Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones

Abstract

To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression inneo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.

Keywords

tumor invasion / prostate carcinoma / antisense RNA / u-PAR

References

[1] WeissB, DavidkovaG, ZhouL W. Antisense RNA gene therapy for studying and modulating biological processes. CMLS, 1999, 55(3): 334-334

[2] WeissB, ZhouS P, ZhouL W. Antisense strategies in dopamine receptor pharmacology. Life Sci, 1997, 60(7): 433-433

[3] VerhuerenH, DekegelD, De BaetselierP, et al.. Development of a monolayer invasion assay for the discrimination and isolation of metastatic lymphoma cells. Invasion Metastasis, 1987, 7(1): 1-1

[4] SumihitoM, AtsuhikoS, HiroshiH, et al.. Cancer cells overexpress mRNA of urokinase-type plasminogen activator, its receptor and inhibitors in human non small cell lung cancer tissue analysis by Northern blotting and in situ hybridization. Int J Cancer, 1998, 78(3): 286-286

[5] KazuyaS, SatoshiT, KenichiS, et al.. Clinicopathologic significance of urokinase receptor- and MMP-9-positive stromal cells in human colorectal cancer: functional multiplicity of matirx degradation on hematogenous metastasis. Int J Cancer, 2000, 86(1): 24-24

[6] YingW, DavidA W, NavaneethaR, et al.. Identification of the urokinase receptor as an adhesion receptor for vitronectin. J Biol Chem, 1994, 269(51): 32380-32380

[7] AndreasenP A, EgelundR, PetersenH. The plasminogen activation system in tumor growth, invasion, and metastasis. CMLS, 2000, 57(1): 25-25