Human prostate cancer cells (DU145, PC3, LNCAP, VCaP) and human prostate epithelial cells (RWPE-1) were available from ATCC (Manassas, VA) and grown under 5% CO2 and 37oC. They were cultured with 10% FBS (Gibco, Rockville, MD) and 1% antibiotics (Gibco) in DMEM culture medium (Gibco).
Total RNA extraction and qRT-PCR
The extracted total RNAs from cultured cells acquired using TRIzol Reagent (Invitrogen, Carlsbad CA) and converted to cDNA by PrimeScript Reverse Transcriptase Kit (Takara, Shiga, Japan). qPCR assay was conducted with SYBR Green PCR Kit (Takara). Expressions of target genes were calculated by 2−ΔΔCt method and standardized to U6 or GAPDH.
The designed shRNAs for LINC01006 or DAAM1, the pcDNA3.1/DAAM1, as well as their relative control shRNAs and vectors, these were all procured from Genechem (Shanghai, China). The miR-34a-5p mimics/inhibitor and NC mimics/inhibitor were designed at GenePharma (Shanghai, China). DU145 and LNCAP cells were transfected in 24-well plates for 48 h by using Lipofectamine 3000 (Invitrogen).
Transfected DU145 and LNCAP cells were added in 6-well plates, with 500 cells in each well. Following culturing for 14 days, resulting colonies were fixed in 4% PFA for 30 min, then stained in 0.5% crystal violet solution for 5 min. Cloned were counted manually.
EdU assay was undertaken in DU145 and LNCAP cells after transfection by use of BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China) with Alexa Fluor 594. Samples were counterstained with EdU medium and DAPI solution, finally visualized under inverted microscope (Olympus, Tokyo, Japan).
Following transfection, the collected DU145 and LNCAP cells were washed using PBS and treated with 4% PFA. TUNEL reagent was purchased from Merck KGaA (Darmstadt, Germany) for staining the apoptotic cell samples. After DAPI staining, samples were analyzed with optical microscopy (Olympus).
Apoptosis of transfected DU145 and LNCAP cells was estimated with application of double Annexin V/PI staining kit (Invitrogen). Cell samples were double-stained in 6-well plates with Binding Buffer for 15 min, then subjected to flow cytometer (BD Biosciences, Franklin Lakes, NJ).
Transfected cells (1 × 105) were placed to the upper chambers of transwell inserts coating with Matrigel (BD Biosciences) for assessing cell invasion. The 100% complete culture medium was added into the lower chambers. Cell migration was assessed without Matrigel. After 24 h, the inserts were fixed and cells invading or migrating through the membrane were stained by crystal violet for counting.
The nuclear and cytoplasmic RNAs were isolated from DU145 and LNCAP cells by subcellular fractionation assay, using PARIS™ Kit (Invitrogen) as guided. GAPDH was examined as the indicator of cell cytoplasm for quantification, and U6 was examined as the indicator of cell nucleus.
The LINC01006-specific FISH probe in this study was designed by Ribobio (Guangzhou, China). Cells of DU145 and LNCAP were hybridized with probe in buffer, then counterstained in Hoechst solution and visualized using confocal laser microscope (Olympus).
Luciferase reporter assay
LINC01006 or DAAM1 fragments covering wild-type and mutant miR-34a-5p binding sites were acquired and inserted into the pmirGLO dual-luciferase vector (Promega, Madison, WI). They were co-transfected with miR-34a-5p mimics and NC mimics in DU145 and LNCAP cells for luciferase assay, then assayed by dual-luciferase reporter assay system (Promega).
RNA pull down
The miR-34a-5p sequence covering LINC01006 or DAAM1 wild-type or mutant binding sites were acquired and biotinylated. Then, the biotin-labelled probes were mixed with cell proteins and magnetic beads (Invitrogen). The final RNA-protein binding mixture was assayed by qRT-PCR.
RNA immunoprecipitation (RIP)
RIP assay was undertaken in DU145 and LNCAP cells with application of Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit, following the protocol (Millipore, Bedford, MA). The cell lysates were treated in RIP buffer with magnetic beads and control IgG antibody or human Ago2 antibody, followed by qRT-PCR analysis.
Data of three individually performed experiments were presented as the mean ± SD. Statistical analyses of this study were undertaken with Student’s t-test or one-way ANOVA by use of GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA). The experimental results were recorded when p < 0.05.