OBJECTIVE: To study the expression of SPOP in patients with acute myeloid leukemia (AML) and its effect on proliferation, apoptosis and cycle of AML cells. METHODS: RT-qPCR was used to detect the expression of SPOP mRNA in bone marrow samples of patients with newly diagnosed AML and normal controls. The stable overexpression of SPOP in AML cell lines THP-1 and U937 were constructed by liposome transfection. The effect of SPOP on cell proliferation was detected by CCK-8, and the effect of SPOP on apoptosis and cell cycle was detected by flow cytometry. The expressions of anti-apoptotic protein Bcl-2 and apoptotic protein Bax, Caspase3 were detected by Western blot. RESULTS: The median expression level of SPOP mRNA in normal control group was 0.993 1(0.6303, 1.433), while that in AML group was 0.522 1(0.242 2, 0.723 7). The expression level of SPOP in AML group was significantly lower than that in normal control group ( P < 0.001). After the overexpression of SPOP, the proportion of apoptotic cells in the U937 overexpression group and THP-1 overexpression group was 10.9%±0.3% and 4.6%±015%, which were higher than 8.9%±0.3% and 3.0%±0.30% in the Empty Vector group, respectively (both P < 0.05). The expression of Caspase3 in U937 overexpression group and THP-1 overexpression group was 1.154±0.086 and 1.2±0.077, which were higher than 1 in Empty Vector group, respectively (both P < 0.05). The ratio of Bax/Bcl-2 in U937 overexpression group and THP-1 overexpression group was 1.328±0.057 and 1.669±0.15, which were higher than 1 in Empty Vector group, respectively (both P < 0.05). In the cell proliferation experiment, the number of cells in the U937 overexpression group and THP-1 overexpression group were both slightly lower than those in the Empty Vector group, but the differences were not statistically significant (P >0.05). In the cell cycle experiment, the proportion of G₁ cells in the U937 overexpression group and THP-1 overexpression group were both slightly higher than those in the Empty Vector group, but the differences were not statistically significant (P >0.05). CONCLUSION SPOP can promote the apoptosis of leukemic cells, and its mechanism may be related to down-regulation of Bcl-2 expression and up-regulation of Bax and Caspase3 expression.
Humans ; Leukemia, Myeloid, Acute/pathology* ; Apoptosis ; Repressor Proteins/genetics* ; Cell Proliferation ; Nuclear Proteins/genetics* ; Cell Cycle ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Caspase 3/metabolism* ; bcl-2-Associated X Protein/metabolism* ; U937 Cells ; Cell Line, Tumor ; RNA, Messenger/genetics*

OBJECTIVE: To investigate the effect of LINC00641 on the viability and apoptosis of acute myeloid leukemia (AML) cells and its mechanism. METHODS: RT-qPCR was applied to detect the relative expression levels of LINC00641, miR-204-5p, and MT1X in human normal bone marrow stromal cell lines HS-5 and AML cell lines, and to screen the optimal cell line THP-1 was screened for subsequent experiments. Bioinformatics, dual luciferase reporter assay, pull down assay, and RIP assay were applied to validate the targeting relationship between LINC00641, MT1X and miR-204-5p. EdU, CCK-8, flow cytometry, and Transwell assay were applied to detect cell proliferation, apoptosis, migration and invasion, respectively. Western blot was applied to detect the expression of MT1X , CyclinD1, Bcl-2, and Bax proteins. RESULTS: Compared with HS-5 cells, the expression of LINC00641 and MT1X was obviously increased in HL60, THP-1, U937, and KG1 cells, while the expression of miR-204-5p was obviously reduced (all P <0.05). THP-1 cells showed the most obvious changes (P <0.05). Silencing LINC00641 or overexpressing miR-204-5p was able to obviously inhibit the proliferation, migration and invasion of THP-1 cells, as well as the expression of CyclinD1 and Bcl-2 proteins, while promote cells apoptosis and Bax protein expression (all P <0.05). Bioinformatics analysis, dual luciferase reporter assay, pull down assay, and RIP assay all confirmed that there were targeted relationships between LINC00641, MT1X and miR-204-5p. Inhibiting miR-204-5p or overexpressing MT1X was able to respectively reverse the inhibitory effect of silencing LINC00641 or overexpressing miR-204-5p on THP-1 cells proliferation, migration and invasion, and reduce cells apoptosis. CONCLUSION LINC00641 is highly expressed in AML, and inhibition of LINC00641 expression can inhibit cell proliferation, migration, and invasion and increase apoptosis by regulating the miR-204-5p/MT1X axis.
Humans ; Apoptosis ; Leukemia, Myeloid, Acute/pathology* ; MicroRNAs ; Cell Proliferation ; RNA, Long Noncoding/genetics* ; Cell Movement ; Cell Survival ; Cell Line, Tumor ; HL-60 Cells

OBJECTIVE: To investigate the role of nucleolin (NCL) in acute myeloid leukemia (AML) Kasumi-1 cells and its underlying mechanism. METHODS: The Kasumi-1 cells were infected with lentivirus carrying shRNA to downregulate NCL expression. Cell proliferation was detected by CCK-8 assay, and cell apoptosis and cell cycle were determined by flow cytometry. Transcriptome next-generation sequencing (NGS) was performed to predict associated signaling pathways, the expression levels of related genes were measured by RT-PCR. RESULTS: Down-regulation of NCL expression significantly inhibited the proliferation of Kasumi-1 cells (P <0.01) and markedly increased the apoptosis rate (P <0.001). Cell cycle analysis showed significant changes in the distribution of cells in the G₁ and S phases after NCL knockdown (P <0.05), while no significant difference was observed in the G₂ phase (P >0.05). Transcriptome sequencing analysis demonstrated that differentially expressed genes in Kasumi-1 cells with low expression of NCL were primarily enriched in key signaling pathways, including ribosome, spliceosome, RNA transport, cell cycle, and amino acid biosynthesis. qPCR validation showed that the expression of BAX, CASP3, CYCS, PMAIP1, TP53 , and CDKN1A was significantly upregulated after NCL downregulation (P <0.05), with CDKN1A exhibiting the most pronounced difference. CONCLUSION NCL plays a critical role in regulating the proliferation, apoptosis, and cell cycle progression of Kasumi-1 cells. The mechanism likely involves suppressing cell cycle progression through activation of the TP53-CDKN1A pathway and promoting apoptosis by upregulating apoptosis-related genes.
Humans ; Leukemia, Myeloid, Acute/pathology* ; Down-Regulation ; Cell Proliferation ; Apoptosis ; RNA-Binding Proteins/genetics* ; Nucleolin ; Cell Line, Tumor ; Phosphoproteins/metabolism* ; Cell Cycle ; Signal Transduction ; RNA, Small Interfering

JMJD1C (Jumonji Domain Containing 1C), a member of the lysine demethylase 3 (KDM3) family, is universally required for the survival of several types of acute myeloid leukemia (AML) cells with different genetic mutations, representing a therapeutic opportunity with broad application. Yet how JMJD1C regulates the leukemic programs of various AML cells is largely unexplored. Here we show that JMJD1C interacts with the master hematopoietic transcription factor RUNX1, which thereby recruits JMJD1C to the genome to facilitate a RUNX1-driven transcriptional program that supports leukemic cell survival. The underlying mechanism hinges on the long N-terminal disordered region of JMJD1C, which harbors two inseparable abilities: condensate formation and direct interaction with RUNX1. This dual capability of JMJD1C may influence enhancer-promoter contacts crucial for the expression of key leukemic genes regulated by RUNX1. Our findings demonstrate a previously unappreciated role for the non-catalytic function of JMJD1C in transcriptional regulation, underlying a mechanism shared by different types of leukemias.
Core Binding Factor Alpha 2 Subunit/genetics* ; Humans ; Leukemia, Myeloid, Acute/pathology* ; Jumonji Domain-Containing Histone Demethylases/chemistry* ; Gene Expression Regulation, Leukemic ; Oxidoreductases, N-Demethylating/genetics* ; Cell Line, Tumor

Glutamine provides carbon and nitrogen to support the proliferation of cancer cells. However, the precise reason why cancer cells are particularly dependent on glutamine remains unclear. In this study, we report that glutamine modulates the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7) to promote cancer cell proliferation and survival. Specifically, lysine 604 (K604) in the sixth of the 7 substrate-recruiting WD repeats of FBW7 undergoes glutaminylation (Gln-K604) by glutaminyl tRNA synthetase. Gln-K604 inhibits SCFFBW7-mediated degradation of c-Myc and Mcl-1, enhances glutamine utilization, and stimulates nucleotide and DNA biosynthesis through the activation of c-Myc. Additionally, Gln-K604 promotes resistance to apoptosis by activating Mcl-1. In contrast, SIRT1 deglutaminylates Gln-K604, thereby reversing its effects. Cancer cells lacking Gln-K604 exhibit overexpression of c-Myc and Mcl-1 and display resistance to chemotherapy-induced apoptosis. Silencing both c-MYC and MCL-1 in these cells sensitizes them to chemotherapy. These findings indicate that the glutamine-mediated signal via Gln-K604 is a key driver of cancer progression and suggest potential strategies for targeted cancer therapies based on varying Gln-K604 status.
Glutamine/metabolism* ; Myeloid Cell Leukemia Sequence 1 Protein/genetics* ; Humans ; Proto-Oncogene Proteins c-myc/genetics* ; Cell Proliferation ; Signal Transduction ; Neoplasms/pathology* ; F-Box-WD Repeat-Containing Protein 7/genetics* ; Cell Survival ; Cell Line, Tumor ; Apoptosis

Objective To investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia (AML) by targeting paired box 9 (PAX9). Methods The relationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis (GEPIA) database and The Cancer Genome Atlas (TCGA) database. Kasumi-1 and AML5 cells were transfected with empty vector (Vector group) or PAX9 (PAX9 group). The proliferation activity was detected by CCK-8 assay, and cells cycle and apoptosis were detected by flow cytometry. Expressions of cyclin-dependent kinase 2 (CDK2), cyclin B1 (CCNB1), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX) were detected by Western blot analysis. The targeted microRNA (miRNA) by PAX9 was predicted by bioinformatics analysis, and the targeted effect was verified by luciferase reporter assay. The level of PAX9 mRNA was detected by real-time quantitative PCR, and expression of PAX9 protein was detected by Western blot analysis. Kasumi-1 and AML5 cells were transfected with miR-NC (miR-NC group) or miR-181b-5p (miR-181b-5p group). The cells were further transfected with PAX9 (miR-181b-5p combined with PAX9 group) in miR-181b-5p group. The proliferation, cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients, which was correlated with poor prognosis. In Kasumi-1 and AML5 cells, compared with Vector group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in PAX9 group. It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p. Compared with miR-NC group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were increased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were decreased in miR-181b-5p group. Compared with miR-181b-5p group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in miR-181b-5p combined with PAX9 group. Conclusion The miR-181b-5p can promote the proliferation of AML cells and delay apoptosis by inhibiting PAX9.
Humans ; Apoptosis/genetics* ; bcl-2-Associated X Protein ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Leukemia, Myeloid, Acute/pathology* ; Luciferases ; MicroRNAs/metabolism* ; PAX9 Transcription Factor/genetics*

OBJECTIVE: To analyze the flow immunophenotype and clinical characteristics of leukemia patients with positive SET-CAN fusion gene. METHODS: A total of 7 newly diagnosed acute leukemia patients with SET-CAN fusion gene admitted to Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from February 2016 to February 2020 were collected. Multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of SET-CAN fusion gene. The immunophenotype was detected by four-color flow cytometry. The case information of 17 literatures published at home and abroad was extracted for statistical analysis. RESULTS: Among the 7 patients, 2 cases were diagnosed as mixed phenotype acute leukemia (MPAL), 2 cases as acute myeloid leukemia (AML), and 3 cases as T-acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL). Leukemia cells in bone marrow specimens of all cases expressed or partially expressed CD34, CD33 and CD7. CD5 and cytoplasmic CD3 were expressed in 5 patients except 2 patients diagnosed with AML. Bone marrow and lymph node specimens were both detected in 2 patients, and the immunophenotypes of the two specimens were not completely consistent, with differences in lineage or maturity related markers. Two patients with MPAL showed differentiated response to treatment. One AML patient gave up treatment, and another AML patient with FLT3-ITD gene mutation had a poor prognosis. All three T-ALL/LBL patients maintained a long duration of remission after induced remission, and one case underwent allogeneic hematopoietic stem cell transplantation. CONCLUSIONS There are common characteristics of immunophenotype in patients with positive SET-CAN fusion gene. Differential expression of immunophenotype in samples from different parts is observed in some cases. The prognosis of these diseases varies.
Humans ; Leukemia, Myeloid, Acute/pathology* ; Bone Marrow/pathology* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Antigens, CD34 ; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ; Immunophenotyping

OBJECTIVE: To investigate the expression and clinical significance of T helper cell 9 (Th9) and its cytokine interleukin 9(IL-9) in peripheral blood of patients with chronic lymphocytic leukemia(CLL). METHODS: A total of 43 newly diagnosed patients with chronic lymphocytic leukemia in the First Affiliated Hospital of Xinjiang Medical University from June 2021 to June 2022 were selected as the case group. The patients were divided into Binet A group (13 cases), Binet B group (20 cases) and Binet C group (10 cases) by Binet staging system, and 20 healthy volunteers who underwent physical examinationin in our hospital in the same period served as control group. The proportion of Th9 cells in peripheral blood was detected by flow cytometry, the expression level of Th9 specific transcription factors PU.1 and IRF4 was detected by Western blot, and the expression level of serum cytokine IL-9 was detected by ELISA. The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in each group were compared, and the correlation between the proportion of Th9, IL-9 and clinicopathological indexes of CLL patients was analyzed. RESULTS: The proportion of Th9, the expression of PU.1, IRF4 and IL-9 in CLL group were significantly higher than those in control group (P<0.05), the proportion of Th9 and the expression of IL-9 in Binet B and C group were higher than those in Binet A group (P<0.05), but there was no significant difference in the proportion of Th9 cells between Binet B group and C group (P>0.05). The expression of IL-9 in Binet C group was significantly higher than that in Binet B group (P<0.05) . The proportion of Th9 cells and IL-9 were highly expression in patients with β2 microglobulin abnormality, IGHV unmutation, P53 abnormality and hepatosplenic lymph node enlargement(P<0.05), but not related to age and sex (P>0.05). The results of Spearman correlation analysis showed that the proportion of Th9 in patients with CLL was negatively correlated with the lymphocytic account and lymphocyte proportion(r_s=-0.32，r_s=-0.34). The proportion of Th9 and IL-9 were positively correlated with Binet stage, Rai stage and CLL-IPI Scoring (r_s=0.79，r_s=0.54，r_s=0.58； r_s=0.72，r_s=0.63，r_s=0.45), but not with WBC, CD4⁺ T cells and CD8⁺T cells (P>0.05). The proportion of Th9 was positively correlated with IL-9 (r_s=0.53). CONCLUSION Th9 cells and IL-9 are abnormally highly expressed in CLL, which is related to the poor prognosis of CLL.
Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/genetics* ; Interleukin-9 ; Clinical Relevance ; T-Lymphocytes, Helper-Inducer/pathology* ; Cytokines

OBJECTIVE: To explore the value of multiple detection methods based on histopathology and supplemented by bone marrow or peripheral blood sample detections in the comprehensive diagnosis of mantle cell lymphoma (MCL). METHODS: The clinical, immunophenotypic, pathologic, cytogenetic and molecular features of 153 newly diagnosed MCL patients admitted to the hematology department of our hospital from May 2009 to September 2022 were analyzed. RESULTS: 144 (96.6%) of the 149 MCL patients who underwent marrow or peripheral blood IGH/CCND1 FISH detection at initial diagnosis were positive, of which 36 cases (24.2%) had a low proportion positive. The immunophenotypes in 115 patients were analyzed by flow cytometry (FCM), 89 cases (77.4%) conformed to MCL while 23 cases (20.0%) were initially diagnosed as B-cell lymphoproliferative disorders (B-LPD). Of the 75 cases who performed bone marrow biopsy, 50 cases (66.7%) had morphological and immunophenotypic characteristics consistent with MCL, 15 cases (20.0%) were classified as B-LPD, and 10 cases with no obvious abnormality. 77 patients underwent histopathology examination, of which 73 cases (94.8%) had typical clinicopathological features of MCL, including 2 CCND1 negative MCL, 2 pleomorphic variants, 5 pleomorphic variants and 4 cases diagnosed as other leukemia or lymphoma. Among 153 cases of MCL, 128 cases were classic MCL(cMCL), and another 25 cases (16.3%) were diagnosed as leukemic non-lymph node MCL (lnnMCL). The incidence of IGHV mutation, TP53 mutation and CD23 expression positive were significantly different between cMCL and lnnMCL. CONCLUSION Histopathology is still the main standard for the diagnosis of cMCL, and detection based on bone marrow or peripheral blood samples is an important means for the diagnosis of lnnMCL. Single marker or examination can cause a certain proportion of misdiagnosis. The accurate diagnosis of MCL depends on a combination of multiple detection methods.
Adult ; Humans ; Lymphoma, Mantle-Cell/genetics* ; Bone Marrow/pathology* ; Leukemia/pathology* ; Mutation ; Immunophenotyping

BACKGROUND: Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. METHODS: Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca 2+ -related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. RESULTS: Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca 2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. CONCLUSIONS Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.
Animals ; Humans ; Mice ; Apoptosis ; Bone Marrow Cells ; Cell Communication ; Connexin 43/genetics* ; Gap Junctions/metabolism* ; Imatinib Mesylate/therapeutic use* ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology* ; Mesenchymal Stem Cells/metabolism* ; Tumor Microenvironment ; Calcium/metabolism*