Contribution of SRF and Nkx2-5 to androgen-dependent gene expression in prostate cancer
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ABSTRACT:
PROVIDER: PRJNA480000 | ENA |
REPOSITORIES: ENA
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Project description:To study and compare the effect of siRNA-mediated SRF or Nkx2-5 silencing on androgen-responsive genes in LNCaP cells.
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Project description:The androgen receptor (AR) is the principal target for treatment of non-organ confined prostate cancer (PCa). Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes and point towards a role for secondary transcription factors (TFs) therein. We identified a novel indirect mechanism of androgen action in which effects of androgens on PCa cells are mediated by Serum Response Factor (SRF). To identify and characterize genes and cellular processes that are androgen-regulated in an SRF-dependent manner in PCa, Affymetrix HG-U133 Plus 2.0 GeneChip Array analysis was performed starting from RNA obtained from LNCaP cells in which androgen stimulation was combined with siRNA-mediated SRF silencing. To this end, LNCaP cells were seeded in 60 mm dishes at a density of 550,000 cells per dish in antibiotic-free medium. The next day, cells were transfected with siGenome SmartPool siRNA targeting SRF (Dharmacon, Lafayette, CO) or a custom-made control SmartPool targeting luciferase (LUC condition) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Forty-two hours after transfection, cells were treated with 5nM R1881 or ethanol vehicle. 3 biological triplicates were included per treatment group. Forty-eight hours later, cells were harvested in Trizol reagent (Invitrogen). RNA was isolated, purified on RNeasy columns (Qiagen, Germantown, MD) and checked for integrity by Agilent testing (Affymetrix, Santa Clara, CA). cDNA was generated and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix) according to the manufacturer’s instructions at the Mayo Clinic Advanced Genomics Technology Microarray Shared Resource core facility. Two-factor factorial design with three biological replicates (12 total samples) using LNCaP or SRF Silenced cells treated with either R1881 or ethanol vehicle.
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Project description:The androgen receptor (AR) is the principal target for treatment of non-organ confined prostate cancer (PCa). Systems and bioinformatics approaches suggest that considerable variation exists in the mechanisms by which AR regulates expression of effector genes and point towards a role for secondary transcription factors (TFs) therein. We identified a novel indirect mechanism of androgen action in which effects of androgens on PCa cells are mediated by Serum Response Factor (SRF). To identify and characterize genes and cellular processes that are androgen-regulated in an SRF-dependent manner in PCa, Affymetrix HG-U133 Plus 2.0 GeneChip Array analysis was performed starting from RNA obtained from LNCaP cells in which androgen stimulation was combined with siRNA-mediated SRF silencing. To this end, LNCaP cells were seeded in 60 mm dishes at a density of 550,000 cells per dish in antibiotic-free medium. The next day, cells were transfected with siGenome SmartPool siRNA targeting SRF (Dharmacon, Lafayette, CO) or a custom-made control SmartPool targeting luciferase (LUC condition) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Forty-two hours after transfection, cells were treated with 5nM R1881 or ethanol vehicle. 3 biological triplicates were included per treatment group. Forty-eight hours later, cells were harvested in Trizol reagent (Invitrogen). RNA was isolated, purified on RNeasy columns (Qiagen, Germantown, MD) and checked for integrity by Agilent testing (Affymetrix, Santa Clara, CA). cDNA was generated and hybridized to Human Genome U133 Plus 2.0 arrays (Affymetrix) according to the manufacturer’s instructions at the Mayo Clinic Advanced Genomics Technology Microarray Shared Resource core facility.
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Project description:The oncofetal mRNA-binding protein IGF2BP1 and the transcriptional regulator SRF modulate gene expression in cancer. In cancer cells, we demonstrate that IGF2BP1 promotes the expression of SRF in a conserved and N6-methyladenosine (m6A) dependent manner by impairing the miRNA-directed decay of the SRF mRNA. This results in enhanced SRF-dependent transcriptional activity and promotes tumor cell growth and invasion. At the post-transcriptional level, IGF2BP1 sustains the expression of various SRF-target genes. The majority of these SRF/IGF2BP1-enhanced genes, including PDLIM7 and FOXK1, shows conserved upregulation with SRF and IGF2BP1 synthesis in cancer. PDLIM7 and FOXK1 promote tumor cell growth and were reported to enhance cell invasion. Consistently, 35 SRF/IGF2BP1-dependent genes showing conserved association with SRF and IGF2BP1 expression indicate a poor overall survival probability in ovarian, liver and lung cancer. In conclusion, these findings identify the SRF/IGF2BP1-, miRNome- and m6A-dependent control of gene expression as a conserved oncogenic driver network in cancer.
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