Decreased Krüppel-like factor 4 in adenomyosis impairs decidualization by repressing autophagy in human endometrial stromal cells

Patient sample collection

Endometrial samples were collected from patients who received in vitro fertilization (IVF) treatment in the reproductive medicine centre of Nanjing Drum Tower Hospital. There were 12 infertile patients with adenomyosis and 12 patients with successful pregnancy in the control group. The detailed information of these patients is listed in Table 1, and there were no significant differences. None of the patients received oral contraceptives 3 months before the operation. Endometrial tissues were obtained by endometrial biopsy 5–7 days after ovulation. The diagnostic criteria for adenomyosis were described in our previous studies [27]. The exclusion criteria were as follows: polycystic ovary syndrome (PCOS), untreated hydrosalpinx, endometrial polyps and endometrial lesions.

Table 1 Demographic details of the participants in this studyIsolation and decidualization stimulation of human endometrial stromal cells (hESCs)

As mentioned in our previous study [11, 20], primary human endometrial stromal cells were isolated from the endometria of infertile patients with adenomyosis and control group patients. hESCs were cultured according to the routine [11, 20]. To induce decidualization, the hESCs were cultured in phenol red-free DMEM/F12 medium (HyClone, Thermo Scientific, South Logan, UT, USA) containing 2.5% (v/v) charcoal/dextran-treated foetal bovine serum (FBS; HyClone, Thermo Scientific, South Logan, UT, USA), 100 IU/ml penicillin, and 100 μg/ml streptomycin supplemented with 0.5 mM 8-Br-cAMP (Sigma, St. Louis, MO, USA) and 1 μM medroxyprogesterone acetate (MPA, Sigma, St. Louis, MO, USA) to induce decidualization in vitro. After stimulation for different times, the culture supernatant was collected to detect decidual prolactin (dPRL) levels and evaluate the degree of decidualization. To overexpress KLF4 or reduce KLF4 expression, hESCs were treated with Ad-His-KLF4 and SiKLF4 for 1–2 days before decidualization stimulation. To explore the effect of autophagy inhibitors on decidualization, cells were pretreated with 3-methyladenine (3-MA, MedChemExpress, Shanghai, China) 2 days before decidualization stimulation.

Oligonucleotide transfection

The siRNA duplexes targeting human KLF4 (SiKLF4: GAGAGACCGAGGAGTTCAA) and siRNA negative control oligonucleotides were synthesized by RiboBio (Guangzhou, China). The siRNA negative control shared a homologous region with the human genome sequences. Oligonucleotide transfection was performed in hESCs using Lipofectamine 3000 (Life Technologies, New York, USA) according to the manufacturer’s instructions. At 48 h posttransfection, the cells were collected or decidualization was induced for the indicated time [27].

RNA isolation and quantitative real-time PCR

Human endometrial stromal cells were lysed with TRIzol reagent (Sigma, St. Louis, MO, USA) and total RNA was extracted in strict accordance with the instructions. Two micrograms of total RNA was added to a 20 μL system and reverse-transcribed into cDNA using 5 × All-In-One RT Master Mix (Abm, Canada). A SYBR Green PCR kit and MyiQ Single Colour Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) were employed for quantitative real-time PCR (qRT-PCR). The primer sequences used were as follows: KLF4, 5′-AGAGTTCCCATCTCAAGGCA-3′ and 5′-GTCAGTTCATCTGAGCGGG-3′; ATG5, 5′-AAAGATGTGCTTCGAGATGTGT-3′ and 5′-CACTTTGTCAGTTACCAACGTCA-3′; dPRL, 5′-CACTACATCCATAACCTCTC-3′ and 5′-ATGCTGACTATCAAGCTCAG-3′; IGFBP-1, 5′-TATGATGGCTCGAAGGCTCTC-3′ and 5′-GTAGACGCACCAGCAGAGTC-3′ and 18S rRNA, 5′-CGGCTACCACATCCAAGGAA-3′ and 5′-CTGGAATTACCGCGGCT-3′. The fold changes in the expression of each gene were measured by the 2−ΔΔCT method. The internal reference gene was 18S rRNA.

Western blot

According to our previous studies [20], we extracted and measured the total protein of endometrial tissue, hESCs and uteri of mice with or without adenomyosis. The protein concentrations were measured by the Bradford assay (Bio-Rad, Hercules, CA, USA). A fixed amount of protein (25–40 μg) was used for SDS polyacrylamide gel (10%-15%) separation electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membrane. Immunoblotting was carried out by incubating with the primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. The specific antibody information was as follows: anti-KLF4 (1:1000; 11,880–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-Beclin-1 (1:1000; 11,306–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-LC3A (1:1000; 18,722–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-LC3B (1:1000; 18,725–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-ATG5 (1:1000; 10,181–2-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-GAPDH (1:10,000; AP0063, GAPDH polyclonal antibody, Bioworld Technology, MN, USA) and goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000; BS13278, Bioworld Technology, St. Louis Park, MN, USA). An enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA) was used for detection and Quantity-one (Bio-Rad Laboratories, Hercules, CA, USA) software was used for density analysis of each band.

Immunofluorescence staining for F-actin filaments

According to our previous research [20], hESCs grown in 8-well chambers (Millipore, Billerica, MA, USA) were treated with adenovirus, siRNA or autophagy inhibitor for 2 days, and decidualization was induced by 8-Br-cAMP + MPA for 3 days. Then, according to our previous research [20], hESCs were fixed with 4% paraformaldehyde (w/v) for 30 min at room temperature. Next, the cells were washed with PBS and permeabilized with 0.5% Triton X-100 in PBS at room temperature. Subsequently, the cells were blocked with 3% BSA in PBS and incubated with fluorescein isothiocyanate-labelled phalloidin (1:300; P5282, Sigma, St. Louis, MO, USA) at 4 °C overnight. Cell nuclei were stained with DAPI (5 μg/ml) on the following day. The final images were taken with a confocal laser microscope (Leica, Wetzlar, Germany).

Luciferase assays

The sequence (-2932 bp to + 100 bp) containing the KLF4 specific binding site in the ATG5 promoter region was inserted into the pGL3-basic luciferase reporter plasmids. At the same time, the sequence (-1859 bp to + 100 bp) without the KLF4-binding site was inserted into the pGL3-basic luciferase reporter plasmid. Human endometrial stromal cells cultured in a 12-well plate were infected with Ad-His-KLF4 and then transfected with 300 ng luciferase reporter plasmid by Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA). After 48 h, a dual luciferase analysis system (Promega, Madison, WI, USA) was used to analyse the luciferase activity. A luminescent counter (Centro xs3 LB 960, Berthold Technologies) was used to measure the luciferase activity.

Chromatin immunoprecipitation (ChIP)/PCR assay

hESCs (70% confluence) were infected with Ad-LacZ or Ad-His-KLF4 (at a multiplicity of infection (MOI) of 50) for 48 h and then maintained in phenol red-free DMEM/F12 medium containing 2.5% charcoal/dextran-treated FBS with 0.5 mM 8-Br-cAMP plus 1 μM MPA. After 72 h, the hESCs were prepared for ChIP as described previously [20, 28]. Crosslinking, cell lysis, and genomic DNA fragment extraction were performed and KLF4 antibody was used for immunoprecipitation.The recovered DNA was analysed by RT-PCR. Specific primers (ATG5 5’-ATGGCCATCGTGAACACGTC-3’ and 5’-CAAATCAGTGGCACTGCAAA-3’) containing the KLF4-binding sequence were used for PCR amplification of ATG5 promoter fragments and a negative control primer (targeting: − 8000 bp to − 7790 bp) was also employed.

Avidin–biotin conjugate DNA precipitation (ABCD) assay

Double-stranded oligonucleotides were designed based on the ATG5 promoter sequence (− 2644 to − 2605 bp). The 5′ end of the sense strand was biotinylated, and a deletion and a mutation were introduced (deletion and mutation of the CACCC sequence) to remove the specific binding site for KLF4. The following primers were designed: human ATG5 wild type: 5′-biotin-GTTCCCAACAGAGAGTCACCCCCAATAAGCTAAAACTTGG-3′; human ATG5 wild-type reverse: 5′-CCAAGTTTTAGCTTATTGGGGGTGACTCTCTGTTGGGAAC-3′; human ATG5 del: 5′-biotin-GTTCCCAACAGAGAGTCCAATAAGCTAAAACTTGG-3′; human ATG5 del reverse: 5′-CCAAGTTTTAGCTTATTGGACTCTCTGTTGGGAAC-3′; human ATG5 mut: 5′-biotin-GTTCCCAACAGAGAGTCTGACCCAATAAGCTAAAACTTGG-3′; human ATG5 mut reverse: 5′-CCAAGTTTTAGCTTATTGGGTCAGACTCTCTGTTGGGAAC-3′. hESCs were infected with Ad-LacZ and Ad-His-KLF4 (50 MOI) for 48 h. The ABCD method was performed as described in our previous research [20, 28]. Cell extracts were harvested and lysed in RIPA buffer. Each double-stranded DNA sample (500 pmol) was incubated with 500 μg of cell extract at 4 °C for 2–4 h, and the protein complexes were pulled down using streptavidin agarose beads (Sigma) in binding buffer (10 mM Tris, pH 8.0; 150 mM NaCl; 0.5% Triton X-100; 0.5 mM DTT; 0.5 mM EDTA; 10% glycerol; and 20 μg/mL poly [dI–dC]) containing a protease inhibitor cocktail. The proteins were eluted, separated by SDS–PAGE, and then probed with KLF4 antibody (1:1000; 11,880–1-AP, rabbit polyclonal antibody, Proteintech Group, USA) and the corresponding secondary antibody.

Immunohistochemistry

Fresh endometrial tissue and mouse uterine tissue were fixed, embedded in paraffin and serially sectioned (5 μm). Formalin-fixed, paraffin-embedded uterine endometria were serially sectioned, dewaxed with xylene and rehydrated through a graded alcohol series, and then endogenous peroxidase activity was blocked using freshly prepared phosphate-buffered saline (PBS) containing 3% hydrogen peroxide for 20 min. Antigen retrieval was conducted by autoclaving the samples at 121 °C for 15 min in the presence of EDTA (pH 9.0), followed by incubation in blocking solution for 30 min. Next, the sections were washed with PBS and incubated with the specific primary antibodies overnight at 4 ℃ [20]. The specific antibody information was as follows: anti-KLF4 (1:500; 11,880–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-Beclin-1 (1:500; 11,306–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-LC3A (1:500; 18,772–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-LC3B (1:500; 18,725–1-AP, rabbit polyclonal antibody, Proteintech Group, USA), anti-ATG5 (1:500; 10,181–2-AP, rabbit polyclonal antibody, Proteintech Group, USA). Subsequently, the sections were rinsed with PBS and incubated with an HRP-conjugated goat anti-rabbit secondary antibody at 37 °C for 20 min. HRP activity was detected using diaminobenzidine (Invitrogen, Carlsbad, CA, USA), and the sections were counterstained with haematoxylin. Control sections were run concurrently with the experimental sections using nonspecific rabbit IgG, and they were similarly pretreated. Nonspecific staining was not detected in the controls.

Mouse model of adenomyosis

All experiments involving animals were approved by the Institutional Animal Care and Use Committee of Nanjing Drum Tower Hospital. ICR mice were purchased from the experimental animal centre of Yangzhou University. On the second day after birth, ICR mice were separated from their mothers in the early morning. After 6 h of starvation, they were treated with 5 μL/g peanut oil/lecithin/condensed milk mixture (2:0.2:3) supplemented with tamoxifen (1.52 mg/(kg.bw)) for three consecutive days. After drip feeding, they were returned to the female cage. From the 22nd day, they were separated from the female and fed freely. Some mice needed to be sacrificed for the study. The method of sacrifice was cervical dislocation after induced anaesthesia and sedation by pentobarbital sodium. The uteri of 2-month old mice were stained with H&E, α-SMA and E-cadherin to observe the invasion of glands into the muscle layer and disordered of muscle layer proliferation.

Statistical analysis

The data are presented as the means ± SEM. All experiments were performed at least three times. Student’s t test was used for comparisons between two groups. Statistical analysis was conducted by ANOVA, followed by the Student–Newman–Keuls test, for experiments involving more than two groups. Pearson correlation analysis was used to assess the relationship between KLF4 and Beclin-1; and LC3-B/LC3-A. P values of less than 0.05 were considered statistically significant.

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