Establishment of urokinase receptor gene antisense RNA transfer system and its application in leukemia research.
- Author:
Xia BAI
1
;
Jian-Xin FU
;
Xiao-Dong XI
;
Chang-Geng RUAN
Author Information
1. Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhow University, Suzhou 215006, China.
- Publication Type:Journal Article
- MeSH:
Flow Cytometry;
Gene Transfer, Horizontal;
Humans;
Leukemia;
pathology;
therapy;
Matrix Metalloproteinase 9;
metabolism;
RNA, Antisense;
genetics;
therapeutic use;
Receptors, Cell Surface;
analysis;
antagonists & inhibitors;
genetics;
Receptors, Urokinase Plasminogen Activator;
Retroviridae;
genetics;
U937 Cells
- From:
Journal of Experimental Hematology
2003;11(6):591-594
- CountryChina
- Language:Chinese
-
Abstract:
Overexpression of urokinase-type plasminogen activator receptor (uPAR) on tumor cell surface is essential for invasion and metastasis in a variety of tumor cells. To establish a retroviral-mediated antisense RNA transfer system of uPAR gene for exploring its function on down-regulation of uPAR expression in leukemia cells, the retroviral vector LaCD87SN was constructed by inserting uPAR gene into LXSN vector in an antisense orientation. An uPAR gene antisense RNA transfer system was established by liposome-mediated transfection in combination with cross infection with retrovirus. Human leukemia cells U937 were transduced with aCD87 amphotropic retrovirus, expressing uPAR antisense RNA, and the U937/aCD87 cells was obtained by G418 selection. The integration and expression of antisense uPAR gene were analyzed by polymerase chain reaction (PCR) assay. The cell surface expression of CD87 and the activities of matrix metalloproteinase (MMP) were assayed by flow cytometry (FCM) and gelatin zymography, respectively. The results showed that the amphotropic retroviral producers Am12/aCD87, which expressed antisense RNA of uPAR gene with a titer of 6.3 x 10(5) cfu/ml in supernatants, were obtained by means of transfection and superinfection. U937/aCD87 cells were established by continuative G418 selection after retrovirus infection. In U937/aCD87 cells, the integrated provirus and the overexpression of antisense uPAR gene was confirmed. Compared with U937/NeoR cells, FCM analysis revealed that CD87 expression on U937/aCD87 cell surface was not downregulated significantly. However, MMP-9 secretion was significantly suppressed in U937/aCD87 cells. In conclusion, although the retroviral-mediated antisense RNA transfer could not efficiently suppress uPAR expression on leukemic cell surface, it may interfere the uPAR-MMP interactions.
