2.1 Isolation, culture and identification of hUCMSCs.
Human umbilical cords were obtained from mothers who had given birth at Shaanxi maternal and Child Health Hospital and were used in accordance with the ethical guidelines and accepted human studies protocols at Northwest A&F University. Written informed consent forms were obtained from the healthy umbilical cord donors. HUCMSCs were prepared as previously described [28]. The surface markers including positive markers CD44, CD29, CD90, and CD105 and negative markers CD34, CD45 of hUCMSCs were analyzed by flow cytometry and CellQuest software (Becton Dickinson). The antibodies against the above surface markers were purchased from BD Biosciences, USA. The induced differentiation of hUCMSCs was conducted in osteogenic (Gibco, A1007201, USA), adipogenic (Gibco, A1007001, USA), or chondrogenic differentiation medium (Gibco, A1007101, USA) according to manufacturer's instructions.
2.2 Cell culture.
Human granulosa-like tumor cell line KGN was obtained from Fenghui Biotechnology (Hunan, China). KGN cells were cultured in DMEM/F12 medium (Hyclone, USA) supplemented with 10% FBS (ExCell Bio, China), 100 IU/mL penicillin and 100 mg/mL streptomycin (Gibco, USA), and incubated at 37°C in a 5% CO2, 95% humidified atmosphere. For the distinction of high and low density, we followed the previous report [21]. In short, at low-density, cells existed as single cells or small colonies (3× 103 cells/cm2). For high-density conditions allows cells to fuse and contact with each other (1.5× 105 cells/cm2).
2.3 Harvest of hUCMSC‑conditioned medium.
The hUCMSCs at passage 3-6 were cultured in175 cm2 flask in 20 mL DMEM/F12 supplemented with 10% FBS, Then collected medium every 24 hours until the cell density reached 90% .Subsequently, the conditioned medium collected was filtered through a 0.22 μm syringe filter and Stored at -80 °C until use. Stem cell conditioned media represented as UCMSCs-CM in this study.
2.4 Cell growth curve analysis
For growth curve analysis, cells were seeded in 24 well plates with complete growth medium or UCMSCs-CM at a density of 5 × 103 cells per well. Three well cells from each group were counted with a haemocytometer after 0.05% trypsin digestion every 24h, and then generated a growth curve.
2.5 Cell viability assay
Cell Counting Kit-8 (CCK-8, Abmole, USA) was used to detect the viability of KGN cells. KGN cells were seeded in 96 well plates and cultured for 24 hours. The culture medium was changed to 100μL fresh complete growth medium or UCMSCs-CM and cultured for 48h, added 10 μL CCK8 solutions in each well. The optical density values were measured by microplate reader (Bio-Rad, California, USA) at 450 nm.
2.6 Flow cytometry analysis
Flow cytometry was performed as previously described with some modifications [29, 30]. Apoptosis was detected by using an Annexin V-FITC apoptosis detection kit (BD Biosciences, USA). Cell cycle was detected by propidium iodide (PI) (50 μg/mL) staining. The results were assessed by FACS Aria flow cytometer (BD Biosciences, USA).
2.7 TUNEL assay
TUNEL assay was performed as previously described with some modifications [31]. After be treating with UCMSCs-CM for 48h, the KGN cells were fixed with 4% paraformaldehyde for 30 min at room temperature. According to the TUNEL kit (green fluorescence, C1088, Beyotime, China) manufacturer's instructions, TUNEL test solution was added to the cells devoid of light at 37°C for 60 min, and then the nuclei were finally stained with DAPI (Sigma, USA) for 10 min. Using a fluorescent inverted microscope (Leica, Germany) to obtain the photos.
2.8 Scratch wound assay
The motility of KGN cells was assessed by the scratch wound assay. Cells were plated into a 6-well cell culture plate at a density of 4 × 105 cells/well in complete growth medium and grown to 100% confluence. Using a 200 μL pipette tip to make the straight scratch and washed with PBS twice to remove the cell debris. At 0, 24 and 48 hours after the scratch, images were captured using an optical microscope at x100 magnification after incubation at 37˚C on complete growth medium or UCMSCs-CM.
2.9 Cell invasion assay
The invasion abilities of KGN cells were evaluated by the Matrigel invasion assay. Briefly, KGN cells in 100 μL DMEM/F12 medium containing 10 g/L HSA(CSL Behring, Australian) were plated on each 24-well transwell filter upper chamber (8 μm pore size, Corning, USA) coated with Matrigel(Corning, 356234, USA). And the lower chamber was filled with 500μL complete growth medium or UCMSCs-CM to stimulate cell invasion. After 16 h of incubation, cells on the upper membrane surface were removed from the bottom of the filter, fixed with 4% paraformaldehyde, and stained with 5% crystal violet, 5-8 random fields were captured under an inverted microscope for quantification of each well.
2.10 Western blot analysis
Following previous description, western blot was conducted [32], Primary antibodies specific against cyclin D1 (60186), p27 (25614), Bax (50599), Bcl-2 (12789), β-actin (20536) ( all from proteintech, USA), YAP1 (ab52771), p-YAP 1(S127) ( ab76252) ( all from abcam, UK), LATS1( 3477T), p LATS1 (S909) (9157S) ( all from Cell Signaling Technology, USA), all the antibodies were used at the concentration 1:1000. Secondary antibody was HRP-labeled donkey anti-mouse/rabbit IgG (H + L) (1:2000) (Proteintech, USA).
2.11 Immunostaining
Immunofluorescence was performed as previously described with some modifications [33]. Cells were fixed in 4% paraformaldehyde and permeabilized with 0.02% Triton X- 100 in PBS, After washing, the cells were blocked with 1% FBS at 25 °C for 20 minutes and then incubated with the primary antibody, rabbit polyclonal anti-YAP antibody (1:200) at 4 °C for 12 hours, after washing, the cells were labeled with a secondary antibody coupled with Aleax-488(1:1000) (Abcam, ab150117, UK) at room temperature for 2 hour. Cells were stained with DAPI (Sigma, USA) at room tempreture for 10 minutes. Then observed under a confocal microscope (Nikon, TiE-A1 plus, Japan).
2.12 Statistical analysis
All data were expressed as the Mean ± SEM from at least three independent experiments. Statistical analysis was carried out by GraphPad Prism 8 software (San Diego, USA). Differences among groups were evaluated by the Student's t test. P-values were calculated, and statistical significance is displayed as *P <0.05, **P <0.01. ***P <0.001, ****P <0.0001, NS: not significant, P>0.05.