Collection of patient-derived tissue specimens and meta-analysis
A total of 60 ovarian tissues and paired adjacent nontumor tissues were collected from patients who received surgical resection at Nanjing Maternity and Child Health Care Hospital. None of the patients had undergone chemotherapy or radiotherapy prior to surgery. Following surgical resection, all tissues were immediately frozen in liquid nitrogen and stored at −80°C until use for total RNA isolation. The present study was approved by the Ethics Committee of Nanjing Maternity and Child Health Care Hospital and was performed in accordance with the Declaration of Helsinki. Written informed consent was provided by all participants before inclusion in the study.
TCGA ovarian cancer dataset were analyzed using UCSC Xena TCGA data browser (https://xena.ucsc.edu).Clinical data were retrieved from the medical records of 60 ovarian cancer patients who received anti-cancer treatment in our facility during 2015-2018. The patients were at the age of 47 ± 5.12, who were diagnosed with the tumor at stage II and received adjuvant chemotherapy after radical tumorectomy. Informed consent in written was obtained from each patient. The patient-derived ovarian cancer tissue specimens and the contralateral,non-cancerous ovarian tissue specimens were obtained during surgery and were stored in liquid nitrogen before analysis. A total of1657 and 1436 cases fromthe Kaplan-Meier Plotter Database werecollected to analyze overall survival and progression-freesurvival rates.
Cell culture and and stable cell line selection
SKOV3, OVCAR-3, Caov-3 and ES-2 human ovarian cancer cell lines as well as the human ovarian surface epithelial cells (HOSEPICs) were purchased from the cell bank of the Chinese academy of science (Shanghai, China). These cells were cultured in a humidified atmosphere at 37 degree with 5% CO2 using RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% of pen/strep stock solution purchased from Thermo Fisher Scientific (Shanghai, China)[15]. LBX2-AS1 knockdown in vitro was achieved by lentiviral-based delivery of two different shRNA (sh-LBX2-AS1#1 and sh-LBX2-AS1#2) into the target cells. A non-targeting shRNA (sh-NC) was used as negative control for LBX2-AS1 knockdown. The lentiviral particles were commercially purchased (Genecopoeia) and were used at a titer of 10^6 U/ml using Lipofectamin 2000 (Thermo Fisher Scientific) as the transcription reagent at a ratio of 1:20 in antibiotic-free cell culture medium. The cells were selected with Puromycin (Thermo Fisher Scientific) 48 hours after transfection for two passages. The efficiency of LBX2-AS1 knockdown was evaluated by qRT-PCR. Augmentation and inhibition of miR-455-5p or miR-491-5p were achieved by transfecting commercially purchased miR-455-5p or miR-491-5p mimic and inhibitor (Ribobio, Guangzhou, China), respectively, at a concentration of 100nM into the target cells using Lipofectamin 2000 (Thermo Fisher Scientific) as the transcription reagentat a ratio of 1:20 in antibiotic-free cell culture medium. Cells were analyzed 24 hours after transfection.
Evaluation of RNA expression levels
Total RNA (>200nt)or total small RNA (<200nt) were extracted using spin column-based isolation kit (Beyotime, Shanghai, China). The cells cultured in vitro and the patient-derived tissue specimens were first lysed using the lysis buffer associated with the kit before RNA extraction. The RNA extraction products were subject to qRT-PCR using commercially purchased cDNA synthesis kit, primers and qPCR mix (Genecopoeia, Guangzhou, China). For RNA >200nt, GAPDH was used as housekeeping gene, and RNU6B was used as housekeeping gene for small RNA. Primers used in this research were as follows: LBX2-AS1 Fw:5’-AGTTTGTCCCAGGTTTGGCA-3’;LBX2-AS1Rev: 5’-CATGCCAGGGTCCTTGTTCT-3’; E2F2 Fw: 5’-GAGCTCACTCAGACCCCAAG-3’; E2F2 Rev: 5’-AACAGGCTGAAGCCAAAAGA-3’; GAPDH Fw: 5’-CCACATCGCTCAGACACCAT-3’; GAPDH Rev: 5’-TGACAAGCTTCCCGTTCTCA-3’; miR-455-5p Fw: 5’-GCCGCCTATGTGCCTTTGGACT-3’; miR-491-5p Fw: 5’-GGAGTGGGGAACCCTTCC-3’; RNU6B Fw: 5’-CTCGCTTCGGCAGCACA-3’; RNU6B Rev: 5’-AACGCTTCACGAATTTGCGT-3’. The reverse primer for the two miRNAs were universal miRNA primer associated with the qPCR kit.
Cell proliferation
The overall cell growth of the SKOV3 and OVCAR-3 ovarian cancer cells was evaluated by CCK-8 cell proliferation assy. Briefly, the cells were seeded on 96-well-plate at a 1000 cells/well ratio; after 24, 48 or 72 hours of cell culture, the cells were incubated with CCK-8 solution at a ratio of 1:10 in the cell culture medium for 1 hour, and the amount of cells in each well was compared by measuring the OD 450 value of each well using a microplate reader.
Cell colony formationassay
The colony forming ability of the SKOV3 and OVCAR-3cells was evaluated by clone formation assay. Briefly, 200 cells resuspended in 10 ml cell culture medium were cultured in a 100 mm petri dish. 2 weeks after cell culture, the clones on the petri dish were fixed with 4% paraformaldehyde, followed by staining with GIMSA solution (Sigma-Aldrich, Shanghai, China). The clones on each petri dish were counted in 3 randomly picked view under microscope.
Transwell invasion assay
Invasiveness and mobility of the SKOV3 and OVCAR-3cells were evaluated by transwell assay using 8μm pore polycarbonate membrane inserts with or without Matrigel coating (Corning, Shanghai, China), respectively. Briefly, 5000 cells resuspended in serum-free medium were added into the transwell chamber, which was placed in cell culture medium supplemented with 5% fetal bovine serum and was cultured overnight. Cells migrated through the membrane were fixed with 4% paraformaldehyde, followed by staining with GIMSA solution and counting in 3 randomly picked view under microscope.
Flow cytometry
Apoptosis of the SKOV3 and OVCAR-3cells in in vitro cell culture was evaluated by Annexin V-FITC/PI staining and flow cytometry. Briefly, the cells were collected by trypsin/EDTA (Sigma-Aldrich) treatment, before they were stained using Annexin V-FITC Apoptosis Detection Kit (Solarbio, Beijing, China) following the manufacturer’s instructions. the Annexin V-FITC/PI staining of these cells were then evaluated by flow cytometry, and the apoptotic cells were determined by Annexin V-FITC+/PI- staining.
Animal studies
Allanimal maintenance and procedures were carried inaccordance with the National Institute of Health Guidefor the Care and Use of Laboratory Animals, with theapproval of the Animal Research Committee of Nanjing Medical University.The tumorigenicity of the SKOV3 cells was evaluated by tumor formation assay using nude mice. Briefly, 10^7 cells resuspended in 0.2 ml of sterile PBS were subcutaneously injected on the back of the nude mice. The tumor volume was measured at day 7, 14, 28 and 35 post injection. The tumor mass was collected for weighting at day 35.
Prediction of lncRNA-miRNA interaction and miRNA-mRNA interaction
Prediction of lncRNA-miRNA and miRNA-mRNA interactions was performed using Encyclopedia of RNA Interactomes (ENCORI) platform (http://starbase.sysu.edu.cn/) with default settings.
Luciferase reporter gene assay
To verify the putative miRNA-mRNA and miRNA-LncRNA interaction, the predicted bindings sites of miR-455-5p and miR-491-5p on LBX2-AS1 or the 3’ untranslated region of E2F2 mRNA were inserted into the 3’ untranslated region of Gaussia luciferase gene on the Luciferase reporter vector, which also carries the secreted alkaline phosphatase (SEAP) gene as the internal reference. The reporter vector was co-transfected with miRNA mimic negative control, miR-455-5p mimic or miR-491-5p mimic into the target cells. 24 hours after transfection, the Gaussia luciferase and alkaline phosphatase secreted into the cell culture medium was evaluated using Secrete-PairGaussia Luciferase Assay Kit (genecopoeia) following the manufacturer’s instructions.
RNA-pulldown assay
To verify the putative miRNA-lncRNA interaction, we used a biotinylated LBX2-AS1 (Ribobio) as probe to enrich LBX2-AS1-interacting miRNAs from the cell lysate of the SKOV3 and OVCAR-3 cells. Briefly, the cells were lysed using a harsh RIPA lysis buffer by pipetting, after which the cell lysate pre-cleared by centrifugation was incubated with the biotinylated LBX2-AS1 for 2 hours at 4 degree with gentle rotation, followed by incubation with streptavidin-conjugated agarose beads (Sigma-Aldrich) for 2 hours under the same condition. miRNAs associated with the beads were then subject to qRT-PCR as described above.
Western blot assay
Protein levels of E2F2 in the SKOV3 and OVCAR-3 cells after various of treatment were evaluated by western blot assay using GAPDH protein as internal reference. The primary antibody for E2F2 (EPR8622, Rabbit monoclonal) and GAPDH (EPR16891, Rabbit monoclonal) as well as the Goat anti-Rabbit secondary antibody (ab205718) were purchased from Abcam (Shanghai, China).
Statistical analysis
Statistical analysis was performed using Graphpad Prism software (Ver. 8.4, GraphPad Software, San Diego, CA, USA). Data were normalized to the mean value of the control group when applicable. Student’s t test was used for two-group comparison. One-way analysis of variance using Dunnett’s method as the post-hoc test was used for comparison of each experimental group to one control group. One-way analysis of variance using Tukey’s method as the post-hoc test was used for multiple group comparison. The p values were marked in histograms and line charts as follows: *, p < 0.05, comparing to the control group; **, p < 0.01, comparing to the control group; ##, p < 0.01, comparing to the LBX2-AS1 knockdown group.