A total of 22 patients were enrolled, including 7 patients in the normal group, 6 patients in the diminished ovarian reserve (DOR) group, and 9 patients in the polycystic ovary syndrome (PCOS) group. Normal group were patients younger than 35 years old with regular menstrual cycles; DOR group were patients affected by secondary infertility older than 40 years ; PCOS patients were younger than 35 years old and were diagnosed according to the Rotterdam 2003 criteria , which require the presence of two of the following three criteria: ultrasound demonstration of polycystic ovaries, chronic anovulation and hyperandrogenism. All the enrolled patients underwent follicular aspiration for the first time and had no history of ovarian surgery.
Primary luteinized granulosa cell isolation and cell culture
Primary human granulosa cells were extracted and purified from the follicular fluid aspirates utilizing density centrifugation (Lymphocyte Separation Medium, LTS1077, Tianjin, China). Cell sedimentation was then washed twice with ice-cold phosphate-buffered saline (PBS), and resuspended in DMEM/F12 (Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco) and 50 U/mL penicillin-streptomycin (Gibco). KGN, a human granulosa-like tumor cell line, was gifted from Prof. Fei Sun (University of Nantong, China). The lentivirus KGN cell line stably overexpressing lnc-GULP1-2:1 (Lv-lnc-GULP1-2:1) and or its control (Lv-EGFP) was constructed using the similar method as in previous studies . KGN, MDA-MB-231 cells were cultured in DMEM/F12 medium (Gibco). 293T, BeWo and HTR-8/SVneo cell lines were cultured in high-glucose DMEM containing glutamax1 (Invitrogen, Paisley, UK). OVCAR3 cells were cultured in Low Glucose DMEM and SKOV3 cells were cultured in RPMI1640 containing glutamax1 (Invitrogen). U87 MG and Hep G2 cells were cultured in MEM (Gibco). JAR was cultured in RPMI-1640 medium (Invitrogen). All the cell lines used in this study have been verified by short tandem repeat (STR) analysis for authenticity, and were routinely cultured at 37℃ in an atmosphere of 5% CO2 in compressed air at high humidity and all media were supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 50 U/mL penicillin-streptomycin (Life Technologies).
Total RNA was extracted by using Trizol reagent (Life technologies, Inc., Gaithersburg, MD, USA). Total RNA (approximately 800 ng) was reverse transcribed into cDNA by using the iScriptTM cDNA Synthesis Kit (Bio-Rad, CA, USA). Briefly, the 20 μl RT reactions (0.8 μg RNA, 5 μl iScript reaction Mix, 1 μl iScript Reverse Transcriptase and ddH2O) was incubated for 5 min at 25℃ and 20 min at 46℃, incubated for 1 min at 95 ℃ and then maintained at 4℃. For real-time PCR, all reactions were performed in triplicate with iTaqTM Universal SYBR® Green Supermix (Bio-Rad) under the following conditions: 30s at 95℃ for initial denaturation, followed by 40 cycles of segments of 95 ℃ for 3s and 60 ° C for 30s in 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference gene. Gene expression was calculated by using the method of 2−△△Ct, △Ct = △Cttarget − △Ctreference, −△△Ct = − (△Ctsample − △Ctcontrol). The sequences of primers for real-time PCR are listed in Supplementary Table 1.
Cell proliferation assays
Cells were inoculated at a density of 1×104 cells in 96-well plates, and cultured for 24 h, followed by corresponding treatment. Cell proliferation was measured by using Cell Counting Kit-8 (Boster, Wuhan, China) at 0, 24, 48 and 72 h after treatment. At the indicated time, fresh 100 μl DMEM/F12 plus 10 μl CCK-8 medium was added to each well, incubated for 2 h in cell incubator. OD value was measured at 450 nm using spectrophotometer (Thermo Fisher, Vantaa, Finland). Each experiment was independently repeated 3 times, and each treatment had 6 replicate wells in each group.
Total Protein Extraction Kit (Sangon Biotech, Shanghai, China) was used for total protein extraction according to production instructions. Protein concentrations were measured by using Dye Reagent (Bio-Rad) with Quick StartTM Bovine Serum Albumin (Bio-Rad) as standard. Samples were then boiled in protein loading buffer (Boster) at 100℃ for 10 min, and equal amounts of protein (40 µg) were loaded into the wells of the SDS-PAGE gel. Following by electrophoresis according to standard procedures, the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) in a wet transfer system (Bio-Rad). Membranes were blocked for 1 h at room temperature in 5% non-fat dry milk in tris-buffered saline supplemented with 0.1% Tween-20 (TBST). After blocking, membranes were incubated with primary antibodies (anti-COL3A1 mouse monoclonal antibody, 1:100, Santa Cruz, Oregon, USA; anti-GAPDH mouse monoclonal antibody, 1:3000, CMCTAG, Milwaukee, USA) in blocking buffer at 4℃ overnight. Then the PVDF membranes were washed 3 times (10 min each) with TBST at room temperature and incubated with secondary antibody (goat polyclonal secondary antibody to mouse IgG-H&L (HRP), 1:5000, Abcam, Cambridge, USA) for 1 h. Finally, immunoreactive bands were detected by using enhanced chemiluminescent substrate in a ChemiDoc MP imaging system (Bio-Rad).
Lentivirus-stabilized KGN cell lines, which stably overexpressing lnc-GULP1-2:1 (Lv-lnc-GULP1-2:1) and its control (Lv-EGFP), were re-inoculated on glass coverslips pre-coated with poly-lysine and cultured for 24 h in an atmosphere of 5% CO2 at high humidity. Cells were treated with 4% paraformaldehyde for 20 min, then gently washed with phosphate buffered saline (PBS) for 3×3 min. Next, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked by using 5% bovine serum albumin (BSA) in TBST for 30 min at room temperature. After blocking, cells were incubated with COL3A1 antibody (1:100, Santa Cruz) or Ki-67 antibody (1:1000, Cell signaling technology, Beverly, USA) at 4℃ overnight; PBS was used as a negative control. Then cells were washed 3 × 3 min with PBS at room temperature and incubated with Alexa Fluor 594-AffiniPure Goat Anti-Rabbit IgG (1:600, Jackson, Pennsylvania, USA) for 30 min. After washing with PBS, the nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) (5 μg/ml). Then the coverslips were washed again with PBS 4 × 5 min. Finally, the coverslips were mounted with anti-fade Mounting Medium (Beyotime Biotechnology, Shanghai, China), and the image was observed and collected under a laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
Fluorescence in situ hybridization
Cells were re-plated on poly-lysine-pre-coated glass coverslips and cultured for 24 h in an atmosphere of 5% CO2 at high humidity, then treated with 4% paraformaldehyde for 20 min. After washing the coverslips, cells were digested by 20μg/ml proteinase K (Servicebio, Wuhan, China) for 5 min. Then the coverslips were washed 3 × 3 min with PBS at room temperature and incubated with prehybridization solution for 1 h at 37℃. Next, the prehybridization solution was discarded, and a hybridization solution (Servicebio) containing lnc-GULP1-2:1 probe (5’-DIG-CATGG CTATTTGATGAACATGACTTT-DIG-3’) or nonsense control probe (5’-DIG GTGTAACACGTCTATACGCCCA-3’) (Genepharma, Shanghai, China) at a concentration of 8 ng/μl, was added dropwise, and hybridization was carried out at 37℃ overnight. Hybridization solution then was washed away. The coverslips were placed in 5% BSA in PBS (Servicebio) for 30 min. The blocking solution was discarded, anti-DIG-cy3 (Jackson, Pennsylvania, USA) was added dropwise, incubated at 37℃ for 50 min, and then washed 3 × 5 min with PBS. The DAPI staining solution was added to the coverslips, incubated for 8 min in the dark, and the anti-fade Mounting Medium (Servicebio) was added after washing. Finally, the slides were observed under a fluorescence microscope (NIKON ECLIPSE CI, Japan).
For gene knockdown analysis, COL3A1 specific siRNA (sc-43062, Santa Cruz) was used to silence the COL3A1 gene, and siRNA control (Silencer™ Select Negative Control, 4390843, Invitrogen) was used as a silence control. Cells were transfected with COL3A1 siRNA or control coupled with Lipofectamine® RNAi-MAX (Invitrogen) according to the product manufacture protocol, and were treated or collected at the indicated time for subsequent analysis.
Cell cycle analysis
Primary granulosa cells treated with 5×1010 PFU/ml adv-control or adv-lnc-GULP1-2:1 (Hanbio Biotechnology, Shanghai, China) for 48h were seeded (70% confluent) in 12-well plates for flow cytometry analysis. Cells were digested with EDTA-free trypsin, washed twice with cold PBS (1000 rpm × 5 min), and then fixed with 70% ethanol at 4 ℃ overnight. The fixed cells were centrifuged (1000 rpm × 5 min) again on the next day, resuspended in 500μl of PI/RNase solution（KeyGen Biotech，Nanjing，China), and then incubated in the dark for 30 minutes at 37 ℃. Flow cytometry studies were performed by using BD Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA). The percentage of cells in different phases of cell cycle were analyzed by using FlowJo 10.4 (BD Biosciences).
Data were expressed as mean ± SD. All statistical analyses were performed using SPSS 22.0 (IBM, Armonk, NY, USA). Unpaired T-test with Welch’s correction was used for the statistical analysis between the two groups, while one-way ANOVA followed by Bonferroni’s post-hoc test was used for the statistical analysis of more than two groups of data. The difference of p <0.05 was considered significant. Each experiment was repeated at least three times independently.