[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.

A rare case of pancytopenia causing- Sheehan's syndrome: Case report and literature review.

We describe the case of a 58-year-old woman who was presented with pancytopenia and hypofibrinogenemia. Treatment with iron supplementation was not satisfactory. Physical findings and a history of a massive postpartum hemorrhage suggested Sheehan's syndrome(SS). After thyroxine and glucocorticoid replacement therapy, the blood cell count improved. SS is a rare etiology of hemocytopenia, of which hematologists need to be aware. We conclude that hormonal therapy can produce full hematological recovery.

[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.

LncRNA NEAT1 regulate diffuse large B-cell lymphoma by targeting miR-495-3p/PD-L1 axis.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) is a common lymphatic tumor in clinic. LncRNAs were reported to play a regulatory role in many cancers, including DLBCL. This study focused on the roles of NEAT1 in DLBCL. METHODS: Real-time quantitative polymerase chain reaction (RT-qPCR) was carried out to detect mRNA expression. StarBase as well as TargetScan was used to predict targeting relationships, which was confirmed by the Dual Luciferase Reporter Assay and RNA pull-down assay. Cell Counting Kit 8 (CCK-8) were applied to measure cell viability. Flow cytometry assay was applied to detect cell apoptosis. Western blotting assay was conduct to determine protein expression. Lactate dehydrogenase (LDH) release assay were applied to evaluated cell cytotoxicity. RESULTS: NEAT1 was overexpressed in DLBCL patients. Knockdown of NEAT1 reduced the viability while enhanced the apoptosis of tumor cells. However, overexpression of NEAT1 exhibited an opposite effect. miR-495-3p was a target of NEAT1 and was decreased in DLBCL cells. However, inhibiting miR-495-3p reversed the effect of NEAT1 knock-down on DLBCL cells and induced the malignant behaviors of DLBCL cells. Moreover, NEAT1 functioned as a sponge of miR-495-3p to upregulate PD-L1. CONCLUSION: Our study demonstrated that a NEAT1/miR-495-3p/PD-L1 axis regulated the development of DLBCL. Therefore, NEAT1 may be a potential biomarker for DLBCL.

Simultaneous Follicular Lymphoma and Myelofibrosis: Report of a Case with Review of the Literature.

BACKGROUND: Myelofibrosis (MF) is often associated with chronic myeloid leukemia, myelodysplastic syndrome and primary myeloproliferative neoplasms (MPN), but few number cases of malignant lymphoma with myelofibrosis was reported, and a few cases about follicular lymphoma with MF were found. Here we described a case of follicular lymphoma (FL) complicated by myelofibrosis. CASE PRESENTATION: A 43-year-old man was diagnosed with follicular lymphoma (FL) complicated by MF, besides, the lymphoma staging of this patient was AnnArbor IV B. The cytokines of plated-derived growth factor (PDGF), basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), interleukin-2 (IL-2), and transforming growth factor-ß (TGF-ß) were positive, while JAK2V617F, MPL, and CALR mutations were negative. After first course of chemotherapy, the peripheral blood and MF improved. The systemic superficial lymph nodes and spleen were significantly narrowed after the third cycle of chemotherapy. CONCLUSION: The production of various cytokines, such as b-FGF, TNF-α, TGF-ß, PDGF, IL-1ß, IL-2, IL-6, IL-10, may contribute to the development of MF.

Dual PI3K/mTOR inhibitor NVP­BEZ235 decreases the proliferation of doxorubicin­resistant K562 cells.

Acute myelogenous leukemia (AML) is frequently accompanied by a poor prognosis. The majority of patients with AML will experience recurrence due to multiple drug resistance. Our previous study reported that targeting the mTOR pathway may increase cell sensitivity to doxorubicin (Doxo) and provide an improved therapeutic approach to leukemia. However, the effect and mechanism of action of NVP­BEZ235 (BEZ235), a dual inhibitor of PI3K/mTOR, on Doxo­resistant K562 cells (K562/A) is yet to be elucidated. Therefore, the aim of the present study was to investigate the effects of BEZ235 on K562/A cell proliferation. K562/A cells was investigated using CCK­8, flow cytometry and western blotting, following BEZ235 treatment. It was observed that BEZ235 significantly decreased the viability of K562/A cells. In addition, BEZ235 arrested K562/A cells at the G0/G1 phase, and reduced the protein expression levels of CDK4, CDK6 and cyclin D1. Apoptotic cells were more frequently detected in K562/A cells treated with BEZ235 compared with the control group (12.97±0.91% vs. 7.37±0.42%, respectively; P<0.05). Cells treated with BEZ235 exhibited downregulation of Bcl­2 and upregulation of Bax. Furthermore, BEZ235 treatment markedly decreased the activation of the PI3K/AKT/mTOR pathway and its downstream effectors. Thus, these results demonstrated that BEZ235 inhibited cell viability, induced G0/G1 arrest and increased apoptosis in K562/A cells, suggesting that BEZ235 may reverse Doxo resistance in leukemia cells. Therefore, targeting the PI3K/mTOR pathway may be of value as a novel therapeutic approach to leukemia.

[Expression of Hsa-miR-9 in Children with Acute Lymphoblastic Leukemia and Its Relationship with CDX2 Gene].

OBJECTIVE: To investigate the expression of Hsa-miR-9 in children with acute lymphoblastic leukemia (ALL) and its relationship with CDX2 gene. METHODS: The clinical data of 130 patients with ALL were collected from nearly five years in our hospital, and in the same period 60 healthy children in the same age were selected as control group. Real-time fluorescent quantitative PCR (qRT-PCR) was applied to examine the expression of Hsa-miR-9 and CDX2 gene of the two groups, the relationship between Hsa-miR-9 expression and ALL patients' clinical characteristics, survival time, CDX2 expression were analyzed. RESULTS: The expression level of Hsa-miR-9 in ALL children was significantly lower than that in the healthy control group (P<0.001). The expression of Hsa-miR-9 in ALL children related with serum WBC, infiltrating lymph nodes, splenomegaly, and risk grade (P<0.05). The median survival time of ALL children with high expression of Hsa-miR-9 was significantly longer than that of the ALL children with low expression (P<0.001). Hsa-miR-9 expression significantly correlated with CDX2 gene expression in ALL children at different treatment stages (P<0.05). CONCLUSION: The expression of Hsa-miR-9 decreases in children with ALL, and ALL children with high expression of Hsa-miR-9 have a longer overall survival time. The expression of hsa-miR-9 in ALL children closely related with CDX2 gene.

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.

[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.