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PDGF induced microRNA alterations in cancer cells.

Shao M, Rossi S, Chelladurai B, Shimizu M, Ntukogu O, Ivan M, Calin GA, Matei D - Nucleic Acids Res. (2011)

Bottom Line: Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells.We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target.We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Indiana University School of Medicine, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.

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Mechanism of miR-146b regulation by PDGF BB. (A) Phosphorylation of PDGFRα and β, Akt and MAPKp42/p44 was measured by western blotting in C272hTert/E7 cells stimulated with PDGF-AA (25 ng/ml) or PDGF-BB (25 ng/ml) for 30 min to 24 h. (B) Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-BB for 2–24 h (upper panel). Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (middle panel). Western blotting quantifies c-fos in C272hTert/E7 cells stimulated with PDGF-BB in the presence or not of PD98059 and LY294002 inhibitors (lower panel). (C) C272Htert/E7 cells were transfected with c-fos targeted siRNA or scrambled siRNA (control) and treated with PDGF BB. MiR-146b was quantified by qPCR (P = 0.007). Statistical significance is marked by asterisks. (D) Schematic representation of the c-fos binding site at position −2300 upstream of the miR-146b and the position of primers chosen for amplification in the ChIP assay. ChIP assay tests binding of c-Fos to the miR-146b promoter region after PDGF-BB stimulation. A 242-bp PCR product was detected by gel agarose electrophoresis from chromatin immunoprecipitated with an antibody against c-fos (lanes 3 and 4) using primers flanking the c-fos binding region of the miR-146b promoter (−2447 to −2205 bp). Positive controls are amplicons from input chromatin (lanes 1 and 2). Negative controls consist of chromatin immunoprecipitated with non-specific IgG and amplified with primers specific to the miR-146b promoter (lanes 5 and 6) and chromatin immunoprecipitated with c-fos antibody and amplified with primers away from the predicted c-fos binding site (lanes 7 and 8).
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Figure 4: Mechanism of miR-146b regulation by PDGF BB. (A) Phosphorylation of PDGFRα and β, Akt and MAPKp42/p44 was measured by western blotting in C272hTert/E7 cells stimulated with PDGF-AA (25 ng/ml) or PDGF-BB (25 ng/ml) for 30 min to 24 h. (B) Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-BB for 2–24 h (upper panel). Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (middle panel). Western blotting quantifies c-fos in C272hTert/E7 cells stimulated with PDGF-BB in the presence or not of PD98059 and LY294002 inhibitors (lower panel). (C) C272Htert/E7 cells were transfected with c-fos targeted siRNA or scrambled siRNA (control) and treated with PDGF BB. MiR-146b was quantified by qPCR (P = 0.007). Statistical significance is marked by asterisks. (D) Schematic representation of the c-fos binding site at position −2300 upstream of the miR-146b and the position of primers chosen for amplification in the ChIP assay. ChIP assay tests binding of c-Fos to the miR-146b promoter region after PDGF-BB stimulation. A 242-bp PCR product was detected by gel agarose electrophoresis from chromatin immunoprecipitated with an antibody against c-fos (lanes 3 and 4) using primers flanking the c-fos binding region of the miR-146b promoter (−2447 to −2205 bp). Positive controls are amplicons from input chromatin (lanes 1 and 2). Negative controls consist of chromatin immunoprecipitated with non-specific IgG and amplified with primers specific to the miR-146b promoter (lanes 5 and 6) and chromatin immunoprecipitated with c-fos antibody and amplified with primers away from the predicted c-fos binding site (lanes 7 and 8).

Mentions: To generate mechanistic insight into PDGF chains specific effects on miR expression, we assessed the differential impact of PDGF-AA versus PDGF-BB on the major downstream signaling pathways. In C272/hTert/E7 cells, PDGF-AA induced phosphorylation of PDGFRα, but not of PDGFRβ within 30 min (Figure 4A). In contrast, PDGF-BB led to phosphorylation of both receptor subunits and its effects were more prominent than those of PDGF-AA. Akt was rapidly phosphorylated after stimulation with each of the two PDGFs. At equivalent doses of PDGF (25 ng/ml), PDGF-BB induced Akt phosphorylation more prominently compared to PDGF-AA. Likewise, MAPK p42/p44 was and more rapidly and robustly activated by PDGF-BB compared to PDGF-AA (Figure 4A), suggesting that the former is a more potent stimulator of this pathway. An important transcription factor induced by PDGF is c-Fos(43). In C272HTERT/E7 cells, c-fos expression was markedly induced by PDGF-BB within 1–3 h, but not measurably by PDGF-AA (Figure 4B, upper and middle panels). As the main mechanism implicated in c-fos engagement by PDGF is the activation of MAPK pathway (44), we measured the effects of its inhibition. The MAPK inhibitor, PD98059, blocked c-fos induction by PDGF-BB, while LY294002 did not affect it (Figure 4B, lower panel), indicating that PDGF-BB engages c-fos more effectively than PDGF-AA, and does so by activating the MAPK pathway.Figure 4.


PDGF induced microRNA alterations in cancer cells.

Shao M, Rossi S, Chelladurai B, Shimizu M, Ntukogu O, Ivan M, Calin GA, Matei D - Nucleic Acids Res. (2011)

Mechanism of miR-146b regulation by PDGF BB. (A) Phosphorylation of PDGFRα and β, Akt and MAPKp42/p44 was measured by western blotting in C272hTert/E7 cells stimulated with PDGF-AA (25 ng/ml) or PDGF-BB (25 ng/ml) for 30 min to 24 h. (B) Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-BB for 2–24 h (upper panel). Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (middle panel). Western blotting quantifies c-fos in C272hTert/E7 cells stimulated with PDGF-BB in the presence or not of PD98059 and LY294002 inhibitors (lower panel). (C) C272Htert/E7 cells were transfected with c-fos targeted siRNA or scrambled siRNA (control) and treated with PDGF BB. MiR-146b was quantified by qPCR (P = 0.007). Statistical significance is marked by asterisks. (D) Schematic representation of the c-fos binding site at position −2300 upstream of the miR-146b and the position of primers chosen for amplification in the ChIP assay. ChIP assay tests binding of c-Fos to the miR-146b promoter region after PDGF-BB stimulation. A 242-bp PCR product was detected by gel agarose electrophoresis from chromatin immunoprecipitated with an antibody against c-fos (lanes 3 and 4) using primers flanking the c-fos binding region of the miR-146b promoter (−2447 to −2205 bp). Positive controls are amplicons from input chromatin (lanes 1 and 2). Negative controls consist of chromatin immunoprecipitated with non-specific IgG and amplified with primers specific to the miR-146b promoter (lanes 5 and 6) and chromatin immunoprecipitated with c-fos antibody and amplified with primers away from the predicted c-fos binding site (lanes 7 and 8).
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Figure 4: Mechanism of miR-146b regulation by PDGF BB. (A) Phosphorylation of PDGFRα and β, Akt and MAPKp42/p44 was measured by western blotting in C272hTert/E7 cells stimulated with PDGF-AA (25 ng/ml) or PDGF-BB (25 ng/ml) for 30 min to 24 h. (B) Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-BB for 2–24 h (upper panel). Western blotting for c-Fos in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (middle panel). Western blotting quantifies c-fos in C272hTert/E7 cells stimulated with PDGF-BB in the presence or not of PD98059 and LY294002 inhibitors (lower panel). (C) C272Htert/E7 cells were transfected with c-fos targeted siRNA or scrambled siRNA (control) and treated with PDGF BB. MiR-146b was quantified by qPCR (P = 0.007). Statistical significance is marked by asterisks. (D) Schematic representation of the c-fos binding site at position −2300 upstream of the miR-146b and the position of primers chosen for amplification in the ChIP assay. ChIP assay tests binding of c-Fos to the miR-146b promoter region after PDGF-BB stimulation. A 242-bp PCR product was detected by gel agarose electrophoresis from chromatin immunoprecipitated with an antibody against c-fos (lanes 3 and 4) using primers flanking the c-fos binding region of the miR-146b promoter (−2447 to −2205 bp). Positive controls are amplicons from input chromatin (lanes 1 and 2). Negative controls consist of chromatin immunoprecipitated with non-specific IgG and amplified with primers specific to the miR-146b promoter (lanes 5 and 6) and chromatin immunoprecipitated with c-fos antibody and amplified with primers away from the predicted c-fos binding site (lanes 7 and 8).
Mentions: To generate mechanistic insight into PDGF chains specific effects on miR expression, we assessed the differential impact of PDGF-AA versus PDGF-BB on the major downstream signaling pathways. In C272/hTert/E7 cells, PDGF-AA induced phosphorylation of PDGFRα, but not of PDGFRβ within 30 min (Figure 4A). In contrast, PDGF-BB led to phosphorylation of both receptor subunits and its effects were more prominent than those of PDGF-AA. Akt was rapidly phosphorylated after stimulation with each of the two PDGFs. At equivalent doses of PDGF (25 ng/ml), PDGF-BB induced Akt phosphorylation more prominently compared to PDGF-AA. Likewise, MAPK p42/p44 was and more rapidly and robustly activated by PDGF-BB compared to PDGF-AA (Figure 4A), suggesting that the former is a more potent stimulator of this pathway. An important transcription factor induced by PDGF is c-Fos(43). In C272HTERT/E7 cells, c-fos expression was markedly induced by PDGF-BB within 1–3 h, but not measurably by PDGF-AA (Figure 4B, upper and middle panels). As the main mechanism implicated in c-fos engagement by PDGF is the activation of MAPK pathway (44), we measured the effects of its inhibition. The MAPK inhibitor, PD98059, blocked c-fos induction by PDGF-BB, while LY294002 did not affect it (Figure 4B, lower panel), indicating that PDGF-BB engages c-fos more effectively than PDGF-AA, and does so by activating the MAPK pathway.Figure 4.

Bottom Line: Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells.We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target.We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Indiana University School of Medicine, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

ABSTRACT
Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells.

Show MeSH
Related in: MedlinePlus