Cell culture
Colon cancer cell lines (SW620, LOVO, HCT-8, HT-29, SW480 and HCT-116) and 293 T cell were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the human normal intestinal epithelial cell line NCM-460 was obtained from Dr. Ying Ying (Shenzhen University, Shenzhen, China). All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin (15,140,122, Gibco) at 37 °C in a 5% CO2 atmosphere. We used 0.25% trypsin (25,200,056, Gibco) to passage the cell lines when they grew to 90% confluence. All cell lines used in this study were authenticated using the short tandem repeat (STR) method (Additional file 1: Table S4) and verified as being free of mycoplasma contamination using polymerase chain reaction (PCR).
RNA extraction, reverse transcription, and qRT-PCR analysis
Total RNA from colon cancer cells was extracted using RNAiso Plus (9109, Takara, Shiga, Japan). Real-time PCR was performed using a Real-Time system (CFX96, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The expression data were normalized to the geometric mean of the expression of the housekeeping gene GAPDH and calculated using the 2 − ΔΔCt method [30]. The primer sequences are listed in Additional file 1: Table S6.
Patients, tissue specimens and immunohistochemistry (IHC) staining
This study was conducted on a total of 148 paraffin-embedded colon cancer specimens, which were acquired from patients diagnosed from 2006 to 2010. Patient consent and approval from the Institutional Research Ethics Committee (No. 2018003) were obtained to use these clinical materials for research purposes. The tumors were staged according to the 7th edition of the Cancer Stage Manual written by the American Joint Committee on Cancer (AJCC) [31]. A section of the excised tumor was analyzed by IHC and H&E staining as described previously [32].
Plasmids, retroviral infection, and transfection
The human SRPK1 cDNA was amplified by PCR and cloned into the pSin-EF2 lentiviral vector to construct the plasmids SW480-pSin-Vector, HCT-116-pSin-Vector, SW480-SRPK1, and HCT-116-SRPK1. Short hairpin RNA (shRNA)-mediated interference was used to knockdown SRPK1 in SW480 and HCT-116 cells that cells by transfection with lentiviral constructs expressing the SRPK1 shRNAs or control shRNA. The constructs (pSin-EF2-puro-Vector, pSin-EF2-puro-SRPK1, pLKO.1-puro-Vector, pLKO.1-puro-SRPK1-sh1#, and pLKO.1-puro-SRPK1-sh2#) were transfected (10 µg of each plasmid) into 2 × 106 293 T cells to generate retroviruses. The indicated stable cell lines were generated via retroviral infection and were selected for 10 days using 0.5 g/mL puromycin (S7417, Selleck, Houston, TX, USA), as described previously [33]. This resulted in SW480-pLKO.1-Vector, HCT-116-pLKO.1-Vector, SW480-SRPK1-sh1#, SW480-SRPK1-sh2#, HCT-116-SRPK1-sh1# and HCT-116-SRPK1-sh2# stable cell lines. The primer sequences can be found in Additional file 1: Table S5.
Western blotting
Western blotting was performed as described previously [33]. Briefly, 50 mg of protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk and anti-SRPK1 (611,072, BD Biosciences, San Jose, CA, USA), anti-Bcl-xL (BCL2 Like 1 long isoform) (ab32370, Abcam, Cambridge, UK), anti-Bcl-xS (BCL2 Like 1 short isoform) (124,266, Genetex, Waltham, MA, USA), anti-pro-PARP1 ( (ab32138, Abcam, Cambridge, UK), anti-cleaved PARP1 (poly(ADP-ribose) polymerase 1) (ab32064, Abcam), anti-p65 (8242, Cell Signaling Technology, Danvers, MA, USA), anti-IKKβ (A301-827A, Bethyl Laboratories, Montgomery, TX, USA), anti-IKKβ pY199 (ab59195, Abcam), anti-IκBα (ab32518, Abcam), anti-IκBα pS36 (ab133462, Abcam), anti-AKT (4694, Cell Signaling Technology), anti-AKT pS473 (4060, Cell Signaling Technology), anti- Histone H3 (4499, Cell Signaling Technology), anti-β-tubulin (ab210797, Abcam) and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (A300-639A, Bethyl Laboratories) overnight at 4 °C. The membranes were then incubated with goat anti-rabbit (ab97051, Abcam) and anti-mouse (ab6789, Abcam) secondary antibodies for 1 h at room temperature, and then visualized and analyzed using the Amersham Imager 600 (Cytiva, Marlborough, MA, USA) software. The quantification of western blotting was performed by Image J (Version1.8.0, NIH, USA, https://imagej.nih.gov/ij/index.html) as pervious described [34]. Primary antibodies against β-tubulin and GAPDH were used as protein loading controls. The AKT inhibitor (S1078, Selleck) was used to verify the phosphorylation of IKK and IκB.
Dual-luciferase reporter assay
Stable cell lines infected with retroviruses were seeded in DMEM supplemented with 10% FBS in triplicate in 24-well plates (5 × 104 cells per well) and cultured for 12 h. The NF-kB luciferase reporter gene (pGL4.32 [luc2 NF-kB-RE Hyrgo], Promega, Madison, WI, USA) and pRL-TK Renilla plasmid were co-transfected into cells using Lipofectamine 3000 reagent (Invitrogen). At 48 h after transfection, dual luciferase reporter gene detection was performed using a Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol [33].
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay
Cells were seeded in DMEM supplemented with 10% FBS in triplicate in 96-well plates (5 × 103 cells per well) and cultured for 12 h. The medium was replaced with medium containing different concentrations of oxaliplatin and incubated for 24 h. According to CellTiter96® AQueous One Solution Cell Proliferation Assay Kit (Promega) instruction, the reagents were added to the culture medium, incubated for 1–4 h, and the absorbance value at 490 nm was read using a microplate reader. IC50 values were calculated using GraphPad Prism software (version 7, GraphPad Software, Inc., San Diego, CA, USA) via nonlinear regression (curve fit) using the oxaliplatin concentration vs. the normalized response (variable slope) method.
Immunofluorescence assay
The immunofluorescence assay was performed as described previously [32]. Briefly, the cells were incubated with a primary monoclonal anti-p65 (1:100) antibody overnight at 4 °C and then incubated with a fluorescein-conjugated goat anti-rabbit secondary antibody (ab150077, Abcam) for 1 h at room temperature. The cells were visualized under a Dragonfly laser scanning confocal microscopy system (Andor, Belfast, UK).
Terminal deoxynulceotidyl transferase nick-end-labeling (TUNEL) assay
Cells (5 × 104) were seeded on coverslips and cultured for 24 h. Briefly, the cells were treated with the indicated concentrations of oxaliplatin for 24 h, fixed for 25 min in 4% paraformaldehyde, and washed twice with phosphate-buffered saline (PBS). The cells were then incubated with blocking solution for 10 min at room temperature. Following further washes with PBS, the cells were incubated on ice with 0.1% Triton X-100 (T8200, Solarbio, Beijing, China) in 0.1% sodium citrate (Tianjin Zhiyuan Chemical Reagent Co., Ltd., Tianjin, China) for 2 min. Subsequently, the cells were washed with PBS and incubated with 50 µL TUNEL reaction mixture containing the rTdT enzyme for 1 h at 37 °C. The TUNEL assay was performed according to the manufacturer’s instructions (G3250, Promega). After washing with PBS three times, the cells were incubated with 200 µL 4′,6-diamidino-2-phenylindole (DAPI) for 15 min in the dark, washed, and analyzed under an inverted light microscope (CKX53, Olympus, Tokyo, Japan).
Nuclear extract preparation
The indicated cells were washed with 5 mL PBS containing a protease inhibitor cocktail (B14011, Bimake, Houston, TX, USA) and a phosphatase inhibitor cocktail (B15001, Bimake) before adding 3 mL ice-cold PBS containing the same protease and phosphatase inhibitor cocktails. The cells were transferred to a pre-chilled 15-mL conical tube and centrifuged for 5 min at 200×g at 4 °C. A Nuclear Extract kit (40,010, Active Motif, Rixensart, Belgium) was then used to isolate the nuclear extracts from the cell pellets, according to the manufacturer’s instructions.
Statistical analysis
All statistical analyses were carried out using SPSS version 22.0 statistical software (IBM Corp., Armonk, NY, USA). Comparisons between groups were performed using the two-tailed Mann–Whitney U-test. The relationship between SRPK1 expression and clinicopathological characteristics was assessed using Spearman’s correlation test. Survival curves were plotted using the Kaplan–Meier method and compared using the log-rank test. Survival data were evaluated using uni- and multivariate Cox regression analyses. Bivariate correlations between variables were calculated using Spearman’s rank correlation coefficients. A p-value of less than 0.05 was considered statistically significant in all cases.