OBJECTIVES: To investigate the role of METTL3 and FOXO3 in anthracycline resistance in acute myeloid leukemia (AML) cells. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptome sequencing (RNA-seq) were performed in anthracycline-resistant and sensitive HL60 and K562 cells with lentivirus-mediated knockdown or overexpression of METTL3 and FOXO3. TCGA and GSE6891 datasets were used for analysis of the clinical and gene expression data of AMI patients. FOXO3 expressions at the mRNA and protein levels in the transfected cells were detected with RT-qPCR and Western blotting, and the changes in cell proliferation and apoptosis were evaluated using CCK8 assay and flow cytometry; the expression of m⁶A-modified mRNA and mRNA stability of FOXO3 was detected analyzed using MeRIP-qPCR and RT-qPCR. Functional enrichment analysis of the differential genes in the transfected cells was performed. RESULTS: Differential gene analysis in anthracycline-resistant versus sensitive AML cells and in cells with METTL3 knockdown revealed the enrichment in FoxO and autophagy pathways (P<0.05), and the anthracycline-resistant cells showed significantly increased m⁶A modification of FOXO3. FOXO3 expression was positively correlated with METTL3 expression. METTL3 knockdown significantly reduced FOXO3 mRNA stability and its protein levels in anthracycline-resistant AML cells, which exhibited higher m6A-modified FOXO3 expression levels than their sensitive counterparts. Database analysis, Kaplan-Meier analysis and RT-qPCR results suggested that a high FOXO3 expression was associated with a poor prognosis of AML patients. In anthracycline-resistant AML cells expressing higher FOXO3 levels than the sensitive cells, lentivirus-mediated overexpression of FOXO3 significantly enhanced cell proliferation and suppressed cell apoptosis. Inhibiting autophagy using an autophagy inhibitor (Baf.A1) obviously enhanced the inhibitory effect of adriamycin on resistant AMI cells and cells overexpressing FOXO3. CONCLUSIONS METTL3 promotes FOXO3 expression via m6A modification, and FOXO3-driven autophagy contributes to anthracycline resistance in AML cells by enhancing cell proliferation and suppressing cell apoptosis.
Humans ; Forkhead Box Protein O3/genetics* ; Leukemia, Myeloid, Acute/genetics* ; Drug Resistance, Neoplasm ; Methyltransferases/genetics* ; Autophagy ; Anthracyclines/pharmacology* ; HL-60 Cells ; Apoptosis ; Cell Proliferation ; K562 Cells

Mitochondrial metabolism is crucial for providing energy and heme precursors during erythroid development. Oxoglutarate dehydrogenase complex (OGDC) is a key enzyme in the mitochondrial tricarboxylic acid (TCA) cycle, and its level gradually increases during erythroid development, indicating its significant role in erythroid development. The aim of the present study was to explore the role and mechanism of OGDC in erythroid development. In this study, we treated erythroid progenitor cells with CPI-613, a novel lipoic acid analog that competitively inhibits OGDC. The results showed that CPI-613 inhibited erythropoietin (EPO)-induced differentiation and enucleation of human CD34⁺ hematopoietic stem cells into erythroid cells, suppressed cell proliferation, and induced apoptosis. The results of in vivo experiments showed that CPI-613 also hindered the recovery of mice from acute hemolytic anemia. Further mechanism research results showed that CPI-613 increased reactive oxygen species (ROS) in erythroid progenitor cells, inhibited mitochondrial respiration, caused mitochondrial damage, and suppressed heme synthesis, thereby inhibiting erythroid differentiation. Clinical research results showed that oxoglutarate dehydrogenase (OGDH) protein expression levels were up-regulated in bone marrow cells of polycythemia vera (PV) patients. Treatment with CPI-613 significantly inhibited the excessive proliferation and differentiation of erythroid progenitor cells of the PV patients. These findings demonstrates the critical role of OGDC in normal erythroid development, suggesting that inhibiting its activity could be a novel therapeutic strategy for treating PV.
Animals ; Humans ; Mitochondria/metabolism* ; Mice ; Ketoglutarate Dehydrogenase Complex/physiology* ; Cell Differentiation/drug effects* ; Cells, Cultured ; Erythropoiesis/drug effects* ; Reactive Oxygen Species/metabolism* ; Cell Proliferation/drug effects* ; Erythroid Precursor Cells/cytology* ; Apoptosis/drug effects* ; Thioctic Acid/pharmacology* ; Caprylates ; Sulfides

Chronic anemia patients (such as thalassemia) often rely on long-term red blood cell transfusion to sustain life. However, alloimmune reactions against blood group antigens can pose serious risks to the patients' clinical treatment and survival. The regulatory mechanisms of transfusion-related alloimmunity are not yet well understood. For example, some patients, despite long-term transfusions, do not develop alloimmune reactions, while others produce alloantibodies against multiple blood group antigens, making transfusion therapy increasingly difficult. Red blood cell blood group alloimmunity involves various immune cells, including antigen-presenting cells and different T cells. Many studies are exploring the regulatory roles and even potential interventions. This article reviews the correlation between cellular immunity and red blood cell blood group antigens in alloimmune responses, and explores the interaction between the two, as well as their impact on immune responses.
Humans ; Immunity, Cellular/immunology* ; Erythrocytes/immunology* ; Blood Group Antigens/immunology* ; Animals ; Isoantibodies/immunology* ; T-Lymphocytes/immunology*

OBJECTIVE: To investigate the metabolic characteristics of ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) in myeloid leukemia by in vitro culture of myeloid leukemia cells and construction of tumor-bearing nude mouse model. METHODS: U937, THP-1, HL60 and K562 cells were cultured in vitro. The cells in logarithmic growth phase (l×10 ⁵ cells/well) were added with ¹⁸F-FDG, and the uptake rate of ¹⁸F-FDG was measured at 15, 30, 60 and 120 min after addation, respectively. The four kinds of cells were inoculated subcutaneously into the hind limbs of nude mice to establish a tumor-bearing nude mouse model. When the tumor size was about 500 mm³, ¹⁸F-FDG was injected through the tail vein of the mice, and positron emission tomography/computed tomography was performed at 60 min after injection. The morphology of tumor-bearing cells was observed by hematoxylin-eosin (HE) staining in serial pathological sections. RESULTS: After co-incubation with ¹⁸F-FDG, the ¹⁸F-FDG uptake rates of U937 cells were significantly higher than THP-1, HL60 and K562 cells at 4 time points (all P <0.05), and THP-1 cells were higher than K562 cells (all P <0.05). The uptake rate of ¹⁸F-FDG by leukemia cells was rapid in the first 60 min, then tended to be stable. Pathological analysis showed that subcutaneous inoculation of U937, THP-1, HL60 and K562 cells could successfully establish tumor-bearing nude mouse models of myeloid leukemia. The ¹⁸F-FDG uptake value in U937 tumor-bearing nude mice was significantly higher than THP-1, HL60 and K562 tumor-bearing nude mice (all P <0.01). The ¹⁸F-FDG uptake values in THP-1 and HL60 tumor-bearing nude mice were significantly higher than that in K562 tumor-bearing nude mice (both P <0.01). CONCLUSION The tumor-bearing nude mouse model of myeloid leukemia can be successfully constructed by subcutaneous inoculation. The ¹⁸F-FDG uptake rate of acute myeloid leukemia (AML) cells is higher in cells cultured in vitro and tumor-bearing nude mouse model. ¹⁸F-FDG may have better clinical application value for AML.
Animals ; Fluorodeoxyglucose F18/metabolism* ; Mice, Nude ; Mice ; Humans ; Leukemia, Myeloid/diagnostic imaging* ; HL-60 Cells ; K562 Cells ; Cell Line, Tumor ; U937 Cells

OBJECTIVE: To investigate the gene carrier rate and genotype distribution characteristics of thalassemia in the population of Kunming, and compare the differences of red blood cell (RBC) parameters between β⁺ heterozygous carriers, β⁰ heterozygous carriers and healthy population, as well as between different sexes of adults aged 18-45 years. METHODS: A retrospective analysis of 3 195 cases of thalassemia gene screened in the First Affiliated Hospital of Kunming Medical University from April 1, 2020 to March 31, 2022 was performed to detect 21 mutations of β-globin genes which was common in Chinese people using fluorescence PCR melting curve method. Patients with single heterozygous carrying β-thalassemia gene were divided into β⁺ heterozygote group and β⁰ heterozygote group, while the control group consisted of 219 healthy individuals. Four indices, including RBC, hemoglobin (Hb), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were collected from all β heterozygous carriers and 219 healthy people, and compared between β⁺ heterozygote group, β⁰ heterozygote group and control group, as well as between β⁺ heterozygous carriers, β⁰ heterozygous carriers and healthy population of different sexes aged 18-45 years. RESULTS: There were 688 cases confirmed thalassemia gene carriers, accounting for 21.53%. Among them, 322 cases were found to have β-globin gene mutations, including 145 cases of β⁺ heterozygote, 151 cases of β⁰ heterozygote, and 14 cases of β⁺ homozygotes as well as β⁺ and β⁰ dual heterozygotes. Additionally, 12 cases were found to have simultaneous mutation or deletion of β-globin and α-globin. The carrier rate of CD26 G>A mutation in β⁺ thalassemia was the highest, accounting for 57.9%, while in β⁰ thalassemia CD17 A>T was the highest, accounting for 46.4%. The erythrocyte parameters of 296 β heterozygous mutation carriers were compared with the normal reference interval, and it was found that 218 cases with RBC value greater than the highest value of reference interval, while 105, 281, and 269 cases with Hb, MCV, and MCH value less than the lowest value of reference interval, respectively. There were significant differences in the 4 erythrocyte parameters between β⁺ heterozygotes, β⁰ heterozygotes and healthy individuals (all P < 0.001), and further comparison between different sexes also showed significant differences (all P < 0.001). CONCLUSIONS The carrier rates of thalassemia gene and β-thalassemia heterozygote are both at high level in Kunming, and there are significant differences in the erythrocyte parameters between β⁺ heterozygous carriers, β⁰ heterozygous carriers and healthy individuals. When genetic counseling, it is necessary to inform and strengthen screening among adults of marriageable age to prevent birth of children with severe thalassemia.
Humans ; beta-Thalassemia/blood* ; Adult ; Heterozygote ; Male ; Female ; beta-Globins/genetics* ; Retrospective Studies ; Middle Aged ; Mutation ; Adolescent ; Genotype ; Erythrocytes ; Erythrocyte Indices ; Young Adult ; China ; Genetic Testing ; Asian People/genetics*

OBJECTIVE: To study the genetic polymorphism of red blood cell blood group among in Shenzhen Han, Indian and Xinjiang Uyghur populations, to provide scientific basis for the demand prediction and collection strategy of rare blood group, and to explore the genetic differences of blood group between Han and Caucasians. METHODS: The haplotypes of antigen coding genes of 10 target blood group systems from 87 Han Chinese and 50 Indian blood donors in Shenzhen, and 49 healthy Uyghur people in Xinjiang were obtained by three-generation sequencing technology, and the polymorphism and frequency characteristics were analyzed. RESULTS: Only a single genotype was detected the Langereis and Vel blood group systems in samples from three different populations. Only one genotype of Dombrock blood group was detected in Shenzhen Han, and Junior blood group in Xinjiang Uygur populations. In the MNS, Duffy, Kidd, Dombrock and Junior blood group systems, the haplotype frequency of Indian and Uyghur people was significantly different from that of Han people. Compared with the Han ethnic group, the rare blood group s-, Fy(a-), Jk(a-b-), and Do(a+b-) have a higher frequency among the Uyghur and Indian populations. CONCLUSION Haplotype frequencies of antigen genes for MNS, Duffy, Kidd, Dombrock and Junior blood group system in Shenzhen Han, Indian and Uyghur populations displayed a polymorphic difference with unique distribution characteristics different from the ethnic groups in other regions.
Humans ; Blood Group Antigens/genetics* ; China/ethnology* ; Erythrocytes ; Ethnicity/genetics* ; Gene Frequency ; Genotype ; Haplotypes ; India/ethnology* ; Polymorphism, Genetic ; White People/genetics* ; Central Asian People/genetics* ; East Asian People/genetics*

OBJECTIVE: To establish a genotyping method for Diego, MNS and Kell blood groups based on quantitative real-time PCR (qPCR) technology, and preliminarily apply it to the screening of rare blood groups in blood donors. METHODS: Blood group gene standards containing heterozygous and homozygous alleles were prepared by blood group serological and PCR-SBT methods. Specific amplification primers and hybridization probes were designed, and explore to establish the qPCR method for detecting Diego, MNS, and Kell blood group genotypes. Then the established qPCR method was used to identify blood group genotypes of 186 blood donor samples. RESULTS: A method based on qPCR technology was established to identify Dia/Dib, S/s and K/k blood group antigens. The genotyping results of the gene standard samples were consistent with the serological testing results and genotypes detected by PCR-SBT. qPCR testing of 186 samples identified 11 cases of DI*A/B heterozygosity and 19 cases of GYPB*S/s heterozygosity, and the rest were DI*B/B, GYPB*s/s, KEL*02/02 homozygosity. No rare blood group genotypes of DI*A/A, GYPB*S/S, KEL*01.01/01.01 were found. CONCLUSION The established qPCR method is suitable for genotyping on Diego, MNS and Kell blood group, and it can be used for batch screening of blood donors and the establishment of rare blood group bank.
Humans ; Genotype ; Genotyping Techniques/methods* ; Real-Time Polymerase Chain Reaction/methods* ; Blood Group Antigens/genetics* ; Kell Blood-Group System/genetics* ; Blood Donors ; Blood Grouping and Crossmatching/methods* ; Erythrocytes ; MNSs Blood-Group System/genetics*

BACKGROUND

Implementation of blood conservation protocol may increase the efficiency of blood utilization and optimize the maximum surgical blood ordering schedule (MSBOS) in our institution. The primary objectives of this study are to evaluate the efficient utilization of a fixed blood request of 4 units of packed red blood cells (PRBCs) and to establish an effective MSBOS on primary isolated elective coronary artery bypass graft (CABG).

This was a retrospective, analytical, observational, cohort type of study conducted through chart review of CABG patients from January 2018 to December 2019. Indices used to evaluate blood utilization efficiency were crossmatch to transfusion (C/T) ratio, transfusion probability (T%), and transfusion index (TI). MSBOS was computed based on the data gathered.

A total of 77 patients were included in this study. Blood transfusion based on C/T ratio was associated with body surface area 3 vessels (P = 0.042). Overall, C/T ratio was 2.44, T% was 70.1%, and TI was 1.69. Unutilized blood was measured at 59%. The computed MSBOS for elective CABG was at 2 units (2.53).

There is efficient PRBC utilization in primary elective CABG when the blood indices were used. Evidence-based MSBOS is established and the previous 4 units PRBC can be reduced to 2 units to promote C/T ratio
Human ; Blood Crossmatching ; Blood Grouping And Crossmatching ; Blood Transfusion ; Coronary Artery Bypass ; Blood Cells, Red ; Erythrocytes

BACKGROUND

Implementation of blood conservation protocol may increase the efficiency of blood utilization and optimize the maximum surgical blood ordering schedule (MSBOS) in our institution. The primary objectives of this study are to evaluate the efficient utilization of a fixed blood request of 4 units of packed red blood cells (PRBCs) and to establish an effective MSBOS on primary isolated elective coronary artery bypass graft (CABG).

This was a retrospective, analytical, observational, cohort type of study conducted through chart review of CABG patients from January 2018 to December 2019. Indices used to evaluate blood utilization efficiency were crossmatch to transfusion (C/T) ratio, transfusion probability (T%), and transfusion index (TI). MSBOS was computed based on the data gathered.

A total of 77 patients were included in this study. Blood transfusion based on C/T ratio was associated with body surface area 3 vessels (P = 0.042). Overall, C/T ratio was 2.44, T% was 70.1%, and TI was 1.69. Unutilized blood was measured at 59%. The computed MSBOS for elective CABG was at 2 units (2.53).

There is efficient PRBC utilization in primary elective CABG when the blood indices were used. Evidence-based MSBOS is established and the previous 4 units PRBC can be reduced to 2 units to promote C/T ratio
Human ; Blood Crossmatching ; Blood Grouping And Crossmatching ; Blood Transfusion ; Coronary Artery Bypass ; Blood Cells, Red ; Erythrocytes

OBJECTIVE: To detect the differential expressions of miR-451, ABCB1 and ABCC2 in drug-sensitive leukemia cell line K562 and drug-resistant cell line K562/A02, and explore the regulatory relationship between miR-451 and the expressions of ABCB1 and ABCC2 , and the mechanism of miR-451 involved in drug resistance in leukemia. METHODS: CCK-8 assay was used to detect the drug resistance of K562/A02 and K562 cells. Quantitative Real-time PCR (qRT-PCR) was used to verify the differential expressions of miR-451 in K562 and K562/A02 cells. MiR-451 mimic and negative control (miR-NC), miR-451 inhibitor and negative control (miR-inNC) were transfected into K562 and K562/A02 cells respectively, then qRT-PCR and Western blot were used to detect the expression levels of mRNA and protein of ABCB1 and ABCC2 in K562 and K562/A02 cells and the transfected groups. RESULTS: The drug resistance of K562/A02 cells to adriamycin was 177 times higher than that of its parent cell line K562. Compared with K562 cells, the expression of miR-451 in K562/A02 cells was significantly higher (P <0.001), and the mRNA and protein expression levels of ABCB1 and ABCC2 in K562/A02 cells were significantly higher than those in K562 cells (P <0.001). After transfected with miR-451 inhibitor, the expression of miR-451 was significantly down-regulated in K562/A02 cells (P <0.001), the sensitivity to chemotherapy drugs was significantly enhanced (P <0.05), and the mRNA and protein expressions of ABCB1 and ABCC2 were significantly decreased (P <0.01). After transfected with miR-451 mimic, the expression of miR-451 was significantly upregulated in K562 cells (P <0.001), and the mRNA and protein expressions of ABCB1 and ABCC2 were significantly increased (P <0.01). CONCLUSION There are significant differences in the expressions of miR-451, ABCB1 and ABCC2 between the drug-sensitive leukemia cell line K562 and drug-resistant cell line K562/A02, which suggests that miR-451 may affect the drug resistance of leukemia cells by regulating the expression of ABCB1 and ABCC2.
Humans ; K562 Cells ; Drug Resistance, Neoplasm/genetics* ; Drug Resistance, Multiple/genetics* ; Doxorubicin/pharmacology* ; MicroRNAs/genetics* ; Leukemia/genetics* ; RNA, Messenger