Therefore, at both the mRNA and protein levels, we demonstrated that miR224-3p regulates autophagy independent of the ERK/AKT/mTOR pathway, SIRT1 and ATG3. To further define the involvement of KRas G12C inhibitor 4 ATG5 and FIP200 in the suppression of autophagy by miR224-3p, both ATG5 and FIP200 overexpressing-plasmids were co-transfected into miR224-3p-overexpressing U251 and U87 cells. normoxia. In addition, we exhibited that miR224-3p inhibited autophagy by directly suppressing the expression of two autophagy-related genes (ATGs), ATG5 and FAK family-interacting protein of 200 kDa (FIP200). Furthermore, = 3. **< 0.01, ***< 0.001, Student's 2-tailed test. To distinguish whether LC3B-II accumulation is due to autophagy induction or to a block in downstream actions, we performed autophagic flux assays. Sequestosome 1 (SQSTM1/p62), a polyubiquitin-binding protein, is selectively incorporated into autophagosomes through direct binding to LC3B and efficiently degraded during autophagy. Thus, the total cellular levels of SQSTM1 reflect autophagic activity [23]. The late autophagy inhibitor bafilomycinA1 (BAF) blocked hypoxia-induced p62 degradation in U251 and U87 cells. BAF treatment significantly increased LC3B-II levels under hypoxia (Physique ?(Figure1B).1B). These data demonstrate that hypoxia induces the autophagic activity of human GBM cells. Hypoxia induces miR224-3p down-regulation in glioblastoma cell lines, and miR224-3p expression is usually low in human glioma Recently, several lines of evidence have directly established miRNAs as key elements in the molecular response of tumor cells to hypoxia. To further understand the miRNA signature of GBM cells under hypoxia, we identified differentially expressed miRNAs using a miRNA microarray (ArrayExpress accession number: E-MTAB-3886). In total, 84 miRNAs were differentially expressed (Supplementary Physique S2A, shown as a Volcano plot), including eight up-regulated (= 3. C. miR224-3p expression in glioma and normal brain tissues was determined by q-PCR analysis and grouped according to WHO I, II grade (= 14), III, IV grade KRas G12C inhibitor 4 (= 16) and normal brain tissue (= 6). The boxes represent the lower and the upper quartiles with medians; the whiskers illustrate the 10 to 90 percentiles of the samples. *< 0.05, **< 0.01, ***< 0.001, Student's 2-tailed test or one-way ANOVA. To further validate the expression of miR224-3p, we measured miR224-3p expression in U251 and U87 cells under hypoxic conditions at 24 h and 48 h by q-PCR. The expression levels of miR210 increased under hypoxic culture conditions (Physique ?(Physique2B,2B, upper panel), indicating effective hypoxia. In contrast, the expression levels of miR224-3p were low under normoxic culture conditions. When exposed to hypoxia, miR224-3p was significantly down-regulated in a time-dependent manner in both GBM cell lines (Physique ?(Physique2B,2B, lower panel). At 48 h after hypoxia treatment, miR224-3p expression decreased more than 5-fold. The consistency between the miRNA microarray data and the results of the q-PCR assay demonstrate the validity of the microarray. To evaluate the clinical significance of miR224-3p, thirty glioma specimens [sixteen high-grade tissues (World Health Organization (WHO); WHO III-IV) and fourteen low-grade tissues (WHO I-II)] and six normal brain specimens were collected to detect miR224-3p expression by q-PCR. MiR224-3p was down-regulated in human glioma tissues compared with normal brain tissues (< 0.001). There was no significant difference between expression in high-grade glioma and low-grade glioma (Figure ?(Figure2C).2C). Therefore, we propose that miR224-3p potentially inhibits hypoxia-induced autophagy and is expressed at low levels in human glioma. MiR224-3p influences glioblastoma cell autophagic activity After screening the hypoxia GBM cell miRNA microarray, we detected miR224-3p as a novel autophagy-related miRNA. To precisely explore the role of miR224-3p in autophagic activity, we repeated LC3 conversion and GFP-LC3 puncta-formation assays in both U251 and U87 cell lines. MiR224-3p inhibitors used to inhibit the level of endogenous miR224-3p were transfected into U251 and U87 cells. The expression of LC3B-II increased and that of p62 decreased (Figure ?(Figure3A),3A), suggesting that the miR224-3p inhibitor enhanced autophagy in the transfected cells. At the same time, we also examined the location of GFP-LC3 by fluorescence microscopy in miR224-3p inhibitor-transfected U251 and U87 cells stably expressing the GFP-LC3 fusion protein. There was a significant increase in GFP-LC3 puncta in miR224-3p inhibitor-transfected cells compared with the negative control cells (Figure 3C, 3D, 3E). In the same way, miRNA224-3p mimic was transfected into both cell lines, and KRas G12C inhibitor 4 autophagy was slightly Rabbit Polyclonal to mGluR8 inhibited, as indicated by the decreased LC3B-II expression and increased accumulation of p62 (Supplementary Figure S3B). Open in a separate window Figure 3 miR224-3p influences glioblastoma cell autophagic activityA..