microRNA-17 regulates the expression of ATG7 and modulates the autophagy process, improving the sensitivity to temozolomide and low-dose ionizing radiation treatments in human glioblastoma cells

Figures & data

Figure 1. Sequence alignment prediction between miR-17 and ATG7 and validation assays. (A) miRanda predicted miR-17 targets (squares) within the 3′-UTR region (with nucleotides coordinates) of human ATG7 sequence (NM_006395). miRanda likelihood of miR-17-mediated mRNA downregulation scores (mirSVRs) are reported. (B) Nucleotide alignments between miR-17 seed sequence and ATG7 wild-type and mutated sequences, respectively cloned into pMIRATG7WT and pMIRATG7MUT vectors (see Materials and Methods) (top); results of the luciferase reporter assay at 24 and 48 h post-transfection (p.t.) in T98G cells are expressed as Relative Luciferase Units (left); results were normalized to pMIR-REPORT vector (vehicle) transfections. (C) Real-time PCR quantitative expression levels of miR-17 and ATG7 in T98G cells, from a RIP-assay using EIF2C2/AGO2-co-immunoprecipitated RNA, described in Materials and Methods; controls are represented by mock (input) and IgG isotype RNA samples. Expression levels are normalized to miR-17 and ATG7 expression levels of input samples. RIP-assay is performed using Protein A (top) and G sepharose matrices (bottom). Results of three independent replicas are indicated with error bars. *P < 0.005, **P < 0.001.

Figure 2. miR-17 modulation of ATG7 and LC3B expression in T98G cells. (A) Relative expression of miR-17 (Real-time PCR) and ATG7 protein (immunoblotting), in T98G cells, transfected with miR-17 specific inhibitor (2.5 μM) and precursor molecules (2.5 μM) evaluated at 72h p.t. Data are normalized using β-Actin mRNA/protein levels. (B) LC3B protein expression after miR-17 inhibitory or precursor treatments (as above) in absence/presence of BafilomycinA1 (10 nM). LC3B-II expression levels were calculated normalizing LC3B-II to β-actin expression, according to the current autophagy guidelines.³³ Protein expression and densitometric analysis using ImageJ tool are evaluated at 72h p.t. Results of three independent experiments are indicated with error bars. *P < 0.005, **P < 0.001.

Figure 3. Combined effects of rapamycin and miR-17 inhibitor/precursor administration in T98G cells on LC3B and ATG7 protein expression. To evaluate the autophagy process activation by miR-17 modulation in T98G cells, rapamycin is administered at different concentrations (50−100 nM), after separate transfection of AmiR-17 or Pmir-17 molecules, at 2.5 µM concentration. LC3B-II and ATG7 protein expression, followed by densitometric analysis using ImageJ software, are evaluated after 72 h p.t. LC3B-II expression levels are calculated normalizing LC3B-II to β-Actin expression, according to the current autophagy guidelines.³³ Data are normalized using β-Actin protein levels and referred to mock untreated samples.

Figure 4. Autophagosomal LC3-FP expression in T98G cells after rapamycin and miR-17 inhibitor/precursor administration. T98G cells are transduced with BacMam LC3B-FP or BacMam LC3B(G120A)-FP viral particles (MOI = 30 each), and after 24 h treated with rapamycin (0−50−100 nM) or transfected with AmiR-17 or PmiR-17 (2.5 µM each). LC3B-FP or LC3B(G120A)-FP expression are analyzed after additional 24h. For DIC phase contrast-fluorescent photographs an inverted microscope (Eclipse TS100, Nikon) with 40× magnification is employed. (Scale bar: 10 μM).

Figure 5. Autophagosomal LC3-FP evaluation in T98G cells after Rapamycyn and miR-17 inhibitor/precursor administration. Ratio percentage between vesicles area and cells areas (Aves/Atot [%]) and number of total vesicles (Ves_tot) are calculated using the AUTOCOUNTER ImageJ Javascript tool as described.³⁵ Results are obtained analyzing different DIC phase contrast-fluorescent photographs for a total of 30 T98G cells for each of the mentioned mock, rapamycin, AmiR-17, and PmiR-17 treatments. *P < 0.005.

Figure 6. Combined effects of TMZ and miR-17 inhibitor (AmiR-17) treatment on short/long-term viability and ATG7/LC3B protein expression in T98G cells. T98G cells are transfected with AmiR-17 (2.5 μM) and, after 24 h, treated with TMZ (0−250−500 μM). (A) Short-term viability in T98G cells is evaluated using MTT assay after 48 h p.t. (B) For T98G long-term viability a clonogenic assay is performed after a two-weeks culturing condition. Statistics (number of clones and mean of each treatment) are reported in Table S1. (C) ATG7 and LC3B expression are evaluated by immunoblotting. For densitometric analysis, LC3B-II expression levels are calculated normalizing LC3B-II to β-actin expression, according to the current autophagy guidelines.³³ Results are normalized to mock untreated samples. **P < 0.001.

Figure 7. Combined effects of IR and miR-17 inhibitor (AmiR-17) treatment on miR-17/ATG7 expression and on long-term viability in U373-MG cells. (A) U373-MG cells are transfected with AmiR-17 (2.5 μM) 48 h before IR administration (0−1.2–5 Gy). miR-17 and ATG7 protein expression are evaluated by means of real-time quantitative PCR and immunoblotting/densitometric analysis, respectively. Results are normalized to mock untreated samples. (B) For U373-MG long-term viability a clonogenic assay is performed after a 2-weeks culturing condition. Statistics (clones number and mean of each treatment) are reported in Table S2. *P < 0.005, **P < 0.001.

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