Small Inhibitory RNA Duplexes for Sp1 mRNA Block Basal and Estrogen-induced Gene Expression and Cell Cycle Progression in MCF-7 Breast Cancer Cells

Maen Abdelrahim,Ismael Samudio,Roger Smith,Robert Burghardt,Stephen Safe

doi:10.1074/jbc.m203828200

Maen Abdelrahim, Ismael Samudio + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m203828200

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Abstract

Small interfering RNA duplexes containing 21-22 nucleotides that mediate sequence-specific mRNA degradation and inhibitory RNA (iRNA) for Sp1 mRNA were used in this study to investigate the role of Sp1 on basal and hormone-induced growth and transactivation in MCF-7 and ZR-75 human breast cancer cells. Transfection of Sp1 iRNA in MCF-7 or ZR-75 cells for 36-44 h decreased Sp1 protein (50-70%) in nuclear extracts, and immunohistochemical analysis showed that the Sp1 protein in transfected MCF-7 cells was barely detectable. In cell cycle progression studies in MCF-7 cells, decreased Sp1 protein was accompanied by a decrease in cells in the S phase and an increase in cells in G(0)/G(1), and estrogen-induced G(0)/G(1) --> S phase progression was inhibited in cells treated with iRNA for Sp1. Sp1 iRNA also specifically blocked basal and estrogen-induced transactivation in cells transfected with a GC-rich construct linked to a luciferase reporter gene (pSp1(3)), and this was accompanied by decreased Sp1 binding to this GC-rich promoter as determined in gel mobility shift and chromatin immunoprecipitation assays. These results clearly demonstrate the key role of the Sp1 protein in basal and estrogen-induced growth and gene expression in breast cancer cells.

Highlights

Sp1 is a member of the Sp and Kruppel-like family of transcription factors that bind GC and CACCC boxes to regulate gene expression [1,2,3]
Cells were transfected with pERE3 and iLMN, iGL2, or iRNA for Sp1 (iSp1) treated with Me2SO or 10 nM E2, and luciferase activity was determined as described under “Materials and Methods.”
This study has used the inhibitory RNA (iRNA) approach for investigating the role of Sp1 protein in the growth and hormone-responsiveness of MCF-7 human breast cancer cells

Summary

Introduction

Sp1 is a member of the Sp and Kruppel-like family of transcription factors that bind GC and CACCC boxes to regulate gene expression [1,2,3]. This study investigates the role of Sp1 protein in mediating hormone-responsiveness in MCF-7 cells using sequence-specific duplexes of 21 nucleotides targeted to Sp1 mRNA as well as Lamin B1 and the heterologous firefly luciferase gene (GL2) mRNAs. Transfection of iRNA for Sp1 (iSp1) decreases (40 – 60%) the expression of nuclear Sp1 protein in ER-positive MCF-7 and ZR-75 human breast cancer cell extracts. Sp1 protein is barely detectable by immunofluorescence, and both basal and estrogen-inducible transactivation is decreased in cells transfected with iSp1 and a GC-rich construct These data, combined with results showing that iSp1 inhibits hormone-induced MCF-7 cell cycle progression from G0/G1 to S phase, demonstrate that ER␣/Sp1mediated transactivation plays a major role in ER-positive breast cancer cell growth

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