ZIP1 mRNA, hZIP1 protein and zinc levels were lower in miR-183-FC cells.
The miR-183 family cluster alters zinc homeostasis in benign prostate cells, organoids and prostate cancer xenografts
Paper
2017
Scientific Reports |
DOI
Background
The miR-183 cluster (miR-183, -96, -182) is overexpressed in prostate cancer and regulates zinc homeostasis. This study overexpressed the entire 4.8 kb miR-183 family cluster, including the conserved intergenic region, in benign prostate cells, primary prostate organoids, and prostate cancer xenografts to mimic physiological cancer-related changes. Overexpression reduced ZIP1 zinc transporter and intracellular zinc levels, altered cancer-related pathways, and identified a secondary transcription start site in the intergenic region that may regulate miR-182. These findings link the miR-183 cluster to zinc dysregulation and carcinogenic pathways in prostate cells.
Key Findings
Overexpression of the full 4.8 kb miR-183 cluster in RWPE-1 and RWPE-2 cells achieved 10–30-fold and 5–10-fold increases in mature miRs, mimicking cancer levels. In benign RWPE-1 cells, 183FC reduced ZIP1 mRNA and protein by ~50% and decreased total zinc by 15% and labile zinc significantly by Zinquin imaging. In primary human prostate epithelial organoids, 183FC reduced organoid size, lowered zinc by X-ray fluorescence, and lacked zinc in central differentiated cells, similar to prostate cancer tissue. In RWPE-2 xenografts, 183FC increased tumor volume and reduced intratumoral zinc. Microarray analysis showed enrichment of adhesion, migration, and wound healing pathways; 183FC increased adhesion in RWPE-1, while miR-183 knockdown reduced PC3 migration. A secondary transcription start site was identified within the intergenic region, with promoter activity in all prostate cell lines and a conserved 1–2 kb region critical for miR-182 regulation. These results demonstrate that physiologically relevant miR-183 cluster expression alters zinc homeostasis and carcinogenic pathways in prostate cells.
Contribution
Creative Biogene cloned a series of pGL4 luciferase reporter plasmids containing deletion mutants of the miR-182 upstream promoter (3.5 kb, 3 kb, 2 kb, 1 kb, and 0.6 kb fragments). These constructs enabled mapping of the secondary transcription start site within the miR-183 cluster intergenic region.
The miR-183 cluster (miR-183, -96, -182) is overexpressed in prostate cancer and regulates zinc homeostasis. This study overexpressed the entire 4.8 kb miR-183 family cluster, including the conserved intergenic region, in benign prostate cells, primary prostate organoids, and prostate cancer xenografts to mimic physiological cancer-related changes. Overexpression reduced ZIP1 zinc transporter and intracellular zinc levels, altered cancer-related pathways, and identified a secondary transcription start site in the intergenic region that may regulate miR-182. These findings link the miR-183 cluster to zinc dysregulation and carcinogenic pathways in prostate cells.
Key Findings
Overexpression of the full 4.8 kb miR-183 cluster in RWPE-1 and RWPE-2 cells achieved 10–30-fold and 5–10-fold increases in mature miRs, mimicking cancer levels. In benign RWPE-1 cells, 183FC reduced ZIP1 mRNA and protein by ~50% and decreased total zinc by 15% and labile zinc significantly by Zinquin imaging. In primary human prostate epithelial organoids, 183FC reduced organoid size, lowered zinc by X-ray fluorescence, and lacked zinc in central differentiated cells, similar to prostate cancer tissue. In RWPE-2 xenografts, 183FC increased tumor volume and reduced intratumoral zinc. Microarray analysis showed enrichment of adhesion, migration, and wound healing pathways; 183FC increased adhesion in RWPE-1, while miR-183 knockdown reduced PC3 migration. A secondary transcription start site was identified within the intergenic region, with promoter activity in all prostate cell lines and a conserved 1–2 kb region critical for miR-182 regulation. These results demonstrate that physiologically relevant miR-183 cluster expression alters zinc homeostasis and carcinogenic pathways in prostate cells.
Contribution
Creative Biogene cloned a series of pGL4 luciferase reporter plasmids containing deletion mutants of the miR-182 upstream promoter (3.5 kb, 3 kb, 2 kb, 1 kb, and 0.6 kb fragments). These constructs enabled mapping of the secondary transcription start site within the miR-183 cluster intergenic region.