Journal: Drug Design, Development and Therapy

Article Title: Exosomes Derived from Hypoxic Glioma Cells Reduce the Sensitivity of Glioma Cells to Temozolomide Through Carrying miR-106a-5p

doi: 10.2147/DDDT.S382690

Figure Lengend Snippet: MiR-106a-5p was upregulated in hypoxic glioma cells. ( A ) The level of miR-106a-5p in HEB, U87MG, SHG44 and U251MG cells was detected by RT-qPCR. ( B ) The level of HIF-1α in hypoxic or normoxia glioma cells was assessed by RT-qPCR. ( C ) The level of miR-106a-5p in hypoxic or normoxia glioma cells was assessed by RT-qPCR. **P< 0.01 compared to HEB or normoxia.

Article Snippet: SHG44 cells were transfected with mimics-ctrl (Invitrogen), miR-106a-5p mimics (Invitrogen) or miR-106a-5p inhibitor (Invitrogen) for 48 h by using lipofectamine 3000 (Invitrogen) in line with the protocol of manufacturer.

Techniques: Quantitative RT-PCR

Journal: Drug Design, Development and Therapy

Article Title: Exosomes Derived from Hypoxic Glioma Cells Reduce the Sensitivity of Glioma Cells to Temozolomide Through Carrying miR-106a-5p

doi: 10.2147/DDDT.S382690

Figure Lengend Snippet: MiR-106a-5p could be transferred from hypoxic glioma cells to glioma cells via exosomes. ( A ) Exosomes were isolated from hypoxic or normoxia glioma cells (H-exo or N-exo), and were observed by TEM. ( B ) NTA was applied for exosome identification. ( C ) The expressions of CD81, CD63 and calnexin in normoxia cells, hypoxia cells, H-exo or N-exo were examined by Western blot. ( D ) The exosomes absorbed by glioma cells were observed by fluorescence staining. ( E ) Glioma cells were treated with N-exo, H-exo or H-exo + miR-106a-5p inhibitor. The level of miR-106a-5p in glioma cells was assessed by RT-qPCR. **P< 0.01 compared to control. ## P< 0.01 compared to hypoxia Exo.

Article Snippet: SHG44 cells were transfected with mimics-ctrl (Invitrogen), miR-106a-5p mimics (Invitrogen) or miR-106a-5p inhibitor (Invitrogen) for 48 h by using lipofectamine 3000 (Invitrogen) in line with the protocol of manufacturer.

Techniques: Isolation, Western Blot, Fluorescence, Staining, Quantitative RT-PCR

Journal: Drug Design, Development and Therapy

Article Title: Exosomes Derived from Hypoxic Glioma Cells Reduce the Sensitivity of Glioma Cells to Temozolomide Through Carrying miR-106a-5p

doi: 10.2147/DDDT.S382690

Figure Lengend Snippet: Exosomes derived from hypoxic glioma cells reduced TMZ sensitivity in glioma cells through carrying miR-106a-5p. Glioma cells were treated with TMZ, TMZ + N-exo, TMZ + H-exo or TMZ + H-exo + miR-106a-5p inhibitor. ( A ) The viability of glioma cells was assessed by CCK8 assay. ( B ) The proliferation of glioma cells was detected by EdU staining. ( C ) The apoptosis of glioma cells was assessed by flow cytometry. **P< 0.01 compared to control. ## P< 0.01 compared to TMZ. ^^ P< 0.01 compared to TMZ + H-exo.

Article Snippet: SHG44 cells were transfected with mimics-ctrl (Invitrogen), miR-106a-5p mimics (Invitrogen) or miR-106a-5p inhibitor (Invitrogen) for 48 h by using lipofectamine 3000 (Invitrogen) in line with the protocol of manufacturer.

Techniques: Derivative Assay, CCK-8 Assay, Staining, Flow Cytometry

Journal: Drug Design, Development and Therapy

Article Title: Exosomes Derived from Hypoxic Glioma Cells Reduce the Sensitivity of Glioma Cells to Temozolomide Through Carrying miR-106a-5p

doi: 10.2147/DDDT.S382690

Figure Lengend Snippet: PTEN was identified to be the downstream target of miR-106a-5p. ( A ) The downstream mRNA of miR-106a-5p was predicted by targetscan. ( B ) The relative luciferase activity in WT/MT-PTEN was assessed by dual luciferase assay. ( C ) Glioma cells were transfected with mimics-ctrl or miR-106a-5p mimics. The level of PTEN in glioma cells was investigated by RT-qPCR. **P< 0.01 compared to control.

Article Snippet: SHG44 cells were transfected with mimics-ctrl (Invitrogen), miR-106a-5p mimics (Invitrogen) or miR-106a-5p inhibitor (Invitrogen) for 48 h by using lipofectamine 3000 (Invitrogen) in line with the protocol of manufacturer.

Techniques: Luciferase, Activity Assay, Transfection, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences

Article Title: CircAKT3 alleviates postoperative cognitive dysfunction by stabilizing the feedback cycle of miR-106a-5p/HDAC4/MEF2C axis in hippocampi of aged mice

doi: 10.1007/s00018-024-05156-9

Figure Lengend Snippet: Increased Neuronal apoptosis and decreased MEF2C expression were observed in the hippocampus of POCD mice. A TUNEL staining showed an elevated apoptosis level in the hippocampus of the POCD group. B Western blot revealed the significantly decreased anti-apoptotic B-cell lymphoma 2 (BCL2) protein and increased pro-apoptotic cleaved caspase 3 protein as well as BCL2-associated X (Bax) protein in POCD mice, in comparison to NPOCD and control group. C Immunofluorescence showed a decrease in the fluorescence intensities in the hippocampal CA3 neurons of POCD mice. Scale bar, 50 μm or 100 μm as presented. The data was shown as mean ± SD. The P values were determined by one-way ANOVA with multiple comparison tests using GraphPad software 9.5 ( B ); *** P < 0.001, **** P < 0.0001, ns (not significant) means P > 0.05

Article Snippet: The overexpression plasmid of pcDNA3.1 containing HDAC4 or MEF2C, miR-106a–5p mimics, negative control, and inhibitor were all synthesized by Ribobio (China).

Techniques: Expressing, TUNEL Assay, Staining, Western Blot, Comparison, Control, Immunofluorescence, Fluorescence, Software

Journal: Cellular and Molecular Life Sciences

Article Title: CircAKT3 alleviates postoperative cognitive dysfunction by stabilizing the feedback cycle of miR-106a-5p/HDAC4/MEF2C axis in hippocampi of aged mice

doi: 10.1007/s00018-024-05156-9

Figure Lengend Snippet: miR-106a-5p inhibited the expression of HDAC4 at the post-transcriptional level, then elevated the level of MEF2C protein. A The potential binding site predicted by Miranda between miR-106a-5p and HDAC4 was shown. B – D There was a decrease in both protein and mRNA of HDAC4 and an increase in MEF2C protein and mRNA in the POCD mice compared to NPOCD and control group. E miR-106a-5p mimics significantly reduced the level of HDAC4 protein and increased the level of MEF2C protein in N2a cells. F HDAC4 mRNA was not decreased after the transfection of miR-106a-5p mimics in comparison to the negative control (NC) group. G miR-106a-5p mimics significantly increased the level of MEF2C mRNA in N2a cells. H Conversely, the inhibitor of miR-106a-5p significantly increased the level of HDAC4 protein but reduced the level of MEF2C protein compared to the NC cells. I Western blot revealed that there was a significantly increased level of Bcl2 protein and a lower level of cleaved caspase 3 as well as Bax protein in N2a cells with MEF2C overexpression. J The relative luciferase activity of firefly luciferase plasmid containing HDAC4-WT segment was inhibited by miR-106a-5p mimics but not NC. Furthermore, there was no significant difference in luciferase plasmid containing HDAC4-Mut segment co-transfected with mimics or NC. The P values were determined by one-way ANOVA with multiple comparison test ( B – D ) or two-tailed unpaired Student’s t test ( E – I ); * P < 0.05, ** P < 0.01, **** P < 0.0001, ns means P > 0.05

Article Snippet: The overexpression plasmid of pcDNA3.1 containing HDAC4 or MEF2C, miR-106a–5p mimics, negative control, and inhibitor were all synthesized by Ribobio (China).

Techniques: Expressing, Binding Assay, Control, Transfection, Comparison, Negative Control, Western Blot, Over Expression, Luciferase, Activity Assay, Plasmid Preparation, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: CircAKT3 alleviates postoperative cognitive dysfunction by stabilizing the feedback cycle of miR-106a-5p/HDAC4/MEF2C axis in hippocampi of aged mice

doi: 10.1007/s00018-024-05156-9

Figure Lengend Snippet: CircAKT3 inhibited the expression of HDAC4 protein and elevated the level of MEF2C protein by increasing the level of miR-106a-5p. A The overexpression of circAKT3 markedly reduced the level of HDAC4 protein and increased the level of MEF2C protein in N2a cells. This effect was blocked by the inhibitor of miR-106a-5p. B There was no significant difference in HDAC4 mRNA between the three groups. C MEF2C mRNA was notably increased after the overexpression of circAKT3, which was further reversed by miR-106a-5p inhibitor. D CircAKT3 was significantly decreased in the hippocampus of mice with anesthesia and surgery (AS). Additionally, there was a significant increase of circAKT3 in the mice that underwent circAKT3 overexpression in hippocampal CA3 neurons following anesthesia and surgery (AS-CircAKT3 OE). E The level of MEF2C mRNA was reduced in the AS group, which was reversed in the AS-CircAKT3 OE group. F The significant increase of HDAC4 protein and reduction of MEF2C protein in the hippocampus of AS mice were reversed by circAKT3 overexpression in vivo. ( G and H ) Immunofluorescence showed that circAKT3 overexpression in vivo with AS reduced the nuclear localization of HDAC4 protein but increased the expression of MEF2C protein in both the nucleus and cytoplasm compared to the AS group. I , J RNA immunoprecipitation showed there was no direct binding between circAKT3 and HDAC4 protein. Compared to the anti-IgG group, the anti-HDAC4 group showed no higher level of circAKT3 ( I ). ns means P > 0.05 (anti-HDAC4 group vs anti-IgG group). Western blot validated the specificity and binding affinity of primary antibodies with magnetic beads for RNA immunoprecipitation ( J ). The scale bar was 20 μm ( G , H ). The P values were determined by one-way ANOVA with multiple comparison tests; * P < 0.05, ** P < 0.01, **** P < 0.0001, ns means P > 0.05

Article Snippet: The overexpression plasmid of pcDNA3.1 containing HDAC4 or MEF2C, miR-106a–5p mimics, negative control, and inhibitor were all synthesized by Ribobio (China).

Techniques: Expressing, Over Expression, In Vivo, Immunofluorescence, RNA Immunoprecipitation, Binding Assay, Western Blot, Magnetic Beads, Comparison

Journal: Cellular and Molecular Life Sciences

Article Title: CircAKT3 alleviates postoperative cognitive dysfunction by stabilizing the feedback cycle of miR-106a-5p/HDAC4/MEF2C axis in hippocampi of aged mice

doi: 10.1007/s00018-024-05156-9

Figure Lengend Snippet: MEF2C promoted the transcription of miR-106a-5p and HDAC4 inhibited the transcription of miR-106a-5p by reducing the level of MEF2C protein. A The level of HDAC4 and MEF2C protein was remarkably elevated in the cells transfected with corresponding plasmids to overexpress HDAC4 or MEF2C. B – D The levels of miR-106a-5p, pre-miR-106a, and pri-miR-106a detected by qPCR were significantly increased by MEF2C overexpression. E The level of MEF2C protein presented a decreased trend in N2a cells with HDAC4 overexpression. F The overexpression of HDAC4 protein inhibited the expression of miR-106a-5p in N2a cells revealed by qPCR. G In comparison to the firefly luciferase reporter containing miR-106a promoter, the relative luciferase activity of miR-106a promoter (PM) was elevated by co-transfection with MEF2C overexpression plasmids, and decreased by co-transfection with HDAC4 overexpression plasmids in 293T cells. H CUT&TAG qPCR unveiled that MEF2C mainly binds to the promoter of miR-106a at the binding site-2 (BS-2), BS-3, and BS-4 in N2a cells (control vs negative control). Furthermore, elevated MEF2C protein induced by circAKT3 overexpression significantly enhanced the level of binding to the miR-106a promoter at BS-2, BS-3, and BS-4. For H in the figure, ## means Negative control vs Control or CircAKT3 OE group and P < 0.01, ### P < 0.001, #### P < 0.0001, ** (means Control vs CircAKT3 OE group) P < 0.01, *** P < 0.001, **** P < 0.0001. The P values were determined by one-way ANOVA with multiple comparison tests ( G , H ) or two-tailed unpaired Student’s t test ( A – F ); * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns means P > 0.05

Article Snippet: The overexpression plasmid of pcDNA3.1 containing HDAC4 or MEF2C, miR-106a–5p mimics, negative control, and inhibitor were all synthesized by Ribobio (China).

Techniques: Transfection, Over Expression, Expressing, Comparison, Luciferase, Activity Assay, Cotransfection, Binding Assay, Control, Negative Control, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: CircAKT3 alleviates postoperative cognitive dysfunction by stabilizing the feedback cycle of miR-106a-5p/HDAC4/MEF2C axis in hippocampi of aged mice

doi: 10.1007/s00018-024-05156-9

Figure Lengend Snippet: The sequence of primers or templates for qPCR or PCR was used in the study

Article Snippet: The overexpression plasmid of pcDNA3.1 containing HDAC4 or MEF2C, miR-106a–5p mimics, negative control, and inhibitor were all synthesized by Ribobio (China).

Techniques: Sequencing, Binding Assay

Journal: Cellular and Molecular Life Sciences

Article Title: CircAKT3 alleviates postoperative cognitive dysfunction by stabilizing the feedback cycle of miR-106a-5p/HDAC4/MEF2C axis in hippocampi of aged mice

doi: 10.1007/s00018-024-05156-9

Figure Lengend Snippet: The top 30 most significant differentially expressed genes of the hippocampus between control and AS aged mice (AS vs control group)

Article Snippet: The overexpression plasmid of pcDNA3.1 containing HDAC4 or MEF2C, miR-106a–5p mimics, negative control, and inhibitor were all synthesized by Ribobio (China).

Techniques: Control

Journal: Cellular and Molecular Life Sciences

Article Title: CircAKT3 alleviates postoperative cognitive dysfunction by stabilizing the feedback cycle of miR-106a-5p/HDAC4/MEF2C axis in hippocampi of aged mice

doi: 10.1007/s00018-024-05156-9

Figure Lengend Snippet: CircAKT3 improved postoperative cognitive dysfunction by stabilizing the feedback cycle of the miR-106a-5p/HDAC4/MEF2C axis. CircAKT3 could elevate the level of miR-106a-5p, which further decreased the level of HDAC4 protein and increased the level of MEF2C protein to inhibit neuronal apoptosis. MEF2C would in turn promote the transcriptional activation of miR-106a and form a feedback loop. However, circAKT3 is reduced in the hippocampal neuron of POCD mice and increased the expression of HDAC4 protein through a decrease in miR-106a-5p. Then the effect of MEF2C on miR-106a promoter and neuronal apoptosis was weakened, which promoted the occurrence and development of POCD. The schematic picture was drawn by BioRender

Article Snippet: The overexpression plasmid of pcDNA3.1 containing HDAC4 or MEF2C, miR-106a–5p mimics, negative control, and inhibitor were all synthesized by Ribobio (China).

Techniques: Activation Assay, Expressing