cell apoptosis of human T-ALL Jurkat cells. The CXCR4 specific siRNA plasmid vector was constructed and then transfected into the cultured Jurkat cell line by DMRIE-C. The expression of CXCR4 mRNA was detected by RT-PCR, the cell distribution in cell cycle and cell apoptosis were determined by flow cytometry. The experiments were divided into 3 groups: group A (blank control), group B (non-silencing dsRNA as negative control) and group C (CXCR4 siRNA). The results showed that the expression level of CXCR4 mRNA in Jurkat cells transfected with CXCR4 siRNA (group C) decreased and cell proportion in G(0)/G(1) phase increased as compared with group A (56.9% +/- 1.4% vs 68.3% +/- 2.4% and 35.8% +/- 1.9% vs 18.1% +/- 1.2% respectively) (p < 0.01), cell proportion in G(2)/M and S phase decreased as compared with group A (19.8% +/- 1.7%, 44.4% +/- 2.1% vs 27.2% +/- 1.5%, 54.7% +/- 2.8% respectively) (p < 0.01). The apoptosis rate of Jurkat cells in group C increased as compared with group A (20.9% +/- 2.0% vs 3.13% +/- 0.9% respectively) (p < 0.01), and the comparison between group A and B showed no statistical difference. It is concluded that the CXCR4 specific siRNA can effectively down-regulate the CXCR4 mRNA expression, which induces the cell apoptosis and cell cycle arrest, thereby inhibits the Jurkat cell proliferation.

[Stem cell factor secretion by bone mesenchymal stem cells stimulated with astragaloside IV].

OBJECTIVE: To study the effect of astragaloside IV on the expression of cytokines in bone mesenchymal stem cells (MSCs) in rats. METHODS: MSCs were isolated from Wistar rats by the method of adhesive cultiration and clone, and then their biological activities were assessed using indirect immunofluorescence. Proliferation of MSCs stimulated with astragaloside IV was ascertained by the MTT method. Expression of cytokines was ascertained using RT-PCR in MSCs with astragaloside IV stimulation or not. RESULTS: MSCs were effectively isolated and purified in vitro, and had expression of many cytokines except IL-3, such as stem cell factor (SCF), thrombopoietin (TPO), granulocyte macrophage colony stimulating factor (GM-CSF) and transforming growth factor (TGF-beta1). Astragaloside IV stimulation promoted MSCs proliferation, and 200 mg/mL astragaloside IV treatment produced a peak effect 72 hrs after culture. The SCF expression in MSCs stimulated with astragaloside IV increased significantly compared with that in MSCs without astragaloside IV stimulation. CONCLUSIONS: Astragaloside IV may promote MSCs proliferation and increase SCF secretion in vitro.