RNAi-mediated downregulation of urokinase plasminogen activator receptor inhibits proliferation, adhesion, migration and invasion in oral cancer cells

doi:10.1016/j.oraloncology.2008.03.004

Oral Oncology

Volume 44, Issue 12, December 2008, Pages 1172-1180

https://doi.org/10.1016/j.oraloncology.2008.03.004 Get rights and content

Summary

RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Overexpression of urokinase-type plasminogen activator receptor (uPAR) has been implicated in progression and metastasis of oral cancer. In our study, RNAi was introduced to downregulate the expression of uPAR in the highly malignant oral squamous cell carcinoma (OSCC) cells. Our data demonstrated that siRNA targeting of uPAR leads to the efficient and specific inhibition of endogenous uPAR mRNA and protein expression as determined by quantitative real-time RT-PCR and Western blotting. Furthermore, simultaneous silencing of uPAR resulted in a dramatic reduction of tumor cell proliferation activity, adhesion, migration and invasion in vitro compared to the controls. These findings provide further evidence for the involvement of uPAR in a variety of cancer key cellular events as a versatile signaling orchestrator, and suggest that RNAi-directed targeting of uPAR can be used as a potent and specific therapeutic tool for the treatment of oral cancer, especially in inhibiting and/or preventing cancer cell invasion and metastasis.

Introduction

Despite the more widespread use of adjuvant radiation therapy or chemoradiation during the last 20 years, oral squamous cell carcinoma (OSCC) continues to portend a relatively unfavorable prognosis with a 5-year overall and disease-free survival estimated to be 56% and 58%, respectively.1, 2, 3 New therapeutic measure are needed to improve the outcome for patients with OSCC.⁴ The recent discovery of RNA interference (RNAi), a more powerful tool for the inhibition of gene expression, has provided new opportunities for cancer therapy.5, 6

The predominant cause of death in patients with OSCC is the ability of cancer cells to invade surrounding tissues and form lymph and distant metastasis. Urokinase-type plasminogen activator receptor (uPAR), as the receptor of serine protease urokinase-type plasminogen activator (uPA), plays an essential role in the proteolytical degradation of extracellular matrix (ECM) and the basement membrane surrounding the primary tumour, which is a major determinant for the cancer invasion and metastasis. Recent studies indicate that uPAR not only functions as a proteinase receptor, but also affects migration, adhesion, angiogenesis, differentiation and proliferation through intracellular signaling pathways.7, 8, 9 Therefore, it is widely accepted that uPAR has a crucial role in the invasion, metastases and progression of cancer including OSCC as a versatile signaling orchestrator.⁸

In previous studies, we and others have previously shown that the overexpression of uPAR was strongly related to the invasiveness, metastases and poor prognosis in OSCC.10, 11, 12, 13

In the present study, we exploited siRNA targeting of uPAR to downregulate the expression of uPAR in OSCC cells, then investigated the inhibition of proliferation activity, adhesion, migration and invasion potential of OSCC in vitro.

Section snippets

Cell line and reagents

The highly malignant oral squamous cell carcinoma (OSCC) cells (the highly malignant human tongue squamous cell carcinoma cell line—Tca8113 cells)¹⁴ were obtained from Key Laboratory for Oral Biomedical Engineering Ministry of Education Sichuan University. The cell line was maintained as a monolayer culture in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS). Linearized RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (pSIREN) was purchased from BD

GFP expression of cells transfected with recombinant vector

To determine whether the recombinant vectors were transfected to OSCC cells, cells infected with EV, pU, pUc per field was assessed by counting 10 random fields under a inverted phase contrast fluorescence microscope (PH + FL) × 100 and a inverted fluorescence microscope (FL) × 100 (Fig. 1), as this vector independently expresses a Zoanthus sp. green fluorescent protein (ZsGreen) allowing to directly monitor the delivery efficiency of the gene silencing construct. The delivery efficiency of EV, pU,

Discussion

The recent discovery of RNA interference (RNAi), is a strong tool for silencing the function of specific genes,16, 17, 18 compared with the conventional gene-suppression technologies such as chemical inhibitors, antisense technology, ribozymes and deoxyribozymes, which often evoke non-specific side effects and/or offer only transient and partial suppression of the gene of interest, RNAi are more potent inhibitors of gene expression with less toxicity.19, 20 More importantly, given the potency

Conflict of interest statement

None declared.

Acknowledgments

This work was partly supported by the grant from National Natural Science Foundation of China (30300391) and Youth Research Foundation of West China College of Stomatology, Sichuan University.

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