Tissue microarrays (TMAs) containing 8 cases normal adults samples and 73 cases patients samples [170 points: 30 normal (16 normal prostate tissue plus14 adjacent normal prostate tissue), 23 Gleason score 3, 71 Gleason score 4, and 46 Gleason score 5] were commercially obtained from Xi’an Alenabio Technology Co., LTD., and the experiments were approved by Research Ethics Committee. Another independent TMAs containing 34 PCa patients samples (68 points: 22 Gleason score 3, 26 Gleason score 4, and 20 Gleason score 5) were obtained from the Department of Pathology of Xijing Hospital with informed consent of the patients, and approval from the Clinical Research Ethics Committee of Xijing Hospital, the Fourth Military Medical University.
Cell lines, antibodies and reagents
Human PCa cell lines LNCaP, C4–2, 22Rv1 and PC3 were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China) and maintained in RPMI-1640 (Invitrogen Grand Island, NY, USA). Human embryonic kidney cell HEK-293 T and human cervical cancer cell line HeLa were purchased from American Type Culture Collection (Manassas, VA, USA), and maintained in DMEM (Invitrogen, Grand Island, NY). All mediums were supplemented with 10% fetal bovine serum (FBS, Gibco, Australia), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 50 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). All cell lines were cultured in incubators with humidified atmosphere of 5% CO2 and 95% air at 37 °C. Anti-MIIP and anti-FLAG antibody were from Sigma-Aldrich (St. Louis, MO, USA). Anti-PP1α was from Sata Cruz Biotechnology (Dallas, Texas, USA). Anti-HA was from Abcam (Cambridge, MA,USA). Anti-PI3K(p110), anti-AKT, anti-p-AKT (Ser473), anti-mTOR, anti-p-mTOR (Ser2448), anti-p-mTOR (Ser2448), anti-AR, anti-GAPDH and anti-α-tubulin antibodies were from Cell Signaling Technology (Beverly, MA, USA). Dihydrotestosterone, Enzalutamide and BKM120 were commercially purchased (MedChemExpress, Princeton, NJ).
Vector construction, cell transfection and lentivirus packaging
The MIIP CDS was amplified by PCR using the primers and cloned into pLEX-HA-MCS vector (Thermo Scientfic, Waltham, MA) with restriction enzymes Spe I and Xho I (New England Biolabs, Ipswich, MA). pFLAG-CMV4-MIIP∆C and pFLAG-CMV4-MIIP∆N were generated by amplifying the fragments encoding MIIP N-terminal 1–219 residues and C-terminal 220–388 residues using PCR from pLEX-HA-MIIP and inserting the fragments into the EcoR I/EcoR V site of pFLAG-CMV4, respectively. pEGFP-N3-MIIP was constructed by subcloning MIIP from pLEX-HA-MIIP into Nhe I/Hind III site of pEGFP-N3. pmCherry-C1-PP1α was generated by PCR and cloned into EcoR I/BamH I site of pmCherry-C1 vector. The primers used for the construction of the above vectors were listed in Additional file 1: Table S1. For lentiviral packaging, we used packaging system (1 μg pLEX-MCS, 0.2 μg VSVG and 1 μg △8.9) to co-transfect HEK-293 T with lipofectamine 2000 (Thermo Scientfic, Waltham, MA). The stable cell lines LNCaP-MIIP, C4–2-MIIP, and PC3-MIIP were obtained by lentivirus infection and puromycin (1 μg/mL) selection. Transient transfection of PCa cell lines were conducted with lipofectamine 2000 according to the manufacturer’s instruction.
Chemically synthesized siRNA duplexes were obtained from GenePharma (Shanghai, China). Negative control siRNA sequence: 5′-UUCUCCGAACGUGUCACGUTT-3′; siPP1α#1: 5′-CUGCUGGCCUAUAGAUCATT -3′, siPP1α#2: 5′-GACGCUACAACAUCAAACUTT -3′, siMIIP#1: 5′-CCAAACCGGAGGAGUGUAUTT -3′, siMIIP#2: 5′-GACCAUGAAUGCGUGUACUTT -3′. SiRNA transfection were conducted with lipofectamine 2000 according to the manufacturer’s instruction.
Immunohistochemistry was performed according to standard procedures. The primary antibodies anti-MIIP (1:200) and anti-p-AKT (Ser473) (1:100), ABC Kit and DAB (Vector, USA) were used for staining. The results were obtained by digital slice scanner (3DHISTECH, Hungary). Protein expression was analyzed by immunoreactivity scored. The total score = cell score × color score. The cell score standard used the number of cells with positive staining (≤5%: 0, 6–25%: 1, 25–50%: 2, 51–75%: 3, ≥75%: 4). The color score standard used the staining intensity (colorless: 0, mild: 1, moderate: 2, strong: 3).
Real-time RT-PCR analysis
Total RNA was prepared using TRIzol (Sigma, USA) and reversely transcribed into cDNA with the PrimeScript™ RT Reagent Kit (TakaRa, Dalian, China). Real-time quantitative PCR was performed using a CFX96™ Real-time system (Bio-Rad, USA) and SYBR Premix Ex Taq (TakaRa, Dalian, China) according to the manufacturer’s instruction. Raw data were normalized to the internal GAPDH and presented as relative expression level calculated by 2△△Ct method. All primers for qRT-PCR are described in Additional file 1: Table S2. All experiments were performed in triplicate.
Western blot analysis
Samples from lysates of cultured cells were resolved on 10–12% SDS-PAGE gels, transferred to nitrocellulose membranes and probed with anti-MIIP (1:2000), anti-AR (1:2000), anti-PI3K (p110) (1:1000), anti-AKT (1:1000), anti-p-AKT (Ser473) (1:2000), anti-mTOR (1:1000), anti-p-mTOR (Ser2448) (1:1000), anti-p-mTOR (Ser2481) (1:1000), anti-PP1α (1:250), anti-HA (1:2000), anti-FLAG (1:2000), anti-α-tubulin (1:1000) and anti GAPDH (1:1000), followed by either anti-rabbit or anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase at a dilution of 1:5000 (Proteintech Group, USA). The signal of the protein bands were detected using the ECL system (Bio-Rad, USA). The band intensity was quantified by using Image J software (NIH, USA), wherein the relative values (GAPDH or tubulin as internal control) of the first bands were designated as 1.
Cell proliferation and colony formation
Cells were seeded at 2000 cells per 96-well in septuplet and cell proliferation was estimated using the CCK-8 kit (Dojindo Laboratories, Japan) according to the manufacturer’s instructions. Colony formation was measured two weeks after seeding 200 cells per well in 6-well plates. Cell colonies were fixed and then stained with Giemsa for 20 to 30 min. The number of colonies was directly reported, and the formation ratio was calculated according to the following formula: colony formation ratio (%) = (colony number / seeded cells number) × 100%. The data were presented as the mean ± SEM.
All the above experiments were repeated at least three times independently.
HEK-293 T cells were transiently transfected with pLEX-HA-MIIP, pCMV4-FLAG-MIIP∆C, or pCMV4-FLAG-MIIP∆N, together with pmCherry-C1-PP1α. LNCaP-HA-MIIP was transfected with pmCherry-C1-PP1α. Fourty-eight hours later, cells were lysed on ice in lysis buffer (30 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100). Protein lysates containing 1 mg total protein were incubated with 2 μg anti-HA, anti-FLAG or 2 μg mouse IgG for 6 h at 4 °C, followed by incubation with protein A + G sepharose IP beads (Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. IP beads were subsequently washed four times with lysis buffer and boiled in SDS sample buffer for 10 min. Samples were then separated by SDS-PAGE followed by immunoblot with anti-PP1α and anti-MIIP or anti-FLAG.
Co-localization analysis and immunofluorescence staining
HeLa cells were plated on cover slips and transfected with pEGFP-N3-MIIP and pmCherry-C1-PP1α, and co-localization analysis were completed by confocal microscopy after culturing for 48 h. LNCaP-HA-MIIP cells were transfected with pmCherry-C1-PP1α and cultured for 48 h before staining. After removal of culture media, cells were washed with PBS, fixed in 4% PFA, permeabilized with 0.2% Triton X-100 for 10 min and then blocked with 2% BSA in PBS for 1 h. Anti-MIIP (1:200) and anti- PP1α (1:100) primary antibodies were diluted in the blocking solution and applied overnight at 4 °C. After PBS wash for three times, secondary antibodies 488 and Cy3 (GeneTex, San Antonio, Texas) were diluted (1:100) in blocking buffer and applied for 1 h at room temperature. After PBS wash for three times, nuclei were stained with DAPI (1:4000) for 10 min. Co-localization analysis were performed by confocal microscopy.
In vivo xenograft experiment
The animal studies were approved by the Animal Ethics Committee of the Fourth Military Medical University. Six-week-old male nude mice were injected subcutaneously in the limb with 1 × 107cells. Tumor growth was monitored by measuring tumor size using vernier calipers every 10 days for 40 days period, and tumor volume calculated using a standard formula: tumor volume (mm3) = width (mm2) × length (mm) × 0.5. Tumor weight was assessed after sacrificing the mice. Tumor cell proliferation were analyzed by Ki67 staining (Abcam, Cambridge, MA). The data were presented as the mean ± SEM.
Statistics and data analyses
Data are expressed as the mean ± SEM, and statistical evaluation was performed using the Student’s t-test for independent groups. Values of p < 0.05 were considered statistically significant.