MicroRNA-93 Regulates Hypoxia-Induced Autophagy by Targeting ULK1

Figure 1

miR-93 targets ULK1 and regulates its expression. (a) Predicted binding sites of mmu-miR-93 to the 3′ UTR of ULK1. The short vertical lines indicate complementary paired bases of miR-93 and ULK1. The 302–309 nucleotides of ULK1 3′ UTR present the miRNA regulatory element (MRE). The underlined bases were mutant MRE. (b) Luciferase reporter assay by the interaction between miR-93 and the predicted MRE in MEFs. Each luciferase construct was cotransfected with scramble NC (NC), scramble IN-NC (IN-NC), miR-93 mimics (93), or miR-93 inhibitor (IN-93). At approximately 24 h after transfection, the luciferase activity was detected. The firefly luciferase activity was normalized to Renilla. Data were shown as the mean ± S.D. from three independent experiments. . (c) miR-93 represses the endogenous ULK1 expression in MEFs. NC, 93, IN-NC, and IN-93 were transfected into MEFs. At 24 h after transfection, qPCR was performed to detect the expression of miR-93 and ULK1. Data were from three independent experiments. ; ; . (d) miR-93 represses the endogenous ULK1 expression in CHO cells. NC, 93, IN-NC, and IN-93 were transfected into CHO cells. At 24 h after transfection, qPCR was performed to detect the expression of miR-93 and ULK1. Data were from three independent experiments. ; . (e) Cell lysates in (c) were prepared and subjected to Western blot analysis by using anti-ULK1 and anti-β-actin antibody. The densitometric ratios of ULK1/actin from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (f) Cell lysates in (d) were prepared and subjected to Western blot analysis by using anti-ULK1 and anti-β-actin antibody. The densitometric ratios of ULK1/actin from the samples were quantified by using ImageJ. Data were from three independent experiments. Representative data are shown. (g, h) MEFs and CHO cells were transfected with miR-93 (93) and miR-93 inhibitor (IN-93) for 24 h, 48 h, and 72 h, individually, setting group NC as a control. Cell lysates were prepared and subjected to Western blot analysis by using anti-ULK1, anti-ATG5, anti-ATG7, anti-Beclin1, anti-Tom20, and anti-Gapdh. Data were from three independent experiments. Representative data are shown.

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