Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: Circ_0004277 expression is remarkably up-regulated in AML. A. Heat map showed the differentially expressed circRNAs (bone marrow mononuclear cells of AML patients v.s. bone marrow mononuclear cells of healthy controls, GSE94591), and the filter criteria was P < 0.05,Log2(Change fold)> 2.5. B. The expression of circ_0004277 in bone marrow mononuclear cells of AML patients was significantly down-regulated compared with that of healthy controls (GSE94591). C. qRT-PCR showed that circ_0004277 expression was lower in the bone marrow samples of 37 AML patients than that in 37 healthy controls. D. qRT-PCR revealed that circ_0004277 expression in AML cells (KG-1a, THP-1, K562 and U937) was down-regulated compared with the human normal stromal cells (HS-5). E. qRT-PCR showed that circ_0004277 was resistant to RNase R. ** P < 0.01, and *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: Circ_0004277 directly targets miR-134-5p. A. Bioinformatics was employed to predict the binding site between circ_0004277 and miR-134-5p. B. Dual-luciferase reporter gene assay assays demonstrated that miR-134-5p could negatively regulate the luciferase activity of circ_0004277-WT, rather than circ_0004277-MUT in U937 and KG-1a cells. C. RIP assay indicated that circ_0004277 and miR-134-5p were enriched in anti-Ago2 group, suggesting circ_0004277 and miR-134-5p were directly interacted. D. RNA pull-down assay showed that circ_0004277 and miR-134-5p could combine with each other. E. qRT-PCR indicated that miR-134-5p expression was decreased in the circ_0004277 overexpression plasmid group compared with the empty plasmid group. F. qRT-PCR indicated that miR-134-5p expression was increased in the bone marrow samples of 37 AML patients compared with that of 37 healthy donors. G. circ_0004277 expression in AML tissues was negatively correlated with miR-134-5p expression in bone marrow samples of the patients with AML. ** P < 0.01, and *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Activity Assay, Pull Down Assay, Quantitative RT-PCR, Expressing, Over Expression, Plasmid Preparation

Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: MiR-134-5p inhibits the proliferation and aggressiveness of KG-1a and U937 cells. A. qRT-PCR showed that miR-134-5p expression was up-regulated in AML cell lines (KG-1a, THP-1, K562 and U937) compared with the human normal stromal cells (HS-5). B. qRT-PCR revealed that miR-134-5p expression was down-regulated in U937 and KG-1a cells transfected with miR-134-5p inhibitor. C. CCK-8 assay showed the viability of KG-1a and U937 cells was significantly decreased after the transfection of anti-miR-134-5p. D. Transwell assay indicated that the migration and invasion of KG-1a and U937 cells were decreased after the transfection of anti-miR-134-5p. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Transwell Assay, Migration

Journal: Bioengineered

Article Title: Circular RNA circ_0004277 Inhibits Acute Myeloid Leukemia Progression Through MicroRNA-134-5p / Single stranded DNA binding protein 2

doi: 10.1080/21655979.2022.2059609

Figure Lengend Snippet: Circ_0004277 modulates the biological behaviors of AML cells by sponging miR-134-5p. A. qRT-PCR showed that the miR-134-5p mimics have no effect on the expression of circ_0004277. B. qRT-PCR indicated that miR-134-5p mimics could up-regulate the expression of miR-134-5p. C. CCK-8 assay showed that miR-134-5p mimics could reverse the effect of circ_0004277 overexpression plasmid on the viability of U937 and KG-1a cells. D. Transwell assay showed that the migration and invasion of U937 and KG-1a cells transfected with circ_0004277 overexpression plasmid was reversed by miR-134-5p mimics. ** P < 0.01, *** P < 0.001.

Article Snippet: Human bone marrow stromal cell line HS-5 and human AML cell lines (KG-1a, THP-1, K562 and U937) were procured from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, CCK-8 Assay, Over Expression, Plasmid Preparation, Transwell Assay, Migration, Transfection

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: Treatment with daunorubicin increases autophagy in K562 cells. ( A ) Schematic representation of the autophagy process with its components and related molecules. ( B ) K562 cells were treated with different indicated doses of daunorubicin (DNR) or vehicle (DMSO) for 24 h. ( C , D ) K562 cells were treated with 2 µM of daunorubicin for 24 h and 10 µM of chloroquine (CQ) for the last 4 h. ( B , C ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated conditions, and in grey: CQ conditions compared with DNR and CQ conditions). ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown.

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Western Blot, Expressing, Control, Software, Labeling, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: Treatment with 3-MA partially prevents the effects of DNR on autophagy and cell growth. K562 were treated with 2 µM of daunorubicin and 3 mM of 3-methyladenine (3-MA). ( A ) Proteins were extracted after 24 h, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. The untreated conditions were set to 1. The normalized means are shown with SEM. (*, p < 0.05, **, p < 0.01, in black: all the conditions compared with the untreated conditions, and in grey: DNR conditions compared with DNR and 3-MA conditions). ( B ) Viable cells were counted in the presence of Trypan Blue after 1, 2, 3, and 4 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (***, p < 0.001).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Western Blot, Expressing, Control, Software

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p inhibits autophagy induced by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h and with 10 µM of chloroquine (CQ) for the last 16 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR. Normalization was completed with the endogenous control RNU44 . ( B ) Proteins were extracted, and the level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3 (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( A , C , D ) The untreated scrambled condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the untreated scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p decreases the downregulation of cell growth by daunorubicin. K562 cells were transiently transfected with miR-15a-5p mimic (miR-15a) or scrambled mimic (Sc) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h ( A ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (*, p < 0.05, ***, p < 0.001). ( B ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. The untreated scrambled condition was set to 1. The normalized means are shown with SEM (**, p < 0.01).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Transfection, Control, Viability Assay, Luminescence Assay

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: Inhibition of mir-15a-5p induces autophagy. ( A – C ) K562 cells were transiently transfected with miR-15a-5p inhibitor (15ai) or scrambled inhibitor (Sci) as control for 48 h, and cells were treated with 2 µM of daunorubicin (DNR) for the last 24 h. ( A ) RNA was extracted and miR-15a-5p expression was measured by RT-qPCR and normalized with the expression of RNU44 . ( B ) Protein were extracted, and level of autophagy was analyzed by Western blot with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Expression of β-actin was also detected as a loading control. ( C ) The Western blotting band intensities of three independent experiments were quantified using the ImageJ software. ( D ) Double-labeling immunofluorescence and representative images of LC3B (in green) and LAMP-2 (in red). Nuclear staining (DAPI, blue) is also shown. LC3B and LAMP-2 immunofluorescence intensities of three independent experiments were quantified using the ImageJ software and divided by the nuclear immunofluorescence. ( D , E ) K562 cells were transiently transfected with miR-15a-5p inhibitor (miR-15ai) or scrambled inhibitor (Sci) as control, and cells were treated with 0.1 µM of daunorubicin (DNR), 0.5 µM of DNR, or vehicle for 72 h. ( D ) Viable cells were counted in the presence of Trypan Blue after 1, 2, and 3 days of culture. ( E ) Cell viability assay (CellTiter-Glo ® Luminescent assay) was performed at 72 h of treatment. ( A – C , E ) The untreated scrambled conditions were set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, all the conditions compared with the untreated scrambled condition). ( D ) The mean of three independent experiments is shown with SEM. The statistical analysis was by a two-way ANOVA followed by Bonferroni’s test (**, p < 0.01).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Inhibition, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Software, Labeling, Immunofluorescence, Staining, Viability Assay, Luminescence Assay

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p directly regulates the expression of autophagy target genes. ( A , B ) K562 cells were transiently transfected with miR-15a-5p mimic (15a) or scrambled mimic (Sc) as control and treated with 2 µM of daunorubicin (DNR) for 24 h before protein and RNA extraction. ( A ) Protein expression of ATG9A, ATG14, and GABARAPL1 was analyzed by Western blotting. Expression of β-actin was also detected as a loading control. ( B ) The expression of ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of a housekeeping gene RPLP0. ( C ) HEK 293T cells were co-transfected with wild-type (WT) or mutated (Mut∆) pMIR-luciferase vector, with either miR-15a-5p mimic or the scrambled oligonucleotide as control, and the pEF-β-galactosidase vector as internal control. After 24 h of transfection, cells were lysed, and the luminescence and β-gal activities were measured. ( B , C ) The normalized means of three independent experiments are shown with SEM. The scrambled condition was set to 1. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, in black: all the conditions compared with the scrambled condition, and in grey: miR-15a conditions compared with scrambled conditions, n.s.: non-significant).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Expressing, Transfection, Control, RNA Extraction, Western Blot, Quantitative RT-PCR, Luciferase, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: MiR-15a-5p Confers Chemoresistance in Acute Myeloid Leukemia by Inhibiting Autophagy Induced by Daunorubicin

doi: 10.3390/ijms22105153

Figure Lengend Snippet: miR-15a-5p regulates autophagy by targeting autophagy genes. K562 cells were transiently co-transfected with miR-15a-5p inhibitor or scrambled inhibitor as control and with either a combination of four siRNA-targets ( ATG9A, ATG14, GABARAPL1, and SMPD1 ) or a siRNA-control for 24 h before protein and RNA extractions. ( A ) Level of autophagy was analyzed by Western blotting with anti-LC3B, anti-LAMP-2, and anti-p62 antibodies. Protein expression of ATG9A, ATG14, and GABARAPL1 was also analyzed, as well as the expression of β-actin as a loading control. ( B ) In all the conditions, the expression of miR-15a-5p, ATG9A, ATG14, GABARAPL1, and SMPD1 was measured by RT-qPCR and normalized with the expression of RNU44 for miR-15a-5p or with RPLP0 for the autophagy target genes. The scrambled inhibitor and siRNA condition was set to 1. The normalized means of three independent experiments are shown with SEM. (*, p < 0.05, **, p < 0.01, ***, p < 0.001, all the conditions compared with the scrambled condition).

Article Snippet: The human AML cell lines K562 and KG1a (purchased from DSMZ, Braunschweig, Germany) were cultured in RPMI 1640 medium (Lonza) supplemented with 10% or 20% FBS, respectively, and with 50 U/mL penicillin and 50 mg/mL streptomycin.

Techniques: Transfection, Control, Western Blot, Expressing, Quantitative RT-PCR

Journal: Blood Cancer Journal

Article Title: Antileukemic activity of the HSP70 inhibitor pifithrin-μ in acute leukemia

doi: 10.1038/bcj.2011.28

Figure Lengend Snippet: IC50 and maximum inhibition values of PFT-μ in leukemic cell lines and primary cells derived from AML patients

Article Snippet: The human cell lines KG-1a (AML), NALM-6 (B-precursor ALL), TOM-1 (B-precursor ALL; BCR - ABL positive), Jurkat, BE-13 (both T-cell leukemia) and K562 (chronic myeloid leukemia, blast crisis) were obtained from the DSMZ (Braunschweig, Germany) and cultured as recommended.

Techniques: Inhibition, Derivative Assay