All manufacturer guidelines were followed when using kits in this investigation unless otherwise specified.
Tissue samples
Patients
The study protocol was approved by the ethics committee of Zhujiang Hospital of Southern Medical University, and all subjects signed an informed consent form before surgery. Participants were divided into a young group (18 ≤ group 1 ≤ 36 years old), middle-aged group (36 < group 2 ≤ 54 years old), and elderly group (group 3 > 54 years old), with three cases in each group. The clinical characteristics of the study participants are summarized in Table 1. Normal ovarian tissues were surgically removed and collected from the Department of Obstetrics and Gynecology, Zhujiang Hospital of Southern Medical University. The criteria for selected ovarian tissues included the exclusion of ovarian diseases, including ovarian cysts, ovarian benign tumors, and ovarian cancer. Postoperative pathology was confirmed by two professional pathologists. The ovarian tissue was placed in a liquid nitrogen tank within 30 min of being isolated until further processing.
Table 1. Characteristics of the study participants
Patient no.
|
Age
(year)
|
Preoperative
diagnosis
|
Postoperative pathology
|
Ovarian pathology
|
Complications
|
1
|
30
|
Cervical cancer
|
Cervical adenocarcinoma,
moderately differentiated
|
No abnormalities in both ovaries
|
None
|
2
|
32
|
Cervical adenocarcinoma
|
Cervical adenocarcinoma,
moderately differentiated
|
No abnormalities in both ovaries
|
None
|
3
|
36
|
Cervical cancer
|
Cervical adenocarcinoma,
moderately differentiated
|
No abnormalities in both ovaries
|
None
|
4
|
50
|
Adenomyosis; uterine fibroids
|
Adenomyosis;
uterine fibroids
|
No abnormalities in both ovaries
|
None
|
5
|
51
|
Cervical squamous
cell carcinoma
|
Cervical squamous cell carcinoma
|
No abnormalities in both ovaries
|
Tricuspid regurgitation
|
6
|
53
|
Endometrium
adenocarcinoma
|
Focal endometrial hyperplasia with atypical, no invasive cancer tissue
|
No abnormalities in both ovaries
|
None
|
7
|
63
|
Pelvic mass
|
Epidermoid cyst of the right ovary with multinucleated giant cell reaction (teratoma)
|
Normal left ovary
|
None
|
8
|
64
|
Abnormal uterine bleeding
|
Cervical adenocarcinoma in situ
|
No abnormalities in both ovaries
|
None
|
9
|
71
|
Cervical intraepithelial neoplasia II
|
Cervical intraepithelial neoplasia II
|
No abnormalities in both ovaries
|
None
|
Animals
The study protocol was approved by the Ethics Committee of the Zhujiang Hospital of Southern Medical University. Kunbai strain healthy female mice (KM mouse) aged 3–5 weeks and 10–11 weeks were purchased from the Experimental Animal Center of Southern Medical University. They were reared in a single cage in a conventional manner under a 14/10 h light/dark environment at a room temperature of 22 °C. For the experiment, 6-week-old and 12-week-old mice were selected, with 5 mice per week. After the mice were sacrificed, both ovaries were immediately removed. One ovary was fixed with 4% paraformaldehyde for 24–48 h. The other ovary and the removed brain tissue, heart, lung, liver, spleen, stomach, small intestine, and muscle were placed in a liquid nitrogen tank.
Hematoxylin and eosin (HE) staining and immunohistochemistry
Fixed KM mouse ovaries were paraffin-embedded. Serial sections of the wax block (4 μm thick) were mounted on a glass slide, and four sets of sections were prepared for HE staining (tissue location control), SCUBE1 immunohistochemical analysis (observation of SCUBE1 distribution), PBS replaces the primary antibody (blank control), and one sheet for standby application. The sections were deparaffinized in xylene, hydrated in gradient alcohol, and then immersed in citric acid-EDTA antigen retrieval solution (Beyotime, Shanghai, China); the mixture was placed in a microwave oven on high heat for 5 minutes, left to rest for 3 minutes, returned to a medium heat for 5 minutes, and then naturally cooled to room temperature. After blocking with endogenous peroxidase, the sections were incubated with an anti-SCUBE1 antibody (1:400, bs-9903R, Bioss, Beijing, China), followed by
incubated the secondary antibody conjugated with biotin and the horseradish enzyme-labeled streptavidin working solution (ZSGB-BIO, Beijing, China) for 1 h at room temperature. The immunoreactivity of SCUBE1 in ovarian cells was then analyzed and scored independently by three observers. The immune response to SCUBE1 was evaluated according to the intensity of the brown color. The immunoreactivity corresponding to the degree of brown color from light to dark was weak, medium, and strong.
Quantitative real time PCR (qRT-PCR)
A TRIzol reagent kit (Takara, Japan) was used to extract total RNA from human ovarian tissue and different organs and tissues of KM mouse, according to the manufacturer’s instruction. The absorbance at 260/280 nm was measured using an ultra-micro high-precision spectrophotometer Nanodrop ND-2000 (Thermo, USA) ratio to determine the concentration and purity of RNA. After reverse transcription of total RNA (1 µg), FastFire qPCR PreMix (SYBR Green) (Tiangen Biotech, Beijing, China) was used for real-time PCR analysis. The primer sequences for SCUBE1 and GAPDH are listed in Table 2.
Table 2. Primers for SCUBE1 and GAPDH used for qRT-PCR
Genes
|
Primer
|
SCUBE1
|
Forward:5’-GAGGATGAGTGCGGCGATGTTC-3’
|
Reverse:5’-CTCTCGTAGGTCTGGCAGGTCTC-3’
|
GAPDH(Human)
|
Forward:5’-CAGGAGGCATTGCTGATGAT -3’
|
Reverse:5’-GAAGGCTGGGGCTCATTT-3’
|
GAPDH(Mice)
|
Forward:5’-GGTTGTCTCCTGCGACTTCA-3’
|
Reverse:5’-TGGTCCAGGGTTTCTTACTCC-3’
|
Cell culture
A granulosa-like tumor cell line, KGN cells, was used in this experiment and was purchased from Wuhan Pu Nuosai Life Technology Co., Ltd., China. Cells were prepared with DMEM/F12 medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (HyClone, USA) at 37 °C in a 5% CO2 atmosphere, and cultivated in an incubator with 95% humidity.
Cell treatment
Cells were seeded in a 6-well plate or Petri dish and divided into three groups (control, model, and experimental groups). Twenty-four hours after cell inoculation, the experimental group was treated with 5 ng/mL SCUBE1 human recombinant protein (rhSCUBE1, Abnova, USA), and the remaining two groups were replaced with the same volume of serum-containing DMEM/F12 medium. After another 24 h, the model and experimental groups replaced the medium with 0.3 mmol/L (mM) H2O2, and the control group replaced the same volume of serum-containing DMEM/F12 medium. After incubation at 37 °C in an incubator with a 5% CO2 atmosphere for 24 h, one step of processing was performed.
Analysis of cell viability
Cells were inoculated in 96-well plates (5000 cells/100 μL/well), incubated at 37 °C in an incubator with a 5% CO2 atmosphere for 24 h, the culture medium was discarded, H2O2 (0.01, 0.1, 0.3, 0.5, 0.8, 1.0, and 1.5 mmol/L) and an equal volume of serum-containing DMEM/F12 medium was added to the control group. Cells were incubated for 24 h, the medium was aspirated, and 100 μL of medium containing 10% CCK-8 solution (Apexbio, USA) was added to each well, with each plate incubated at 37 °C in an incubator with a 5% CO2 atmosphere for 3 h. A microplate reader was used to measure the optical density (OD) of each well at a 450 nm wavelength. All experiments were repeated three times, analyzed, and graphed using GraphPad Prism 8 software. The concentration of H2O2 was determined based on the half-maximal inhibitory concentration (IC50). The inhibition rate of cell proliferation (%) = (mean OD value of control group - mean OD value of experimental group) / (mean OD value of control group - mean OD value of blank group) × 100.
The protective effect of rhSCUBE1 on KGN cells was determined by CCK-8 assay according to the manufacturer’s instructions. Cells were seeded in 96-well plates and divided into eight groups: control group (DMEM medium containing serum), model group (0.3 mmol/L H2O2), low concentration group (5 ng/mL rhSCUBE1), medium concentration group (20 ng/mL rhSCUBE1), high concentration group (40 ng/mL rhSCUBE1), low concentration pretreatment group (5 ng/mL rhSCUBE1 + 0.3 mmol/L H2O2), medium concentration pretreatment group (20 ng/mL rhSCUBE1 + 0.3 mmol/L H2O2), and high concentration pretreatment group (40 ng/mL rhSCUBE1 + 0.3 mmol/L H2O2). Twenty-four hours after cell inoculation, the recombinant protein treatment groups and the pretreatment groups were added with the corresponding concentrations of rhSCUBE1, with the remaining groups replaced with the same volume of serum-containing DMEM. After 24 h, the medium was changed in the pretreatment groups to 0.3 mM H2O2, and in the rest of the groups it was changed using the same volume of serum-containing DMEM medium. These groups were all incubated at 37 °C in an incubator with a 5% CO2 atmosphere for 24 h, with the respective culture media discarded, and the cells tested using the CCK-8 assay.
Determination of intracellular ROS level
Cells were washed three times with PBS and incubated with DCFH-DA (10 µmol/L)(Beyotime, Shanghai, China) in a 37 °C incubator for 20 min. Cell culture medium was washed 3 times to remove excess DCFH-DA, followed by an analysis of cells fluorescence intensity with a flow cytometer (BD Biosciences, USA).
Measurement of mitochondrial membrane potential
The changes in mitochondrial membrane potential (ΔΨm) were measured using the fluorescent dye rhodamine123 (Rh123, Beyotime, Shanghai, China). Cells were digested and collected by trypsin (Gibco, USA), washed twice with PBS, with Rh123 added at a final concentration of 10 mmol/L. Cells were, incubated at 37 °C for 30 min, followed by washing with PBS three times. Immediately after, flow cytometry was performed to measure the average fluorescence intensity of the cells.
Analysis of apoptosis by flow cytometry
Treated cells were collected, washed, centrifuged, and resuspended in Binding Buffer, and then 5 μL Annexin V-FITC and 10 μL PI (CWBIO, Jiangsu, China) were added, mixed, and incubated at room temperature for 15 min in the dark for analysis with flow cytometry.
Western blotting
Total protein extract was obtained from human ovarian tissues or cultured KGN cells. The BCA protein quantification kit (CWBIO, Jiangsu, China) was used to detect the protein concentration and adjust it to the same level. The protein sample (20 μg) was loaded onto an SDS-polyacrylamide gel for electrophoresis and then transferred to a PVDF membrane. After blocking non-specific protein binding sites with 5% skimmed milk for 1.5 h, they were combined with the corresponding primary antibodies [β-actin (1:2000, Servicebio, Wuhan, China), SCUBE1 (1:500, Bioss, Beijing, China), Bax 1:1000, Servicebio, Wuhan, China), Bcl-2 (1:1000, Proteintech, Wuhan, China), Caspase-3 (1:500, Proteintech, Wuhan, China) and p53 (1:1000, Servicebio, Wuhan, China)] overnight at 4 °C. They were then incubated with a secondary antibody (1:3000, Servicebio, Wuhan, China) for 1 h at room temperature. The bands were visualized using an ECL detection reagent (Merck Millipore, USA).
SCUBE1 (c.1169C>G, p.P390R) pathogenicity and stability analysis
A variety of silicon prediction tools, including Polyphen-2 [13], SIFT, Mutation Taster [14], LRT, SNAP, and PANTHER, were used to analyze the pathogenicity of SCUBE1 (c.1169C>G, p.P390R).
According to the Exome Aggregation Consortium (ExAC) PLI (loss-intolerance) [15] the score used to evaluate the possibility of SCUBE1 loss of function (LOF) mutation ranges from 0 to 1. The higher the score, the lower the tolerance.
Align-GVGD [16] is a free online tool for the physical and chemical characterization of mutations. It combines the GVs and GDs for the prediction. The predicted categories range from C0 to C65, and the higher the level, the greater the probability that the mutation will interfere with protein function.
iStable [17] is an integrated prediction tool that predicts the results of MUPRO and I-Mutant2.0. Using the sequence of the protein, we predicted the changes in stability due to specific substitutions.
PyMol software was used to analyze the local spatial configuration changes in the wild-type and mutant-type SCUBE1 models.
Statistical analysis
The experiments were repeated at least three times. Data are expressed as mean ± SD, and SPSS software (Version 23.0, SPSS, Inc., Chicago, USA) was used for analysis. One-way analysis of variance was used to determine the statistical differences. Non-normally distributed data were analyzed with a non-parametric Kruskal-Wallis test. A P value < 0.05 was considered statistically significant (* P < 0.05, **P < 0.01).