IFN induces miR-21 through a signal transducer and activator of transcription 3-dependent pathway as a suppressive negative feedback on IFN-induced apoptosis

Abstract

The microRNA miR-21 is overexpressed in many human cancers, wherein accumulating evidence indicates that it functions as an oncogene. Here, we report that the cytokine IFN rapidly induces miR-21 expression in human and mouse cells. Signal transducer and activator of transcription 3 (STAT3) was implicated in this pathway based on the lack of IFN effect on miR-21 expression in prostate cancer cells with a deletion in the STAT3 gene. STAT3 ablation abrogated IFN induction of miR-21, confirming the important role of STAT3 in regulating miR-21. Chromatin immunoprecipitation analysis showed that STAT3 directly bound the miR-21 promoter in response to IFN. Experiments in mouse embryo fibroblasts with a genetic deletion of the p65 NF-κB subunit showed that IFN-induced miR-21 expression was also dependent on NF-κB. STAT3 silencing blocked both IFN-induced p65 binding to the miR-21 promoter and p65 nuclear translocation. Thus, IFN-induced miR-21 expression is coregulated by STAT3 and NF-κB at the level of the miR-21 promoter. Several cell death regulators were identified as downstream targets of miR-21, including PTEN and Akt. Functional experiments in prostate cancer cells directly showed that miR-21 plays a critical role in suppressing IFN-induced apoptosis. Our results identify miR-21 as a novel IFN target gene that functions as a key feedback regulator of IFN-induced apoptosis.

©2010 AACR.

Figures

Figure 1. IFN induces miR-21 in a variety of cell types

(A) Human U87 glioma, DU145 and PC3 prostate cancer cells, and skin fibroblasts, and B16 mouse melanoma were treated with IFN (1000 IU/ml, for 6 h) prior to RNA extraction. DU145 cells were treated with IFN (1000 IU/ml) for the indicated times, or with the indicated concentrations of IFN for 6 h prior to RNA extraction and miR-21 (B) or ISG15 (C) expression was determined by qPCR. Expression was normalized to expression of U6 RNA for miR-21 and B-actin for ISG15, respectively. Data represent the mean +/− SD of at least three experiments performed in duplicate.

Figure 2. The roles of STAT3 and NF-κB in the induction of miR-21 by IFN

DU145 cells were (A) transfected with DN-F705 STAT3 (F705) or (B) transduced with lentivirus encoding shRNA for STAT3, and treated with human IFNα (1000 IU/ml for 6 h). (C,D) MEFs from wild type mice or mice in which the p65 subunit of NF-κB was knocked out (p65−/−) were treated with murine IFN (1000 IU/ml) or LPS (40 ng/ml) as indicated for 6 h. (C) miR-21, or (D) miR-296 or miR-351 expression was determined by qPCR and expressed relative to U6 RNA levels. Data represent the mean +/− SD of at least three experiments performed in duplicate.

Figure 3. The IFN-induced binding of STAT3 and the p65 subunit of NF-κB to the miR-21 promoter

(A) ChIP analyses of STAT3 binding to the STAT3-I, -II and -III within the miR-21 promoter in DU145 mock treated or treated with IFNα (1000 U/ml) for 1 h. (B) ChIP analysis of p65 binding to κB site nearby the STAT3-I site was performed on DU145 cells and DU145 cells in which STAT3 levels were knocked down by shRNA for STAT3 (STAT3-KD). The ChIP-enriched DNA levels analyzed by Q-PCR were normalized to input DNA, followed by subtraction of non-specific binding determined by control IgG. (C) Immunoblot analysis for p65 in nuclear extracts prepared from control and STAT3-KD DU145 cells treated with IFN (1000 IU/ml) for the indicated times. Lamin serves as a loading control for nuclear extracts.

Figure 4. miR-21 suppresses IFN-induced apoptosis

(A) PC3 cells and PC3 cells overexpressing miR-21 (+miR-21) were treated with IFN (1000 IU/ml for 24 h) and analyzed for apoptosis by TUNEL assays. Phase contrast and fluorescent images are on the left and right, respectively. Apoptotic cells are fluorescent and appear green in the photomicrograph. (B) PC3 cells, PC3 cells overexpressing miR-21, DU145 cells, and DU145 cells in which miR-21 was knocked down (−miR-21) were treated for 24 hr with IFN (1000 IU/ml), camptothecin (CP, 1 μM) or staurosporine (ST, 1 μM) and analyzed for apoptosis by cell death detection ELISA assays. Data represent the mean +/− SD of at least three experiments performed in duplicate.

Figure 5. The effect of miR-21 on potential apoptotic effectors

(A and B) PC3 cells transduced with control vector and PC3 cells overexpressing miR-21 (+miR-21) were treated with IFN (1000 IU/ml) for the indicated times, and cell lysates were immunoblotted for total and phosphorylated Akt levels (A) or p85 and actin (B). (C) DU145 cells transduced with anti-miR control (EV) and DU145 cells in which miR-21 was knocked down (Anti-miR-21) were immunoblotted for the indicated proteins. For analysis of STAT3 phosphorylation cells were treated with IFN (1000 IU/ml, 2 hr)