Journal: Molecular Medicine Reports

Article Title: Effect of miR‑29a‑3p in exosomes on glioma cells by regulating the PI3K/AKT/HIF‑1α pathway

doi: 10.3892/mmr.2023.12959

Figure Lengend Snippet: miR-29a-3p inhibits HIF-1α expression by targeting the PI3K/AKT pathway. (A) A schematic diagram of the binding site of miR-29a-3p to the PI3K gene and the PI3K mutation site was predicted by TargetScan. (B) Dual luciferase gene reporter assay was used to verify the targeting negative regulatory effect of miR-29a-3p on PI3K, ****P<0.0001. The expression level of PI3K was analyzed by (C) quantitative PCR and (D) western blotting after U251 cells were transfected with miR-29a-3p mimics and inhibitor, *P<0.05, ***P<0.001. (E) Effect of miR-29a-3p on the expression levels of the PI3K downstream molecules AKT, p-AKT and HIF-1α, *P<0.05, **P<0.01. All data were expressed as the mean ± standard deviation (n=3). miR, microRNA; HIF-1α, hypoxia-inducible factor 1; NC, negative control; UTR, 3′ untranslated region; wt, wild-type; mu, mutant; p-, phosphorylated.

Article Snippet: The miR-29-3p-5p mimic/inhibitor was synthesized by Shanghai Jima Co., Ltd., the double luciferase gene reporter assay was commissioned from Tianjin Sheweisi Biotechnology Co., Ltd., the CCK-8 cell viability assay kit was purchased from Bestbio, Annexin V-phycoerythrin (PE) and 7-AAD were purchased from Becton, Dickinson and Company, rabbit anti-human PI3K (cat. no. 4249), AKT (cat. no. 4685) and phosphorylated (p-)AKT (cat. no. 4060) antibodies were purchased from Cell Signaling Technology, Inc., rabbit anti-human HIF-1α (cat. no. 20960-1-AP) antibodies were purchased from Proteintech Group, Inc. and rabbit anti-human Bax (cat. no. 41162) antibody was purchased from Cell Signaling Technology, Inc., rabbit anti-human Bcl-2 (cat. no. ab32124), pyruvate dehydrogenase kinase (PDK)-1 (cat. no. ab202468), PDK2 (cat. no. ab154549) from Abcam, β-actin (cat. no. TA-09), goat anti-rabbit secondary antibody (cat. no. ZB-5301) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., the PVDF membrane was purchased from MilliporeSigma (cat. no. ISEQ00010), the ECL luminescent solution was purchased from Jiangsu KGI Biotechnology Co., Ltd. (cat. no. KGP1127) and the glycolysis assay kit was purchased from Agilent (cat. no. 103020-100).

Techniques: Expressing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Standard Deviation, Negative Control

Journal: Molecular Medicine Reports

Article Title: Effect of miR‑29a‑3p in exosomes on glioma cells by regulating the PI3K/AKT/HIF‑1α pathway

doi: 10.3892/mmr.2023.12959

Figure Lengend Snippet: The effect of miR-29a-3p via the PI3K/AKT/HIF-1α pathway on glioma cells' proliferation and apoptosis levels. (A) The proliferation level was detected by CCK-8 assay, **P<0.01, ***P<0.001, ****P<0.0001 vs. miR-NC + DMSO; # P<0.05, ### P<0.001 vs. miR-NC. (B) The expression levels of HIF-1α, Bcl-2, Bax, AKT, PI3K and the ratio of p-AKT/AKT, were detected by western blotting, **P<0.01, ****P<0.0001. (C) The levels of apoptosis in each group were detected by flow cytometry, **P<0.01, ****P<0.0001. All data were expressed as the mean ± standard deviation (n=3). miR, microRNA; HIF-1α, hypoxia-inducible factor 1; NC, negative control; HIF-1α, hypoxia-inducible factor 1; p-, phosphorylated; PE, phycoerythrin.

Article Snippet: The miR-29-3p-5p mimic/inhibitor was synthesized by Shanghai Jima Co., Ltd., the double luciferase gene reporter assay was commissioned from Tianjin Sheweisi Biotechnology Co., Ltd., the CCK-8 cell viability assay kit was purchased from Bestbio, Annexin V-phycoerythrin (PE) and 7-AAD were purchased from Becton, Dickinson and Company, rabbit anti-human PI3K (cat. no. 4249), AKT (cat. no. 4685) and phosphorylated (p-)AKT (cat. no. 4060) antibodies were purchased from Cell Signaling Technology, Inc., rabbit anti-human HIF-1α (cat. no. 20960-1-AP) antibodies were purchased from Proteintech Group, Inc. and rabbit anti-human Bax (cat. no. 41162) antibody was purchased from Cell Signaling Technology, Inc., rabbit anti-human Bcl-2 (cat. no. ab32124), pyruvate dehydrogenase kinase (PDK)-1 (cat. no. ab202468), PDK2 (cat. no. ab154549) from Abcam, β-actin (cat. no. TA-09), goat anti-rabbit secondary antibody (cat. no. ZB-5301) was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., the PVDF membrane was purchased from MilliporeSigma (cat. no. ISEQ00010), the ECL luminescent solution was purchased from Jiangsu KGI Biotechnology Co., Ltd. (cat. no. KGP1127) and the glycolysis assay kit was purchased from Agilent (cat. no. 103020-100).

Techniques: CCK-8 Assay, Expressing, Western Blot, Flow Cytometry, Standard Deviation, Negative Control