4.1. Bioinformatics analysis of the clinical significance of SPP1
FPKM gene expression data, somatic mutation, survival data of Pan-cancer analyzed in The Cancer Genome Atlas (TCGA) project were downloaded from University of California Santa Cruz (UCSC) Xena website (https://xenabrowser.net/datapages/) 27. Tumor mutational burden (TMB) was computed according to total number of mutations referring to all non-synonymous mutations in the coding region and mutations at splice sites28. The R package survivalROC was utilized to study the time-dependent prognostic value of SPP1. A two‐sided log‐rank P < 0.05 was considered significance for survival analysis. The relationship of SPP1 expression level with individual cancer stages and lymph node metastasis status was analyzed by UALCAN database (http://ualcan.path.uab.edu/) 29, which is a portal for the relationship among gene expression, tumor subgroup and survival analyses. SPP1 expression in different metastatic sites was analyzed in Human Cancer Metastasis Database (HCMDB, http://hcmdb.i-sanger.com/)30, which is an integrated database of Gene Expression Omnibus (GEO) and TCGA designed to reserve and process large scale expression data in tumor metastasis. GSE32269 from GEO database totally including hormone sensitive prostate cancer and CRPC samples, were processed using the DESeq package in R.
4.2. Cell culture and transfections
PC-3, DU-145, LNCaP and 22Rv1 were obtained from Cell Bank of Peking Union Medical College Culture Collection (Beijing, China). LNCaP and 22Rv1cell lines were cultured in RPMI-1640 medium with 10% fetal bovine serum and 100 mg/mL penicillin, 100 mg/mL streptomycin were used to culture the cells. PC-3 and DU-145 cell line was cultured in F-12K Medium and Eagle's Minimum Essential Medium, respectively. Cell culture was at 37℃ with 5% CO2-humidified atmosphere. RNA interference was applied to knockdown SPP1 in cell lines. In our study, three different SPP1siRNAs and lentiviral vector were synthesized by JTS scientific (Wuhan, China). All these siRNAs were transfected into PCa cell lines by Lipofectamine 8000 based on the product manual (Invitrogen). The following siRNA sequences were finally used: SPP1(H)-508 (Sense oligo: 5’-GAGUUGAAUGGUGCAUA-CATT-3’, Antisense oligo: 3’-UGUAUGCAC CAUUCAACUCTT-5’ ). SPP1(Target sequence, NP_001035149.1) were cloned into the lentiviral vector JLVO-CAG-GFP-Apuro. Lentiviral vector was transfected into PC-3 and LNCaP cell lines with Polybrene, and cell lines with stable SPP1 expression were selected and amplified.
4.3. RNA isolation and qRT-PCR
Easy pure RNA Kits (TransGen Biotech, BJ, China) was applied for total RNA extraction. TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, BJ, China) was used for reverse transcription according to the manufacturer’s protocol. The mRNA expression level was acquired through cyclic threshold method. SPP1 primers were used: SPP1 (forward: 5′-CGAGGTGATAGTGTGGTTTATGG-3′, reverse: 3′-GCACCATTCAACTCCTCGCTTTC − 5′). PCR reactions were performed in triplicates with following context: After an initial hot start at 95°C for 5 min, PCR amplification was conducted for 40 cycles of 95°C/10s and 60°C/30s, denaturation at 95°C/15 s, and annealing and extension at 60°C for 60s. The relative level of gene expression was detected via the cyclic threshold method.
4.4. Western blot
The protein expression levels of SPP1, Vimentin, E-cadherin, N-cadherin, MAPK p38, ERK1/2, PI3K p110α, PI3K p85, AKT, pAKT, AR and β-actin were evaluated by Western blot. 2.5x105 cells were plated in 6-well plates at 37˚C with 5% CO2 for 24 h, and were washed thrice in ice‑cold PBS, harvested and cell pellets were resuspended in NP-40 lysis buffer (Beyotime, P0013F) containing protease inhibitors and subjected to sonication. Protein concentrations were assessed using a Pierce bicinchoninic acid protein assay kit (Beyotime, P0006.). Equal amounts of proteins (20 µg per sample) were separated by SDS‑PAGE and transferred onto PVDF membranes. After blocked with with 5% milk in TBST, the membrane was incubated at 4˚C overnight with the primary antibody (1:1,000). The following primary antibodies were used: anti-SPP1 (Abcam, Cambridge, UK), anti-Vimentin (Cell Signal Tech, Danvers, USA), anti E-cadherin (Cell Signal Tech, Danvers, USA), anti-N-cadherin (Cell Signal Tech, Danvers, USA), anti-MAPK p38(Cell Signal Tech, Danvers, USA), anti-ERK1/2(Cell Signal Tech, Danvers, USA), anti-PI3K p110α(Cell Signal Tech, Danvers, USA), anti-PI3K p85(Cell Signal Tech, Danvers, USA), anti-AKT(Cell Signal Tech, Danvers, USA), phopho-AKT(Ser 473)( Cell Signal Tech, Danvers, USA), anti-AR(Cell Signal Tech, Danvers, USA) and anti-β-actin (Proteintech, Chicago, USA). Subsequently, the membranes were washed thrice with PBS and incubated with horseradish peroxidase‑conjugated secondary antibodies (anti‑rabbit or anti‑mouse IgG; 1:5,000; Cell Signaling Technology, Inc.). Bands were visualized with BeyoECL Plus (Beyotime, P0018S).
4.5. Immunohistochemical staining
Immunohistochemical (IHC) staining was performed to determine the expression of SPP1 in primary PCa and bone metastasis CRPC. The details of IHC staining procedure are similar to those described in our previous study13. The slices were immersed in methanol with 3% hydrogen peroxide, after dewaxing in xylene and rehydrating in ethanol, then washed in tap water, and then immersed in distilled water. The slides were incubated with anti-SPP1 antibody (Abcam, Cambridge, UK) in PBS at a dilution of 1:400 at 4°C overnight, and then incubated with the secondary antibody.
4.6. Cell proliferation assay
Through the manufacturer’s protocol, cell proliferation test was performed by using the Celltiter-Glo assay (Promega P/N G7570). In this assay, the luminescence value is directly proportional to the amount of ATP, and ATP is positively related to the number of living cells, so cell viability can be obtained by detecting the ATP content. The cell lines were transfected with siRNA SPP1 or siRNA negative control (NC), and disposed by enzalutamide. Every 24 h, viable cells counts were detected, and both SPP1 siRNA and siRNA-NC groups were conducted independently in triplicate.
4.7. Cell apoptosis assay
22Rv1 and its siRNA SPP1 transfected cell lines were plated in 6-well plates at 3×105/well. Four groups were involved in this experiment: the control group disposed with DMSO, enzalutamide treatment group, siRNA SPP1 transfected group, and SPP1 knockdown with enzalutamide treatment group. All the groups were stained with Annexin V and PI (BD Biosciences, CA, US). Afterwards, the stained cells were examined by flow cytometer (BD Biosciences) and data were processed by Cell Quest software. All the experiments were carried out in triplicates.
4.8. Migration and adhesion assay
Migration ability of cells with or without SPP1 transfected was evaluated by wound healing assays. Monolayers of PCa cell lines and their transfected cells were cultured in 24-well plates. Cell layer was scratched with 200 µL pipette’s tip and washed several times using medium to wash dislodged cells. DU-145, 22Rv1 and their transfected cell lines migrated into wound area were photographed at the 48h and 72h, respectively.
After being transfected with SPP1 siRNA or siRNA NC, the cell lines were cultured with 1% fetal bovine serum and plated into the top chamber, cell invasion abilities was evaluated by matrigel transwell assays. 5×104 cells were added into the top chamber of a polycarbonate transwell filter chamber covered with matrigel. After 48h, the cells in the upper of membrane were wiped off, and fixed with 4% paraformaldehyde and stained with 0.5% crystal violet for counting the number of cell. All experiments were carried out in triplicates.
4.9. Statistical analysis
GraphPad Prism 8.0 (GraphPad Software, Inc., CA, USA) was applied for statistical analysis. Data are showed as the mean ± S.E.M. Two groups were compared using Student’s t-test. P value < 0.05 was defined as significant.