1.The Inhibitory Effects of an Antisense u-PAR Vector on Invasion by Highly Invasive Human Prostate Carcinoma PC-3M Cell Subclones
Guoning LIAO ; Qingfen LI ; Zhuoya LI ; Feili GONG ; Yaozu DENG ; Youme FENG
Chinese Journal of Cancer Biotherapy 1996;0(04):-
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
2.Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones.
Guoning LIAO ; Qingfen LI ; Youmei FENG ; Yaozu DENG ; Zhuoya LI ; Feili GONG ; Ding MA
Journal of Huazhong University of Science and Technology (Medical Sciences) 2003;23(4):369-372
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.
Cell Line, Tumor
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Cloning, Molecular
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Humans
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Male
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Neoplasm Invasiveness
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Plasmids
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Prostatic Neoplasms
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metabolism
;
pathology
;
RNA, Antisense
;
biosynthesis
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genetics
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Receptors, Cell Surface
;
biosynthesis
;
genetics
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Receptors, Urokinase Plasminogen Activator
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Reverse Transcriptase Polymerase Chain Reaction
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Transfection
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Urokinase-Type Plasminogen Activator
;
antagonists & inhibitors
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genetics
;
metabolism
3.The inhibitory effects of an antisense u-PAR vector on invasion of highly invasive human prostate carcinoma PC-3M cell subclones.
Guoning LIAO ; Qingfen LI ; Youmei FENG ; Yaozu DENG ; Zhuoya LI ; Feili GONG ; Ding MA
Journal of Huazhong University of Science and Technology (Medical Sciences) 2003;23(2):101-104
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.
Animals
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Antisense Elements (Genetics)
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genetics
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pharmacology
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Cell Division
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drug effects
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Cloning, Molecular
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Humans
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Male
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Matrix Metalloproteinase 9
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genetics
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metabolism
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Mice
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Mice, Inbred BALB C
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Mice, Nude
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Neoplasm Invasiveness
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Prostatic Neoplasms
;
metabolism
;
pathology
;
RNA, Antisense
;
Receptors, Cell Surface
;
genetics
;
metabolism
;
Receptors, Urokinase Plasminogen Activator
;
Reverse Transcriptase Polymerase Chain Reaction
;
Transfection
;
Tumor Cells, Cultured
4.Up-regulation of VLDL Receptor Expression and Its Signaling Pathway Induced by VLDL and β-VLDL
LIU ZHIGUO ; LI HE ; LI YINGHONG ; WANG YAN ; ZONG YIQIANG ; FENG YOUMEI ; FENG ZONGCHEN ; DENG YAOZU ; QU SHEN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2009;29(1):1-7
Very low density lipoprotein receptor (VLDLR) is thought to participate in the patho-genesis of atherosclerosis induced by VLDL and β-VLDL. The present study was undertaken to elu-cidate the effects of VLDL and β-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and β-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway in-volving VLDLR expression regulation. The results showed that VLDL and β-VLDL stimulated ERKI/2 activity in a PKC-dependent manner. VLDL or β-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERKI/2 or PKC. Our findings revealed that VLDL or β-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.