mRNA and miRNA expression profiles download
We compared genes and miRNAs expression between patients with primary prostate and breast cancer with bone metastatic prostate and breast cancer. The gene expression profiles and the miRNA expression profile were obtained from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). One dataset, with GEO accession number GSE32269, was deposited by Cai C et al[20], which contains 22 primary prostate cancer (hormone-dependent) versus 29 metastatic prostate cancer. Another dataset named GSE137842 was submitted by Lefley D[21] which contains 3 primary breast cancer and 3 metastasized to bone breast cancer. The two datasets were obtained from Affymetrix Human Genome U133 Plus 2.0 Array. The miRNA microarray dataset, GSE26964[22], was composed entirely of 6 primary prostate cancer samples and 7 bone metastatic prostate cancer samples (platform: Capitalbio mammal microRNA V3.0).
Identification of differentially expressed miRNAs and differentially expressed gene
Herein, the gene and miRNA expression profile data preprocessing included background correction, quantile normalization, and probe summarization[23]. Then, R software and limma package in Bioconductor were used to extract differentially expressed miRNAs (DE-miRNAs) and differentially expressed genes (DEGs)[24] following criterion P-value < 0.05, |log2fold change| > 1.
GO and KEGG pathway annotation
The identified common DEGs and DE-miRNAs were further subject to Gene Ontology consortium (GO, http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) functional enrichment using online tool of DAVID (Database for Annotation, Visualization and Integrated Discovery)[25]. GO analysis was performed for the cellular component (CC), biological process (BP), and molecular function (MF) categories[26] and KEGG pathway enrichment analysis for the selected genes (hypomethylated genes with high expression and hypermethylated genes with low expression)[27]. All parameters were set as default, p value < 0.01 was considered statistically significant.
mRNA-miRNA regulation network construction
Four databases including miRDB, TargetScan, miRanda, miTarbase, mirwalk was used to identify the number of miRNA-regulated target gene pairs. The threshold of correlation coefficient was set as -0.3 and significance P value was set as 0.05. The pairs supported by two or more databases were further processed and retained. Regulatory network visualization for the regulatory relationship between miRNA-mRNA was conducted using Cytoscape[28].
PPI network and hub genes analysis
The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to detect the interactions of the DEGs, with confidence scores >0.7 were considered significant. PPI network further was visualized by Cytoscape. The cytoHubba plug-in and Molecular Complex Detection (MCODE) plug-in were used to identify hub genes and screen modules of the PPI network. All of the parameters of plug-in were left as the defaults. The genes in modules and hub genes were also analyzed by Metascape.
Prognostic analysis of hub genes
The survival data were extracted from each sample that was acquired from the clinical information of the above samples downloading from the TCGA, and combined with the previously obtained expression profiling data, which were applied to the K-M survival curve drawing for each node by using R survival40, and the prognostic differences of the hub gene-MEF2C genes were analyzed.
Cell culture
The human prostate cancer cell lines DU145, LNCAP and breast cancer cell line MCF7 were obtained from COWELDGEN SCIENTIFIC (Coweldgen scientific Co.,LTD, Shanghai, China). DU145 and MCF-7 cells were grown in Minimum Essential medium (MEM, Life Technologies, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies, USA). LNCAP were cultured in RPMI 1640 medium (Life Technologies, USA) containing 10% FBS. All medium used in the study were supplemented with penicillin (100 U/ml) and streptomycin(100 mg/ml) (Biosharp, China). All cell lines were grown under a humidified atmosphere of 5% CO2 at 37 °C.
Cell transfection
Cells were seeded on 6-well plate, transfection was performed until cell density reached 70-80% at room temperature. MiR-524-5p mimics and normal control (NC) were designed through the reference of miRbase Database (www.miRbase.org and ) and synthesized in GenePharma (Shanghai, China). Cells were transfected with 50nM miR-524-5p mimics or negative control using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.
RNA extraction and RT-PCR analyses
Total RNA was extracted from cell lines with Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. 2.0 μg total RNA was reverse transcribed in a final volume of 20 μL using the PrimeScriptTM RT reagent Kit (Takara, Japan) according to the manufacturer’s protocol. 0.5 μL complementary DNA (cDNA) was amplified and quantified on the LightCycler® 96 system (Roche, Switzerland) using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus)(Takara, Japan). miRcute Plus miRNA First-Strand cDNA Kit (TianGen Biotech, Beijing, China) and SYBR Green (TianGen Biotech, Beijing, China) were used to quantify mature miRNA levels. U6 or beta-actin was used as the internal controls. Relative fold expressions were calculated with the comparative threshold cycle (2-ΔΔCt) method. The primers used are provided in supplementary Table 1.
Western blot
Total protein was extracted using the cell lysate for determining protein expression. Protein sample was quantified by bicinchoninic acid (BCA), separated by SDS-PAGE gel electrophoresis, and blocked with 5% skim milk. Membranes were then incubated with the primary antibody (Proteintech Group, Inc., Wuhan, China) and the corresponding secondary antibody (Beyotime Biotechnology, Shanghai, China). Band exposure was developed by chemiluminescence.
Dual-luciferase reporter gene assay
The MEF2C 3' UTR sequence containing wild-type and mutant binding sites was cloned into the pmirGLO luciferase vector. After the MEF2C 3'UTR WT (or MUT) were co-transfected with miR-524-5p mimic or negative control by Lipofectamine 3000 respectively. After 72 h, the cells were lysed and centrifuged at 12,000 rpm for 5 min to perform Dual-Lumi Luciferase Assay (Beyotime Biotechnology, Shanghai, China). The luciferase activity of the cells was determined by EnVision Multifunctional Microplate Reader (PerkinElmer, Germany). pmirGLO vector transfected into cells served as internal control.
Cell Counting Kit-8 (CCK-8) assay
100 μL of cell suspension containing 1×104 cells was added in each well of the 96-well plate. At 6 hrs, 24 hrs, 48 hrs, 72 hrs, and 96 hrs, 10 μL of CCK-8 reagent (Beyotime Biotechnology, Shanghai, China) was supplied, respectively. After cell culture for 2 hrs, the absorbance value of each well at 450 nm wavelength was measured by a microplate reader for plotting a growth curve.
Transwell assay
Cell suspension with 1×105 cells/mL was prepared with serum-free medium. 100 μL of the suspension was supplied into the chamber and 600 μL of complete medium was added in the basolateral chamber. At the other day, un-penetrating cells above the chamber were wiped off. Subsequently, the chamber was fixed in 4% paraformaldehyde for 30 mins and dyed with 1% crystal violet for another 10~15 mins. Five randomly selected fields in each sample were captured using an inverted microscope (magnification 100×).
In intro 3D model of prostate and breast cancer metastasis
All mouse experiments were approved by The Institutional Animal Care and Use Committee of ShangHai Sixth People’s Hospital. To simulate in vivo bone metastasis microenvironment, one-day-old neonatal CD-1 mice were used to build an in vitro 3D model. In details, after CD-1 mice were sacrificed, their calvaria bone were separated under sterile condition and cut in the occipital lobe to produce an arch structure[29]. Then the calvarial bones were washed with PBS (pH 7.4) and were co-cultured with DU145 cells or MCF7 cells and DU145 cells or MCF7 cells transfected with miR-524-5p mimic in the 48-well plate at a density of 5 × 105 cells per well. The cranium bones without culturing with cells was set as negative control. After incubated at 37 °C for 4 days, the crystal violet staining experiment was performed: the bone fragments were taken out, fixed with 95% alcohol for 10 minutes, washed with PBS 3 times, and stained with 5 mg/mL crystal violet for 15 minutes. Wash the stained bone slices with PBS 3 times and observe under an inverted microscope. Each bone slice is randomly taken from 3 different fields to count the adhered cells (magnification 400×)[30, 31].
Statistical processing
SPSS 22.0 software (SPSS lnc., Chicago, IL, US) was utilized for statistical analysis. The quantitative data were represented as mean ± SD (x¯± s). The independent t-test was used for analyzing the measurement data. Chi-square test was applied for analyzing the categorical data. P<0.05 was considered statistically significant.