Plasmids, cells, and tissue lysates
A T7-SRp20 expression vector (plasmid pJR17) was constructed by swapping the T7-SRp20 coding region from a pCG-T7-SRp20 expression vector (Tom Misteli of NCI and Javier Caceres of Edinburgh) into the pRevTRE vector to put T7-SRp20 under the control of tetracycline.
Immortal rodent fibroblast NIH 3T3 and MEF 3T3 tet-off cells (Clontech) and human C33A, 786-O, U2OS, HeLa, CaSki, WI-38, and MRC-5 cells were grown in Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) or calf serum (HyClone), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. The B-cell lymphoma-derived cell lines BCBL-1, JSC-1, and SUDHL-6 were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS. Primary human bronchial epithelial cells (HBEpiC) and primary human renal epithelial cells (HREC) were obtained and grown in medium from ScienCell Research Laboratories. HBEpiC were grown in bronchial epithelial cell medium, and HREC were grown in DMEM supplemented with 10% FBS and Epithelial Cell Growth Supplement (ScienCell Research Laboratories). Primary newborn human foreskin keratinocytes (HFKn) were purchased from Invitrogen and grown in calcium-free Medium 154CF plus human keratinocyte growth supplement (Invitrogen). Peripheral blood mononuclear cells were obtained from healthy blood donors at the NIH Clinical Center blood bank. MEF 3T3 tet-off cells were transfected with plasmid pJR17 (T7-SRp20) by Lipofectamine 2000 (Invitrogen), and selected with 200 μg/mL hygromycin and 1 μg/mL doxycycline for stable transfection. A cell line stably transfected with the empty vector, pRevTRE, was also established as a control. To express T7-SRp20, the stably transfected MEF3T3 tet-off cells were grown in doxycycline-free DMEM growth medium.
Tissue lysates of various pairs of tumor and matched normal tissues from different organs were purchased from Protein Biotechnologies.
Synthetic, double-stranded siRNAs were obtained from Dharmacon, Inc. SRp20 and hnRNP U siRNAs were purchased as an siGenome SMARTpool (human SRp20, cat. No. M-030081-00; human hnRNP U, cat. No. J-013501-00). Human YB-1 siRNA is a mixture of siRNAs 393, 394, and oJR1, as described 11
. The nonspecific (NS) siRNA has 52% GC content (cat. No. D-001206-08-20). The SRp20 siRNA s12732, targeting a splice junction of SRp20 exon 2 and exon 3, was purchased from Ambion and SRp20 siRNA D-03 was obtained separately from Dharmacon. RNAi was conducted by two or three separate transfections, at intervals of 48 h, with 10 nM (Ambion) or 40 nM (Dharmacon) siRNA in the presence of Lipofectamine 2000. BCBL-1 and JSC-1 lymphoma cell lines were transfected with 40 nM siRNA using the HiPerfect transfection reagent (Qiagen) by three separate transfections in accordance with the instructions of the manufacturer. The cells with knocked-down SRp20, YB-1, or hnRNP U expression were then analyzed for cell number by trypan blue exclusion, for cell cycle by flow cytometry, for RNA splicing by RT-PCR, for protein expression by Western blotting, and for tumor induction by nude mouse injection.
HEKn cells at 2 × 105 cells per well in 12-well plates were transfected 4 h after passage 3 (day 0) with 40 nM SRp20 siRNA or NS siRNA by using Lipofectamine 2000. Cells were transfected again on days 2 and 4 without passage and were counted on day 6. HBEpiC cells at 2.5 × 105 cells per well in 6-well plates were transfected on days 1 and 3 with siRNA as described above for HFKn and were counted on day 5. HREC cells at 2 × 104 cells per well in 24-well plates were transfected on days 1 and 3 with siRNAs as described above for HFKn and were counted on day 5.
Protein samples in 2× SDS sample buffer were denatured by boiling for 5 min, separated by NuPAGE Bis-Tris gel electrophoresis (Invitrogen), transferred onto a nitrocellulose membrane, and blotted with the following antibodies: mouse monoclonal antibodies against SRp20 (7B4, American Type Culture Collection), beta-tubulin (BD PharMingen), hnRNP K (Santa Cruz Biotech), and PARP (Calbiochem), or rabbit antibodies against PLK1 (Millipore/Upstate), N-terminal FoxM1 (K19), and Cdc25B (Santa Cruz Biotech).
RNA preparation and RT-PCR
Total cell RNA was prepared from the cells using TRIzol (Invitrogen) following the manufacturer's instructions. After DNase I treatment, 1 μg of RNA was reverse transcribed at 42o
C using random hexamers and then amplified using the following gene-specific primer pairs: oJR85 (5'-CGGCTGCCCTACCTACGGA-3') and oJR87 (5'-GGGAGGGCAGCTATTAGGA-3') for human PLK1; oJR104 (5'-CGGCTGCCCTACCTACGAACG-3') and oJR105 (5'- GGCACCAGTCCGAAGGAGAGA-3') for mouse PLK1; oJR111 (5'-TGAAGCCACTGCTACCACG-3') and oJR112 (5'-AGGGCTCTCCACTTTGATGG-3') for human FoxM1; oJR113 (5'-TCGGCCCCGCGTGGAGCAGAC-3') and oJR114 (5'-TAACCCGATTCTGCTCCAGGTGAC-3') for mouse FoxM1; oJR100 (5'- GGGCAAGTTCAGCAACATCGTGGA-3') and oJR101 (5'-GTAGCCGCCTTTCAGGATATACATC-3') for both human and mouse Cdc25B 22
Quantitative RT-PCR (qRT-PCR)
Total RNA purified from cells with or without SRp20 knockdown was quantified by qRT-PCR by using Applied Biosystems TaqMan probes for human PLK1 (#HS00983227) and 18S rRNA (#4333760F) in accordance with the manufacturer's instructions. qRT-PCR was performed in a Cepheid Smart cycler. Briefly, 1 μg of total RNA treated with RNase-free DNase I was reverse transcribed at 42o
C using random hexamers, followed by a 20-μL PCR reaction that included 2 μL RT product, 1 μL TaqMan Gene Expression Assays (20×), and 10 μL TaqMan Universal PCR Master mix (2×). The PCR reactions were performed at 50̊C for 2 min and 95̊C for 10 min, followed by 40 cycles of 95̊C for 15 s and 60̊C for 10 min. The relative expression level of PLK1 in each sample was calculated by the 2-ΔΔCT
after it was normalized to 18S rRNA.
Cervical, soft tissue, and epithelial tumors and normal tissue sections were purchased from US BioMax. Immunohistochemistry was performed with a Vectastain ABC kit (Vector Laboratories) in accordance with the manufacturer's protocol. Sections were deparaffinized, rehydrated, and microwaved for 15 min in the presence of 1× Antigen Retrieval Citra Plus buffer (BioGenex). Endogenous peroxidases were quenched using 3% hydrogen peroxide for 15 min. Sections were incubated with an anti-SRp20 7B4 antibody overnight at 4̊C, followed by a secondary antibody for 30 min and ABC reagent for 30 min. The specific signal was developed by using a DAB substrate kit (Vector Laboratories).
Colony formation assay
NIH 3T3 cells transiently transfected with plasmid pT7-SRp20 (pJR17) or empty vector pFLAG-CMV5.1, or MEF 3T3 tet-off cells stably transfected with plasmid pJR17 or empty vector pRevTRE, were harvested, adjusted to 1 × 104 cells in DMEM containing 0.35% agar and 10% doxycycline-free FBS, and then laid onto a bottom layer containing 0.5% agar and 10% FBS in DMEM in 6-well plates. The plates were stained 3-4 weeks later with 0.005% crystal violet for colony counting.
Tumor induction in nude mice
For tumor induction with HeLa cells, cells transfected twice with SRp20 siRNA or nonspecific siRNAs were collected 24 h after the second transfection and implanted by dorsal subcutaneous inoculation of 1 × 106 cells into both sides of nude mice, with 10 mice in each group. Tumor sizes were measured at 14, 17, and 19 days after implantation. Tumor weight was recorded when the animals were sacrificed on day 22.
For tumor induction with MEF/3T3 tet-off cells, 3 × 106 cells stably transfected with a T7-SRp20 vector or with the empty vector (pRevTRE) were grown in doxycycline-free medium and implanted as described above. Tumor sizes were measured every 4 to 5 days, and tumor weight was recorded when the animals were sacrificed on day 50.
Flow cytometric cell cycle analysis
Cell cycle distribution was determined by flow cytometry. U2OS or HeLa cells transfected twice with SRp20 siRNA or NS siRNA at an interval of 48 h were trypsinized 96 h after the first siRNA transfection, washed twice with PBS, resuspended in Vindelov's propidium iodide buffer, and analyzed with a CYAN MLE cytometer (Dako-Cytomation). Ten thousand events were collected per sample, and data were analyzed with Modfit LT software (Verity Software House).
To analyze the synchronized cells for cell cycle progression, MEF 3T3 tet-off cells stably transfected with T7-SRp20x were seeded in a 6-well plate at 5 × 104 cells per well in DMEM containing 10% doxycycline-free FBS overnight, synchronized by starvation for 48 h in DMEM containing 0.1% FBS, and then collected at the indicated times after serum stimulation (with 10% FBS) for cell cycle analysis by flow cytometry as described above.
Oncomine cancer microarray database analyses
The Oncomine cancer microarray database [http://www.oncomine.com
] was used to analyze expression profiles of SRp20 in a variety of human cancerous and normal tissues and in association with tumor progression (grade) and 5-year survival rate. Statistics from individual studies were also obtained from the Oncomine cancer database (January 15 or April 15, 2010, version) and were combined for a Fisher's meta-analysis. Overexpression of SRp20 was defined as significantly higher expression (P
< 0.05) in tumor tissues than in the corresponding normal tissues, in high-grade tumors than in low-grade tumors, or in shorter-living (<5 years) cancer patients than in longer-living (>5 years) cancer patients.
Southern blot and semi-quantitative PCR analysis
Southern blot analysis was conducted by using ~5 μg of EcoRI-digested genomic DNA extracted from paired normal and cancerous lung tissues (BioChain Institute, Hayward, CA) and hybridized with an SRp20 DNA probe, a 649-bp PCR fragment amplified with a 5' primer (oJR53, 5'-AAGCCGTCCCGATCCTTCTC-3') and a 3' primer (oJR57, 5'-GACTGCTTGTTCAACTATAGCTGCA-3') from HeLa genomic DNA and randomly labeled with 32P. The blot was reprobed for cyclophilin as a loading control by using a cyclophilin probe, a 660-bp fragment derived from a Hind III-digested 1.1-kb PCR product amplified with a 5' primer (oSB21, 5'-CCAAAGCATTGTACCGCAGAG-3') and a 3' primer (oSB22, 5'-TTGCATATACTGCCTTCTCTTTATC-3') from HeLa genomic DNA and randomly labeled with 32P.
For semi-quantitative PCR analysis of SRp20 gene amplification in paired normal and cancerous cervical or lung tissues, genomic DNA isolated from the cervical or lung tissues was serially diluted and analyzed by PCR by using a primer pair of oJR56 (5'-TCTCTTGAAACAGTGACACAAAGGTG-3') and oJR57 for SRp20 gene detection. Cyclophilin PCR with a primer pair of oSB21 and oSB22 served as loading control.
Statistical data for the paired microarray datasets in Fig. , were obtained directly from the Oncomine cancer database (www.oncomine.com
). Statistics from individual studies with significantly higher expression of SRp20 (P
< 0.05) in tumor tissues than in the corresponding normal tissues were also obtained from the Oncomine cancer database (January 15 or April 15, 2010, version) and were combined for Fisher's meta-analysis. All two-group statistical comparisons of means in Fig. and Fig. were calculated with two-tailed student's t
test using Excel (Microsoft).
Fig 3 SRp20 association with tumor progression and prognosis. (A) Increased expression of SRp20 correlates with tumor grade in breast cancer in studies by Schmidt et al. [left; 25] and Sotiriou et al. [right; 26] as obtained from the Oncomine cancer database (more ...)
Fig 6 Human diploid fibroblasts and primary human epithelial cells express minimal amounts of SRp20. (A) MRC-5 and WI-38 fibroblasts express less SRp20 than U2OS and HeLa cells by Western blot analysis. Tubulin served as a control for sample loading. (B) Ectopic (more ...)
Fig 10 SRp20 overexpression is tumorigenic in nude mice. (A-B) HeLa cells with reduced SRp20 expression are less competent at inducing tumors. HeLa cells (1 × 106) with or without SRp20 knockdown were implanted subcutaneously, and the tumor diameter (more ...)