Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 2 Reduced expression of PMCA4 in RBCs of MM. (A, B) Indirect cellular immunofluorescence labeling of the surface of RBCs with PMCA4 (blue), RBC membrane with Did labeling (red), PMCA4 expression in MM RBCs is significantly reduced (P = 5.1E-03). The white arrow indicates the stacking of RBCs (rouleaux formation) in MM (magnification, 60×). (C) Relative mRNA expression of ATPase plasma membrane Ca2+ transport 4 (ATP2B4) in nucleated RBCs of MM bone marrow is significantly lower than that in the normal control group (GAPDH was used as an internal control) (P = 5.1E-03). (D, E) The expression of PMCA4 in MM RBCs was significantly reduced (P = 5.1E-03), and CD138 was expressed. (F) After the antibody-labeled RBCs reacted with the substrate BCIP/NBT, the dark blue NBT-formazan deposits in the erythrocyte membrane of MM reacting with PMCA4 protein content were significantly lower than those of the normal control group. (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Labeling, Membrane, Clinical Proteomics, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 3 MiRNA-4261 targeting the ATP2B4 gene is highly expressed in MM. (A) Screening miRNAs by Venn diagram. The conditions were the following: expression in myeloma cells; lack of expression in normal RBCs; and target gene of ATP2B4. (B) qRT–PCR confirms that miR-4261 is highly expressed in MM RBCs (P = 9.2E-03). (C–G) qRT–PCR was used to measure the content of miR-4261 in various components in blood samples (PRBC = 2.3E-02, PPBMC = 2.3E-02, Pplatelet = 0.66, Pplasma = 3.7E-02, Pexosome = 2.8E-02). (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 5 MiR-4261 can target and regulate the ATP2B4 gene and reduce the expression of PMCA4 protein. (A) Schematic diagram of the predicted miR- 4261 binding site in the 3’-UTR of ATP2B4 mRNA. (B) Dual-luciferase reporter assay. The 3’-UTR fragments (wild-type and mutant sequences) of ATP2B4 were cloned into the psiCHECK-2 vector and then cotransfected with miR-4261 mimics or NC. Comparison of relative firefly/Renilla luciferase activity between the wild-type (WT) and negative control (NC) groups (PWT+mimics vs. WT+NC = 1.9E-4, and PWT+mimics vs. MUT+mimics = 1.4E-3). (C) The content of miR-4261 in HEK293T cells 36, 48, and 72 hours after transfection of miR-4261 mimics (P = 2.7E-2) and the endogenous content of miR-4261 in H929 cells and RPMI8226 cells. (D) The mRNA level of ATP2B4 was reduced by miR-4261, and the relative expression decreased most significantly at 72 hours after transfection (GAPDH was used as an internal control) (P = 3.6E-2). (E, F) MiR-4261 reduced the protein level of ATP2B4 (GAPDH was used as an internal control) (P = 4.7E-2). (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Clone Assay, Plasmid Preparation, Comparison, Activity Assay, Negative Control, Transfection, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 7 Functional analysis of PMCA4 in maintaining calcium homeostasis in RBCs. RBCs were pretreated with different concentrations of Omega- Agatoxin IVA for 10 minutes and then treated with 0.10 mM or 0.25 mM t-BHP for 0, 1, 2, 4, 8 and 10 minutes prior to the detection of the ROS and free calcium levels in RBCs. (A) ROS levels in RBCs after treatment with 0.10 mM t-BHP. (B) Free calcium levels in RBCs after treatment with 0.10 mM t-BHP. (C) ROS levels in RBCs after treatment with 0.25 mM t-BHP. (D) Free calcium levels in RBCs after treatment with 0.25 mM t- BHP. (Error bars represent the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the 0 nM control group. Refer to Supplementary Tables 5-8 for P values. One-way ANOVA was used for statistical analysis).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Functional Assay, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 8 Schematic diagram of RBC calcium overload mediated by MM exosomal miR-4261. Exosomes secreted by MM cells transfer miR-4261 to erythroid precursor cells, resulting in RBC calcium overload by downregulating the expression of ATP2B4. Calcium overload can cause crosstalk that leads to an increase in intracellular ROS levels, forming a vicious cycle that causes further damage to the activity of PMCA4 in RBCs.

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Activity Assay

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 2 Reduced expression of PMCA4 in RBCs of MM. (A, B) Indirect cellular immunofluorescence labeling of the surface of RBCs with PMCA4 (blue), RBC membrane with Did labeling (red), PMCA4 expression in MM RBCs is significantly reduced (P = 5.1E-03). The white arrow indicates the stacking of RBCs (rouleaux formation) in MM (magnification, 60×). (C) Relative mRNA expression of ATPase plasma membrane Ca2+ transport 4 (ATP2B4) in nucleated RBCs of MM bone marrow is significantly lower than that in the normal control group (GAPDH was used as an internal control) (P = 5.1E-03). (D, E) The expression of PMCA4 in MM RBCs was significantly reduced (P = 5.1E-03), and CD138 was expressed. (F) After the antibody-labeled RBCs reacted with the substrate BCIP/NBT, the dark blue NBT-formazan deposits in the erythrocyte membrane of MM reacting with PMCA4 protein content were significantly lower than those of the normal control group. (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Labeling, Membrane, Clinical Proteomics, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 3 MiRNA-4261 targeting the ATP2B4 gene is highly expressed in MM. (A) Screening miRNAs by Venn diagram. The conditions were the following: expression in myeloma cells; lack of expression in normal RBCs; and target gene of ATP2B4. (B) qRT–PCR confirms that miR-4261 is highly expressed in MM RBCs (P = 9.2E-03). (C–G) qRT–PCR was used to measure the content of miR-4261 in various components in blood samples (PRBC = 2.3E-02, PPBMC = 2.3E-02, Pplatelet = 0.66, Pplasma = 3.7E-02, Pexosome = 2.8E-02). (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 5 MiR-4261 can target and regulate the ATP2B4 gene and reduce the expression of PMCA4 protein. (A) Schematic diagram of the predicted miR- 4261 binding site in the 3’-UTR of ATP2B4 mRNA. (B) Dual-luciferase reporter assay. The 3’-UTR fragments (wild-type and mutant sequences) of ATP2B4 were cloned into the psiCHECK-2 vector and then cotransfected with miR-4261 mimics or NC. Comparison of relative firefly/Renilla luciferase activity between the wild-type (WT) and negative control (NC) groups (PWT+mimics vs. WT+NC = 1.9E-4, and PWT+mimics vs. MUT+mimics = 1.4E-3). (C) The content of miR-4261 in HEK293T cells 36, 48, and 72 hours after transfection of miR-4261 mimics (P = 2.7E-2) and the endogenous content of miR-4261 in H929 cells and RPMI8226 cells. (D) The mRNA level of ATP2B4 was reduced by miR-4261, and the relative expression decreased most significantly at 72 hours after transfection (GAPDH was used as an internal control) (P = 3.6E-2). (E, F) MiR-4261 reduced the protein level of ATP2B4 (GAPDH was used as an internal control) (P = 4.7E-2). (Error bars represent the mean ± SEM, *P < 0.05).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Mutagenesis, Clone Assay, Plasmid Preparation, Comparison, Activity Assay, Negative Control, Transfection, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 7 Functional analysis of PMCA4 in maintaining calcium homeostasis in RBCs. RBCs were pretreated with different concentrations of Omega- Agatoxin IVA for 10 minutes and then treated with 0.10 mM or 0.25 mM t-BHP for 0, 1, 2, 4, 8 and 10 minutes prior to the detection of the ROS and free calcium levels in RBCs. (A) ROS levels in RBCs after treatment with 0.10 mM t-BHP. (B) Free calcium levels in RBCs after treatment with 0.10 mM t-BHP. (C) ROS levels in RBCs after treatment with 0.25 mM t-BHP. (D) Free calcium levels in RBCs after treatment with 0.25 mM t- BHP. (Error bars represent the mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the 0 nM control group. Refer to Supplementary Tables 5-8 for P values. One-way ANOVA was used for statistical analysis).

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Functional Assay, Control

Journal: Frontiers in oncology

Article Title: Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

doi: 10.3389/fonc.2022.978755

Figure Lengend Snippet: FIGURE 8 Schematic diagram of RBC calcium overload mediated by MM exosomal miR-4261. Exosomes secreted by MM cells transfer miR-4261 to erythroid precursor cells, resulting in RBC calcium overload by downregulating the expression of ATP2B4. Calcium overload can cause crosstalk that leads to an increase in intracellular ROS levels, forming a vicious cycle that causes further damage to the activity of PMCA4 in RBCs.

Article Snippet: PMCA4 protein on the RBC membrane was labeled with PMCA4 antibody (NB300-569, NOVUS) at a dilution ratio of 1:400.

Techniques: Expressing, Activity Assay