2.1 Patients and tissue specimens

In this study, we analyzed 350 endometrial tissue samples, which consisted of 269 cases of type I endometrial carcinoma (EC), 26 cases of atypical endometrial hyperplasia (AEH), and 55 samples from normal endometrium (NE). The samples were collected between January 2006 and December 2013 at the Women’s Hospital, School of Medicine, Zhejiang University. Table 1 shows a group of 26 patients with atypical endometrial hyperplasia specimens. The second group consists of 269 patients with type I endometrial carcinoma who underwent total hysterectomy, removal of both tubes and ovaries and pelvic lymph node dissection without any anticancer therapy or hormonal therapy before their surgery. Table 2 shows the clinicopathological characteristics of the patients with type I endometrial carcinoma. The last group enrolled 55 patients with normal endometrium with hysterectomies due to benign gynecologic diseases.

2.2 Follow-up

The patients with type I EC were followed up postoperatively by either in-person interviews at the clinic or phone call. Regional tumor recurrence, distant metastasis, and patient survival were recorded and disease-free survival (DFS) and overall survival (OS) were calculated from the day of the surgery until recurrence or death. The last day of follow-up was May 2020. The median follow-up was 72 months (5–120 months). During follow-up, 35 patients (13.0%) had a clinical recurrence and 30 (11.2%) died of progressive disease.

2.3 Immunohistochemical staining

The tissues were immersed in paraffin, cut into sections that were 4 μm thick, placed on slides, treated with xylene for 15 minutes to remove wax, subjected to a series of solutions containing varying amounts of water and alcohol, and then rehydrated with distilled water. To restore antigen reactivity, the sections were heated in a 0.1 mol/L pH 6 citrate buffer for 20 minutes in a water bath at a temperature of 95 °C. After heating, the sections were cooled and left at room temperature for 30 minutes. The slices were incubated with diluted normal goat serum for 30 minutes to inhibit nonspecific binding.

The avidin-biotin-peroxidase method was used to perform the immunohistochemical staining using a SLABC kit (K5007, DAKO, Glostrup, Denmark). The slides were kept at room temperature for 2 hours and treated with CSAD (1:200, Invitrogen, PA5-58811, Carlsbad, CA, USA). The sections were then developed for 2 minutes with the enzyme-substrate 3,3′-diaminobenzidine chromagen (K3468, DAKO, Glostrup, Denmark) and counterstained with Mayer’s hematoxylin for 30 s. After dehydration, the tissues were cover slipped with permount and subsequently examined.

2.4 Evaluation of immunoreactivity

Two experienced pathologists with no clinicopathological data information independently assessed immunohistochemical staining in five fields. If there was a difference of opinion, the pathologists engaged in discussions until they reached a consensus on the outcome. CSAD immunoreactivity was assessed by staining intensity and the distribution of positively stained tumor cells. Intensity was scored as negative (0), weakly positive (1), moderately (2) or strongly positive (3). The staining distribution was scored as 0 for <25%, 1 for 25 to <50%, and 2 for 50 to <75%, 3 for 75 to <100% positively stained cells. The product of both parameters yielded the immunohistochemical (IHC) score, which ranged from 0 to 9 points. A total score of <4 was considered as decreased expression and ≥4 as increased expression.

2.5 Cell culture

Human endometrial carcinoma cell lines HEC1A and Ishikawa were purchased from Wuhan Procell Life Technology (Wuhan, China). Cells were cultured in McCoy’s 5a Medium (HEC1A) and dulbecco’s modified eagle medium (DMEM) (for Ishikawa) supplemented with 10% fetal bovine serum (FBS).

2.6 Transfection with small interfering RNA (siRNA)

siRNAs were used to inhibit the CSAD expression in HEC1A and Ishikawa cells. Following the manufacturer’s instructions, the cells were transfected with CSAD-specific siRNA using Lipofectamine™ RNAiMAX (13778150, Invitrogen, Carlsbad, CA, USA). The construction and sequences for the siRNAs were as follows:

si-CSAD-1:forward: 5′-CAGAAAGGAACCAGUGUCUTT-3′;

reverse: 5′-AGACACUGGUUCCUUUCUGTT-3′;

si-CSAD-2:forward: 5′-GUGAUUCGCUACAGUGUCATT-3′;

reverse: 5′-UGACACUGUAGCGAAUCACTT-3′

The siRNAs were synthesized at Tsingke Biotechnology Co., Ltd. (Hangzhou, China). Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and western blot determined the transfection efficiency of CSAD.

2.7 Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was extracted using total RNA extractor (TRIzol) Reagent (15596018CN, Invitrogen, Carlsbad, CA, USA), and amplification of the complementary DNA (cDNA) (1 μg per sample) was performed using the Prime Script Kit (RR014A, TaKaRa Bio Inc., Otsu, Japan) according to the manufacturer’s indication. Synergy Brands (SYBR) Green fluorescent-based assay (638320, TaKaRa Bio Inc., Otsu, Japan) was used to run real-time PCR in triplicate on a ViiATM7 RT-PCR system (Applied Biosystems, Carlsbad, CA, USA). The primers for real-time PCR were as follows:

CSAD: forward: 5′-CTTCTCCAGGATACCTCGAACC-3′;

reverse:5′-CAGAGCCACATCGTAGAACTTG-3′;

glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: 5′- GGAGCGAGATCCCTCCAAAAT-3′;

reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′;

Relative mRNA expression levels were calculated by the 2^−(ΔΔCt) method and were normalized to the internal control (GAPDH), ΔCt = Ct (targeting gene) − Ct (GAPDH), and ΔΔCt = ΔCt (treated) − ΔCt (control).

2.8 Western blot analysis

Cells containing protease and phosphatase inhibitor cocktail were lysed in radio immunoprecipitation assay (RIPA) lysis (P0013B, Beyotime, Shanghai, China). Protein concentration was determined using the Bicinchoninic Acid Assay (BCA) protein assay kit (P0012S, Beyotime Biotechnology, Shanghai, China). Protein samples were separated by 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.2 μm Polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA) under a constant 300 mA. The membrane was then incubated with the primary antibodies at 4 °C overnight after being blocked in TBST (Tris-buffered saline (TBS) with 0.5% Tween) containing 5% skim milk at 37 °C for 1 h.

After incubating with a Horseradish Peroxidase (HRP)-conjugated secondary antibody for 1 h at 37 °C, the membrane was washed in TBST and prepared for signal detection. The signals were automatically visualized using the FUSION FX6 (vilber, Eberhardzell, BW, Germany) and quantitatively analyzed with Image Lab software (6.1, Bio-Rad, Hercules, CA, USA). GAPDH protein expression was used as the internal control. The following antibodies were used: CSAD (1:2000, A13845, Wuhan, Hubei, China), HRP Goat Anti-Rabbit IgG (H + L) (1:5000, AS014, Wuhan, Hubei, China), HRP Goat Anti-Mouse IgG (H + L) (1:5000, AS003, Wuhan, Hubei, China) from ABclonal; BCL2 (1:1000, 26593-1-AP, Wuhan, Hubei, China), BAX (1:1000, 50599-2-Ig, Wuhan, Hubei, China) from Proteintech; GAPDH (1:5000, db11729, Hangzhou, Zhejiang, China) from Diagbio.

2.9 Cell proliferation

The cells were placed in 96-well plates for 24 h and transfected with siR-CSAD and siR-control. The proliferation was measured by the cell counting kit (CCK)-8 kit (40203ES60, Yeasen Biotech, Shanghai, China) according to the manufacturer’s protocol.

2.10 Apoptosis assay

Cell apoptosis was examined using a FACSVerse (BD Biosciences, Franklin Lakes, NJ, USA) flow cytometer. Yeasen Biotech’s Annexin V-FITC/PI Apoptosis Detection Kit (40302ES20, Yeasen Biotech, Shanghai, China) was used to evaluate apoptosis following the manufacturer’s instructions. The following steps were taken to prepare the cells for analysis: 1 × 10⁵ cells/mL of cells were resuspended in 1× binding buffer. Then, 5 μL of Annexin V-FITC reagent and 10 μL of PI reagent were added to a tube. The mixture was incubated at room temperature in the dark for 15 minutes.

2.11 Statistical analysis

Statistical analyses were performed using IBM SPSS 24.0 for Windows (IBM Corp., Armonk, NY, USA). The correlation of CSAD expression with clinicopathological characteristics and the significance of differences in CSAD expression were determined by Pearson’s chi-square test. Survival curves were generated using the Kaplan-Meyer method, and survival rates were compared using the log-rank test. The Cox proportional hazard method was employed in univariate and multivariate studies to identify independent determinants of survival. In vitro assay, a student’s test was used to determine the statistical differences between the two groups. All statistical tests were two-sided, and p-values < 0.05 were considered statistically significant.

3.1 The Evaluation of CSAD expression in endometrial tissue samples

Three hundred fifty (350) endometrial tissue samples were collected for immunohistochemistry to evaluate CSAD expression, comprising 269 type I EC, 26 AEH and 55 NE. The mean age of patients was 54.39 years for type I EC, 48.38 years for AEH, 46.58 years for NE. According to the 2009 report by the International Federation of Obstetrics and Gynecology, 185 individuals with type I endometrial carcinoma were in Stage I, while 84 were in Stage II–IV. 214 cases were well-moderately differentiated (G1/G2), and 55 cases were poorly differentiated (G3). Of 269 cases, 28 (10.41%) showed evidence of lymph node metastasis (LNM). Lymphatic vascular space invasion (LVSI) was detected in 31 (11.52%) of 269 cases. Immunohistochemical staining of CSAD expression is shown in Fig. 1 and described below.

Fig. 1.Representative immunohistochemical staining showing CSAD expression in normal endometrium (NE), Atypical endometrial hyperplasia (AEH) and Type I Endometrial carcinoma (Type I EC) tissues using serial section technique. Increased CSAD expression was observed in one representative case with type I EC. Magnifications, ×200. CSAD, Cysteine sulfinate decarboxylase.

3.2 CSAD expression

Most type I EC tissue samples exhibited uniform and intense CSAD staining, mainly localized in the cytoplasm and cell membrane. CSAD immunoreactivity was less observed in either the cytoplasm or cell membrane of NE tissues. Table 1 demonstrates that 30.86% (83/269) of type I EC samples had a significant level of CSAD expression (score ≥4). However, increased expression of CSAD was seen in 15.38% (4/26) AEH specimens and 8 of 55 NE specimens (14.55%). The expression of CSAD in Type I EC was significantly higher than in NE and AEH (NE vs. Type I EC, p = 0.014; AEH vs. Type I EC, p = 0.099), although there was no statistical difference compared to the AEH group. Additionally, there was no statistically significant difference in CSAD expression between NE and AEH (NC vs. AEH, p = 0.921).

3.3 The correlation of CSAD expression with clinicopathological characteristics in Type I EC

The correlation of CSAD expression with clinicopathological characteristics of Type I EC tissues is shown in Table 2. Tissue samples from 83 Type I EC patients (30.86%) exhibited an elevated CASD (score ≥4), while the remaining 186 samples (69.14%) showed reduced staining (score <4). Increased CSAD expression was significantly associated with FIGO stages (p = 0.044), lymph vascular space invasion (LVSI) (p = 0.025) and lymph node metastasis (LNM) (p = 0.006). In contrast, a decrease in CSAD expression showed a strong correlation with larger tumor sizes (p < 0.05).

3.4 Survival analysis

Kaplan-Meier analysis was used to investigate the prognostic value of the CSAD expression in type I EC. The survival curves in Fig. 2 illustrate the association of CSAD expression with disease-free survival (DFS) and overall survival (OS) in 269 patients diagnosed with type I EC. Significance was tested in univariate and multivariate Cox regression models. The Kaplan-Meier analysis showed that increased CSAD expression was significantly associated with shorter DFS (p = 0.044) and OS (p = 0.045). In addition, Cox univariate proportional hazards analysis showed that menopause status, International Federation of Gynecology and Obstetrics (FIGO) stage, histological differentiation, myometrial invasion, LVSI, LNM and CSAD expression were significant predictors of shorter DFS and OS. There was also an association between poor OS and peritoneal lavage cytology. Furthermore, multivariate analysis showed that only myometrial invasion, LVSI and LNM were independent predictors of shorter DFS. LVSI and LNM indicated poor OS in patients with type I EC (Table 3).

Fig. 2.Kaplan-Meyer curves showing the association of aberrant expression of CSAD and patient disease-free survival (DFS) and overall survival (OS). Increased CSAD was significantly associated with shorter DFS and OS. CSAD, Cysteine sulfinate decarboxylase.

3.5 Down-regulation of CSAD expression using siRNA targets CSAD, inhibits Endometrial carcinoma cell growth and induces cell apoptosis

The siRNAs were used to inhibit CSAD expression in Endometrial carcinoma cells. The results from qRT-PCR and Western blot analysis demonstrated that siRNA targeted at CSAD could significantly reduce mRNA and protein levels of CSAD in HEC1A and Ishikawa (IK) cells (Fig. 3). Further studies found down-regulation of CSAD expression markedly inhibited cell growth (p < 0.05) in both HEC1A and Ishikawa cells (Fig. 4). Additionally, deceased CSAD expression markedly downregulated apoptosis-related protein BCL-2 and upregulated BAX expression. This indicates that silence CSAD suppressed cell growth by regulating apoptosis progression (p < 0.05) (Fig. 3A,B). Flow cytometry was used to measure apoptosis rates after reduced expression of CSAD. The results showed that the silence of CSAD could induce apoptosis compared with negative controls in HEC1A and IK cells (Fig. 5) (p < 0.05).

Fig. 3.Western-Blot and qRT-PCR showing the expression of CSAD were inhibited by siRNAs. (A) Western Blot Analysis of CSAD, BCL-2, and BAX Protein Expression in HEC1A Cells. (B) Western Blot Analysis of CSAD, BCL-2, and BAX Protein Expression in IK Cells. (C) qRT-PCR Analysis of CSAD mRNA Expression in HEC1A Cells. (D) qRT-PCR Analysis of CSAD mRNA Expression in IK Cells. (****, p < 0.0001). CSAD, Cysteine sulfinate decarboxylase; IK, Ishikawa; si, small interfering; BCL-2, affect B-cell lymphoma-2; BAX, BCL2-Associated X; GAPDA, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; HEC1A, human endometrial adenocarcinoma cell stage ⅠA.

Fig. 4.Silence of CSAD markedly suppressed the cell growth in both HEC1A and IK cells. (A) Effects of CSAD Silencing on the Proliferation of HEC1A Cells. (B) Effects of CSAD Silencing on the Proliferation of IK Cells. (ns, p > 0.05; **, p = 0.0014 (HEC1A), p = 0.0038 (IK); ****, p < 0.0001). CSAD, Cysteine sulfinate decarboxylase; IK, Ishikawa; si, small interfering; OD, optical density; HEC1A, human endometrial adenocarcinoma cell stage ⅠA; NC, negative control.

Fig. 5.The silence of CSAD could induce apoptosis compared with negative controls in HEC1A and IK cells. (**, p = 0.0030 (HEC1A, nc vs. si-CSAD-1), p = 0.0021 (HEC1A, nc vs. si-CSAD-2); ****, p < 0.0001 (IK)). CSAD, Cysteine sulfinate decarboxylase; IK, Ishikawa; si, small interfering; NC, negative control; V-FITC-A, Annexin V-FITC; HEC1A, human endometrial adenocarcinoma cell stage ⅠA.