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

Abstract

Exosomes secreted by glioma cells can carry a number of bioactive molecules. As the most abundant noncoding RNA in exosomes, microRNAs (miRNAs) are involved in signaling between tumor cells in a number of ways. In addition, hypoxia is an important feature of the microenvironment of most tumors. The present study investigated the effect of miR‑29a‑3p in glioma exosomes on the proliferation and apoptosis levels of U251 glioma cells under hypoxia. Qualitative PCR results showed that the expression level of miR‑29a‑3p in plasma exosomes of glioma patients was lower than that of normal subjects. By conducting hypoxia experiments in vitro on U251 glioma cells, it was found that the expression level of miR‑29a‑3p decreased following hypoxia, while overexpression of miR‑29a‑3p significantly decreased the proliferation of U251 glioma cells and promoted apoptosis by inhibiting the expression of the antiapoptotic marker Bcl‑2 and increasing the expression of the proapoptotic marker Bax The potential targets of miR‑29a‑3p were predicted by online tools and validated by a dual‑luciferase gene reporter assay. miR‑29a‑3p was found to target and regulate PI3K, which in turn inhibited the activity of the PI3K‑AKT pathway, thereby reducing the expression of hypoxia inducible factor (HIF)‑1α protein. Furthermore, the effects of miR‑29a‑3p on proliferation and apoptosis in glioma cells in those processes could be reversed by the PI3K‑AKT agonist Recilisib. In addition, the inhibitory effect of miR‑29a‑3p on the PI3K/AKT/HIF‑1α regulatory axis could cause a decrease in the expression levels of pyruvate dehydrogenase kinase‑1 and pyruvate dehydrogenase kinase‑2 and eventually lead to a reduction in glycolysis in U251 glioma cells. Similarly, Recilisib slowed the inhibitory effect of miR‑29a‑3p on glycolysis and glycolysis‑related molecules. The results of this study tentatively confirm that miR‑29a‑3p carried by exosomes can be used as a novel diagnostic marker and a potential inhibitory molecule for glioma cells, providing a new theoretical and experimental basis for the precise clinical treatment of glioma.

Keywords: exosome; glioma; hypoxia inducible factor 1α; microRNA 29a‑3p.

Figure 1.

Plasma exosome expression of miR-29a-3p in healthy controls and glioma patients. Identification and analysis of particle size by transmission electron microscopy of peripheral blood exosomes. (A) Exosomes by transmission electron microscopy. (B) Peak diagram of exosome particle size. (C) Reverse transcription-quantitative PCR showed the expression levels of miR-29a-3p in plasma exosomes in healthy controls (n=20) and glioma patients (n=20). Data were expressed as the mean ± standard deviation and the experiment was repeated three times for each group, **P<0.01. miR, microRNA.

Figure 2.

Effect of miR-29a-3p on the proliferation and apoptosis levels of glioma cells under normoxic and hypoxic conditions. (A) The expression level of miR-29a-3p by reverse transcription-quantitative PCR, ***P<0.001 vs. miR-NC under hypoxia. (B) The effect of miR-29a-3p on the proliferation level of glioma cells U251, ^####P<0.0001 vs. miR-NC under normoxia; ***P<0.001, ****P<0.0001 vs. miR-NC under hypoxia. (C) Effect of miR-29a-3p on apoptotic levels in glioma cells U251, *P<0.05 vs. miR-NC under hypoxia, ***P<0.001 vs. miR-NC under normoxia. (D) Effect of miR-29a-3p on the expression levels of Bax, Bcl-2 and HIF-1α. *P<0.05, **P<0.01. All data were expressed as the mean ± standard deviation (n=3). miR, microRNA; NC, negative control; HIF-1α, hypoxia-inducible factor 1.

Figure 3.

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.

Figure 4.

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.

Figure 5.

The effect of miR-29a-3p on glycolysis levels in glioma cells. (A) The expression levels of HIF-1α, PDK1 and PDK2 were detected by western blotting, *P<0.05, **P<0.01, ***P<0.001. (B) The level of glycolysis in each group was detected by Seahorse energy metabolism assay; *P<0.05 vs. miR-NC, ^##P<0.01 vs. miR-29a-3p mimics. All data were expressed as the mean ± standard deviation (n=3). miR, microRNA; HIF-1α, hypoxia-inducible factor 1; PDK, pyruvate dehydrogenase kinase; NC, negative control.