1.1 Tissue Samples
A total of 96 epithelial ovarian cancer tissues were obtained from the Department of Gynecology at the Fourth Hospital, Hebei Medical University, China (November 2011–June 2015). The informed consent of each subject was obtained, and this study was approved by the Medical Ethics Committee of the Fourth Affiliated Hospital of Hebei Medical University. The detailed clinicopathological features of the patients are summarized in Table 1. The inclusion criterion for cases was histologically confirmed primary EOC. The exclusion criteria was a history of chemotherapy therapy before surgery. According to the NCCN guidelines, recurrent disease was identified clinically (i.e., pelvic pain and weight loss), biochemically (i.e., elevated CA-125 levels), and/or with imaging [18]. Based on the platinum-free interval (PFI), which was calculated from the date of the last platinum compound treatment to the date of disease progression, all the study participants were divided into a platinum-sensitive group (n=92) and a platinum-resistant group (n=62). PFI of less than 6 months is widely used to clinically define platinum-resistant disease, whereas a PFI greater than 6 months is often used to define platinum-sensitive disease [19]. The participants were regularly followed-up for 5 years.
Table 1
Clinical characteristics of 96 EOC patients
Characteristics
|
Stage
|
Patients(n)
|
Median
|
Percentage/Range
|
Age
|
<50 Years
|
34
|
58Years
|
35.4%
|
|
≥50 Years
|
62
|
58Years
|
64.6%
|
Histology
|
Serous
|
60
|
62.5%
|
|
Endometrioid
|
21
|
|
21.9%
|
|
Mucinous
|
6
|
|
6.25%
|
|
Clear cell
|
3
|
|
3.1%
|
|
Mixed type
|
6
|
|
6.25%
|
FIGO stage
|
I-II
|
21
|
|
21.9%
|
|
III-IV
|
75
|
|
78.1%
|
Grade
|
1
|
24
|
|
25.0%
|
|
2
|
42
|
|
43.8%
|
|
3
|
30
|
|
31.2%
|
tumor residual size
|
0
|
25
|
|
26.0%
|
|
<1cm
|
48
|
|
50.0%
|
|
>1cm
|
23
|
|
24.0%
|
Platinum-based
|
Cisplatin
Carboplatin
|
26
70
|
|
27.1%
72.9%
|
Follow-up time
|
|
96
|
37.2months |
2-60 months
|
1.2 Genomic DNA extraction and MALDI-TOF mass spectrometry
High-quality DNA was isolated from 40 EOC tissue samples using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wisconsin), as described by the manufacturers. MALDI-TOF mass spectrometry (Sequenom, San Diego, California, U.S.), described by Breitling et al. [20], was used to detect the methylation level of the MGRN1 promoter region. This experiment was conducted at CapitalBio Co., Ltd. (Beijing, China).
1.3 RNA extraction and quantitative real-time reverse transcriptase-PCR (RT-qPCR)
Total RNA was isolated from 96 epithelial ovarian cancer tissue samples using the TRIzol-chloroform extraction method (Generay Biotech Co., Ltd., Shanghai, China), as described by the manufacturers. The total cDNA was reverse-transcribed using the Revert Aid First-Strand cDNA Synthesis Kit (Thermo Scientific, USA). The specific primers for the target genes that were used in RT-qPCR were designed using Primer Premier 5.0 and produced by Sangon Biotech Co., Ltd. (Shanghai, China). GAPDH was used as the housekeeping gene. The primer sequences for PCR amplification were as follows: MGRN1 forward, 5'-TACAAAGACGATG CCGACAG-3'; MGRN1 reverse, 5'-GCCTGGCAGTAGATGGTGAT-3'; GAPDH forward, 5'-AATCCCATCACCATCTTCCA-3'; and GAPDH reverse, 5'-TGGACTCCACGACGTACTCA -3'. The reactions were run with the QuantiNova TMSYBR® Green PCR Kit (Qiagen, Hilden, Germany) in an Mx3005P instrument. The comparative quantification of each target gene was performed based on the cycle threshold (Ct) and normalized to GAPDH using the 2-ΔCt method.
1.4 MGRN1 immunohistochemical (IHC) study of the clinical samples
Of the 96 epithelial ovarian cancer samples, 86 paraffin-embedded epithelial ovarian cancer tissue samples collected in the pathology department of the Fourth Hospital of Hebei Medical University were used for immunohistochemical (IHC) staining of MGRN1. MGRN1 immunostaining was performed using a primary antibody, namely rabbit antihuman MGRN1 (RNF156, 1:500 dilution; Proteintech, China). Briefly, 4-μm thick sections were dewaxed in xylene and dehydrated through a graded series of ethanol. After blocking endogenous peroxidase and non-specific binding, the sections were incubated overnight at 4°C with primary antibodyand then with biotinylated secondary antibody and streptavidin-peroxidase complex. After the sections were washed in PBS, they were incubated with DAB reagent and counterstained with haematoxylin. Negative control sections were incubated with PBS instead of primary antibody. The immunohistochemical staining was evaluated using a previously reported scoring method [21]. The immunoreactivity of MGRN1 was considered to be positive in tumor cells showing cytoplasmic staining without nuclear staining. The sections were independently examined by two pathologists, who were blinded to the clinicopathological information.
2.1 Cell culture
The human serous ovarian cancer SKOV3 cell line was purchased from the iCell Bioscience Inc. (Shanghai, China). The SKOV3 cell line was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc.) The medium was always supplemented with 10% (w/v) fetal bovine serum, 100 U penicillin, and 100 µg/L streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were maintained in a 95% humidified and 5% CO2 atmosphere at 37°C. All the experiments were performed in triplicate.
2.2 Stable cell lines
MGRN1 expression plasmids and lentiviral packaging reagents and shRNA were purchased from Genecopoeia Inc. (MD, USA). The designed three target sequences in the MGRN1 gene were 5′-GGAAACTACTTTGCTTCGCAC-3′ (shRNAa); 5′-GCGTGTTTCCAGTAGTCATC C-3′ (shRNAb) and 5′- GGCATTGAGAACAAGAACAAC-3′ (shRNAc). The most effective construct, recombinant plasmid inserted with MGRN1 gene shRNA expression vector shRNAa was selected for the study. A random sequence of shRNA (shNC) was used as the negative control. SKOV3 cells were seeded in a six-well plate at a density of 4×105 cells/mL in a volume of 2mL/well. When the SKOV3 cells reached 70–80% confluence, they were transfected with shRNA. Transfection of the SKOV3 cell line was performed according to the manufacturer’s protocol.
2.3 Detection of changes in MGRN1 via Western blotting
Proteins were isolated using RIPA lysis buffer. The total proteins were extracted, and a BCA protein assay kit (Thermo) was used to quantify protein concentration. Rabbit anti-human MGRN1 antibody (RNF156, Proteintech, China) and β-actin (ab8226, Abcam, Cambridge, UK) were used as the primary antibody. Anti-rabbit IgG was used as the secondary antibody (diluent ratio of 1:5000; Proteintech, China). The antigen-antibody reaction was visualized by detection with an odyssey assay (ECL, Millipore, Billerica, MA).
2.4 Cell viability assays
The cells were inoculated in 96-well microplates in medium containing 10% fetal bovine serum and penicillin/streptomycin. After overnight incubation, the cells were treated with cisplatin (Pfizer), returned to the incubator for 24 h, and then analyzed. Cell Counting Kit-8 (CCK-8) was used to measure cell activity. Ten microliters of CCK-8 was added to each well and incubated for 3 h (37°C; 5% carbon dioxide). Then, the absorbance was measured at 492 nm with a microplate reader. Each experiment was repeated three times.
2.5 apoptosis assays
The cells were collected after 24h of cisplatin treatment. The Annexin V Apoptosis Detection kit I (BD Biosciences, Franklin Lakes, NJ, USA) was used to analyze the apoptosis of the SKOV3 cells. Briefly, cells were seeded into 6-well plates. After treatment with drugs, the adherent cells were trypsinized without EDTA and collected by centrifugation. After washing with PBS two times, the cells were resuspended in 100μL of 1×binding buffer and were subsequently incubated with 5μL of Annexin V staining solution at room temperature for 30min in the dark. Then, 400μL of 1×binding buffer was added, and the fluorescence intensity was evaluated on a FACS Aria™ (BD Biosciences) flow cytometer. Each assay was performed in triplicate.
2.6 RNA sequencing
This experiment was conducted at Differential Gene Technology Co., Ltd. (Anhui, China). EdgeR was used to identify the top ten enriched annotation terms among the differentially expressed genes (1.5-fold in either direction, P<0.05) between the SKOV3 sh-NC group and the SKOV3 sh-MGRN1 group.
3. Statistical analysis
The statistical analyses were performed using SPSS 21.0 (Chicago, IL, USA). The Wilcoxon rank sum test was used to compare the methylation level and mRNA expression of MGRN1 between the two groups. The χ2 test was used to compare the protein expression of MGRN1 in each group. Spearman correlation analysis was performed to analyze the correlation between MGRN1 expression and methylation status. A t test was used to analyze the cell activity and apoptosis data.