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. 2019 Mar 21;10(5):1051–1059. doi: 10.1111/1759-7714.13038

Figure 4.

Figure 4

MiR‐194 directly inhibits the expression of NFAT5 through its 3’untranslated region (UTR). (a) The miR‐194 binding site predicted in the 3’UTR of NFAT5 messenger RNA (mRNA). (b) Mutant was generated at the seed region of NFAT5 3’UTR, indicated by the underline. A 3’UTR fragment of NFAT5 mRNA containing wild‐type or mutant of the miR‐194 binding sequence was cloned into the downstream of the luciferase gene in pMIR vector. (c) A549 cells were transfected with pMIR reporter vectors containing either wild‐type or mutant NFAT5 3’UTR (NFAT5‐3’UTR‐wt and NFAT5‐3’UTR‐mut) with either miR‐194 mimics (miR‐194) or miR‐194 mimic control (NC). Luciferase activity was determined 48 hours after transfection. (d) The expression level of NFAT5 mRNA was examined by real‐time PCR. Inline graphic NC, Inline graphic miR‐194. (e) The NFAT5 protein was examined by Western blot. (f) The relative gray values of each band (normalized to β‐actin). Protein bands from three independent Western blot assays were quantified using Quantity One software (Bio‐Rad, USA). Inline graphic NC, Inline graphic miR‐194. Data are reported as mean ± standard deviation of three independent experiments (*P < 0.05, t‐test).