Study on Apoptosis-Inducing Effect of XIAP Antisense Oligonucleotides on Glioblastoma Cells in Vitro

Main materials

The A-172 human glioblastoma cells were bought from the Shanghai Cytology Research Institute. The cell-transfection kit was bought from the GIBICO, USA. The one-step RT-PCR kit was bought from the TaKaRa BioTech Co. Ltd., Japan. In combination with literature, an ASODN containing 19 basic groups was artificially synthetized with the coding regions at No.251-269, and the nucleotide of XIAP mRNA was 5’-GCT GAG TCT CCA TAT TGC C-3’. At the same time, the non-relevant serial oligodeoxynucleotid (N-ODN) was designed with the sequence of 5’-AGA GAT TCC GAT CCG T-3’, with phosphorothioate oligonucleotide and PAGE purification. The N-ODN was synthetized by the Beijing AuGCT Bioengineering Co., China. The homology wasn’t found between the ASODN and human gene after computer retrieval. XIAP primer‘s upstream was 5’-CAC TTG AGG TTC TGG TTG CAG-3’; and its downstream 5’-CTT-GTCAACTGCTTCAGCACA-3’, of which amplified fragment was 204 bp. The β-actin primer’s upstream was 5’-GAT AAA GTA AAG TGC TTT CAC TGT-3’ and its downstream was 5’-GTA GTT CTT ACC AGA CAC TCC TCA A-3’, of which amplified fragment was 308 bp.

Methods

The cell culture, grouping and transfection: The refrigerated A-172 human glioblastoma cell lines was inoculated in a 25-cm² culture flask after anabiosis at a water temperature of 37°C. A DMEM medium added with a 15% heat-inactivated fetal bovine serum was used. Then the cells were put into the incubator containing 5% CO₂ with ample saturated concentration at 37°C. The medium was changed on alternate days until the bottom of culture flask was besprent with the cells. The trypsin digestion and cell counting were conducted 1 day before the treatment, and the cells were inoculated on a 96-hole plate and in a 25-cm² culture flask respectively, based on the quantity of 5 × 10³/hole and 1 × 10⁵/flask. Then the cells were put into the incubator with 5% CO₂ at 37°C for an overnight. The cell transfection was conducted strictly based on instructions of the transfection kit.

Overall RNA extraction and RT-PCR

Cell dissociation was conducted using 0.2% trypsin digestion and centrifugated 72 h after treatment. Uni-cell suspensions in each group were respectively prepared, and cell counting was conducted. A 1 × 10⁶ cell was put into a new 0.5-ml EP-tube, and a 3000-rpm centrifugation was performed at 4°C for 10 min, forming cell assemblies. Supernatant was carefully removed, and 1 ml of Trizol was added in the tube. Repeated blow using a transferpettor tip was carried out, and after the vibration, a static culture of the cell was conducted at room temperature for 5 min. After adding a 200 μl of chloroform, vibrate the tube for 15 sec so as to make the cultured cells in the tube were sufficiently mixed and then left the tube at 4°C for 3 min followed by the procedure of centrifugation at 12,000 rpm for 15 min.

The supernatant was carefully moved into a 1.5-ml sterilized, centrifuge tube without RNase in it. After the isovolumic pre-cooling isopropanol (-20°C) was added in and mixed, the tube had an ice bath at 4°C for 10 min and then was centrifugated at 1,2000 rpm for 15 min. Then the supernatant was discarded, and the liquid was absorbed using the absorbent. A 1-ml 75% glacial alcohol (prepared by DEPC water) was added in and mixed at 4° C followed by the centrifugation at 8000 rpm for 5 min, and then the supernatant was discarded. The liquid was trickled out from absorbent paper, with airing at room temperature for 20 min. A 20-30 μl of DEPC water was added in, and the available solution was the right RNA sample.

One-step method was used for RT-PCR, with 25-μl reaction system. The treatment was conducted strictly based on specifications of the RT-PCR kit. The amplification includes the following procedures: i) 50°C, 30 min and initial denaturation at 94°C, 2 min; ii) denaturation at 94°C, 30 sec, renaturation at 58°C, 30 sec, with a phase of extension in the PCR at 72°C for 1 min. The above procedures were repeated for 30 cycles.

Western blot assay

In the following procedures of collection and treatment of A-172 cells, the protein samples were prepared by boiling and centrifugalization after clearage, and then transferred to nitrocellulose filter (NC filter) after gelatum separation using 6% polyacrylamide gel electrophoresis. Finally chemiluminescence reagents were used for detection of the XIAP protein. Biogel imaging analytical system was used for scanning to determine the grey-scale value, and then the specific values of the experiment groups/cell control group were calculated, so as to obtain the relative expression of proteins.

MTT method for assaying the activity of cell proliferation

A 96-hole plate was used for the treatment of the cells, which lasted for 72 h. Under a circumstance of reserving the DMEM, a 20 μl of MTT solution was added in each hole, and then the cell culture was conducted in the incubator with a 5% culture solution at 37°C for 4 h. The supernatant cultured in the holes of DMEM was carefully extracted and discarded, after that, a 150 μl of dimethyl sulfoxide (DMSO) was added in each hole, followed by the vibration for 10 min so as to allow the crystals fully dissolved. The 550-nm wavelength of photoabsorption value in each hole was detected on a microplate scanning spectrophotometer, and the outcomes were recorded. The inhibition of succinate dehydrogenase (SDH) activity in cells was regarded as the outcome of determination in this assay. Computational formula is that the inhibition ratio of SDH activity (%) = (1− the absorption value of experiment groups/absorption value of control group) × 100%.

Detection of apoptosis by flow-cytometry (FCM)

A-172 cells were prepared to be the cell suspension after the digestion process, and were determined using the Annexin V-FITC and PI double-staining methods. The judgment standards in the FCM chart were as follows: Q1 zone: PI (+) Annexin V (-) regarded as necrotic cell zone; Q2 zone: PI (+) Annexin V (+) as late apoptosis or dead cell zone; Q3 zone: PI (-) Annexin V (-) as living cell zone; Q4 zone: PI (-) Annexin V (+) as viable apoptotic cell zone. The result from the statistical analysis of the apoptosis was Q2 + Q4.

Statistical analysis

The statistical data were tested using mean ± SD deviation, and the SPSS13.0 software was sued for χ² test of the correlated data. The value of P < 0.01 was considered as statistically significant.