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Double-stranded RNA inhibits the growth and migration of malignant gliomas through the activation of the PKR/eIF2α pathway

Qun YAN, Sha-sha BIAN, Min-feng SHU

Fudan University Journal of Medical Sciences, 2024, 51(03): 295-305. DOI: 10.3969/j.issn.1672-8467.2024.03.002

Fig 2 Zeocin inhibited the survival ability of D54 and U251 glioma cells A-B: Plate cloning experiment tested the survival ability of D54 glioma cells treated with different concentrations of Zeocin; C-D: Plate cloning experiment tested the survival ability of U251 glioma cells treated with different concentrations of Zeocin; E-F: CCK8 experiment tested the survival ability of D54 and U251 glioma cells treated with different concentrations of Zeocin. All P values were compared with 0 μg/mL group. n=3, ⁽¹⁾P < 0.05, ⁽²⁾P < 0.001, ⁽³⁾P < 0.000 1.

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Fig 1 Zeocin inhibited the migration ability of D54 and U251 glioma cells A-B: Scratch experiment tested migration ability of D54 glioma cells treated with different concentrations of Zeocin; C-D: Scratch experiment tested migration ability of U251 glioma cells treated with different concentrations of Zeocin. n=3, ⁽¹⁾P < 0.001, ⁽²⁾P < 0.000 1.
Fig 3 Zeocin induced endogenous dsRNA production in glioma A: J2 immunofluorescence imaging and analysis tested the endogenous RNA levels in D54 glioma cells treated with different concentrations of Zeocin; B: J2 immunofluorescence imaging and analysis tested the endogenous RNA levels in U251 glioma cells treated with different concentrations of Zeocin; C-D: Image J counted the number of dsRNAs in D54 and U251 glioma cells; MOI: multiplicity of infection. HSV-1 was positive control, and all P values were compared with 0 μg/mL group. n=3, ⁽¹⁾P < 0.000 1.
Fig 4 Zeocin induced endogenous dsRNA production in glioma and activated the PKR pathway A-C: Western blot detected the PKR and eIF2α protein expression in D54 cells treated with different concentrations of Zeocin, the dose-dependent upregulation effect of p-PKR and p-eIF2α was shown in the red box; D-F: Western blot detected the PKR and eIF2α protein expression in U251 cells treated with different concentrations of Zeocin, the dose-dependent upregulation effect of p-PKR and p-eIF2α was shown in the red box; HSV-1 was positive control, and all P values were compared with 0 μg/mL group. n=3, ⁽¹⁾P < 0.01, ⁽²⁾P < 0.001, ⁽³⁾P < 0.000 1, ns: Not significant.
Fig 5 Zeocin decreased the m6A levels of malignant glioma cells A-B: Dot blotting assay detected the m6A level of D54 and U251 cells treated with different concentrations of Zeocin; C-D: Image J counted grayscale scan values of m6A levels in D54 and U251 glioma cells. n=3, ⁽¹⁾P < 0.01, ⁽²⁾P < 0.001, ⁽³⁾P < 0.000 1.
Fig 6 Zeocin inhibited the protein expression of methyltransferase of METTL14 A-B: Western blot assay detected METTL14 protein levels in D54 glioma cells treated with different concentrations of Zeocin, the dose-dependent downregulation effect of METTL14 was shown in the red box; C-D: Western blot assay detected METTL14 protein levels in U251 glioma cells treated with different concentrations of Zeocin, the dose-dependent downregulation effect of METTL14 was shown in the red box; E-F: Western blot detected the METTL14 protein expression at different time in D54 cells treated with the same concentrations of Zeocin; G-H: Western blot detected the METTL14 protein expression at different time in U251 cells treated with the same concentration of Zeocin; HSV-1 was positive control, and all P values were compared with 0 μg/mL group. n=3, ⁽¹⁾P < 0.000 1;ns: Not significant.
Fig 7 The mRNA level of methyltransferase in glioma cells which were treated with Zeocin A: RT-qPCR tested the mRNA level of METTL14 in D54 glioma cells which were treated with different concentrations of Zeocin; B: RT-qPCR tested the mRNA level of METTL14 in U251 glioma cells which were treated with different concentrations of Zeocin; HSV-1 was positive control, and all P-values were compared with 0 μg/mL group.⁽¹⁾P < 0.01; ns: Not significant.

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