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Background:
Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is involved in drug resistance in ovarian
cancer and inhibits cellular apoptosis .Recently, ultrasound mediated microbubbles delivery system carrying RNA interference
(RNAi) has been reported in ovarian cancer cells. Untargeted microbubbles delivery systems, however, have a major limitation
of poor gene silence efficiency due to lack of the native receptor, Luteinizing Hormone-Releasing Hormone Receptor (LHRHR)
on the surface of ovarian cancer cells. We hypothesize that the gene transfection efficiency of the pshRNA- survivin would be
enhanced by adding targeting micro bubbles, thus increasing the knockdown effect of survivin expression in ovarian carcinoma
cells.
Methods:
Two microbubbles (pshRNA-survivin loaded lipid microbubbles and LHRHa-targeted pshRNA-survivin loaded
lipid microbubbles) were created using biotin-streptavidin bridge recombination. A2780/DDP cell lines were used to perform
experiments. The cells were divided into seven groups: control group,pshRNA-survivin group,pshRNA-survivinloaded lipid
microbubblesgroup,LHRHa-targeted pshRNA-survivin loaded lipid microbubblesgroup,US+pshRNA-survivin group, US+
pshRNA-survivin loaded lipid microbubblesgroup,US+ LHRHa-targeted pshRNA-survivin loaded lipid microbubblesgroup and
were treated respectively.The RNAi knockdown efficiency was determined by Q-PCR and Western blot.The cellular proliferation
was detected by MTT Assay and apoptosis was detected by FACS analysis and Hoechst staining.
Results:
QPCR at 48 hours revealed survivin mRNA knockdown induced by US+ pshRNA-survivin loaded lipid microbubbles
and US+ LHRHa-targeted pshRNA-survivin loaded lipid microbubbles was 56% and 64% respectively compared to negative
control. Western blot revealed translational inhibition induced by both groups.MTT assay indicated increased cell death in
US+LHRHa-targetedpshRNA-survivin loaded lipid microbubbles, the ratio of inhibition was 42.08%,54.60%, and 74.25%.After
transfaction 24h,48h,72h, respectively, which were higher than other groups(p<0.05),FACS analysis revealed increased Annexin
V positivity cell and Hoechst 33258 staining revealed increased hyperchromatic and compact at condensed or granular state
of apoptotic nuclei cells in US+LHRHa-targetedpsh RNA-survivin loaded lipid microbubbles group was more significant than
other groups, which suggest higher rate of apoptosis.Western blotting revealed enhanced caspase-9 and caspase-3 expression in
US+LHRHa-targetedpshRNA-survivin loaded lipid microbubbles than in other groups.
Conclusions:
Ultrasound mediate LHRHa targeted pshRNA-survivin loaded lipid microbubbles resulted in survivin expression
knockdown and apoptosis in A2780/DDP cells
in vitro
and shows that LHRHa targeted lipid microbubbles as a potential vehicle
application in future gene therapy models of RNAi in ovarian cancer.
Biography
Shufang has completed her Ph.D at the age of 27 years from Chongqing. She is a Gynecological oncologist of the Second Affiliated Hospital of
Chongqing Medical University. She has published more than 40 papers in journals.
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