Cell lines and cell culture
The human PCa cell lines 22RV1, PC-3, VCaP, DU145, LNCaP and normal prostate epithelial cells RWPE-1 were obtained from Procell (Wuhan, China). RWPE-1 cells were grown in defined keratinocyte-SFM (1×) (Invitrogen). PC-3, LNCaP and 22Rv1 cells were cultured in RPMI-1640 medium (Life Technologies, Carlsbad, CA, US) supplemented with penicillin G (100 U/ml), streptomycin (100 mg/ml) and 10% fetal bovine serum (FBS, Life Technologies). DU145 and VCaP cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS. The C4-2B cell line was purchased from the MD Anderson Cancer Center and maintained in T-medium (Invitrogen) supplemented with 10 % FBS. All cell lines were grown under a humidified atmosphere of 5 % CO2 at 37°C.
Plasmids, Transfection And Generation Of Stable Cell Lines
The human MIR154 gene was PCR-amplified from genomic DNA and cloned into a Ubi-MCS-SV40-EGFP-IRES-puromycin lentiviral vector (GV369, Genechem, Shanghai, China). The 3'UTR of IGF1R, EGFR and FGFR1 were PCR-amplified from genomic DNA and cloned into pmirGLO vectors (Promega, USA), and the list of primers used in cloning reactions is shown in Additional file 1- Table S1. Agomir-154-3p, agomir-154-5p, antagomir-154-3p, antagomir-154-59, the siRNA of EGFR and FGFR1 were purchased from RIBOBIO Company (Guangzhou, China). Cells were treated with MK-2206 (Selleck Chemicals, Houston, TX, USA) at the concentrations (1 µM). Transfection of miRNA, siRNAs, and plasmids was performed as previously described .
Rna Extraction, Reverse Transcription, And Real-time Rt-pcr
Total RNA from tissues or cells was extracted as described previously . Messenger RNA (mRNA) and miRNA were reverse transcribed from total mRNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was amplified and quantified on the CFX96 system (BIO-RAD, USA) using iQ SYBR Green (BIO-RAD, USA). The primers are provided in Additional file 2- Table S2. Real-time PCR was performed according to a standard method, as described previously . Primers for U6, miR-154-3p and miR-154-5p were synthesized and purified by RiboBio (Guangzhou, China). U6 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the endogenous controls. Relative fold expressions were calculated with the comparative threshold cycle (2− ΔΔCt) method.
Patients And Tumor Tissues
A total of 285 PCa tissues, including 203 PCa tissues without bone metastasis and 82 PCa tissues with bone metastasis, and 46 benign prostate hyperplasia tissues (BPH) were obtained during surgery or needle biopsy between January 2014 and December 2018 at the Jiangmen Key Laboratory of Clinical Biobanks and Translational Research, The Affiliated Jiangmen Hospital of Sun Yat-sen University (Guangdong, China). Patients were diagnosed based on clinical and pathological evidence, and the specimens were immediately snap-frozen and stored in liquid nitrogen tanks. For the use of these clinical materials for research purposes, prior patient’ consents and approval from the Institutional Research Ethics Committee were obtained. The clinicopathological features of the patients are summarized in Additional file 3- Table S3. The median of miR-154-5p expression in PCa tissues was used to stratify high and low expression of miR-154-5p.
Cells were co-transfected with HA-Ago2, followed by HA-Ago2 immunoprecipitation using anti-HA-antibody. Real-time PCR analysis of the IP material was performed to test the association of the mRNA of EGFR, FGFR1, IGF1R and MET with the RISC complex. The specific processes were performed as previously described .
Western blot was performed according to a standard method, as previously described . Antibodies against EGFR, FGFR1, IGF1R, p-AKT (S473), p-AKT (T308) and AKT were purchased from Cell Signaling Technology. As a loading control, membranes were stripped and reprobed with an anti-α-tubulin antibody (Sigma-Aldrich, USA).
Luciferase Reporter Assay
Cells (4 ×104) were seeded in triplicate in 24-well plates and cultured for 24 h and performed as previously described . Luciferase and Renilla signals were measured 36 h after transfection using a Dual Luciferase Reporter Assay Kit (Promega).
Akt Activity Assay
To measure Akt kinase activities of in cells or tumor tissues, Akt activity assay was performed as previous described . The immune complexes were then incubated with a biotinylated peptide substrate that became phosphorylated in the presence of activated Akt. The phosphorylated substrates, which reflected the activity of Akt kinase in the extract, was then quantified with the K-LISA Akt Activity Kit (Calbiochem, Darmstadt, Germany) that comprises a primary antibody recognizing the phosphorylated substrate peptides.
All mouse experiments were approved by the Institutional Animal Care and Use Committee of Guangdong Medical University, and the approval-No. was GDY2102160. For the bone metastasis study, BALB/c-nu mice ((5–6 weeks old, 18–20 g)) were anaesthetized and inoculated into the left cardiac ventricle with 1 × 105 PC-3 cells in 100 µl of PBS. After two days of cell injection, animals were injected with 100 µl agomir scramble or agomir-154-5p through the lateral tail vein every three days for 4 weeks. Bone metastases were monitored by bioluminescent imaging (BLI) as previously described . Osteolytic lesions were identified on radiographs as radiolucent lesions in the bone. The area of the osteolytic lesions was measured using the Metamorph image analysis system and software (Universal Imaging Corporation), and the total extent of bone destruction per animal was expressed in square millimeters. Each bone metastasis was scored based on the following criteria: 0, no metastasis; 1, bone lesion covering < 1/4 of the bone width; 2, bone lesion involving 1/4 ~ 1/2 of the bone width; 3, bone lesion across 1/2 ~ 3/4 of the bone width; and 4, bone lesion > 3/4 of the bone width. The bone metastasis score for each mouse was the sum of the scores of all bone lesions from four limbs. For survival studies, mice were monitored daily for signs of discomfort, and were either euthanized all at one time or individually when presenting signs of distress, such as a 10% loss of body weight, paralysis, or head tilting.
In Situ Hybridization
In situ hybridization (ISH) was performed on PDAC tumors using locked nucleic acid (LNA) probes for miR-154-5p (Exiqon, Vedbaek, Denmark). Briefly, Paraffin-embedded tumors were deparaffinized, treated with proteinase K, and fixed in paraformaldehyde. The digoxigeninlabeled LNA probe was hybridized overnight. Slides were rinsed and incubated with anti-digoxigenin, a horseradish peroxidase (HRP)-linked antibody (Zsbio, China), for 2 hr. The detection reaction was performed using the DAB Ready-to-Use Kit (ZLI-9018, Zsbio, China). For each sample, the whole fields of each slide were analyzed by optical microscope. The ISH scores given by the two independent investigators were averaged for further comparative evaluation of the miR-154-5p expression. Tumor cell proportion was scored as follows: 0 (no positive tumor cells), 1 (< 10% positive tumor cells), 2 (10–35% positive tumor cells), 3 (35–70% positive tumor cells), and 4 (> 70% positive tumor cells). The staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellow brown), and 3 (strong staining, brown). The ISH score was calculated as the product of staining intensity score and the proportion of positive tumor cells. Using this method of assessment, we evaluated miR-154-5p expression in pancreatic cancer samples by determining the staining intensity (SI), with scores of 0, 1, 2, 3, 4, 6, 8, 9, or 12. ISH score 4 was the median of all sample tissue SI scores. High and low expression of miR-154-5p were stratified by the follow criteria. An ISH score of 4 was used to define tumors with high expression of miR-154-5p and SI < 4 as tumors with low expression of iR-154-5p.
Invasion And Migration Assays
The invasion and migration assays were performed using Transwell chamber consisting of 8µm membrane filter inserts (Corning) with or without coated Matrigel (BD Biosciences) respectively, and was carried out as previously described . Briefly, the cells were trypsinized and suspended in serum-free medium. Then, 1.5x105 cells were added to the upper chamber, and lower chamber was filled with the culture medium supplemented with 10% FBS. After incubation for 24–48 h, cells passed through the coated membrane to the lower surface, where cells were fixed with 4% paraformaldehyde and stained with haematoxylin. The cell count was performed under a microscope (×100).
Cells were seeded into 96-well plates in triplicate at the initial density of 0.2 × 104 cells/well. At various time points, groups of cells were incubated with 100 µl of 0.5 mg/ml sterile MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide; Sigma] for 4 h at 37ºC. The culture medium was then removed, and 150 µl of DMSO (Sigma) was added. The absorbance values were measured at 570 nm using 655 nm as the reference wavelength.
Colony Formation Assay
Cells (0.2 × 103) were plated into six well plates and cultured for 10 days. Colonies were then fixed for 15 min with 10% formaldehyde and stained for 30s
with 1.0% crystal violet. Plating efficiency = number of colonies (≥ 50 cells per colony) per input cells x 100%. Different colony morphologies were captured under a light microscope (Olympus).
All values are presented as the mean ± standard deviation (SD). Significant differences were determined using the GraphPad 5.0 software (USA). One-way ANOVA was used to determine statistical differences between multiple testing and the post hoc test after ANOVA is Tukey. Unpaired or paired t-test was used to determine statistical differences between two groups. The chi-square test was used to analyze the relationship between miR-154-5p expression and clinicopathological characteristics. Survival curves were plotted using the Kaplan Meier method and compared by log-rank test. P < 0.05 was considered statistical significant. All experiments were repeated three times.