Co-Silencing The Expressions Of Survivin And GRP78 Is More Efficient To Induce Apoptosis In Hepatoblastoma Cells Than Single Gene Interference

[Objective] Survivin is an attractive target to induce cancer cell apoptosis because it is selectively overexpressed in the majority of tumours but not in the normal tissues. High expression of Survivin protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival rate. GRP78 is an important endoplasmic reticulum (ER) residential chaperone and an ER stress hallmark which promotes tumor cell proliferation, survival and protects tumor cells against apoptosis. To date, all methods that silence Survivin or GRP78 only have not had ideal results. In this study, we detecte the expression of Survivin and GRP78 gene in human gastric cancer and hepatic cell carcinoma samples and knock down both Survivin and GRP78 at same time to induce strong apoptosis of tumor cells.[Methods]1. Clinical gastric cancer and Hepatic cell carcinoma sample arrays and Immunohistochemical AnalysisTumor and adjacent non-tumor tissues of 12 gastric cancer patients and 31 hepatic cell carcinoma patients were surgically collected for immunohistochemical analysis.2. The expression of survivin and GRP78 of tumor cell lines and L02 cells detected by Flow CytometryCells were incubated with rabbit anti-Survivin, or goat anti GRP78. The antigen-antibody complexes were detected using FITC-conjugated anti-rabbit IgG, or anti-goat IgG, and the mean fluorescence intensities were determined by flow cytometry and analyzed with Cell Quest software. 3. The expression of survivin and GRP78 of HepG2 and L02 cells observed by Fluorescence MicroscopyCells were incubated with rabbit anti-human Survivin or goat anti-human GRP78 antibody overnight and then exposed to the secondary antibody (FITC-conjugated goat anti-rabbit IgG and FITC-conjugated donkey anti-goat IgG) for 1 hour. Cells were photographed under Olympus BX51 microscope.4. DNA vector based siRNA was designed to knockdown Survivin or GRP78Design of specific siRNA to survivin or GRP78 was performed using Ambion Target finder. All plasmids include enhancing green fluorescent protein (GFP) gene as a fluorescence marker. Plasmids were named pgsiRNA-sur,pgsiRNA-grp and pgsiRNA-sur2+grp. The control siRNA plasmid was named as pgsiRNA-C.5. Cell transfectionpgsiRNA-C,pgsiRNA-sur,pgsiRNA-grp and pgsiRNA-sur2+grp were transfected in HepG2 cells by LipofectamineTM 2000.6. Transfection Rate Determination by FCM and fluorescence microscopeCells were collected at 24h after transfection.The transfection rate was determined by flow cytometry and analyzed with Cell Quest software.The expression of GFP in cells were observed withⅨ70 inverted fluorescence microscope.7. Real-Time PCRRNA was purified from HepG2 cells using Trizol reagent at 48h after transfection. mRNAwas reversely transcribed into cDNA using Revert Aid. cDNA ofβ-actin, Survivin,or GRP78 was amplified with the SYBR Green Master Mix.8. Western BlotWhole cell lysates at 48h after transfection were applied on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. The membrane was incubated with rabbit anti-Survivin, and goat anti GRP78 respectively. The antigen-antibody complexes were detected using horseradish peroxidase-conjugated anti-rabbit IgG, or anti-goat IgG, and visualized using ECL system. (3-actin (rabbit polyclonal antibody) signals were used to normalize the GRP78 and Survivin signals.9. Proliferation AssayEffects of transfection on cellular proliferation were determined using the MTT staining method. The cell proliferation rate (PR) was calculated using the following equation:PR= (OD value in the treated samples/OD value in the control samples)×100%.10. Apoptosis Analysis by Flow CytometryThe transfected HepG2 cells were incubated for 48h. Then cells were collected, and resuspended in the APC-labeled Annexin V and PI. Cell-associated fluorescence was analyzed using WinMDI2.8 software.[Results]1. The proportion of survivin and GRP78 positive cells in hepatic cell carcinoma tissue were significantly higher than tissue beside liver tumor in the cytoplasmic type staining(P<0.01).2. mRNA of GRP78 was not different between L02 and K562,Raji,HepG2. GRP78 mRNA shows high expression compared with L02 in AGS,HeLa,7721 and MCF-7. GRP78 were found high expression in the membrane of MCF-7, but little or no in other cell lines. In HepG2 survivin is highly expressed in cytoplasmic.3. Apoptosis increased remarkbly in HepG2 following transfection with pgsiRNA-surl and pgsiRNA-sur2. pgsiRNA-sur2 inducing apoptosis and inhibiting cell proliferation is more significant.4. The ER stress protein GRP78 in HepG2 transfected with pgsiRNA-sur was significantly increased compared to that of HepG2 transfected with pgsiRNA-C or without transfection, while GRP78 in HepG2 transfected with pgsiRNA-C and non-transfected HepG2 did not display Statistical difference.5. pgsiRNA-sur2+GRP78 inducing apoptosis and inhibiting cell proliferation is the most significant among pgsiRNA-sur2,pgsiRNA-grp and pgsiRNA-sur2+grp(P<0.05). [Conclusion]Survivin and GRP78 are overexpressed in liver carcinoma tissue, mainly located in the cytoplasm.Survivin is highly expressed in liver cancer cell line HepG2, mainly located in the cytoplasm. Expression of GRP78 between HepG2 and L02 have no Statistical difference which suggest that liver cancer cell surfer ER stress in tumor circument and induce the increase of GRP78. siRNA to Survivin can silence the expression of survivin of HepG2 cells and increase the expression of ER stress protein GRP78. Further study proves that pgsiRNA-sur2+grp inhibiting HepG2 proliferation and inducing apoptosis is the best among pgsiRNA-sur2,pgsiRNA-grp and pgsiRNA-sur2+grp.These results show that increased expression of GRP78 following silence of Survivin maybe anti-apoptosis of tumour cells and co-silencing the expressions of Survivin and GRP78 is more efficient to induce apoptosis in tumor cells than single gene interference.