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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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Related in: MedlinePlus

MiR-146b targets the EGFR. (A) Western blotting for EGFR in U118MG cells transfected with miR-146b precursor or control (left) or with LNA targeting miR-146b or scrambled LNA (right). (B) Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-BB for 3–24 h (upper panel). Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (lower panel). (C) Western blotting assessed EGFR expression in C272/hTERT/E7 cells treated with PDGF-BB for 6 h after transfection of miR-146b precursor or control leading to miR-146b over-expression. Densitometry quantifies EGFR expression levels relative to GAPDH in independent experiments. Statistical significance is indicated by asterisks (upper panels, NS, not significant). Reporter assay measured the activity of an EGFR 3′-UTR-luciferase construct before and after stimulation with 50 ng/ml of PDGF-BB (lower panels). Reporter activity of wild-type EGFR 3′-UTR (left) and mutated 3′-UTR (right) were measured and normalized to renilla activity. Statistical significance is marked by asterisks; NS, non-significant. (D) Phosphorylation of PDGFRβ, EGFR, Akt and MAPK p42/p44 were measured by western blotting (upper panel) in C272hTert/E7 cells stimulated for 30 min with PDGF BB (5 ng/ml, lane 2 and 25 ng/ml, lane 3), EGF (20 ng/ml, lane 4 and 150 ng/ml, lane 5) or a combination of PDGF and EGF (pre-treatment with PDGF for 3 h, followed by EGF; lane 6; or concomitant stimulation with PDGF and EGF for 30 min; lane 7). Phosphorylation of EGFR and total expression of EGFR were measured by Western blotting in C272hTert/E7 cells (lower panel) transfected with LNA targeting miR-146b or control. Cells were then treated with EGF (20 ng/ml, lanes 2 and 5) or with PDGF for 3 h prior to stimulation with EGF (lanes 3 and 6). Densitometry quantifies EGFR phosphorylation and total expression relative to GAPDH. (E) EGFR and miR-146b relative expression plot obtained by using relative expression data after standardization of EGFR relative expression values and miR-146b. F(x) is the estimated linear trend function.
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Figure 5: MiR-146b targets the EGFR. (A) Western blotting for EGFR in U118MG cells transfected with miR-146b precursor or control (left) or with LNA targeting miR-146b or scrambled LNA (right). (B) Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-BB for 3–24 h (upper panel). Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (lower panel). (C) Western blotting assessed EGFR expression in C272/hTERT/E7 cells treated with PDGF-BB for 6 h after transfection of miR-146b precursor or control leading to miR-146b over-expression. Densitometry quantifies EGFR expression levels relative to GAPDH in independent experiments. Statistical significance is indicated by asterisks (upper panels, NS, not significant). Reporter assay measured the activity of an EGFR 3′-UTR-luciferase construct before and after stimulation with 50 ng/ml of PDGF-BB (lower panels). Reporter activity of wild-type EGFR 3′-UTR (left) and mutated 3′-UTR (right) were measured and normalized to renilla activity. Statistical significance is marked by asterisks; NS, non-significant. (D) Phosphorylation of PDGFRβ, EGFR, Akt and MAPK p42/p44 were measured by western blotting (upper panel) in C272hTert/E7 cells stimulated for 30 min with PDGF BB (5 ng/ml, lane 2 and 25 ng/ml, lane 3), EGF (20 ng/ml, lane 4 and 150 ng/ml, lane 5) or a combination of PDGF and EGF (pre-treatment with PDGF for 3 h, followed by EGF; lane 6; or concomitant stimulation with PDGF and EGF for 30 min; lane 7). Phosphorylation of EGFR and total expression of EGFR were measured by Western blotting in C272hTert/E7 cells (lower panel) transfected with LNA targeting miR-146b or control. Cells were then treated with EGF (20 ng/ml, lanes 2 and 5) or with PDGF for 3 h prior to stimulation with EGF (lanes 3 and 6). Densitometry quantifies EGFR phosphorylation and total expression relative to GAPDH. (E) EGFR and miR-146b relative expression plot obtained by using relative expression data after standardization of EGFR relative expression values and miR-146b. F(x) is the estimated linear trend function.

Mentions: We next searched for possible targets of miR-146b by using bioinformatic predictive tools (http://www.diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi). Having learned that in other systems EGFR is a miR-146b target (45), we tested whether changes in miR-146b expression alter EGFR expression. Transfection of a commercial miR-146b mimic led to repression of EGFR protein expression level (Figure 5A, left panel). Conversely, transfection of a LNA targeting miR-146b caused its knock down (not shown) and subsequent up-regulation of EGFR protein expression level (Figure 5A, right panel). However, as expected, the variation in miR-146b expression levels resulting from ectopic overexpression or LNA-mediated knock down of miR-146b achieved in these experiments exceeded the range of PDGF-BB induced changes in cancer cells. Therefore, to test whether by altering miR-146b’s expression, PDGF-BB regulates the expression level of EGFR; we measured the receptor’s expression after PDGF-BB treatment and noted down-regulation of EGFR protein level 6–24 h after PDGF-BB stimulation (Figure 5B, upper panel). In contrast, PDGF-AA did not decrease measurably EGFR expression, in concordance with the known lack of induction of miR-146b by PDGF-AA (Figure 5B, lower panel).Figure 5.


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)

MiR-146b targets the EGFR. (A) Western blotting for EGFR in U118MG cells transfected with miR-146b precursor or control (left) or with LNA targeting miR-146b or scrambled LNA (right). (B) Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-BB for 3–24 h (upper panel). Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (lower panel). (C) Western blotting assessed EGFR expression in C272/hTERT/E7 cells treated with PDGF-BB for 6 h after transfection of miR-146b precursor or control leading to miR-146b over-expression. Densitometry quantifies EGFR expression levels relative to GAPDH in independent experiments. Statistical significance is indicated by asterisks (upper panels, NS, not significant). Reporter assay measured the activity of an EGFR 3′-UTR-luciferase construct before and after stimulation with 50 ng/ml of PDGF-BB (lower panels). Reporter activity of wild-type EGFR 3′-UTR (left) and mutated 3′-UTR (right) were measured and normalized to renilla activity. Statistical significance is marked by asterisks; NS, non-significant. (D) Phosphorylation of PDGFRβ, EGFR, Akt and MAPK p42/p44 were measured by western blotting (upper panel) in C272hTert/E7 cells stimulated for 30 min with PDGF BB (5 ng/ml, lane 2 and 25 ng/ml, lane 3), EGF (20 ng/ml, lane 4 and 150 ng/ml, lane 5) or a combination of PDGF and EGF (pre-treatment with PDGF for 3 h, followed by EGF; lane 6; or concomitant stimulation with PDGF and EGF for 30 min; lane 7). Phosphorylation of EGFR and total expression of EGFR were measured by Western blotting in C272hTert/E7 cells (lower panel) transfected with LNA targeting miR-146b or control. Cells were then treated with EGF (20 ng/ml, lanes 2 and 5) or with PDGF for 3 h prior to stimulation with EGF (lanes 3 and 6). Densitometry quantifies EGFR phosphorylation and total expression relative to GAPDH. (E) EGFR and miR-146b relative expression plot obtained by using relative expression data after standardization of EGFR relative expression values and miR-146b. F(x) is the estimated linear trend function.
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Figure 5: MiR-146b targets the EGFR. (A) Western blotting for EGFR in U118MG cells transfected with miR-146b precursor or control (left) or with LNA targeting miR-146b or scrambled LNA (right). (B) Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-BB for 3–24 h (upper panel). Western blotting for EGFR in C272/hTert/E7 cells treated with PDGF-AA for 2–24 h (lower panel). (C) Western blotting assessed EGFR expression in C272/hTERT/E7 cells treated with PDGF-BB for 6 h after transfection of miR-146b precursor or control leading to miR-146b over-expression. Densitometry quantifies EGFR expression levels relative to GAPDH in independent experiments. Statistical significance is indicated by asterisks (upper panels, NS, not significant). Reporter assay measured the activity of an EGFR 3′-UTR-luciferase construct before and after stimulation with 50 ng/ml of PDGF-BB (lower panels). Reporter activity of wild-type EGFR 3′-UTR (left) and mutated 3′-UTR (right) were measured and normalized to renilla activity. Statistical significance is marked by asterisks; NS, non-significant. (D) Phosphorylation of PDGFRβ, EGFR, Akt and MAPK p42/p44 were measured by western blotting (upper panel) in C272hTert/E7 cells stimulated for 30 min with PDGF BB (5 ng/ml, lane 2 and 25 ng/ml, lane 3), EGF (20 ng/ml, lane 4 and 150 ng/ml, lane 5) or a combination of PDGF and EGF (pre-treatment with PDGF for 3 h, followed by EGF; lane 6; or concomitant stimulation with PDGF and EGF for 30 min; lane 7). Phosphorylation of EGFR and total expression of EGFR were measured by Western blotting in C272hTert/E7 cells (lower panel) transfected with LNA targeting miR-146b or control. Cells were then treated with EGF (20 ng/ml, lanes 2 and 5) or with PDGF for 3 h prior to stimulation with EGF (lanes 3 and 6). Densitometry quantifies EGFR phosphorylation and total expression relative to GAPDH. (E) EGFR and miR-146b relative expression plot obtained by using relative expression data after standardization of EGFR relative expression values and miR-146b. F(x) is the estimated linear trend function.
Mentions: We next searched for possible targets of miR-146b by using bioinformatic predictive tools (http://www.diana.pcbi.upenn.edu/cgi-bin/miRGen/v3/Targets.cgi). Having learned that in other systems EGFR is a miR-146b target (45), we tested whether changes in miR-146b expression alter EGFR expression. Transfection of a commercial miR-146b mimic led to repression of EGFR protein expression level (Figure 5A, left panel). Conversely, transfection of a LNA targeting miR-146b caused its knock down (not shown) and subsequent up-regulation of EGFR protein expression level (Figure 5A, right panel). However, as expected, the variation in miR-146b expression levels resulting from ectopic overexpression or LNA-mediated knock down of miR-146b achieved in these experiments exceeded the range of PDGF-BB induced changes in cancer cells. Therefore, to test whether by altering miR-146b’s expression, PDGF-BB regulates the expression level of EGFR; we measured the receptor’s expression after PDGF-BB treatment and noted down-regulation of EGFR protein level 6–24 h after PDGF-BB stimulation (Figure 5B, upper panel). In contrast, PDGF-AA did not decrease measurably EGFR expression, in concordance with the known lack of induction of miR-146b by PDGF-AA (Figure 5B, lower panel).Figure 5.

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