Cell lines and animals
The human bladder epithelium immortalized cell line SV-HUC-1 and BC cell lines T24, RT4, 5637, J82, and SW780 were purchased from ATCC (MD, USA). The cells were cultured in DMEM containing 10% fetal bovine serum in an incubator with 5% CO2 at 37 °C.
Babl/c mice (20 ~ 24 g, 6 weeks) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). The animals were housed in a laboratory with temperature control of 24 ± 2 °C and a humidity of 40% ~ 60%. The animal experiment was approved by the medical ethics committee of The Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University.
BC tissue sample
Sixty clinical BC tissues and paired paracancerous tissues were from the resected specimens of BC patients from January 2006 to May 2012 in the Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University. The samples were confirmed pathologically by two pathologists independently and stored in liquid nitrogen. This study was approved by the Institutional Ethical Review Committee of the Affiliated Huaian No.1 People’s Hospital of Nanjing Medical University. All patients enrolled in the study signed written informed consent.
Vectors construction and transfection
Over-expression of LINC00662 and VEGFA were achieved by using a pcDNA3.1 vector (GenePharma, Shanghai, China) transfection. The shRNA against LINC00662 (5'-GTGATTCATACATCCCTATGG-3') was synthesized to knockdown of LINC00662 (Gene-Pharma) according to the BLOCK-iT™ RNAi Designer (Thermo). The miR-195-5p mimics and miR-195-5p inhibitor were designed and synthesized by Gene-Pharma. The empty vectors were used as control. LipofectamineTM 2000 kit was used for transfection operations.
Cell proliferation activity
Cell proliferation was measured using the CCK-8 kit (Beyotime). Briefly, after transfection, the cells were digested and collected, washed with PBS, and then resuspended in DMEM medium to 5×104 cells/ml. The cell suspensions were added to a 96-well culture plate at 100 μl/well. The cells were incubated for 24, 48 and 72 h, respectively, and then 10 μl of CCK-8 solution was added to each well and incubated for 2 h. The absorbance was determined at 450 nm using an Infinite M200 spectrophotometer (TECAN). Six duplicates for each group.
TUNEL staining assay
An in situ cell death detection kit (Roche) was conducted to measure the apoptosis. Briefly, after transfection, the cells were digested and collected, washed with PBS, and then resuspended in DMEM medium to 5×104 cells/ml. The cell suspensions were added to a 24-well culture plate at 500 μl/well. The cells were incubated for 24 h. The cells were washed with PBS and then fixed with 4% paraformaldehyde for 30 min. After washing with PBS once again, the PBS containing 0.3% Triton X-100 was added and incubated for 5 min at room temperature. Then the cells were washed with PBS twice and 50 μl of TUNEL assay solution (prepared according to the instructions) was added and incubated at 37 ° C for 60 min in the dark. The cells were washed with PBS three times and sealed with the antifade mounting medium and observed under a fluorescence microscope (EX: 450-500 nm, EM: 515-565 nm).
The migration activity of BC cells was evaluated by wound healing assay. Briefly, after transfection, the cells were digested and collected, washed with PBS, and then resuspended in DMEM medium to 1×106 cells/ml. The cell suspensions were added to a 6-well culture plate at 1 ml/well. After the cells were covered with the bottom of the plate, the cell layers were scratched by a perpendicular tip to form a wounded gap, and the scraped cells were washed away with PBS. The serum-free medium was added and incubated for 24 h. Finally, the gap width was measured.
The invasion activity of BC cells was determined by the Transwell chamber assay. First, the matrigel was diluted to 1 mg/ml and 100 μl diluted matrigel was added to the upper chamber. Then, after transfection, the cells were digested and collected, washed with PBS, and then resuspended in serum-free DMEM to 1×104 cells/ml. Then 400 μl cell suspension was added to the upper chamber and 1 ml DMEM containing 10% serum was added to the lower chamber. The cells in the chamber were incubated in an incubator for 24 h, then the cells were fixed with methanol for 20 min, and stained with crystal violet for 20 min. The cells on the chamber membrane were wiped with a cotton swab and then observed and photographed under the microscope.
The total RNAs of BC tissues and cells were extracted with Trizol reagent (Invitrogen), the total miRNAs were extracted with miRcute miRNA isolation kit (Genconway) according to the manufacturer's instructions. For miRNA assay, the reverse transcription of the first strand of cDNA was performed by miRNA first-strand cDNA synthesis kit (TIANGEN). Briefly, the poly A tail was added to the 3' end of miR-195-5p, and then the Poly(A) modified miR-195-5p was subjected to reverse transcription to generate the first strand of cDNA according to the manufacturer's instructions. The qRT-PCR assay of the miR-195-5p cDNA was performed by miRcute miRNA fluorescence quantification kit (TIANGEN). Reaction system: 20 μl, reaction conditions: 94 °C for 2 min, (94 °C for 20 s, 60 °C for 30 s, 72 °C for 30 s) × 45 cycle. U6 was used as an internal control. For mRNA assay, the reverse transcription of the first strand of cDNA was performed by iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer's instructions. Then the cDNAs were subjected to qRT-PCR assay with an SYBR Green Realtime PCR kit (TOYOBO). Reaction system: SYBR Green Realtime PCR Master Mix 7.5 μl, upstream and downstream primers each 0.25 μl, RT product 1 μl, and PCR water supplemented to 15 μl. Reaction conditions: 94 °C for 2 min, (94 °C for 15 s, 55 °C for 30 s, 72 °C for 30 s) × 45 cycles. GAPDH was used as an internal control. The relative expression levels were calculated using 2-ΔΔCT method. The corresponding primers were in Table S1 in the supplemental material.
The BC tissues or cells were lysed with pre-cooled RIPA lysate (Sigma-Aldrich) and protease inhibitor cocktail (Beyotime). Then the tissue or cells were homogenized on ice, and the homogenates centrifuged at 12 000 rpm for 10 min at 4 °C. The supernatants were collected and the protein concentration was measured by the BCA method. Then the loading buffer was added to the proteins and boiled for 10 min in a water bath. The proteins were subjected to SDS-PAGE gel electrophoresis at 20 μg/well, and then the proteins were transferred to a PVDF membrane. The transferred PVDF membrane was washed in TBST for 5 min and blocked with 5% skim milk for 2 h at room temperature. Then the PVDF membrane was placed in a hybridization bag and probed with primary anti-VEGFA (#ab1316, Abcam), anti-Ras (#ab16907, Abcam), anti-phospho-Raf-1 (#ab173539, Abcam), anti-Raf-1 (#ab50858, Abcam), anti-phospho-ERK1/2 (#ab214362, Abcam), ERK1/2 (#ab54230, Abcam), anti-phospho-MEK1/2 (#2338, Cell Signaling Technology), anti-MEK1/2 (#4694, Cell Signaling Technology), and GAPDH (#ab8245, Abcam) antibody that diluted with the blocking solution were added and incubated at 4 °C overnight. The PVDF membrane was washed with TBST buffer three times for 10 min each time, then the secondary antibody diluted in blocking solution (HRP goat anti-mouse) was added and incubated at 37 °C for 1.5 h. The immunoreactive protein bands were visualized by chemiluminescence using an Amersham prime ECL Plus detection system (GE Healthcare Life Sciences).
Subcellular fractionation assay
The cytoplasmic and nuclear were extracted from BC cells using the PARIS™ Kit (Invitrogen). The extracts of cytoplasmic and nuclear were subjected to qRT-PCR assay. U6 as nuclear control, GAPDH as the cytoplasmic control.
Dual-luciferase reporter assay
The binding was determined by dual-luciferase reporter assay system (Promega). The target sequences were amplified by PCR and inserted into the pmirGLO vector (Promega) to construct the luciferase reporter vectors (pmirGLO-LINC00662-wt, pmirGLO-LINC00662-mut, pmirGLO-VEGFA-wt, pmirGLO-VEGFA-mut). Then these reporter vectors were co-transfected with or without miR-195-5p mimics (NC mimics as a control) into HEK-293T cells, respectively. The luciferase activity was determined on an illuminometer (Berthold, Germany).
RNA pull-down assay
Firstly, the RNA was biotinylated with Biotin RNA Labeling Mix (Roche) and transcribed with T7 polymerase (Promega). Then the DNA was digested with RNase-free DNase I (Promega) and purified by RNeasy Mini Kit (QIAGEN). Three μg of biotinylated RNA was added to RNA structure buffer (10 mM Tris pH 7.0, 0.1 M KCl, 10 mM MgCl2) and treated at 90 °C for 2 min, then placed on ice for 2 min, and stood at room temperature for 30 min. The T24 or RT4 cells (1×107 cells) were suspended in 2 ml of PBS, then 2 ml of RIPA lysis buffer was added, and the cells were lysed for 1 h at 4 °C. The lysate was centrifuged at 12 000 g for 10 min at 4 ° C, and the supernatant was collected and transferred to an RNase-free tube. Then 400 ng biotinylated RNA and 500 μl RIP buffer were added to the tube and incubated for 1 h, then 50 μl of streptavidin agarose beads were added and incubated for 1 h. The beads were washed with RIP buffer 5 times and washing solutions were collected for RT-PCR or western blot assay.
RIP assay was conducted in T24 and RT4 cells using Magna RIP RNA-binding protein immunoprecipitation kit (Millipore). Briefly, the cells were collected after washing with cold PBS and the RIP lysis buffer was added. Then the suspension was centrifugated and 100 μl cell lysates were transferred to the RIP immunoprecipitation buffer which contained Ago2-conjugated magnetic beads, the mouse IgG as a negative control (Millipore, MA, USA). The magnetic beads washed with RIP wash buffer and then incubated with proteinase K for 30 min at 55°C. The RNA was extracted for qRT-PCR assay.
Fluorescence in situ hybridization (FISH) assay
The presence of LINC00662 was determined by using a FISH kit (Boye Biological) and fluorescein isothiocyanate (FITC)-conjugated LINC00662 DNA probe (5'-CCAGGAGGCAGACCTTGT-3'). Briefly, the cells were seeded in a 24-well culture plate placing a slide. After the cells were covered, the cells were washed with cold PBS twice and fixed for 15 min with 4% RNase-free PFA. Then the cells were washed with PBS and incubated with 0.5%Triton X100 for 5 min, and then treated with gradient ethanol (80%-90%-100%). Then hybridized with the probe and acquired image under a confocal laser microscope (Leica, Solms, German).
Tumor growth in vivo
Balb/c mice were depilated on the back, and then 100 μl of T24 cells successfully transfected with sh-LINC00662 were subcutaneously injected into the left dorsal (1×107 cells/mouse). The tumor volumes were monitored weekly for 5 weeks. On day 35, the mice were sacrificed and the tumors were excised and weighted.
First, the sections were dewaxed and hydrated, then stained by SABC method, and the antigen was repaired by heat and blocked by 5% BSA blocking solution. Then, the sections were incubated with Ki-67 (#ab156956, Abcam) or LINC00662 probe (5'-CCAGGAGGCAGACCTTGT-3') primary antibody overnight at 4 ℃ and incubated with secondary goat anti-rabbit IgG antibody (#ab6721, Abcam) and horseradish-streptavidin working solution for 1.5 h. Then the sections were subjected to DAB staining and hematoxylin counterstaining, dehydration, xylene transparent, neutral gum seal. Images were taken with a microscope.
All data were expressed as means ± SD. Student’s t test, One-way ANOVA, Kaplan-Meier’s method, and the log-rank test were performed by SPSS 13.0. All data from at least three independent experiments. Statistical significance was accepted at P values < 0.05.