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Down-Regulation of CXCR4 by Inducible Small Interfering RNA Inhibits Breast Cancer Cell Invasion in Vitro¹

Division of Medical Genetics, Department of Medicine, University of Washington, Seattle, Washington 98195 [Y. C., G. S., C-Z. S.], and Department of Biochemistry, Zhongshan Medical College, Sun Yat-sen University, Guangzhou, China [Y. C.]

	ABSTRACT

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RNA interference (RNAi) is a powerful tool for studying gene function. Here, we describe an inducible small interfering RNA expression system that allows a tight control of the specific gene silencing by RNAi. Using this system, we demonstrated the inducible RNAi effect on the gene expression in mammalian cells. We further showed that inducible knockdown of endogenous CXC chemokine receptor-4 (CXCR4) gene expression in breast cancer cells resulted in significant inhibition of breast cancer cell migration in vitro. This system should be useful for both basic researches on gene function and therapeutic applications of RNAi.

	Introduction

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RNAi,³ a cellular mechanism in which double-stranded RNA triggers the silencing of the corresponding cellular gene, has become a powerful tool for studying gene function (1, 2, 3, 4, 5) . A major breakthrough in the application of RNAi technology in mammalian cells came from the observation that synthetic siRNA of 21 nt in length that mimic Dicer cleavage products efficiently induced sequence-specific gene silencing when transiently transfected into mammalian cells (6 , 7) . More recently, another important technical advance came from the demonstration that double-stranded RNA of 19–29 nt expressed endogenously using RNA Pol III promoter induced target gene silencing in mammalian cells (8, 9, 10, 11, 12, 13, 14, 15) . The endogenous expression of siRNA from DNA templates offers several advantages over the exogenous siRNA delivery (16 , 17) . However, the down-regulation of essential genes by RNAi will result in an arrest of cell growth or in cell death, thus imposing significant limitations on the applications of RNAi to long-term studies on the function of these genes in vitro using cultured cells or in vivo using animals. The availability of an inducible RNAi system will overcome this limitation.

Here, we describe the establishment of a Dox inducible RNAi system in mammalian cells. Using this system, we showed that the inducible down-regulation of endogenous CXCR4 gene expression in breast cancer cells resulted in inhibition of cancer cell invasion. This inducible RNAi system will be useful both for basic researches on gene function and therapeutic applications using RNAi.

	Materials and Methods

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Constructs.
The human U6 small nuclear RNA promoter was amplified by PCR from the genomic DNA of HeLa cells and cloned into the pBluescript II KS (+) to generate the vector pU6B for expression of shRNA. The BplI site is designed to encompass 5 nt of the U6 promoter from -4 to +1 relative to the transcription start site at the 5' end and a run of 5Ts as the termination signal of Pol III at the 3' end. The RNAi cassette was annealed from synthetic oligos containing inverted 27-nt stem and 8-nt loop. After annealing, the top and bottom strands of the double-stranded oligos will have the protruding sequences of 5 Ts (Pol III termination signal) and 5'-CGGTG-3' (U6 promoter from -4 to +1) at their 3' ends, respectively. The annealed RNAi duplex was inserted into the pU6B vector that has been cut with BplI to generate the pU6RNAi construct. This cloning strategy allows the convenient cloning of RNAi cassette immediately downstream of the U6 promoter.

The inducible U6 promoter was generated by replacing the corresponding sequences of the U6 promoter with the TetO sequences using the pU6B vector as the backbone. The TetO sequences are 5'-ACTCTATCATTGATAGAGT-3' and 5'-CTCCCTATCAGTGATAGAGAT-3'. A KpnI and Xho fragment containing seven copies of TetO elements was isolated from pTRE2 (Clontech) and inserted into the KpnI and XhoI sites immediately upstream of the U6 promoter to generate constructs E and H. The annealed RNAi duplex can be conveniently inserted into BplI site of the pU6I vector for Dox inducible RNAi. The constructs are depicted in Fig. 1A .

View larger version (35K): [in this window] [in a new window]   Fig. 1. Tet inducible shRNA expression system. A, schematics of the modified U6 promoter constructs. B, expression of siRNA from these modified U6 promoters. 293T cells in 24-well plates were transfected with 0.075 ng of pRL-SV40, 0.75 ng of pGL3 control, and either 15 ng of the empty vector pU6B (V) or constructs A to H that contain the shRNA expression cassette targeting the firefly luciferase from the pGL3 control as illustrated in A. C, Dox inducible RNAi from different U6 promoter constructs. Constructs B and F were not included in this assay because they were impaired in siRNA expression as shown in B. 293T cells cultured in 24-well plates were transfected with 0.075 ng of pRL-SV40, 0.75 ng of pGL3 control, 225 ng of pTet-tTS, and 15 ng of constructs A, C, D, E, G, or H that contain the RNAi-expressing cassette (RNAi +) or the same set of constructs that contain no RNAi-expressing cassette (RNAi -) as indicated. The cells were treated with or without Dox at 1 µg/ml as indicated. D, dose response of Dox induction of RNAi. Cell culture and assays are the same as in C except that different concentrations of Dox were used to induce the RNAi effects with constructs A (Lane 1) and H (Lanes 2–7). Lanes 2–7 received 0, 0.001, 0.01, 0.1, 0.5, and 1 µg/ml Dox, respectively. Dual luciferase assays were carried out 72 h after transfection for all of the assays. Results are the ratio of firefly to Renilla luciferase activity (mean ± SD, n = 3). RLU, relative light unit.

RT-PCR and Western Blot Analysis.
Total RNA was isolated with TRIzol Reagent (Invitrogen). RT-PCR was carried out using the one-step RT-PCR system (Promega). Cell lysates from breast cancer cells were prepared after cell lysis in radioimmunoprecipitation assay buffer. Protein concentration was determined using the Bradford dye-binding assay (Bio-Rad). Eighty micrograms of protein were electrophoresed on a 12% SDS-PAGE and transferred to membrane. CXCR4 and actin proteins were detected using anti CXCR4 antibody (R&D Systems) and anti-actin antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology), respectively, and chemiluminescence (enhanced chemiluminescence; Amersham Pharmacia Biotech). Each experiment was repeated at least twice with similar results.

In Vitro Invasion Assay.
Breast cancer cell invasion was assayed in 24-well Biocoat Matrigel invasion chambers (8 µm; Becton Dickinson) according to the manufacturer’s protocol. Briefly, cells (1 x 10⁴) were plated in the top chamber. The bottom chamber contained SDF-1 (100 ng/ml in 0.1% BSA/DMEM) as a chemoattractant. After 22-h incubation, the noninvasive cells were removed with a cotton swab. The cells that have migrated through the membrane and stuck to the lower surface of the membrane were fixed with methanol and stained with hematoxylin. For quantification, cells were counted under a microscope in five predetermined fields. Assays were performed in triplicates. Data are expressed as the percentage of invasion through the Matrigel Matrix and membrane relative to the migration through the control membrane according to the manufacturer’s manual.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Design of a Simple and High Efficient Vector System for shRNA Expression in Mammalian Cells.
In an effort to facilitate the construction of shRNA expression cassette for RNAi quickly and efficiently, we have designed a vector system that allows a one-step cloning of shRNA expression cassette from synthetic oligos directly downstream of the U6 promoter. The main feature of this cloning strategy is the use of the restriction enzyme BplI that generates 3' protruding ends of 5 nt in length that can be any nucleotides. Therefore, sequences of 5'-CACCG-3' and TTTTT that correspond to the sequences from -4 to +1 relative to the U6 transcription start site and pol III termination signal, respectively, can be conveniently incorporated into the BplI site, allowing direct cloning of RNAi duplex oligos immediately under the transcription initiation site of the human U6 promoter.

Development of Tetracycline-inducible RNAi System.
An inducible system that allows the regulation of RNAi effect at will in mammalian cells will significantly facilitate the use of RNAi to basic research and clinical application. Our inducible system consists of three components, i.e., a tetracycline-controlled transcription repressor Tet-tTS, an inducible U6 promoter containing multiple TetO sequences, and the inducing agent Dox. The human U6 small nuclear RNA promoter contains the core promoter (the PSE) and the TATA box) and the distal promoter element (DSE; Ref. 18 ). The repressor for the Tet inducible system, Tet-tTS, binds to the TetO in the absence of Dox to repress gene expression. In the presence of Dox, the dissociation of Tet-tTS from TetO leads to gene expression.

A series of constructs were tested for their regulation of shRNA expression by Tet-tTS and Dox. We first used the firefly luciferase reporter as the target for our inducible RNAi system. Construct F (Fig. 1A) containing two TetO elements that are placed between the PSE and the TATA box, and between the TATA box and transcription start site, completely impaired the activity of the U6 promoter as demonstrated by its inability to drive the expression of functional siRNA (Fig. 1B) . Insertion of one copy of TetO between the TATA box and transcription start site (construct B; Fig. 1B ) also resulted in complete abrogation of shRNA expression, demonstrating that the sequences between the TATA box and transcription start site of the U6 promoter are required for expression of the shRNA (Fig. 1B) . Insertion of one copy of TetO between the PSE and TATA element (construct C; Fig. 1A ) does not disrupt U6 promoter activity (Fig. 1B) . However, it is unable to mediate Tet-tTS repression of U6 promoter activity (Fig. 1C) . Therefore, we designed and tested more constructs for responsiveness to Dox and Tet-tTS. Although insertion of one copy of TetO between the DSE and PSE (constructs D) or seven copies of TetO elements upstream of the U6 promoter (construct E; Fig. 1A ) did not affect the U6 promoter-driven shRNA expression (Fig. 1B) , these two constructs were incapable of mediating Tet-tTS repression of shRNA production from the U6 promoter (Fig. 1C) . Because multiple binding sites promote synergistic DNA binding to a promoter by DNA binding proteins, we postulate that the observed nonrepression of the construct C and D by the strong repressor Tet-tTS might be caused by an insufficient recruitment of Tet-tTS to the promoter by one copy of TetO. The seven copies of TetO in construct E are expected to be able to efficiently recruit Tet-tTS to the upstream of U6 promoter. Nevertheless, no repression of siRNA expression by Tet-tTS was observed, suggesting that binding of Tet-tTS to the upstream of U6 promoter alone was not sufficient to repress transcription from the U6 promoter. The regulatory elements of DSE, PSE, and the TATA box act cooperatively to activate transcription from the U6 promoter (18) . Therefore, our strategy was to place multiple TetO sequences between the DSE, PSE, and TATA box without destroying the integrity of the U6 promoter. This design was expected not only to enhance the cooperative binding of Tet-tTS repressors to the TetO sequences in the U6 promoter but also to simultaneously block the communication among the DSE, PSE, and TATA box elements by Tet-tTS. Construct G containing two TetO elements, one between the DSE and PSE and another between the PSE and TATA box (Fig. 1A) , was as active as the wild-type U6 promoter in driving the expression of shRNA (Fig. 1B) . Although it can mediate Tet-tTS and Dox regulation to a certain extent, significant levels of background expression of shRNA were observed from construct G (Fig. 1C) .

To eliminate the leaky expression from construct G, we placed seven copies of TetOs upstream of U6 promoter of construct G to generate construct H (named pU6I hereafter). Although the seven copies of the upstream TetOs were unable to confer repression by themselves (Fig. 1A , construct E), we reasoned that they might work together with the two downstream TetOs to enhance their recruitment of Tet-tTS. The promoter activity of pU6I is similar to that of the wild-type U6 promoter without cotransfected repressor Tet-tTS (Fig. 1B) . As anticipated, pU6I has no background RNAi effect when cotransfected with the Tet-tTS repressor in the absence of Dox (Fig. 1C) . The addition of Dox leads to RNAi effect that is comparable with that of wild-type pU6RNAi. We further showed that the induction of RNAi is Dox dose dependent (Fig. 1D) . This Dox dose-dependent RNAi effect will allow an additional layer of regulation of gene silencing. Therefore, by adjusting the amount of Dox, the degree of gene silencing can be controlled. These results demonstrate that these TetO elements act in synergy to confer optimal Tet-tTS binding and repression of shRNA expression in the absence of Dox.

Inducible Knockdown of Endogenous CXCR4 Gene Expression.
We next determined whether the inducible system could be used to regulate RNAi effect on endogenous gene expression. The CXCR4-SDF-1 axis plays pleiotropic roles in organogenesis, angiogenesis, host immune response, homing, tumor metastasis, and HIV-1 infection. Current studies suggested that chemokines and chemokine receptors might be involved in tissue preferential metastasis of cancers. CXCR4 expression was significantly up-regulated in the highly invasive MDA-MB-231 human breast cancer cells (19) . The high expression of CXCR4 in breast cancer cells and peak levels of expression of its ligand CXCL12 (SDF-1) in organs representing the destinations of breast cancer metastasis indicate that this pair of chemokine and receptor may play a role in the organ-specific metastasis of breast cancer cells (19) . We, therefore, chose the MDA-MB-231 cells and CXCR4 gene as a model for the application of our inducible RNAi system. MDA-MB-231 cells were stably transfected with the empty vector pU6I or pU6I-CXCR4 vector that expresses shRNA targeting endogenous CXCR4 under the control of the inducible U6 promoter and pTet-tTS. Stable cells were selected with G418 and pooled. Although stable cells containing the pU6I empty vector showed no effect on the expression of endogenous CXCR4 in the presence or absence of Dox, cells stably integrating the pU6I-CXCR4 showed a Dox-dependent RNAi effect on the endogenous CXCR4 silencing at both RNA and protein levels (Fig. 2, A and B) .

View larger version (51K): [in this window] [in a new window]   Fig. 2. Inducible silencing of endogenous CXCR4 expression by Dox. MDA-MB-231 cells that harbor stably integrated pU6I empty vector (RNAi -) or pU6I-CXCR4 (RNAi +) that expresses inducible shRNA targeting CXCR4 were cultured in the presence or absence of Dox for 72 h. A, inducible down-regulation of CXCR4 mRNA. The expression of CXCR4 was measured by RT-PCR using total RNA isolated from these cells. GAPDH was used as control. B, inducible down-regulation of CXCR4 protein. The expression of CXCR4 was determined using Western blot using anti-CXCR4-specific antibody. Actin protein was used as a control.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Inducible silencing of CXCR4 expression in MDA-MB-231 breast cancer cells decreased cell invasion in vitro. MDA-MB-231 cells that harbor stably integrated pU6I empty vector (RNAi -) and pU6I-CXCR4 (CXCR4 +) were used in the study. Cell invasion was examined using Matrigel invasion chamber. Data are shown as the percentage of invasion (mean ± SD, n = 3).

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a grant from the NIH (to C-Z. S.).

² To whom requests for reprints should be addressed, at Division of Medical Genetics, Department of Medicine, University of Washington, 1705 NE Pacific Streets, Seattle, WA 98195-7720. Phone: (206) 616-2814; E-mail: czsong{at}u.washington.edu

³ The abbreviations used are: RNAi, RNA interference; shRNA, short hairpin RNA; siRNA, small interfering RNA; nt, nucleotide; Dox, doxycycline; TetO, tetracycline operator; Pol III, polymerase III; SDF, stromal cell-derived factor; RT-PCR, reverse transcription-PCR; TetR, tetracycline repressor.

Received 4/ 7/03. Revised 5/15/03. Accepted 6/11/03.

	REFERENCES

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