Effects Of Survivin And CHAF1A On Glioma Cells

Background:Glioma is one of the most common intracranial tumors. It has great harm to human health. Multiple steps and factors are involved in the process of glioma development. In addition to the proto-oncogene activation and anti-oncogene inactivation, the abnormal expression of some apoptosis related gene also plays an important role in this process. Therefore, study the role of apoptosis regulating genes in gliomas and define the mechanism will be helpful for the diagnosis and treatment of cerebral glioma.Survivin is a newly discovered apoptosis inhibitor gene. It is high expressed in most tumor and embryonic tissues, but not in normal tissues. The product of survivin has a unique structure. It can inhibit cell apoptosis and accelerate cell division by activating a series of signaling pathways to promote the proliferation of tumor cells. Thus, it plays an important protective role in the survival of tumor cells. Due to the high and specific expression in tumor tissues, it is very likely that survivin is the bridge directly linked to the anti apoptosis and cell malignant transformation. Therefore, survivin was found to be an important target of anti-tumor treatment. In addition, in recent years, with the continuous application of RNA interference technology, its high specificity and effectiveness have made it become a very important means of studying gene function. Then, as an apoptotic inhibitor, whether RNA interference targeting survivin could inhibit the growth of glioma, and what is the mechanism?Chromatinassembly factor 1 subunit A (CHAF1A), also known as CAF1, is a member of histone chaperone. CHAF1A has been shown to play important role in cell proliferation, DNA repair and epigenetic regulation of embryonic stem cell. Tumor cells are featured with over proliferation and oncogene/tumor suppressor dysregulation caused by DNA repair. It has been reported that over-expression of CHAF1A is associated with breast cell transformation, development. CHAF1A is also a neuroblastoma and colorectal cancer prognosis biomarker. Hence, CHAF1A is speculated to be a cancer driver gene. However, the expression profile and biologic role of CHAF1A in glioblastoma remains largely unidentified.To solve these problems, we firstly used a technique called RNA interference to construct the survivin shRNA recombinant adenovirus (survivin shRNA adenovirus vectors, Ad-survivin-shRNA). Then, the vectors were amplification and purified. To investigate the role of blocking survivin expression in glioma cells both in vitro, cultivate human glioma cell line U251 cells were used and then transfected with recombinant adenovirus. Firstly, to detect the effect of Ad-survivin-shRNA on U251 cell apoptosis, both hoechst33342 staining and flow cytometry were performed. Then, the proliferation and application of U251 cells were determined by CCK-8 assay. PCR and Western blot were used to investigate the survivin gene and protein expression after transfection of Ad-survivin-shRNA in U251 cells. To investigate the involvement of Ad-survivin-shRNA in the glioma in vivo, U251 glioma subcutaneous model was established using nude mice. After the injection of recombinant adenovirus into the glioma in the nude mice, the tumor growth curve was observed. In addition, immunohistochemistry was used to detect the expression of survivin protein. Cell apoptosis was detected by TUNEL method. Our result showed that injection of Ad-survivin-shRNA in the tumor tissue could inhibit the proliferation and growth of tumor cells, decrease survivin expression and increase cell apoptosis.To investigate the effect of CHAF1A on glioma cells,122 gliomablst samples were chosen for this study. We firstly detected the expression of CHAF1A using immunohistochemistry. In addition, cell cycle and apoptosis were detected after knockout of CHAF1A in glioblastoma cells by CRISPR/CAS9 lentvirus. Furthur more, we investigate the signaling pathway associated with biological function of CHAF1A. We found that high CHAF1A protein expression level is significantly associated with the poor overall survival in 122 glioblastoma patients. CHAF1A plays important role in glioblastoma proliferationOur study will help further understanding the effect of surviving and CHAF1A on the glioma cells. It will provide theoretical and experimental basis for the clinical treatment of glioma.Objective:To investigate the involvement of survivin and CHAF1A in glioma cells both in vivo and in vitro.Methods:1. Cell cultureHuman glioma U251 or U87 cell line (provided by Shanghai Institutes for Biological Sciences) was maintained in DMEM containing 10% fetal bovine serum (FBS). Cells were incubated with 5% CO2 incubator at 37℃. Cells were incubated in culture bottle in the concentration of 105/ml. Cells were infected by the virus until they reached 70%-80% confluence.2. Construction of vectorsSurvivin shRNA adenovirus vectors (Ad-survivin-shRNA) were constructed by Shanghai Genechem Company (sense,5’-GGACCACCGCATCTCTACA-3’; antisense,5’-TGTAGAGATGCGGTGGT CC-3’). U251 cells were harvested overnight in a density of 105 cells/ml before transfection.Lenti-cas9 and Lenti-sgRNA were purchased from Genechem (Shanghai, CHN). Cells were firstly infected with lenti-cas9 and selected by puromycin. The stable sub-lines were then infected with lenti-sgRNA to specifically knockout target genes. The sgRNA used were sg-EGFP: sg-EGFP:5’-GGTGAACCGCATCGAGCTGA-3’; sg-CHAFlA:5’-CCGACCTGTGGCGGCTCCCC-3’3. Transfection of vectorsAdenovirus vectors (AD) were diluted in non serum DMEM and placed in the incubator.4h later, they were maintained in DMEM containing 10% FBS. At 24h and 48h the expression of green fluorescent protein (GFP) carried by recombinant adenovirus vector was observed using inverted fluorescence microscope separately. Then the ratio of GFP positive cells to total cell number was calculated to determine the multiplicity of infection (MOI). In the negative control group, empty vectors were used to transfect U251 cells. Cells were infected with Ad-survivin-shRNA or empty vector at the appropriate MOI in the DMEM solution with fetal bovine serum-free.4h later, the medium was changed with 5% CO2 incubator at 37℃.24h,48h,72h, and 96h later, cell morphology was observed using inverted fluorescence microscope. In the blank control group, U251 cells were used without any treatment.Lenti-CAS9 virus infected U251 cells were incubated for 12h and then the medium was changed for continuing starving.3 days later, these cultured cells were infected with lenti-sgRNA virus.12h later, the medium was changed. In the negative control group, cells were infected with empty vectors. The MOI of U251 cells was 1. The MOI of U87 cells was 5.4. Real-time PCRRNA was extracted from cultured cells. Then RNA concentration was measured. At the same time, the ratio of OD260/280 was recorded to analyze the quality of RNA. RNA was reversely transcribed. After the PCR reaction, the amplified product was separated by 1.5% agarose gel. The relative expression of survivin mRNA in the treatment group, the negative control group, and the blank control group were normalized for each well to the β-actin mRNA levels.5. Western blotCells were collected and resuspended in RIPA lysate.30min later, cells were centrifuged. 1xSDS sample buffer was added in the supernatant. To detect protein levels, cell lysates were collected for immunoreaction with primary antibodies followed by the secondary antibodies immunoblot, respectively. For densitometric analyses, immunoreactive bands were scanned and intensity quantitated using Meta-Morph software.6. Hoechst 33342 stainingAd-survivin-shRNA or negative adenovirus vector infected U251 cells were incubated for 4h with 5% CO2 incubator at 37℃ and then the medium was changed for continuing starving.96h later, the cells were fixed with cold 4% paraformaldehyde. Afterwards, the specimens were incubated with Hoechst 33342 dye 500 ul (final concentration lOug/ml) for 5min at 4℃ followed by three washes with PBS. In the blank control group, U251 cells did not receive any process. All images were obtained with a Zeiss LSM780 inverted fluorescence microscope.7. Cell proliferation detected by CCK-8 assayU251 cells, grown overnight at a density of 2 x 105cells/ml in 96-well plates, were infected with Ad-survivin-shRNA or empty vector at MOI 1000 in culture medium.10 ul CCK-8 was added into each plate at 24,48,72, and 96h respectively, which contains 100 ul U251 cells. Only active cells could be dyed by CCK-8.2h later, color change was measured in an enzyme mark instrument at 450 nm. According to the results of the experiment data, the cell proliferation inhibition rate was calculated, and the cell proliferation inhibition curve was drawn to analyze the effect of drug. The cell proliferation inhibition rate (%)= (control group-experimental group)/control group X 100%.8. Flow cytometric detection of apoptosisAdenovirus or lentivirus vector infected cells were cultured in 24-well plates. Cells were washed three times with PBS followed by incubation in 195 ul Annexin V-PE binding solution and 5 ul Annexin V-PE for 20min. In the blank control group, cells were treated as above. Cell apoptosis was measured by detecting the positive rate of V-PE Annexin according to the manufacturer’s instructions and analyzed using flow cytometry.9. Tumorigenicity assays in nude miceSix-week-old male nude mice were injected in the right armpit region with 1×107 U251 cells subcutaneously. The tumor size was measured daily until the tumor diameter was up to about 5 mm. Then, mice were divided into three groups (n= 6/group), which were injected with the same volume of saline, Ad-survivin-shRNA (1010pfu), or empty vector every three days. Tumor volume was caculated by the tumor length x tumor width2 x 0.5 (tumor length, the tumor’s longest diameter; tumor width, the longest diameter perpendicular to the tumor length) every two days to draw the tumor growth curve. Three days after the forth injection, mice were sacrificed and tumors were collected for the following immunohistochemistry and TUNEL staining.10. ImmunohistochemistryTumors were fixed for 24h at room temperature. After paraffin embedding, automated paraffin microtome was used to obtain 4 um sections. Sections were air-dried overnight at room temperature and baked for 20min in a 68℃ oven. Sections were deparaffinized by three xylene washes following gradient ethanol rehydration for immunohistochemical staining. Sections were incubated for 15 min in 0.3% hydrogen peroxide in absolute methanol to quench endogenous peroxidase, rinsed (3×, PBS), and repaired antigen in boiled 0.1 mol/1 citric acid buffer solution for 15 min. Subsequently, sections were blocked in 5% normal goat serum in PBS at room temperature for 20min. Sections were then incubated with primary antibodies at 4℃ overnight followed by HRP conjugated goat anti-rabbit antibody incubation. Bound secondary antibody was then amplified with the Vector Elite ABC kit and visualized by diaminobenzidine reaction. Sections were then stained with hematoxylin for 3 min. Microphotographs were captured using a light microscope. The integral optical density (IOD) of all the microphotographs was analyzed with the aid of Leica Qwin V3 analysis software.11. TUNEL stainingSections were prepared as above. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), using an HRP-based kit (Roche) and counterstaining with hematoxylin. Slides were viewed at 400 x magnification. Cell apoptosis was analyzed as percentage of TUNEL/ hematoxylin-labeled nuclei (apoptotic index, AI) from ten fields in digital images.12. MTT assayCells were infected with CHAF1A sgRNA lentivirus over night and then plated in six-well plates at density of 105cells/well. Cell growth was determined using MTT colorimetric growth assay for 5 days. Each day, cell growth was determined by adding MTT solution (50mg/well) for 4 h. Cellular MTT was solubilized with acidic isopropanol and optical density was measured at 570 nm. The doubling time was calculated for the exponential growth phase. All experiments were performed 3 times in triplicates. The viability of the cells was assessed from three replicates in three independent experiments by the MTT.13. Colony formation assayAfter lentivirus infection overnight, the cells were seeded in 6-well plates at 5X102 per well and incubated for 2 weeks for the colony formation assay. The cells were then washed twice with PBS, fixed with methanol/acetic acid (3:1, v/v), and stained with 0.5% crystal violet. The number of colonies was counted under the microscope.14. Cell cycle assayAfter infection, the cells were collected by trypsinization, fixed in 70% ethanol, washed in PBS, re-suspended in 200 ml of PBS, incubated for 30 min at 37℃ in the dark, and analyzed immediately using a FACSCalibur instrument. The data were analyzed using the CellQuest Pro software.Results1. Effect of adenovirus-mediated transfer of survivin shRNA on glioma cells1.1 MOI determinationIn U251 cells transfected with Ad-survivin-shRNA or empty vetors, high transfection efficiency was observed and no difference was found. However, the mean fluorescence intensity was linearly correlated with MOI. Meanwhile, the fluorescence intensity increased at 48h compared with 24h at MOI 1000 both in the Ad-survivin-shRNA group and negative control group. No effect was found in U251 cell morphology after transfection at MOI 1000 and no difference was found in the fluorescence intensity between the two groups at the same time after transfection. Therefore, the MOI 1000, which has high transfection efficiency but no effect on the morphology of U251 cells, was selected as the optimal multiplicity of infection for the following experiments.1.2 Cell Morphology observation after Ad-survivin-shRNA transductionThere was a marked reduction in the U25I cells infected with Ad-survivin-shRNA at 48h compared with blank control group and negative control group. Moreover, the synapse contacts were reduced, and the cells became round and suspended in the cells transfected with Ad-survivin-shRNA. There was no obvious morphological change in the negative control group.1.3 Ad-survivin-shRNA inhibited survivin expressionRT-PCR showed that compared with the empty vector infected U251 cells and the blank control U251 cells, Ad-survivin-shRNA infected cells showed reduced survivin mRNA 48h later.We next investigated whether the reduced survivin mRNA would lead to survivin protein level decrease after Ad-survivin-shRNA infection using western blot. We found decreased survivin protein at 48h,72h, and 96h after Ad-survivin-shRNA infection compared with blank control and negative control group.1.4 Ad-survivin-shRNA attenuated cell growth potential in vitroTo further investigate the influence of Ad-survivin-shRNA to the number of cells, we initially investigate the cell proliferation using CCK-8 assay. Compared with the negative control group, Ad-survivin-shRNA infection obviously inhibited the proliferation of U251 cells.1.5 Ad-survivin-shRNA elicited apoptosis in vitroHoechst 33342 staining showed that Ad-survivin-shRNA infection induced karyopyknosis of U251 cells and high fluorescence intensity, which indicate the increased apoptosis of U251 cells. In addition, flow cytometric detection showed that the positive rates of V-PE Annexin in the Ad-survivin-shRNA infection group were dramatically higher than the blank control group and negative control group. These results indicated that survivin down-regulation could inhibit the proliferation of U251 cells.1.6 U251 cells infected with Ad-survivin-shRNA attenuated tumor growthWe next investigated the changes in tumor growth in vivo by injection of U251 cells following Ad-survivin-shRNA injection. From day 9, mice of the Ad-survivin-shRNA-treated group showed a significantly smaller tumor size compared with the other two groups, which suggest that inhibiton of survivin could inhibit tumor growth.1.7 Effect of Ad-survivin-shRNA on survivin protein expression and cell apoptosis in vivoTo investigate the role of Ad-survivin-shRNA in survivin expression and tumor cell apoptosis in vivo, immonuhistochemistry (IHC) and TUNEL assay were performed using tumor tissues. Immonuhistochemistry staining of tumor sections showed marked decreased expression of survivin in the Ad-survivin-shRNA treatment tumor. TUNEL test showed a significant increase of TUNEL-positive cells in those tumors injected with Ad-survivin-shRNA, as compared with the blank control group and negative control group. These results suggest that Ad-survivin-shRNA participates in tumor cell apoptosis.2. Effect of CHAF1A on glioma cells2.1 CHAF1A is a predictor of survival122 patients underwent surgical resection for primary glioblastoma over the study period. Immunohistochemical staining analysis revealed that patients with low levels of CHAF1A had significantly longer overall survival relative to those with high levels of CHAP 1 A. There was no correlation between the expression level of CHAF1A protein and patient age or gender. In addition, an analysis date from GEO publicly available microarray, which is capable to assess the effect of 22277 genes on survival in 205 glioblastoma patients, has shown a significant correlation between low expression of CHAF1A and high overall survival.2.2. Knockout of CHAF1A by CRISPR/CAS9 suppresses glioblastoma cell proliferationTo determine whether CHAF1A play a role in cell proliferation, CHAF1A was knockout via CRISPR/CAS9 in U251 and U87 cells. Ttransfection of U251 and U87 cells with sgCHAF1A lentivirus dramatically decreased CHAF1A protein level. CHAF1A knockout with sg-CHAFIA notably suppressed the proliferation of U251 and U87 cells through MTT assays. In addition, CHAF1A knockout resulted in a significant decrease in the number of colonies in both glioblastoma cells. Our dates suggested that CHAF1A is able to regulate glioblastoma cell proliferation.2.3. Knockout of CHAF1A induce cell cycle and altered the apoptosis of glioblastoma cellsTo further investigate the mechanism of CHAF1A induced cell proliferation inhibition, cell cycle states were exam. As expected, knockout of CHAF1A induces G1-phase arrest of glioblastoma cells. In addition, the number of apoptotic cells in CHAF1A sgRNA infected cells was significantly higher than that in control sgRNA infected cells. Moreover, cleavages of both caspase 3 and PARP were markedly increased in CHAF1A knockout cells. Taken together, these data suggest that CHAF1A may acts to regulate survival through cell cycle and apoptosis regulation in glioblastoma cells.2.4. FOXO3a activity was altered in CHAF1A knockout glioblastoma cellsTo clarify the signal transduction pathways involved in CHAF1A induced cell proliferation inhibition, the expression of Bcl-2 family proteins were examined in glioblastoma cells. The expression of Bcl-2, Mcl-1 or Bax was not altered in the CHAF1A knockout cells, whereas the expression of Bim was significantly increased. The CHAF1A sgRNA infected U251 and U87 cells exhibited decreased FOXO3a (Ser253) phosphorylation but not total FOXO3a. In addition, the phosphorylation level of Akt was downregulated by knockout of CHAF1A in U251 and U87 glioblastoma cells, suggesting a potential role of Akt in the signaling cascade that mediates FOXO3a phosphorylation/inactivation in response to CHAF1A regulationConclusion:1. Transfection of adenovirus-mediated transfer of survivin shRNA can inhibit the proliferation and promote the apoptosis of cultured U251 cells.2. Injection of adenovirus-mediated transfer of survivin shRNA into the glioma tissue in the nude mice could inhibit the growth of tumor tissue, reduce the survivin expression, and promote the apoptosis of glioma cells.3. Over-expression of CHAF1A promotes cell proliferation and apoptosis resistance in glioblastoma cells via AKT/FOX03a/Bim pathway.Significance:Our study will help further the understanding of the precise regulation of survivin and CHAF1A in glioma cells. Moreover, it will provide an important experimental basis for the treatment of brain glioma with survivin or CHAF1A as the target in the future.