ABSTRACT
Objective: To observe the inhibiting effects of an antisense u-PAR vector on invasion by highly invasive PC-3M cell subclones. Methods: The effects of an antisense vector on invasion by highly invasive PC-3M cell subclones were observed and compared in vitro by monolayer invasion assay and soft agar clone. Then, both a quantitative RT-PCR and zymography were used to exam the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones. Furthermore, the tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. Results: It is found that the speed of growth in vitro was slowing down by highly invasive PC-3M cell subclones transfected with the antisense u-PAR, and the ability of anchorage-independent growth of those cell subclones was also decreasing sharply,and the inhibiting rate was 79% and 60%, respectively. Although the antisense u-PAR didn′t change MMP-9 gene transcription, but they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups reached statistical significance ( P
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 in neo-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.
Subject(s)
Humans , Male , Cell Line, Tumor , Cloning, Molecular , Neoplasm Invasiveness , Plasmids , Prostatic Neoplasms , Metabolism , Pathology , RNA, Antisense , Genetics , Receptors, Cell Surface , Genetics , Receptors, Urokinase Plasminogen Activator , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Urokinase-Type Plasminogen Activator , Genetics , MetabolismABSTRACT
To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.