Human normal breast epithelial cell line (MCF-10A), human TNBC cell lines (MDA-MB-231, MDA-MB-436, BT-549, HCC1937) as well as human embryonic kidney cell line (HEK-293 T) were all procured from the American Type Culture Collection (ATCC; Manassas, VA, USA) and preserved at 37 °C with 5% CO2. Leibovitz’s L-15 medium (Thomas Scientific, Swedesboro, NJ, USA) was applied to incubate MDA-MB-231 and MDA-MB-436 cells; ATCC-formulated RPMI-1640 medium (ATCC) was used for cultivating BT-549 and HCC1937 cells; Mammary Epithelial Cell Growth Medium (MEGM; Gibco, Grand Island, NY, USA) was adopted for cultivating MCF-10A cells; Dulbecco’s modified Eagle medium (DMEM; Gibco) was used for incubating HEK-293 T cells. 10% fetal bovine serum (FBS; Gibco) together with 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO, USA) was also applied to all media.
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA in cultured cells was first extracted by using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). PrimeScript™ II Reverse Transcriptase (Takara, Kusatsu, Japan) was used for complementary DNA (cDNA) synthesis. CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) together with SYBR Green PCR Kit (Takara) was applied to conduct PCR. All results were calculated by 2−ΔΔCt method. The experiment was conducted for three times in an independent manner.
Confluent TNBC cells at 80–90% confluence were seeded into 6-well plates at 1 × 106 cells/well for 48-h transfection with the specific short hairpin RNAs (shRNAs; GenePharma, Shanghai, China) against SNHG8 (sh/SNHG8#1 and #2), and nonspecific shRNAs (sh/NC) were used in negative control (NC) group. Besides, miR-335-5p mimics/inhibitor together with their relative NCs (miR-NC) were procured form RiboBio (Guangzhou, China). Overexpression of SNHG8 or PYGO2 was achieved by recombinant plasmids pcDNA3.1-SNHG8 or pcDNA3.1-PYGO2, and empty vectors were used as NC. Lipofectamine 3000 reagent (Invitrogen) was applied for cell transfection for 48 h.
TNBC cells after transfection were seeded into 6-well cell culture plates (500 cells/well) and cultured for 14 days of colony formation. After that, colonies were immobilized by 4% paraformaldehyde, dyed by 0.5% crystal violet for 20 min and counted manually. The experiment was conducted for three times in an independent manner.
5-Ethynyl-2′ -deoxyuridine (EdU) assay
After the 48-h transfection, TNBC cells after transfection were laid in 96-well plates (1 × 104 cells/well) and labeled using BeyoClick™ EdU Cell Proliferation Kit (Beyotime, Shanghai, China). Cell nuclei visualization was conducted with the aid of 4′,6-diamidino-2-phenylindole (DAPI) staining solution. Eventually, a fluorescence microscope (Olympus, Tokyo, Japan) was used for observing positively labeled cells. The experiment was conducted for three times in an independent manner.
Transwell migration assay
TNBC cells after transfection were planted in the upper chamber of Transwell chambers (24-well; Corning Incorporated, Corning, NY, USA). Complete culture medium, meanwhile, was filled in the lower chamber which was cultivated with PBS. Twenty-four hours later, cells migrating to the lower chamber were removed with caution by a cotton swab and then fixed in methanol solution for 15 min. Crystal violet was adopted to stain the membranes for 10 min, and the invaded or migrated cells were observed and counted under a microscope (Olympus) at the magnification of 10 × 10. The experiment was conducted for three times in an independent manner.
Subcellular separation assay
Cytoplasmic & Nuclear RNA Purification Kit was commercially acquired from Norgen Biotek Corp (Thorold, ON, Canada) for conducting subcellular separation assay in TNBC cells according to the instruction from the provider. Cells were treated in cell fractionation buffer to isolate cell cytoplasm. SNHG8 levels in cytoplasmic and nuclear fractions were separately detected by RT-qPCR, using GAPDH and U6 as controls. The experiment was conducted for three times in an independent manner.
Fluorescence in situ hybridization (FISH) assay
RNA FISH probe specific to SNHG8 was designed and synthesized at RiboBio and utilized as per the instruction. Air-dried cells were incubated with probes in hybridization buffer, and DAPI was adopted to observe nuclei. Images were obtained using a fluorescence microscope (Olympus). The experiment was conducted for three times in an independent manner.
RNA immunoprecipitation (RIP) assay
Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) was used as per the instruction. Cell lysates and Ago2 antibody (1:50; MA5–14861; Invitrogen) conjugated on magnetic beads were co-cultured in RIP buffer overnight at 4 °C through rotation, while IgG antibody (1:500; 31,786; Invitrogen) was utilized as NC. Finally, the RNA precipitates were purified and extracted using the Imprint® RNA Immunoprecipitation Kit (RIP-12RXN, Sigma-Aldrich, USA) and RT-qPCR was then applied for relative RNA enrichment examination. The experiment was conducted for three times in an independent manner.
RNA pull down assay
Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific, Waltham, MA, USA) was applied for RNA pull down assay according to the standard method from the provider. Protein extracts were collected and incubated with biotinylated miR-335-5p probe, followed by the addition of streptavidin magnetic beads. Then RNAs were eluted down the beads and analyzed using RT-qPCR. The experiment was conducted for three times in an independent manner.
Luciferase reporter gene assay
SNHG8 sequence or the 3′-untranslated region (3′-UTR) of PYGO2 with the predicted binding sequence for miR-335-5p was cloned into pmirGLO dual-luciferase reporter gene vectors (Promega, Madison, WI, USA), and pmirGLO/SNHG8-WT and pmirGLO/3’UTR-WT plasmids were thus obtained. Meanwhile, pmirGLO/SNHG8-Mut and pmirGLO/3’UTR-Mut plasmids were constructed using SNHG8 or the PYGO2 3’UTR sequence with mutant binding sequence for miR-335-5p. Luciferase reporter plasmids and other specific plasmids were co-transfected into cells for 48 h, and Dual-Luciferase® Reporter Assay System (Promega) was then applied to examine relative luciferase activity (firefly/Renilla). Assay was performed three times.
All quantitative assays were bio-repeated thrice, and experimental results were displayed as mean ± standard deviation (SD). A p-value less than 0.05 was considered as statistically significant. GraphPad PRISM 6 (GraphPad, La Jolla, CA, USA) was used for analyzing data. Group difference comparison was conducted with Student’s t-test or one-way/two-way analysis of variance (ANOVA).