[Effect of MiR-424-5p on the Drug Resistance of Diffuse Large B-Cell Lymphoma Cells by Regulating PD-1/PD-L1 Signaling Pathway].

OBJECTIVE: To explore the effect of microRNA-424-5p (miR-424-5p) on the drug resistance of diffuse large B-cell lymphoma (DLBCL) cells by regulating the programmed death receptor-1 (PD-1)/programmed death ligand-1 (PD-L1) signaling pathway. METHODS: Human DLBCL cell line CRL2631 cells were induced to construct CRL2631-CHOP resistant cell line. RT-qPCR and Western blot were used to detect the expression levels of MiR-424-5p, PD-L1 mRNA and protein, and multidrug resistance gene-1 (MDR-1) protein in CRL2631 cells and CRL2631-CHOP cells, respectively. The target genes of MiR-424-5p was verified by dual luciferase reporter assay. The miRNA simulation/interference technology and thiazole blue (MTT) method were used to detect the resistance of CRL2631 cells and CRL2631-CHOP cells to CHOP. RESULTS: Compared with CRL2631 cells, the drug resistance of CRL2631-CHOP cells to CHOP and the levels of MDR-1 protein (P<0.05), PD-L1 mRNA and protein in the cells were significantly increased (both P<0.001), while the relative level of MiR-424-5p was significantly reduced (P<0.001). The result of the dual luciferase reporter assay showed that PD-L1 was the direct downstream target gene of MiR-424-5p (P<0.001). After transfection of MiR-424-5p inhibitor, the resistance of CRL2631 cells to CHOP drugs increased, and the expression level of MDR-1 protein (P<0.01), PD-L1 mRNA and protein also increased significantly (both P<0.01). After transfection of MiR-424-5p mimics, the resistance of CRL2631-CHOP cells to CHOP drugs decreased, and the expression level of MDR-1 protein (P<0.001), PD-L1 mRNA and protein also decreased significantly (both P<0.001). Overexpression of PD-L1 could reverse the inhibitory effect of upregulating MiR-424-5p on PD-L1 (P<0.001). CONCLUSION: Down-regulation of MiR-424-5p enhances the drug resistance of DLBCL cells by regulating the PD-1/PD-L1 signaling pathway.

[Gene Mutation and Clinical Characteristics of Patients with Acute Leukemia].

OBJECTIVE: To investigate the characteristics of gene mutation, clinical characteristics and significance in acute leukemia (AL) patients. METHODS: The clinical data of 102 AL patients in Hebei General Hospital from September 2016 to September 2020 were collected and analyzed retrospectively, including the characteristics of gene mutation, age, peripheral blood cells, bone marrow blasts, leukemia subtypes and myeloperoxidase (MPO). RESULTS: The total gene mutation rate was 87.25% (89/102) in all 102 patients. A total of 275 gene mutations were detected, with an average of 2.70 gene mutations per patient. The most frequent mutations of 102 patients were as follows: CEBPA (6.91%), NPM1 and ASXL1(6.18%), TET2 (5.82%), DNMT3A (5.45%), IDH2 and FLT3-ITD (5.09%). Gene mutations often occurred simultaneously. CEBPA mutation occurred in 10 cases of M2 subtype, while TET2 mutation occurred in 9 cases of M2 subtype. Among the most common gene mutations in MPO low expression group, mutation rates of NPM1, DNMT3A, IDH2, SF related gene mutation and RUNX1 were significantly different than those in MPO high expression group (all P<0.05). Univariate analysis showed that age, NPM1, DNMT3A and FLT3-ITD had significant effects on leukocyte level. Logistic regression analysis showed that patients with positive NPM1 mutations may had higher leukocyte levels (p=0.038), and those with positive DNMT3A mutations may had higher platelet levels (p=0.042). CONCLUSION: The incidence of gene mutation in patients with AL is high, and it often occurs simultaneously. CEBPA and TET2 gene mutations are more common in M2 subtype. In patients with MPO low expression, the most common gene mutations are NPM1, DNMT3A and IDH2. AL patients with NPM1 gene mutation had higher white blood cell levels, while with DNMT3A gene mutation had higher platelet levels.

[Visceral Vein Thrombosis of Myeloproliferative Neoplasm --Review].

Classical myeloproliferative neoplasm (MPN) related thrombosis mainly affects elderly patients and often involves arterial circulation, while, MPN-visceral venous thrombosis (SVT) mainly affects young women, and is closely associated with JAK2V617F mutation but not closely with CALR mutation. The pathogenesis of MPN-SVT is not only related to JAK2V617F mutation and vascular endothelial damage, but also needs further research to determine the machanism. JAK2V617F mutation is the most common in MPN-SVT clinically. Patients with non-cirrhotic SVT need to detect MPN mutation, while the detection of CALR or MPL mutation needs to be combined with clinical judgment. At present, the main treatment strategies of MPN-SVT are JAK inhibitors, supplementation of anticoagulants and treatment of portal hypertension. This article reviews the latest research progress on the epidemiology, pathogenesis, diagnosis and treatment strategies of MPN-SVT.

Pulmonary tuberculosis presenting as henoch-schönlein purpura: Case report and literature review.

INTRODUCTION: Henoch-Schönlein purpura (HSP) is an extremely rare condition in patients with pulmonary tuberculosis, with only a few reported cases. Compared to patients with typical clinical symptoms, it is difficult to make a definitive diagnosis when HSP presents as an initial manifestation in pulmonary tuberculosis patients. Herein, a case of pulmonary tuberculosis that showed HSP at first was reported, and the related literatures were reviewed. PATIENT CONCERNS: A 24-year-old man presented with palpable purpura on the extremities, accompanied by abdominal pain, bloody stools, and knee pain. DIAGNOSES: The patient was diagnosed with pulmonary tuberculosis based on the results of interferon gamma release assays, purified protein derivative test, and computed tomography. INTERVENTIONS: The patient was treated with vitamin C and chlorpheniramine for 2 weeks, and the above-mentioned symptoms were relieved. However, 3 weeks later, the purpura recurred with high-grade fever and chest pain during the inspiratory phase. The patient was then treated with anti-tuberculosis drugs, and the purpura as well as the high fever disappeared. OUTCOMES: The patient recovered well and remained free of symptoms during the follow-up examination. CONCLUSION: Pulmonary tuberculosis presenting with HSP as an initial manifestation is not common. Therefore, it is difficult to clinically diagnose and treat this disease. When an adult patient shows HSP, it is important to consider the possibility of tuberculosis to avoid misdiagnosis and delayed treatment.

[Effect of Arsenic Trioxide on K562 Cell Proliferation and Its Mechanism].

OBJECTIVE: To investigate the molecular mechanism of arsenic trioxide(ATO) inhibiting K562 cell proliferation, and explore the new targets for treating chronic myeloid leukemia(CML). METHODS: human CML cell line K562 cells were cultured in vitro, and were treated with different concentrations of ATO; MTT was used to detect the cell proliferation; flow cytometry(FCM) was used to determine cell apoptosis, cell cycle and the expression of CD44; Transcriptional levels of ß-catenin and cyclin D1 were assayed by RT-PCR. RESULTS: 2 µmol/L ATO could inhibit the cell proliferation obviously in a time-and-dose-dependent manner. With drug concentration increasing and time prolonging, the expression rate of CD44 was declined gradrually. FCM with AnnexinV/PI double staining showed that K562 cells were induced to apoptosis after exposure to 2.5-10 µmol/L ATO for 48 hours and in dose-dependent manner. Treating with different concentration ATO for 48 hours, cell ratio of G0/G1 phase increased and cell ratio in S phase decreased gradually. RT-PCR showed that the expression of ß-catenin and CyclinD1 decreased with increasing of drug concentration. CONCLUSION: ATO in certain concentration range can inhibit K562 cell proliferation, and induce the cell apotosis, the mechanismin influencing the Wnt/ß-catenin pathway may be the downregulation of CD44 expression, arresting K562 cells in G0/G1 phase, and affecting the gene transcription, thus inhibiting K562 cell proliferation.

[Effect of Anti-CD44 Monoclonal Antibody A3D8 on Expression of AP-1 in HL-60 Cells].

OBJECTIVE: To explore the effect of anti-CD44 monoclonal antibody A3D8 on expression of transcription factor AP-1 in acute myeloid leukemia cells. METHODS: After acute leukemia cell line HL-60 was treated by different concentrations of A3D8, the proliferation and cell cycle were detected by MTT and FCM respectively. The expressions of c-JUN and c-FOS at mRNA and protein level were detected by RT-PCR and Western Blot respectively. RESULTS: The proliferation of HL-60 was inhibited by A3D8. The A3D8 treatment increased the percentage of G0/G1 cells. The expressions of c-JUN at mRNA and protein level were both decreased in HL-60 cells treated with A3D8. The expressions of c-FOS at mRNA and protein level in rapamycin treatment groups showed no statistically significant difference as compared with that in control group. CONCLUSIONS: A3D8 can affect the activity of AP-1 through inhibiting the expressions of c-JUN at mRNA and protein level.

[Research Progress on CALR Mutation in the Myeloproliterative Neoplasm -Review].

There is no gold diagnostic standard for BCR-ABL fusion gene negative chronic myeloproliterative neoplasm(cMPN). The following detection methods such as comprehensive bone marrow cell morphology, bone marrow pathology, genetic mutation, flow cytometry and immunohistochemical are needed to diagnose the BCR-ABL fusion gene positive cMPN. The JAK2 mutation can be used as a specific diagnostic criteria for polycythemia vera (PV), but there is no specific and sensitive indication for the JAK2 mutation-negative MPN. CALR mutation would be an indication in a certain extent. In this review, the CALR mutation detection, detection mean and its correlation with disease diagnosis and prognosis etc were summarized.

Inhibition of multiple myeloma cell proliferation by ginsenoside Rg3 via reduction in the secretion of IGF-1.

Ginsenoside Rg3 (Rg3) is one of the primary constituents isolated from ginseng, and has been found to exhibit cytotoxic effects against cancer cells. The present study aimed to investigate the effects of Rg3 on human multiple myeloma cell proliferation and apoptosis, and to examine its underlying molecular mechanisms. Cell viability was detected using a Cell Counting kit­8 assay, and cell cycle arrest and cell apoptosis were analyzed using flow cytometry. In addition, the expression levels of cell cycle­associated markers and apoptosis­associated proteins, and the release of cytochrome C were determined using western blot analysis. The effects of Rg3 on the insulin­like growth factor (IGF)-1/AKT/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase signaling pathways were also investigated using western blot analysis. The results showed that Rg3 inhibited cell viability in U266, RPMI8226 and SKO­007 cells in a time­ and dose­dependent manner, and caused cell cycle arrest in the G1 phase by regulating the cyclin­dependent kinase pathway. Furthermore, Rg3 induced multiple myeloma cell apoptosis, and was involved in B cell lymphoma-2 (Bcl2)/Bcl2-associated X protein imbalance, caspase activation and the release of cytochrome C from the mitochondria into the cytoplasm. Mechanistically, it was found that the inhibitory effects of Rg3 on multiple myeloma cell proliferation were essential for secretion of IGF­1 and inactivation of the Akt/mTOR pathway. Collectively, these findings demonstrated that Rg3 effectively inhibited cell proliferation and induced apoptosis of multiple myeloma cells. These data broaden the clinical investigation of Rg3 in the treatment of multiple myeloma, associated with the inactivation of IGF-1/AKT/mTOR signaling.

[Effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of HL-60 cells and its mechanism].

This study was aimed to investigate the effect of COX-2 inhibitor celecoxib on proliferation, apoptosis of human acute myeloid leukemia cell line HL-60 and its mechanism. HL-60 cells were cultured with different concentrations of celecoxib for 24 h. Cell proliferation was analyzed by CCK-8 assay, cell apoptosis and cell cycle distribution were detected by flow cytometry. Cyclin D1, cyclin E1 and COX-2 mRNA expressions were determined by RT-PCR. The results showed that after the HL-60 cells were treated with different concentrations of celecoxib for 24 h, the cell growth was significantly inhibited in a dose-dependent manner(r = 0.955), IC50 was 63.037 µmol/L of celecoxib. Celecoxib could effectively induce apoptosis in HL-60 cells also in dose-dependent manner(r = 0.988), blocked the HL-60 cells in the G0/G1 phase. The expression of cyclin D1, cyclin E1 and COX-2 mRNA were downregulated. It is concluded that celecoxib can inhibit the proliferation of HL-60 cells in dose-dependent manner, celecoxib causes cell G0/G1 arrest and induces cell apoptosis possibly through down-regulation of the cyclin D1 and cyclin E1 expression, and down-regulation of COX-2 expression respectively.

[IL-23 alone or with IL-2 induces the killing effect of hPBMNC on K562 cells].

This study was aimed to explore the killing effect of PBMNC induced by IL-23 alone or combined with IL-2 on K562 cells and its mechanism. The PBMNC were induced in vitro by IL-23 (50 ng/ml) alone or IL-23 combined with IL-2 (100 U/ml) for 72 h, and then were co-cultured with leukemia cell line K562. The CCK-8 method was used to detect the effect of PBMNC induced at different times on K562 cells, the ELISA was performed for detecting IFN-γ level in culture supernatant, and the perforin and granzymes B were detected by RQ-PCR. The results showed that the killing effect of PBMNC induced by IL-23 alone or IL-23 combined with IL-2 on K562 cells was observed, and obviously enhanced with prolonging of time, moreover, there was statistical difference among different time points (P < 0.05). The IFN-γ level in supernatant of PBMNC cultured with cytokines significantly increased, and the IFN-γ levels in group of IL-23 combined with IL-2 were higher than that in other groups (P < 0.05). The mRNA expressions level of perforin and granzymes B of the expanded PBMNC in groups cultured with cytokines were higher than that in control group (P < 0.05), and the mRNA expressions of perforin and granzymes B in group of IL-23 combined with IL-2 were significantly higher than that in others (P < 0.05). It is concluded that IL-23 can promote the killing effect of PBMNC on K562 cells. The combination of IL-2 with IL-23 displays synergic effect and a time-dependent manner. IL-23 also enhances the expression of IFN-γ, perforin and granzyme B in PBMNC. Its combination with IL-2 displays synergistic effect, suggesting that the anti-leukemic activity of IL-23 may be realized through inducing PBMNC to express IFN-γ, perforin and granzyme B.

Expression levels of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and focal adhesion kinase in patients with multiple myeloma and their relationship to clinical stage and extramedullary infiltration.

We explored the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and focal adhesion kinase (FAK) mRNA and protein, and analyzed the relationship between expression levels and clinical staging and extramedullary infiltration in patients with multiple myeloma (MM). The expression levels of mRNA and protein were measured by fluorescence quantitative polymerase chain reaction (FQ-PCR) and Western blotting. Expressions of PTEN and FAK mRNA were significantly different between patients with MM and controls. Spearman bivariate correlation analysis showed that PTEN mRNA was significantly negatively correlated with FAK mRNA. PTEN and FAK mRNA expressions were significantly different between patients with stage I + II MM and stage III MM. No difference was found in PTEN mRNA expression, whereas FAK mRNA expression was significantly different between patients with MM with and without extramedullary infiltration. PTEN protein was higher and total FAK (T-FAK) protein was significantly lower in six controls than in 12 patients with stage III MM. Phosphorylated FAK (p-FAK) protein was measured as 0.082 ± 0.040 in 11 patients with MM, but not detected in six controls. No significant difference of PTEN and T-FAK protein was found, while p-FAK protein was significantly different between patients with MM with and without extramedullary infiltration. These results indicate that abnormal expression of PTEN and FAK in patients with MM may be associated with disease progression and extramedullary infiltration.

mitochondrial ceramidase overexpression up-regulates Bcl-2 protein level in K562 cells, probably through its metabolite sphingosine-1-phosphate.

Recently, a mitochondrial ceramidase has been identified and cloned, whose mitochondrial localization strongly suggests the existence of an unexpected mitochondrial pathway of ceramide metabolism that may play a key role in mitochondrial functions, especially in the regulation of apoptosis. To explore the biological effect of mitochondrial ceramidase on cells, pcDNA 3.1/His-CDase plasmid, containing mitochondrial ceramidase cDNA sequence, was transducted into K562 cells mediated by liposome, and G418 was used to screen for positive colonies. A stable transfected K562 cell line was established and named as 'K562TC'. The difference between K562 and K562TC cells in chemotheraputic cytotoxicity response and serum-withdrawal resistance and Bcl-2 protein expression were evaluated by MTT assay, annexin V/PI test, flow cytometry or Western blotting, respectively. The results showed that although survival was comparable between K562 and K562TC cells after exposed to adriamycin, etoposide or arsenious acid, K562TC cells with elevated Bcl-2 protein expression level as identified by FCM or Western blotting revealed stronger resistance to apoptosis induced by serum withdrawal than their parental cells. Inhibition of mitochondrial ceramidase expression in K562TC cells by its specific antisense oligodeoxynucleotide was correlated with a decrease in Bcl-2 protein level. N, N-dimethylsphingosine, a sphingosine kinase inhibitor, depleted intracellular sphingosine-1-phosphate production, also abrogated Bcl-2 protein expression in K562TC cells, while Bcl-2 protein level in K562 cells was up-regulated by exogenous sphingosine-1-phosphate. It is concluded that mitochondrial ceramidase overexpression in K562 cells leads to markedly elevated level of Bcl-2 protein and results in more resistance to serum withdrawal. This effect is initiated not by sphingosine, the direct metabolite of mitochondrial ceramidase, but via sphingosine-1-phosphate, its phosphorylated form. This is the first evidence that mitochondrial ceramidase, through its sphingoid metabolite sphingosine-1-phosphate, up-regulates Bcl-2 protein expression in K562 cells.

[Mutation analysis of SHIP gene in acute leukemia].

OBJECTIVE: The SH2 domain containing inositol 5'-phosphatase (SHIP) is predominately expressed in hematopoietic cells, and is a crucial negative regulator in the development of hematopoietic cells. This paper is to evaluate the role of the SHIP gene in human leukemogenesis. METHODS: Expression of SHIP gene in bone marrow and/or peripheral blood from 32 patients with acute myeloid leukemia (AML), 9 with acute lymphoblastic leukemia (ALL), as well as human hematopoietic cell lines was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and DNA sequencing. RESULTS: RT-PCR showed that all samples expressed SHIP gene. Mutations of SHIP gene were detected in 7 (22%) of 32 AML patients and one (12%) of 9 ALL patients. Interestingly, two missense mutations that had been observed in a AML patient at diagnosis disappeared after complete remission (CR). In addition, in vitro Akt phosphorylation was prolonged and increased following IL-3 stimulation of this patient's cells. CONCLUSION: Our data demonstrate for the first time the mutation of SHIP gene in acute leukemia and suggest a possible role of the mutation of this gene in the development of acute leukemia. SHIP may serve as a tumor suppressor by negatively regulating the PI3K/Akt signaling pathway in hematopoietic cells.

Mutation analysis of SHIP gene in acute leukemia.

The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145 kD protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. SHIP is predominately expressed in hematopoietic cells, and is a crucial negative regulator in the development of hematopoietic cells. To evaluate the role of the SHIP gene in human leukemogenesis, expression and mutation of SHIP gene in bone marrow and/or peripheral blood from 32 patients with acute myeloid leukemia (AML), 9 patients with acute lymphoblastic leukemia (ALL), as well as human hematopoietic cell lines were analyzed by reverse transcription-polymerase chain reaction (RT-PCR), single strand conformational polymorphism (SSCP) and sequencing. The RT-PCR showed that all samples expressed SHIP gene. Mutations of SHIP gene were detected in 7 out of 32 AML patients (22%) and one out of 9 ALL patients (12%). Interestingly, two missense mutations that had been observed in one AML patient at diagnosis disappeared after complete remission (CR). In addition, Akt phosphorylation was prolonged and increased following IL-3 stimulation in this patient sample. In conclusion, data of this study demonstrate the mutation of the SHIP gene in acute leukemia for the first time and suggest a possible role of the mutation of this gene in the development of acute leukemia. SHIP serves as a tumor suppressor by negatively regulating the PI3K/Akt signaling pathway in hematopoietic cells.

Mutation analysis of hematopoietic cell phosphatase gene in acute leukemia.

The hematopoietic cell phosphatase (HCP or SHP-1), the SH2 domain contain protein tyrosine phosphatase, is a crucial negative regulator in the process of hematopoietic cell development, proliferation and receptor-mediated mitogenic signaling pathways, and its mutation is responsible for the over-expansion and inappropriate activation of myelomonocytic population in motheaten mice. The aim of the study was to evaluate the role of the HCP gene in leukemogenesis. Bone marrow and/or peripheral blood from 32 acute myeloid leukemia (AML) patients, 9 acute lymphocytic leukemia (ALL) patients, 8 leukemia cell lines and 50 normal controls were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) based on single strand conformation polymorphism (SSCP) and sequencing. RT-PCR showed that all samples expressed HCP gene, only one missense mutation at codon 225 (AAC to AGC, Asn to Ser) within N-terminal SH2 domain was found in an ALL patient. In addition, four polymorphic base substitutions were detected in codon 69, 85, 86 and 266, respectively. In conclusion, mutation of HCP gene is an infrequent genetic aberration which may only play a role in pathogenesis of a small part of leukemia, however, its significance needs to be further clarified.

[Effect of panax notoginseng saponin on procoagulant activity and differentiation induction in NB4 cells].

OBJECTIVE: To investigate the effect of Panax notoginseng saponin (PNS) on procoagulant activity (PCA) and differentiation induction in NB4 cells. METHODS: After NB4 cells were treated with PNS, the recalcification time, PCA and TF-mRNA expression in NB4 cells were tested by RT-PCR. The inhibitory effect of PNS on NB4 cell proliferation was analysed by MTT method, NBT assay, cell morphological observation and flow cytometry. RESULTS: (1) PNS of all concentrations could significantly prolong the recalcification time and lower the PCA level in NB4 cells in time-concentration-dependent manner. Simultaneously it down-regulated the expression of TF-mRNA. (2) PNS could partially inhibit the NB4 cell proliferation. (3) PNS could raise the NBT reducing capability of NB4 cells (P < 0.05). And morphological examination showed the differentiating tendency of monocyte and macrophage. CONCLUSION: PNS could reduce the procoagulant activity and TF-mRNA expression in NB4 cells, and partially induce the differentiation of NB4 cells, therefore, it is hopeful to be a new anti-coagulant agent.

Hyperhomocysteinemia and the MTHFR C677T mutation in Budd-Chiari syndrome.

Hyperhomocysteinemia (HH) is a factor that predisposes individuals to thrombosis, and the C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) is known to give increased plasma homocysteine. However, little is known about their roles in Budd-Chiari syndrome (BCS). This study evaluated the roles of HH and the MTHFR C677T mutation in patients with BCS. We compared 41 BCS patients with 80 sex- and age-matched healthy controls. The mean plasma homocysteine level was significantly higher in patients with BCS (20.15 +/- 5.78 micromol/L) compared with normal controls (15.80 +/- 6.58 micromol/L), P < 0.01. HH (>19.5 micromol/L in men and >15.0 micromol/L in women) was detected in 15 (36.59%) patients and in 14 (17.5%) controls (odds ratio [OR], 2.72; 95% confidence internal [CI], 1.17-6.32). The prevalence of the mutated MTHFR 677TT genotype and the 677T allele in normal controls was 10.0% and 31.3%, respectively. The mutant 677T homozygotes and alleles were more frequent in patients with BCS than in controls (22.0% vs. 10.0%, 0.025 < P < 0.05; 45.1% vs. 31.3%, 0.025 < P < 0.05). The relative risk of BCS among the carriers of 677TT was significantly increased (OR, 3.3; 95% CI, 1.1-10.0). The mutant MTHFR heterozygous 677C/T carriers were not significantly increased in patients with BCS compared with controls (46.3% vs. < 2.5%, P > 0.05). The relative risk OR of BCS among carriers of 677C/T was 1.6 (95% CI, 0.7-3.6). This study suggests that both HH and the homozygous C677T mutation in the MTHFR gene are important risk factors of BCS.