Clinical Human Samples
A total of 73 pairs of frozen HGSOC tissues were obtained in the Central Hospital of Xinxiang from March 2017 to August 2019. The mean age of these patients was 54.58 ± 11.49 years, and none received radiotherapy or chemotherapy before surgery. After surgical resection of the tumors, all HGSOC tissue samples were determined by histopathological examination. Normal ovarian tissues were collected from cervical cancer surgery patients. Written informed consent obtained from all patients. Tissues were snap frozen in liquid nitrogen and stored at -80℃ for use. The study was approved by the Ethics Committee of the Central Hospital of Xinxiang. All patients provided written informed consent form prior to their inclusion. The written informed consent was obtained from each patient. All the clinical specimens were collected in accordance with the Declaration of Helsinki.
Cell cultures and treatment
FTSEC, OVCAR3, OVCAR4, SNU119, CAOV4 and CAOV3 cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). They were cultured in 1640 or DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum at 37℃ in tmosphere containing 5% CO2. As for detecting half-life, cells were treated with 50 µM α-amanitin (Sigma-Aldrich) for 0–24 h in the article.
Lentivirus, siRNA, miR-1301-3p mimic or inhibitor and transfection
The pLV-shPVT1 Lentivirus plasmid was constructed based on pLV-shNC as backbone. siRNAs of PVT1 and miR-1301-3p mimic and inhibitor were purchased from Suzhou Synbio Technologies. The ovarian cell lines were transfected with plasmids or siRNA via into Lipofectamine 2000 transfection reagent (Thermo, Shanghai, China). The sequences were in Table 1.
Western blot and quantitative real-time PCR
Cell sample was lysed with RIPA buffer and then boiled for denaturation. The sample were loading in SDS-PAGE gel, and transferred to PVDF membrane, followed by blocking with 5% BSA for 2 h at room temperature. The primary antibodies for HER2 (ab194976, Abcam), HER3 (ab255607, Abcam) and GAPDH (ab181602, Abcam) for overnight at 4 ℃, the secondary antibodies for 2 h at room temperature.
Total RNA was obtained from cells and tissues using RNAzol (Invitrogen), RNA was reversely transcribed into first-strand cDNAs by using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa). Real-time qPCR was performed using TaqMan Human microRNA assay (Applied Biosystems) for miRNA analysis and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) for mRNA analysis.
The primer sequences were as follows:
PVT1 forward primer: 5’- TGGTACCGAGCTCGGATCCTC − 3’,
reverse primer: 5’- CCGCCACTGTGCTGGATGATA-3’;
HER2 forward primer: 5’- TGACTGCCTGGCCTGCCTCCA − 3’,
reverse primer: 5’- GGCAGACGAGGGTGCAGGATC − 3’;
HER3 forward primer: 5’- ACCGAGATGCTGAGATAGTG − 3’,
reverse primer: 5’- GCACACTCATCATGGCAGCA − 3’;
miR-1301-3p:
forward primer: 5’- GCCCGC TTGCAGCTGCCTGGGAG-3’,
reverse primer: 5’- GTGCAGGGTCCGAGGT − 3’,
ꞵ-actin gene as internal reference, forward primer: 5’- AGGCCAA CCGCGAGAAGATG-3’, reverse primer: 3’- CACACGGAGTACTTG CGCTCAG-5’.
Cell proliferation assays
Cells (1000 per well) were seeded into 96-well plates for proliferation assay. CAOV3 cells were transfected with overexpression plasmid, OVCAR3 cell lines were transfected with siRNAs. Cell viability was measured by using the CCK-8 kit (Dojindo, Japan) according to the manufacturer’s protocol. All experiments were performed for 3 times. Detection of absorbance every 24 hours. The cell proliferation curves were plotted using the absorbance at each time point.
Transwell assay
Cell migration assay was performed using Transwell chamber inserts (8.0 um, Millipore, USA) in a 24-well plate. First, 2 × 104 cells were counted to seed into the upper chamber. Culture medium containing 20% FBS was placed in the bottom chamber. Then cells were incubated at 37 °C for 1–2 day. After incubation, the cells on the upper surface were scraped and washed away gently, then fixed with methanol. Lastly, cells were stained with 0.5% crystal violet. The numbers of sample cells were counted in five randomly selected fields examined by microscopy. The experiments were repeated independently in triplicate.
RNA immunoprecipitation (RIP-MS2) assays
The pcDNA3.1-MS2 or pcDNA3.1-MS2-PVT1 was co-transfected with pMS2-GFP (Add gene) into OVCAR3 cells. After 48 h, cells were used to perform RIP experiments using a GFP antibody (3 µg per reaction; ab290, abcam) and the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions.22
As for HER2 mRNA pull down: HER2 mRNA was transcription in vitro and labeled using the Biotin RNA Labeling Mix (Thermo Fisher, USA). Next, the biotinylated RNAs were incubated with cell lysis buffer from the OVCAR3 cells at 4 °C for 4 h, afterwards, add 30 µl streptavidin beads to sample gently and incubated on a rotator overnight according to the manufacturer’s protocol (Thermo Fisher, Waltham, USA). The coprecipitated RNAs were detected by real-time PCR
RBP immunoprecipitation and RIP-qPCR assays
We performed RIP experiments using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, MA, USA) according to the manufacturer’s instructions. A anti-HER2 antibody was used for the experiments. The lysates were incubated with the antibody overnight at 4 °C. The coprecipitated RNAs were detected by real-time PCR.
Chromatin Immunoprecipitation
ChIP assays were carried out as described.23 OVCAR3 cell lines were digested by trypsin. Then cells were harvested after 13000 rpm centrifugation, dissolved in PBS for 4% formaldehyde to fix 10 min at room temperature, added 0.25 M glycine to quench at room temperature for 5 min. The cell pellet through centrifuging was resuspended in 1 mL lysis buffer followed by sonication to achieve 300–500 bp DNA fragments.The lysates were incubated with 2 mg anti-HER2 antibody and beads overnight at 4℃. The beads were washed with 600 mL wash buffer four times. Then the reverse crosslinking was carried out by adding proteinase K (10 mg/ml) at 55℃ for 2 hr. DNAs were purified by phenol/chloroformextraction extraction and ethanol precipitation to dissolve in 100 mL ddH2O for qRT-PCR. Primers were listed as follows:
D1 (0-500 bp) forward primer: 5’- gccgcagtcgcccaagcc-3’,
reverse primer: 5’- gctctgcccggaggagcgcg-3’;
D2 (500–1000 bp) forward primer: 5’- ctgtaggtcaactctgcc-3’,
reverse primer: 5’- gactgagctccagctccagt-3’;
D3 (1000–1500 bp) forward primer: 5’- gacagagtgaaacttcatct-3’,
reverse primer: 5’- gtggacactggcctcccatgt-3’;
D4 (1500–2000 bp) forward primer: 5’- ttaagcagtgaaaggattaa-3’,
reverse primer: 5’- ggctctgagagggactgggc-3’;
D5 (2000–2500 bp) forward primer: 5’- atcgcttaaacccaggagg-3’,
reverse primer: 5’- tttaatattcctaccatcc-3’;
D6 (2500–3000 bp) forward primer: 5’- aattgcaactatgagacatt-3’,
reverse primer: 5’- tatcagccagaaataattt-3’;
D7 (3000–3500 bp) forward primer: 5’- cccgggagtaagccaggcatg-3’,
reverse primer: 5’- acaggatctcactctgtcat-3’;
Capture of Nascent RNAs
OVCAR3 cells were seeded into 48-well plates and treated with siRNA (Fig. 3B: siNC or siHER2; Fig. 4A: siNC or siPVT1). After 2 days, 0.2 mM EU was incorporated into the cells for 12hr to capture nascent RNAs,. EU-labeled RNAs were biotinylated and captured by using the Click-iT Nascent RNA Capture Kit (Invitrogen) in accordance with the manufacturer’s instructions.
Luciferase reporter assay
PVT1-wt, PVT1-mut, HER3-wt or HER3-mut sequences was subcloned into pGL3 vector. Then the plasmids were co-transfected with miR-1031-3p mimic into OVCAR3 cell lines using Lipofectamine 2000. After 2 days, luciferase activities were tested using a PmirGLO Dual Luciferase Expression Vector (Promega, Madison) according to the manufacturer's instructions. Luciferase activity in each group was tested and normalized to Renilla.
Animal experiment
The animal studies were approved by the Animal Ethics Committee of the Central Hospital of Xinxiang. Twelve female BALB/C nude mice (5 weeks of age) were randomly divided into two groups. Mice were anesthetized and injected subcutaneously 2*10^6 cells (dissolved in 100 ul PBS) (pLV-NC or pLV-PVT1 stable expression CAOV3 cell lines) into the left or right flanks. Tumor size was monitored and measured once per week via vernier caliper and the tumor volume was calculated as 1/2 LW2, where L and W are the largest and the smallest perpendicular tumor diameter, respectively. Euthanize mice after four weeks. Tumor tissues were snap frozen in liquid nitrogen and stored at -80℃ for use. All experiments were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals of the Central Hospital of Xinxiang and approved by the Animal Ethics Committee of “Animal Ethical and Welfare Committee (AEWC).
Statistical Analysis
Statistical analysis was performed using Graphpad Prism software (La Jolla, CA, USA). The measurement data were expressed by the mean ± SD. The differences between groups were analyzed by t-test. A P value < 0.05 indicated a significant difference. When representative figures are shown, these are representative of three independent repeats.