Reagents and antibodies
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), TRIzol® Reagent and Lipofectamine™ 2000 were purchased from Invitrogen (Carlsbad, CA); 3-(4,5-dimethylthylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) (Dingguo biology, Shanghai, China); Dimethylsulfoxide (DMSO) (Shanghai Sibas Biotechnology Development Co., Ltd., China); 5-Bromo-2-deoxyuridine (BrdU) Cell Proliferation ELISA kit was purchased from Roche Applied Sciences (Indianapolis, IN); Giemsa was purchased from Chemicon International (Temecula, CA); Propidium Iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO); Bicinchoninic acid (BCA) Protein assay was purchased from HyClone-Pierce (South Logan, UT); M-MLV Reverse Transcription was purchased from Promega (Madison, WI); Oligo-dT was purchased from Sangon Biotech (Shanghai, China); SYBR green Master Mixture was purchased from Takara (Otsu, Japan); pFH-L vector and virion-packaging elements (packing plasmid mix) were obtained from Holybiol (Shanghai, China). Mouse anti-STIM1, mouse anti-GAPDH, p21Waf1/Cip1, cyclin D1, cyclin-dependent kinase 4 (CDK4) and goat anti-mouse IgG were purchased from Santa Cruz biotechnology (Santa Cruz, CA), mouse anti-STIM2 was purchased from Abcam plc (Abcam, UK), mouse anti-Orai1 was purchased from Sigma biotechnology (Sigma- -Aldrich, US). All other chemicals were of analytical grade.
Human kidney cell line HEK293,human glioblastoma cell lines, U251, U87 and U373, were all obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM containing 10% FBS, 100U/mL penicillin and 100 μg/mL streptomycin at 37°C in a humidified atmosphere containing 5% CO2.
siRNA design and construction of recombinant lentiviral vector
Recombinant lentiviral vector was constructed as described previously . The candidate sequence (5′-CCTGGATGATGTAGATCATAA-3′) in the STIM1 cDNA sequence (GenBank accession number NM_003156) was selected for siRNA and blasted against the human genome database to eliminate cross-silence phenomenon with non-target genes. Scrambled siRNA (5′-TTCTCCGAACGTGTCACGT-3′) that does not target any genes was used as the negative control. Short hairpin RNA (shRNA) was constructed by annealing the synthetic DNA oligonucleotide primers, then naturally cooled to room temperature, and inserted between the NheI and PacI sites of lentiviral expression vector pFH-L which contains the green fluorescent protein (GFP) gene as a reporter with an internal CMV promoter. Clone identity was verified by sequencing.
Considering STIM1 CDS > 2 kb and inefficient expression of construct RESC lentiviral vector, another shRNA targeting the same gene STIM1 (NM_003156.3) was chosen to construct to get comparable results. The sense siRNA sequences were CGGCAGAAGCTGCAGCTGA and antisense siRNA sequences were TCAGCTGCAGCTTCTGCCG.
Recombinant lentiviral vector was produced by co-transfecting HEK293FT cells with lentiviral expression vector and packing plasmid mix using Lipofectamine™ 2000, according to the manufacturer’s instructions. Infectious lentiviral particles were harvested at 48 h post-transfection, centrifuged to get rid off cell debris, and then filtered through 0.45 μm cellulose acetate filters. The virus was concentrated by spinning at 4,000 g for 15 min following by a second spin (<1,000 g, 2 min). The titer of recombinant lentivirus was determined by serial dilution on 293 T cells.
Recombinant lentivirus transfection in U251 cells
For lentivirus transduction, U251 cells were subcultured at 5 × 104 cells/well into 6-well culture plates. After grown to 30% confluence, cells were transducted with STIM1-siRNA-expressing lentivirus (si-STIM1) or control-siRNA-expressing lentivirus (si-CTRL) at a multiplicity of infection (MOI) of 50. Cells were harvested at 72 h after infection and the transduction efficiency was evaluated by counting the percentage of GFP-positive cells.
Quantitative real-time RT-PCR analysis
Total RNA from infected cells was isolated using TRIzolReagent as recommended by the manufacturer. The quantity and purity of RNA were determined by UV absorbance spectroscopy. cDNA preparation was performed according to standard procedures using oligo-dT primer and M-MLV Reverse Transcriptase. Quantitative real-time PCR was performed by SYBR Green Master Mixture and analyzed on TAKARA TP800-Thermal Cycler Dice™ Real-Time System. The following primers were used for STIM1: 5′-AGCCTCAGCCATAGTCACAG-3′ (Forward), 5′-TTCCACATCCACATCACCATTG-3′ (Reverse); for p21Waf1/Cip1, 5′-GGGACAGCAGAGGAAGACC-3′ (Forward), 5′-GACTAAGGCAGAAGATGTAGAGC-3′ (Reverse); for cyclin D1, 5′-GGTGGCAAGAGTGTGGAG-3′ (Forward), 5′-CCTGGAAGTCAACGGTAGC-3′ (Reverse); for CDK4, 5′-GAGGCGACTGGAGGCTTTT-3′ (Forward), 5′-GGATGTGGCACAGACGTCC-3′ (Reverse). Housekeeping gene GAPDH was used as internal control and the primers are: 5′-AGGTCGGAGTCAACGGATTTG-3′ (Forward), 5′-GTGATGGCATGGACTGTGGT-3′ (Reverse). Thermal cycling conditions were subjected to 15 s at 95°C and 45 cycles of 5 s at 95°C and 30s at 60°C. Data was analyzed with TAKARA Thermal Dice Real Time System software Ver3.0. The gene expression change after lentivirus transduction was presented as relative expression (fold over the negative control or percentage of the negative control) after normalizing to GAPDH, and calculated using the 2-ΔΔCt method as described previously .
Western blot analysis
Lentivirus-transduced cells were washed twice with ice-cold PBS and suspended in a lysis buffer (2% Mercaptoethanol, 20% Glycerol, 4% SDS in 100 mM Tris-HCl buffer, pH 6.8). After 15 min of incubation on ice, cells were disrupted by ultrasound on ice. Total cell lysates were then centrifuged (12,000 g, 15 min, 4°C) and the supernatants were employed for further processing. The protein concentration was determined by BCA protein assay kit. Equal amount of proteins was loaded and separated by SDS-PAGE, and then transferred onto PVDF membrane (Schleicher&Schuell Co., Keene, NH) using an electro-blotting apparatus (Tanon, Shanghai, China). The membrane was blocked with 5% nonfat milk in TBST solution for 1 h at room temperature, and incubated overnight at 4°C with specific antibody to STIM1, p21Waf1/Cip1, STIM2, Orai1, cyclin D1 and CDK4 at the dilution 1:800, 1:1000, 1:800, 1:1000, 1:1500, and 1:1000, respectively. After three washes in TBST solution, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody diluted with TBST solution at room temperature for 2 h. The signals of detected proteins were visualized on ECL plus Western blotting detection system (Amersham Biosciences, Inc., Piscataway NJ). GAPDH protein levels were used as a loading control.
MTT cell viability assay and direct cell counting method
Cell viability was determined by a colorimetric MTT assay which described previously . Briefly, lentivirus-transduced or TRPC entryway paralysed cells were seeded in 96-well plates at a density of 2 × 103 cells/well. Ten microliters of MTT solution (5 mg/mL) was added into each well once daily for 5 days, and plates were incubated for 4 h at 37°C. After removal of the supernatant, 100 μL of DMSO was added to dissolve the crystals. The absorbance at 490 nm was detected with a microplate reader (Bio-Rad 680). Growth curve was performed according to the absorbance values (A) of 490 nm.
On the other hand, direct cell counting method was also used to cross-checking the results of MTT assay. Double target RNAi U251 cells were seeded in 96-well plates at a density of 1 × 104 cells/well. After that, number of cells at 24 h and 48 h after seeding would be counted by blood cell counting plate. Besides, we count 3 wells for reduce error every time point. Growth curve was made according to the average number of cells in 3 wells.
BrdU incorporation assay
Cell proliferation was also quantified by measuring BrdU incorporation during DNA synthesis using the BrdU Cell Proliferation ELISA kit. The experiment was performed according to the manufacturer’s protocol. Briefly, 10 μL/well of BrdU labeling solution was added to cells at 24 h and 72 h after culture. After overnight incubation, cells were fixed with 200 μL/well of fix solution for 30 min in the dark at room temperature, and then incubated with peroxidase-conjugated anti-BrdU antibody for 90 min in the dark at room temperature. A substrate solution was then added into each well, and absorbance was measured using microplate reader (Bio-Rad 680) at a wavelength of 450 nm with a reference wavelength of 630 nm. The number of proliferating cells is represented by the level of BrdU incorporation which directly correlates to the absorbance values. Growth rate (R) was calculated by the following equation:
where A72h and A24h indicate the absorbance at 450 nm after 24 and 72 hours of incubation.
Colony formation assay
Lentivirus-transduced glioblastoma cells (200 cells/ well) were seeded in 6-well plates. Culture medium was changed at regular time intervals. After 14 days of culture, adherent cells were washed twice with PBS, fixed with 4% paraformaldehyde for 30 min at room temperature. The colonies were stained with Giemsa solution for 15 min, then washed with water and air-dried. Cell colonies were counted using a light microscopy. The experiment was performed in triplicate.
Cell cycle analysis
The effect of STIM1 on cell cycle distribution was determined by flow cytometry . Briefly, lentivirus-transduced U251 cells (1 × 105 cells/dish) were seeded at 6-cm dishes. Cells were harvested when they reach 80% confluence, and fixed at least 1 h with 70% ice-cold ethanol at 4°C. Cells were washed with PBS and resuspended in 1 mL of PBS containing 50 μg/mL PI and 100 μg/mL RNase A. After following incubation for 1 h in the dark at room temperature, cells were analyzed by flow cytometry using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA) at 24, 48 and 72 hrs after transduction. The fractions of cells in G0/G1, S, and G2/M phases were analyzed using dedicated software.
Xenograft tumor model
The antitumor effects of siSTIM1 were evaluated in viv o using the U251 human glioma xenograft model in nude mice. All animal procedures were performed according to the guidelines of Peking Union Medical College Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences. Briefly, U251 cells were infected with si-STIM1-expressing lentivirus or control-siRNA-expressing lentivirus at MOI of 50. When an apparent efficiency of 80%-90% GFP-positive cells was observed, cells were harvested and suspended at a density of 2 × 107/mL DMEM (without serum), and 5 × 106 cells were subcutaneously injected into the right dorsal flank of male BALB/c nu/nu athymic nude mice (SLAC Laboratory Animal Co. Ltd., Shanghai, China, 4-6 week-old) (n = 10 per group). The mice were housed and maintained under specific-pathogen free (SPF) condition. Tumor size was measured every 10 days using microcaliper, and tumor volume was calculated according to the following formula: tumor volume = (width2 × length)/2. The animals were sacrificed and tumors were excised after 30 days after injection. Tumor size was measured, and then the tumor was immediately frozen in liquid N2 for protein extraction.
Protein extraction of xenograft tumors
Tumor tissue (approximate 5 mg) frozen in liquid N2 was manually homogenized on ice, lysed in lysis buffer supplemented with protease inhibitors, and then incubated on ice for 2 h. The supernatants were cleared by centrifugation (12,000 rpm, 20 min, 4°C). Protein extracts were used for assessing expression of STIM1 protein in the tumor samples by Western blot which described above.
Data were expressed as the mean ± standard deviation (SD) of at least three independment experiments. The results were analyzed by Student’s t-test, and P < 0.05 was considered statistically significant.