[Construction of adenovirus expressing anti-vascular endothelial growth factor hairpin ribozyme and its inhibition of growth of colorectal cancer HT-29 cells].
OBJECTIVE
Oversexpression of vascular endothelial growth factor (VEGF) is correlated to poor prognosis of colorectal cancer. This study was to construct replication deficient adenovirus carrying anti-VEGF hairpin ribozyme and investigate its inhibitory effects on VEGF expression and growth of colorectal cancer HT-29 cells.


METHODS
Anti-VEGF hairpin ribozyme was cloned into the transfer vector pAdTrack-CMV, and recombined with adenoviral backbone vector pAdEasy-1 in BJ5183 bacteria. The recombinant plasmids were packaged and amplified in 293 cells (named Ad-Rz). The expression of anti-VEGF ribozyme in HT-29 cells was examined by RT-PCR. Inhibition of Ad-RZ on VEGF mRNA and protein expressions were detected by real-time PCR and ELISA, respectively. Inhibition of cell growth was measured by MTT assay. HT-29 cell xenografts were established in nude mice and the microvessel density (MVD) was determined by CD34 staining.


RESULTS
The anti-VEGF ribozyme was successfully inserted into the adenoviral vector. Anti-VEGF ribozyme was effectively expressed in HT-29 cells. After infection by Ad-RZ, the relative expression of VEGF mRNA was decreased to about(45+/-0.01)% of that of PBS control group in HT-29 cells (P<0.05); the amount of VEGF protein in the supernatant of Ad-Rz treated cells [(OD=(0.46+/-0.35)/million cells] was lower than that of PBS treated cells [(OD=(0.80+/-0.35)/million cells] (P<0.05). Although Ad-Rz did not significantly inhibit cell growth of HT-29 cells (P>0.05), it significantly inhibited tumor angiogenesis of HT-29 cell xenografts in nude mice, compared with PBS group (P<0.05). The proliferation rate of xenografts treated by Ad-Rz was lower than that treated by PBS, but the difference was not significant (P>0.05).


CONCLUSIONS
Anti-VEGF hairpin ribozyme mediated by adenovirus could inhibit the expression of VEGF and the tumor angiogenesis in colorectal cancer HT-29 cells.
Chang
Shu-Jian
SJ
Department of Gastroenterology, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, 215006, PR China.
Chen
Wei-Chang
WC
Yang
Ji-Cheng
JC
Xie
Yu-Feng
YF
Sheng
Wei-Hua
WH
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D049109:Cell Proliferation; D005260:Female; D019073:HT29 Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010957:Plasmids; D016337:RNA, Catalytic / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D011897:Random Allocation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D047368:Tumor Burden; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic; D012333:RNA, Messenger; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; C418481:hairpin ribozyme