Human normal cell line (RWPE-1) and four human PCa cell lines, including DU145, PC3, LNCAP and VCaP, were all obtained from ATCC (Manassas, VA). All cells were cultured under 37oC in 5% CO2 in DMEM culture medium (Gibco) containing 10% FBS (Life Technologies, Carlsbad, CA).
Total RNA extraction and quantitative real-time polymerase chain reaction (RT-qPCR)
Using TRIzol Reagent (Invitrogen, USA), total RNA was isolated and cDNA was synthesized by PrimeScript RT master mix (Takara, Tokyo, Japan). To evaluate the expression of genes, SYBR Green PCR Kit (Takara) was applied to conduct qPCR. The quantitative analysis was determined by 2−ΔΔCt method and all results were normalized to U6 or GAPDH. The experiments were performed at least three times.
Plasmid construct and transfection
The designed shRNAs targeting LINC01006 or DAAM1, as well as their corresponding control shRNA plasmids, were all synthesized by Genechem (Shanghai, China). Besides, pcDNA3.1 vectors (Invitrogen) were subcloned with DAAM1 or LINC01006 for overexpression, and empty pcDNA3.1 vector was used as the negative control. Mimics/inhibitor of miR-34a-5p and NC mimics/inhibitor were procured from RiboBio (Shanghai, China). All cell transfection were performed using Lipofectamine 3000 (Invitrogen).
Colony formation assay
Cells were seeded into 6-well plates at a density of 500 cells per well for 2 weeks of incubation. After 30 min fixation with 4% PFA, 0.5% crystal violet solution was used for cell staining for 5 min. In the end, the visible colonies were manually recorded. The experiments were performed at least three times.
Ethynyl-2-deoxyuridine (EdU) incorporation assay
Using BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China) with Alexa Fluor 594, EdU proliferation assay was conducted. In brief, transfected cells were cultured in 96-well plates with 1 × 104 cells/well. After double-stained using EdU and DAPI solution, cells were finally observed under laser confocal microscopy (Olympus, Tokyo, Japan). The experiments were performed at least three times.
In brief, transfected DU145 and LNCAP cells were treated with 4% PFA and then added with TUNEL reagent (Merck KGaA, Darmstadt, Germany). After being stained with DAPI dye, optical microscopy (Olympus) was used to analyze labeled samples. The experiments were performed at least three times.
Double Annexin V/PI staining kit (Invitrogen) was applied to estimate cell apoptosis. After 15 min of double-staining with binding buffer, cell samples were then determined with a flow cytometer (FACScan, BD Biosciences). The experiments were performed at least three times.
In brief, 2 × 104 cells were plated into the upper chambers of transwell plates. Then the bottom chamber was added with 100% complete culture medium. The chambers were added with Matrigel (BD Biosciences San Diego, CA, USA) for invasion assay and no Matrigel for migration assay. 24 h later, cells were stained by crystal violet for counting. The experiments were performed at least three times.
Western blot analysis
Total protein samples were separated with SDS-PAGE and later transferred to PVDF membranes (Thermo fisher, IL, USA). Afterwards, membranes went through incubation with primary antibodies at 4℃ overnight after being blocked in milk without fat. After washed with PBS×3, the blots were then incubated with secondary antibody for 1 h at dark room. The antibodies against DAAM1 and β-actin were bought from Santa Cruz Biotechnology (Dallas, USA). The quantification analysis of protein was conducted by Chemiluminescence system (GE Healthcare, Chicago, USA). The experiments were performed at least three times.
Subcellular fractionation assay was carried out with PARIS™ Kit (Invitrogen) to isolate the nuclear and cytoplasmic RNAs from PCa cells. GAPDH was treated as cytoplasmic control for quantification, while U6 was the nuclear control. The experiments were performed at least three times.
After fixation with 4% PFA for 15 min at 37°C and then permeabilized with 0.5% Triton X-100, PCa cells were hybridized with LINC01006-specific FISH probe in buffer, followed by counterstaining with Hoechst solution. With a confocal laser microscope (Olympus), images were obtained. The experiments were performed at least three times.
Luciferase reporter assay
Fragments of LINC01006 or DAAM1 containing wild-type and mutant binding sites of miR-34a-5p were cloned into the pmirGLO dual-luciferase vector (Promega, Madison, WI) to respectively construct LINC01006-Wt/Mut and DAAM1-Wt/Mut. DU145 and LNCAP cells were co-transfected with above-mentioned plasmids and miR-34a-5p mimics or NC mimics. Luciferase activity was analyzed using dual-luciferase reporter assay system (Promega). The experiments were performed at least three times.
RNA pull down
MiR-34a-5p wild type or mutant type sequence was labeled with biotin. Then, biotin-labeled RNA was bound to the magnetic beads (Invitrogen). The bound RNAs were purified for RT-qPCR analysis. The experiments were performed at least three times.
RNA immunoprecipitation (RIP)
Using RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA), RIP assay was performed with control IgG antibody or human Ago2 antibody. After lysed in RIPA buffer, cell lysates were mixed with antibody-bound magnetic beads. Subsequently, the immunoprecipitated RNAs were evaluated by RT-qPCR analysis. The experiments were performed at least three times.
BALB/c nude mice (at age of 5–6 weeks) were acquired from SLRC Laboratory Animals (Shanghai, China). For the in vivo tumor formation assay, DU145 cells transfected with sh-NC and sh-LINC01006#1 were suspended in PBS. After that, cells were subcutaneously injected into mice. The tumor volume was measured every 5 days. After one month, the mice were euthanized. Then mice tumors were excised and weighed.
For the tumor metastasis assay, DU145 cells transfected with sh-NC and sh-LINC01006#1were injected into the tail vein of nude mice. The mice were euthanized 50 days later. Then the lungs were removed and fixed with dampen formaldehyde solution for hematoxylin-eosin (H&E) staining. Finally, the lung metastases were observed by microscope. All animal studies were approved by the Ethic committee of Southwest Shandong Hospital Co., Ltd. The experiments were performed at least three times.
All experimental data of three individually performed experiments were presented as the mean ± SD and analyzed by GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA). Student’s t-test or one-way ANOVA analysis was used to analyze differences between groups. The experiments were conducted at least three times. The experimental results were considered statistically significant when p value was less than 0.05.