Rapamycin induces miR-21 expression via an AKT-independent mechanism.

<p><b>A</b>) Western blot analysis of 621-101 cells treated with DMSO (lane 1), Rapamycin (20 nM, 24 h - lane 2), the AKT inhibitor MK2206 (10 nM, 24 h - lane 3), and Rapamycin and MK2206 (lane 4). Rapamycin treatment induces AKT phosphorylation at S473 and MK2206 abrogates Rapamycin's effect on phosphor-Akt. <b>B</b>) Expression of miR-21, 24, 29b, and 221 in 621-101 cells treated as in A). miR-21 levels are induced by Rapamycin, however the addition of MK2206 has no effect suggesting an AKT-independent mechanism.</p

Exiqon miRNA microarray confirms 8 Rapamycin-dependent miRNA.

<p>621-101 cells were treated with Rapamycin 20 nM or DMSO for 24 hours. Total RNA was isolated and applied to the Exiqon platform, which assays 946 human miRNA. <b>A</b>) Heat map of miRNA dysregulated by Rapamycin >1.5-fold, log₂ scale. RNA from three biologic replicates per condition was pooled; each miRNA was assayed in quadruplet on the array. <b>B</b>) miRNA dysregulated by Rapamycin >1.5-fold (normalized to RNU44). Highlighted miRNA (except miR-31 and 210) are common to both the Exiqon and Signosis platforms. miR-21 is circled.</p

miR-21 is mTOR-dependent and may be TSC2-independent.

<p><b>A</b>) Stable downregulation of tuberin in C3H-10T1/2 pre-pericytes results in increased phosphorylation of ribosomal protein S6, as expected. Treatment with Rapamycin (20 nM, 24 h) inhibits phosphorylation of S6. <b>B</b>) Downregulation of TSC2 in C3H-10T1/2 cells does not affect miR-21 expression. Inhibition of mTORC1 with Rapamycin induces ∼2-fold increase in miR-21 expression in both control shRNA and TSC2 shRNA cells. Bars represent the mean of two biologic replicates +/− SD. * p<0.05. <b>C</b>) LAM patient-derived cells (621-101), TSC2-null rat uterine leiomyoma-derived cells (ELT3), TSC2-null mouse embryonic fibroblasts (MEFs), HEK293 and lung adenocarcinoma (A549) cells were treated with Rapamycin 20 nM vs Control for 24 h. Relative MiR-21 expression was determined by qRT-PCR. Human cells were normalized to RNU44, mouse cells to snora202 and rat cells to U87, which are all small nucleolar RNA molecules. For all charts, bars represent the mean of three biologic replicates +/− standard error. * p<0.05. ** p<0.01.</p

qRT-PCR confirmation of Rapamycin-dependent miRNA in TSC2-deficient cells.

<p>TSC2−/− cells were treated with Rapamycin 20 nM or DMSO for 24 hr and miRNA expression was assessed by qRT-PCR. <b>A</b>) miRNA expression is similar in 621-101 cells using RNU44 (left panel) or RNU48 (right panel) for normalization. <b>B</b>) miRNA expression in 621-101 cells normalized to RNU44. Highlighted results are significant using a Bonferroni correction.</p

Rapamycin-regulated miRNA in LAM patient-derived cells identified by the Exiqon Array (Fold Change >1.5, normalized to RNU44).

<p>Rapamycin-regulated miRNA in LAM patient-derived cells identified by the Exiqon Array (Fold Change >1.5, normalized to RNU44).</p