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. 2019 Jul 15;129(8):3310–3323. doi: 10.1172/JCI127166

Figure 5. CASTOR1 transcript is directly targeted by miR-K4-5p and probably -K1-5p.

Figure 5

(A) Deletion of the miRNA cluster relieved KSHV suppression of the CASTOR1 3′-UTR. Reporter activity was examined in mock (MM), WT (KMM), and ΔmiR-mutant cells transfected with pGL3-CASTOR1 3′-UTR and pRL-TK–expressing Renilla luciferase for normalization. (B) Knockdown of miR-K1-5p or -K4-5p increased CASTOR1 3′-UTR activity in KMM cells. Cells transfected with pGL3-CASTOR1 3′-UTR and the miRNA suppressor LNA-K1-5p, LNA-K4-5p, or scrambled control (NC) were examined. (C) Pre–miR-K1 or -K4 inhibited the CASTOR1 3′-UTR. 293T cells were transfected with pGL3-CASTOR1 3′-UTR and pre–miR-K1 or -K4 plasmid or vector control, and reporter activity was assessed. (D) Pre–miR-K1 suppressed a reporter containing a 35-bp fragment from the CASTOR1 3′-UTR. 293T cells were transfected with a reporter containing a 35-bp fragment from the CASTOR1 3′-UTR and pre–miR-K1 plasmid or vector control, and the reporter activity was examined. (E) Pre–miR-K4 suppressed a reporter containing a 26-bp fragment from the CASTOR1 3′-UTR. 293T cells were transfected with a reporter containing a 26-bp fragment from the CASTOR1 3′-UTR and pre–miR-K4 plasmid or vector control, and the reporter activity was assessed. (F) Putative targeting sequence of miR-K1-5p in the CASTOR1 3′-UTR. (G) Putative targeting sequence of miR-K4-5p in the CASTOR1 3′-UTR and mutagenesis of the putative binding site. (H) Mutation of the miR-K4-5p binding site in the CASTOR1 3′-UTR fragment abolished reporter suppression by miR-K4-5p. 293T cells were transfected with a mutated reporter plasmid and pre–miR-K4 plasmid or vector control, and the reporter activity was examined. All experiments were independently repeated 3 times with similar results, and results from 1 representative experiment with at least 3 biological replicates are shown as the mean ± SEM. Data for 2 groups were analyzed by 2-tailed Student’s t test. Data for more than 2 groups were analyzed by 1-way ANOVA followed by Tukey’s post hoc test for P values of less than 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001.