Human cancer specimens and cell lines
We created a library of 126 primary gastric tumors and their matched non-neoplastic mucosa tissues from 126 Asian patients that were originally stored at the SingHealth Tissue Repository, an institutional resource of National Cancer Centre of Singapore and Singapore General Hospital. All patient samples were obtained with informed patient consent and approvals from Institutional Review Boards and Ethics Committees. GC cell lines HGC-27, KATO III, AGS cells were purchased from the American Type Culture Collection. MKN7, TMK1 and IM95 cells were obtained from the Japan Health Science Research Resource Bank. Cell lines were maintained in a humidified atmosphere containing 5% CO2 at 37°C. HGC-27, IM95 cells were cultured in DMEM medium supplemented with 10% FBS (Sigma); AGS, TMK1 and KATO III and MKN7 were cultured in RPMI 1640 medium supplemented with 10% FBS.
Microarray Profiling & Pre-processing
Genomic DNA was extracted from flash-frozen tissues using a Qiagen genomic DNA extraction kit. Total RNAs was extracted using Trizol (Invitrogen, CA), digested with RNase free DNase (RQ1 DNase, Promega), and subsequently purified using an RNeasy Mini kit (Qiagen,CA). Genome-wide mRNA expression was profiled using Affymetrix GeneChip Human Genome U133 Plus 2.0 array. Copy number analysis was profiled using Affymetrix Human SNP array 6.0. DNA methylation analysis was profiled using Illumina Infinium methylation assay. Profiling on the individual microarray platforms was done according to manufacturer’s specifications.
The raw microarray data was pre-processed using the respective platform software from the manufacturer: mRNA and SNP data using Affymetrix’s Genome Studio and Genotyping Console respectively, and DNA methylation using Illumina’s BeadStudio. For SNP data, the normal gastric samples were used as the reference for normalization. The copy number segmentation was generated using Circular Binary Segmentation (CBS) from the R package DNA copy
 using default settings. Gene-based copy number alterations were obtained by averaging the segments within each gene. Amplification and deletion were called using the threshold of 0.3 and −0.3 respectively
The microarray data has been deposited into the National Centre for Biotechnology Information’s (NCBI) Gene Expression Omnibus (GEO) website, series accession number GSE31168 (SNP6) and GSE15460 (mRNA).
PIK3R3 co-regulated gene and IPA pathway analysis
Co-regulated genes with PIK3R3 were elucidated using Pearson correlation on the mRNA data. We focused on probes that were specifically targeting the genes (i.e., probes with suffix ‘_at’ and ‘_a_at’) and significant correlation after correcting for multiple testing (p < 1e-5). Pathways involving the probes were assessed using Ingenuity Pathway Analysis software (
http://www.ingenuity.com). All statistical analyses were computed using the R statistical package.
RNA Extraction and RT-PCR
Total cellular RNA was isolated with the High Pure RNA isolation Kit (Roche) following the manufacturer's protocol and total RNA was quantified with a Nanodrop ND-1000 spectrophotometer. Total RNA (1 μg) was reverse-transcribed using iSCRIPT cDNA synthesis kit (Bio-rad) under condtions defined by the supplier. cDNA was quantified by real-time PCR on the Rotor-Gene Q System (Qiagen). PIK3R3 forward primer: GAGAGGGGAATGAAAAGGAGA, and reverse primer: ATCATGAATCTCACCCAGACG
. β-actin forward primer: AGAGCCTCGCCTTTGCCGAT, and reverse primer: TTGCACATGCCGGAGCCGTT; GAPDH forward primer: TCTTTTGCGTCGCCAGCCGA, and reverse primer: CCAGGCGCCCAATACGACCA. PCR was done using QuantiFast SYBR Green PCR Kit (Qiagen) according to manufacturer’s instructions.
To test the effect of LY294002 on PIK3R3 expression, the complete medium was replaced by serum free medium and LY294002 (20 μM/L, Sigma) was added. The total RNA was extracted 24 h after LY294002 treatment.
PIK3R3 siRNA transfection
Two PIK3R3 siRNAs ordered from Santa Cruz (sc-39124) or Invitrogen (s16152) were used to knock down PIK3R3 in GC cells. HGC-27 or TMK1 cells were seeded into 12 or 6-well plates in complete medium and cultured overnight. Then the medium was replaced with opti-MEM medium (Invitrogen) containing PIK3R3 or control siRNA and Lipofectamine RNAimax (Invitrogen) according to the manufacturer’s recommendations. 48 h after transfection, cell lysates were prepared for Western blot analysis for detection of PIK3R3 expression.
Cell proliferation assay
Cell proliferation assay was done as previously described
[20, 21]. Briefly, 24 h after siRNA transfection, the HGC27 cells were trypsinized, resuspended in 1.1 ml complete culture medium, and re-seeded into 4 new 12-well plates at 250 μl per well. The cells were fixed 1 to 4 days after re-seeding, respectively, and stained with crystal violet. Using 10% acetic acid, dye was extracted, and absorbance at 595 nm was measured using a multiwall spectrophotometer (Bio-Rad).
Measurement of BrdU incorporation
48 h after siRNA transfection, HGC27 cells cultured in 6 well-plates were incubated with BrdU (10 μg/ml) for 7 min. The cells were detached, fixed and stained using FITC BrdU Flow Kit (BD Pharmingen) following the manufacturer's protocol. DNA synthesis was determined by flow cytometer.
Annexin-V/propidium iodide staining
Annexin V staining was detected by flow cytometry using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen). 48 h after siRNA transfection, Both floating and adherent HGC27 cells were collected. Then the cells were washed and stained following the manufacturer's protocol. Apoptotic cells were determined by flow cytometer.
Cell cycle analysis
48 h after siRNA transfection, HGC-27 cells were harvested, washed twice with cold phosphate buffered saline (PBS, pH 7.4), and fixed with 70% ethanol/30% PBS at 4°C overnight. The fixed cells were incubated with 0.5 ml PBS containing 10 μg/ml RNase for 30 min at 37°C, then stained with 20 μg/ml propidium iodide (PI, Sigma) for 30 min in the dark at room temperature, and finally analyzed on a FACS cytometer (Beckman FC5000). A minimum of 1 × 104 cells/sample was evaluated.
Western blot analysis
Cells were harvested, and lysed 48 h after siRNA transfection. Proteins were separated by SDS–PAGE under reducing conditions and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% tween 20 (PBST), and the membranes for PI3KR3 blot were blocked with 2% gelatine, 0.2% FBS in PBST
. The blots were incubated overnight at 4°C with the following antibodies: anti-PIK3R3 antibody, anti-beta actin antibody (Santa Cruz), anti-phospho-RB (Ser 780), anti-cyclin D1, anti- phospho-p38 MAPK antibody (Thr180/Tyr 182), anti-phospho-AKT (Ser 473), anti-phospho-AKT (Ser 308), anti-phospho-m-TOR (Ser 2448) (Cell signaling Technology), anti-PCNA antibody (Millipore). Immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare).