1.1 Cell lines and patients
OVC cell lines (HO-8910, HO-8910PM, CoC1, Caov-3, Caov-4) were purchased from cell resource center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (3111C0001CCC000280; 3111C0001CCC000281; 3111C0001CCC000368; 3111C0001CCC000339; 3111C0001CCC000367). Human normal ovarian cell line HOSEpiC was accessed from the cell bank of Chinese Academy of Sciences (Shanghai, China). All cell lines were grown in RPMI 1640 (Gibco, 11875093) medium containing 10% fetal bovine serum (FBS; Gibco, 10099141C) in 5% CO2 at 37 ℃.
Paired clinical OVC tumor (n = 30) and adjacent normal tissue samples (n = 30) were collected from OVC patients in The Second Affiliated hospital of Zhejiang University School of Medicine, and the tissue samples were immediately frozen in liquid nitrogen at -80 ℃ after surgical excision. All patients had not received preoperative chemotherapy or radiotherapy, and had signed the informed consent. This study was approved by Ethics Committee of The Second Affiliated hospital of Zhejiang University School of Medicine.
1.2 Bioinformatics Analysis
Hh signaling pathway related genes were obtained from Kyoto Encyclopedia of Genes and Genomes database (KEGG, https://www.kegg.jp/kegg/pathway.html). Associated analysis was performed on the identified Hh-related genes by means of constructing a PPI network on STRING database (https://string-db.org/). TargetScan database (http://www.targetscan.org/vert_71/) was used to predict the upstream regulatory miRNAs of Gli2. OVC miRNA expression microarray GSE58517 (5 normal tissue samples and 5 OVC tissue samples) was downloaded from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). Normal tissue samples were used as control, and differential analysis was performed by using R package “limma”, with the threshold set as |logFC|>2 and p value < 0.05.
1.3 Overexpression and knockdown of genes
miR-636 mimic, mimic NC, miR-636 inhibitor, inhibitor NC, agomiR-636 and agomiR-NC were all purchased from Shanghai GenePharma Co., Ltd. Short hairpin RNA (shRNA) targeting Gli2 was synthesized by Sangon Biotech Co., Ltd (Shanghai). pEGFP1 overexpression vector was used to establish pEGFP1-Gli2 recombinant plasmid. Transfection was carried out by Lipofectamine®3000 (Invitrogen company, USA) according to instructions.
1.4 Real-Time fluorescence quantitative PCR (qRT-PCR)
Total RNA was extracted from tissues and cells using Trizol (Invitrogen), and then cDNA was synthesized by reverse transcription kit (Invitrogen). qRT-PCR was performed on ABI 7900HT instrument (Applied Biosystems, USA) with miScript SYBR Green PCR Kit (Qiagen, Germany) under the following thermal cycling conditions: pre-denaturation at 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ 2 min, 95 ℃ 5 s and 60 ℃ 30 s. Smo, Gli2, Snail, Vimentin, Tgfb, E-cadherin were normalized with GAPDH as internal reference, and miR-636 was normalized with U6 as internal reference. The expression differences of target genes in control group and test group were compared by 2−ΔΔCt value. The experiment was performed three times. All primers used were shown in Supplement Table 1.
1.5 Western blot
After transfection for 48 h, cells in different treatment groups were washed with precooled Phosphate Buffered Saline (PBS, Thermo fisher, USA) 3 times. Transfected cells were lysed on ice with whole cell lysate for 10 min, and protein quantitation was determined using BCA protein assay kit (Thermo Fisher, USA). The protein samples were then treated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V after boiled for 10 min at 95 ℃ with 10 µl loading buffer, after which the proteins were transferred onto nitrocellulose membrane at 100 mA for 120 min. After being blocked with 5% bovine serum albumin (BSA) or Tris-Buffered Saline Tween (TBST) for 60 min, the membrane was incubated overnight at 4 °C with primary antibodies. The membrane was washed with 1 × TBST (Solarbio, Beijing, China) on a shaking table three times with 5 min per time and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody goat anti-rabbit IgG for 120 min at room temperature. TBST was used to wash the membrane three times, and an electrochemiluminescence kit (ECL; Solarbio, Beijing, China) was employed to visualize the protein bands. All antibodies used were detailed in Supplement Table 2.
1.6 Cell proliferation assay
MTT method was implemented for the detection of cell proliferation. HO-8910PM cells were seeded into 96-well plates at a density of 5 × 103 cells/well, and each treatment was run in triplicate. After 1, 2, 3, 4 and 5 d, sterile MTT solution (Beyotime) was added to cells for assessment of cell proliferation according to instructions. The absorbance at 490 nm was measured by an enzyme-labeled instrument (Molecular Devices, Sunnyvale, CA, USA).
1.7 Wound healing assay
For wound healing assay, HO-8910PM cells (1 × 106) were planted into 6-well plates, and then cell monolayers were wounded with a 200 µl sterile pipette tip when cells grew to 80% in confluence. Isolated cells were removed with mediums, and the cells remained were cultured in fresh mediums for 24 h. Images were photographed at 0 h and 24 h and the wound closure rate was measured.
1.8 Flow cytometry
For cell cycle detection, treated cells (48 h after transfection) of different groups were collected and digested with 0.25% trypsin. Cells were washed with PBS and re-suspended in 70% ice-cold ethanol (1 ml) for 24 h at 4 °C. Then the cells were stained with propidium iodide (PI) and ribonuclease in dark for 30 min (4 ℃). Flow cytometry was used according to standard procedure. The results were analyzed using ModFit.
1.9 Dual-Luciferase Assay
To verify whether miR-636 can directly targeted bind to Gli2 3’UTR, wild-type (WT) and mutant-type (MUT) Gli2 3'UTR were inserted into psiCHECK luciferase reporter vector (Sangon Co., LTD, Shanghai, China). Subsequently, HO-8910PM cells were seeded into 48-well plates for 24 h of incubation, and miR-636 mimic/mimic NC and psiCHECK-WT/MUT were then co-transfected into cells. Finally, the luciferase activity was determined by luciferase assay kit (Promega, Fitchburg, WI, USA).
1.10 Mice experiment
A total of 20 male nude mice (6-week-old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd, and then housed in sterile conditions (12 h/12 h, dark/light; 25℃; 60%-70% humidity). HO-8910PM cells (1 × 106) were inoculated into the abdomen of mice. The mice were divided into two groups with 10 mice in each group when tumor volume reached to 70 mm3. Thereafter, the mice were injected with agomiR-636 (10 nmol/50 ml) or agomiR-NC twice a day, and once a week for 4 weeks. Tumor volume measurement method: Volume (cm3) = (length × width 2) /2. The nude mice were euthanized after 4 weeks, and the tumors were isolated, weighed and photographed. Mice care and laboratory procedures were approved by the ethics committee for laboratory animals.
1.11 Immunohistochemistry (IHC)
Transplanted tumor tissues from mice were placed in 4% paraformaldehyde for fixation in a refrigerator at 4 ℃. After gradient dehydration with different concentration of ethanol, the tissues were transparently treated with dimethylbenzene and embedded in paraffin. Immunochemical staining was performed after the tissue blocks were cut into slices14. Thereafter, the slices were treated with dimethylbenzene and finally sealed using neutral balsam for slides preparation. All antibodies used were detailed in Supplement Table 2.
1.12 Statistical analysis
All data were processed using SPSS 22.0 software. Mean ± standard deviation (SD) was used to express measurement data, while t test was used for comparison between two groups and one-way ANOVA was performed for comparison among more than two groups. * P < 0.05 was considered lowly significant, ** P < 0.01 was considered median significant, while *** P < 0.001 was considered highly significant.