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

Figure 4. KSHV miR-K1-5p and -K4-5p inhibit CASTOR1 expression and activate mTORC1.

Figure 4

miRNA suppressors reduced the level of miR-K1-5p (A) and -K4-5p (B) in KSHV-transformed cells. RT-qPCR examination of miR-K1-5p (A) and miR-K4-5p (B) in untransfected cells or in KMM cells transfected with a LNA-based scrambled control (NC), miRNA suppressor LNA-K1-5p (A), or LNA-K4-5p (B). Three independent experiments were repeated with similar results, and results from 1 representative experiment with 3 biological replicates are shown as the mean ± SEM. (C and D) Knockdown of either miR-K1-5p or -K4-5p increased CASTOR1 expression in KMM but not MM cells. Untransfected cells or cells transfected with different concentrations of LNA-based NC, LNA-K1-5p, or LNA-K4-5p were examined for CASTOR1 protein (C) and mRNA (D) levels. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. mRNA results from 3 biological replicates in D are shown as the mean ± SEM. (E) Knockdown of miR-K1-5p and -K4-5p additively increased CASTOR1 mRNA levels. Untransfected cells or cells transfected with different concentrations of LNA-based NC, LNA-K1-5p, LNA-K4-5p, or LNA-K1-5p plus LNA-K4-5p were examined for CASTOR1 mRNA expression. Three independent experiments were repeated with similar results, and results from 1 representative experiment with 3 biological replicates are shown as the mean ± SEM. Knockdown of miR-K1-5p (F) or -K4-5p (G) inhibited mTORC1 activation in KMM cells but not MM cells. Cells transfected with LNA-based NC or LNA-K1-5p (F) or LNA-K4-5p (G) were examined for p-S6K and p-4EBP1 by Western blotting. Three independent experiments were repeated with similar results, and results from 1 representative experiment are shown. For the Western blot analysis of KMM cells in G, the same set of samples were run in different gels but with the same loading calibration. Data were analyzed by 1-way ANOVA followed by Tukey’s post hoc test for P values below 0.05. *P < 0.05, **P < 0.01, and ***P < 0.001.