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. 2021 Jul 5;17(11):2795–2810. doi: 10.7150/ijbs.60509

Figure 2.

Figure 2

miR-21a-5p downregulates the expression of Cntfr α by targeting the 3'UTR. miR-21a-5p mimic, inhibitor, and negative controls were transfected into astrocytes. (A, B) qRT-PCR was used to detect the mRNA level of Cntfr α, normalized by GAPDH. (C) Western blotting was used to detect the protein level of CNTFR α, normalized by β-actin; the results were analyzed by Image J, GraphPad, and SPSS. (D) Prediction of targeting sequence between miR-21a-5p and Cntfr α; Dual-luciferase reporter assays were performed to determine the targeting sequence of miR-21a-5p and Cntfr α. (E) An astrocyte lysate used for RNA pulldown assay, after which the expression of miR-21a-5p was assessed by qRT-PCR; relative levels of miR-21a-5p were normalized by Input. (F) miR-21 inhibitor was used to down-regulate the expression of miR-21a-5p in astrocytes, after which naïve astrocytes were induced into A1s. The expression of Cntfr α was detected by qRT-PCR and normalized by GAPDH. (G, H) Astrocytes were transfected with miR-21a-5p mimic, inhibitor, and negative control, then treated with CNTF for 0, 15 30, 60 minutes; Western blotting was used to detect the expression of p-STAT3, STAT3, and β-actin. The results were analyzed with Image J, GraphPad, and SPSS. The data are expressed as mean ±SD, n=3. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.