Conditional inhibition of cancer cell proliferation by tetracycline-responsive, H1 promoter-driven silencing of PLK1

Abstract

RNA interference (RNAi) is a powerful tool for studying gene function. We developed an inducible genetic element for short interfering RNA-mediated gene silencing. This system uses a tetracycline (Tet)-responsive derivative of the H1 promoter and the Tet repressor (TetR) for conditional expression of short hairpin RNA (shRNA) in HeLa cells. Promoter constructs were generated, which contain the Tet operator (TetO) derived from a prokaryotic Tet resistance transposon upstream and/or downstream of the TATA box. To quantify the response of controllable transcription units for shRNA expression, we examined the functional activity of polo-like kinase 1 (PLK1), a key component of mitotic progression, that is overexpressed in many human tumors. Cotransfection of plasmids for the expression of TetR and shRNA/PLK1 under the control of an H1 promoter-variant carrying TetO upstream of the TATA box did not alter PLK1 expression and proliferation properties of HeLa cells in the absence of doxycycline. Addition of the antibiotic led to marked downregulation of endogenous PLK1 accompanied by strong inhibition of cellular proliferation. Our data indicate that an inducible transcription system for shRNAs based on the human H1 promoter could be a versatile tool for controlled gene silencing in vitro.

Knockdown of SMYD3 by RNA interference inhibits cervical carcinoma cell growth and invasion in vitro

“…A variety of systems allowing controllable gene knockdown have been developed (21)(22)(23). Dox-controlled gene-knockdown is one of the most popular (11,24,25).…”

Polo-Like Kinase 1: Target and Regulator of Transcriptional Control

“…The p53-dependent, 5-FU-induced changes in gene expression include the suppression of the Plk1 promoter activity without any detectable binding of p53 to the Plk1 promoter. 23 Since Plk1 knockdown causes inhibition of proliferation and induction of apoptosis in cancer cells, [24][25][26] it can be assumed that one of the mechanisms leading to p53-induced apoptosis includes the transcriptional repression of the Plk1 gene.…”

Polo-like Kinase 1-mediated Phosphorylation Stabilizes Pin1 by Inhibiting Its Ubiquitination in Human Cells

“…Regarding this observation, we subsequently tested whether down-regulation of Plk1 by RNA interference translates to reduced levels of Pin1 protein in cells. For this purpose, we used our recently developed inducible genetic system for shRNA-mediated gene silencing (45). This system uses the Tet repressor and a tetracycline-responsive derivative of the H1 promoter for the conditional expression of shRNA targeted to Plk1 in HeLa cells.…”

“…T-Rex HeLa cells were transfected with different types of designed expression plasmids, pUS, pDS, and pUS/DS (45). FuGENE 6 (Roche Applied Science) was used as transfection reagent according to the manufacturer's instructions.…”