Circadian gene Rev-erbα influenced by sleep conduces to pregnancy by promoting endometrial decidualization via IL-6-PR-C/EBPβ axis

Mice

All C57 BL/6 mice (6–8 weeks) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Mice were bred in a room of 22–25 °C, 40–60% relative humidity, 12 h light-12 h dark cycles with the same time of light-on every day and fed with food and water ad libitum. The mouse vagina was rinsed with physiological saline to detect estrus cycle at nine o'clock every day. The mice with normal estrus cycle were used in the following experiments. For sleep disturbance model, the mice were raised in room of 12 h light-12 h dark cycles with different time of light-on. The time of light-on (referred to ZT0) was advanced 6 h every four days for 3 months. For rhythmic oscillation test, uterus was collected from mice at diestrous phase and frozen on dry ice immediately. For in vivo decidualization, the female mice and vasectomized male mice were caged together at 19:00, and the vaginal plugs were detected at next 7:00, which referred to pseudopregnancy 0.5 days (PE0.5). Unilateral uterine horn was injected with 25 μL sesame oil at PE3.5, and the decidual level was analyzed at PE7.5. For pregnancy outcomes assay, the female mice and male mice were caged together at 19:00, and the vaginal plugs were detected at next 7:00, which referred to embryonic 0.5 days (E0.5). The mice with normal sleep were injected with physiological saline. Some mice with sleep disturbance were injected with 50 mg/kg SR9009 (HY-16989, MedChemExpress) once daily or 10 mg/kg anti-IL6 (504513, Biolegend) every three days. All mice were sacrificed at E13.5 to observe the pregnancy outcomes. All experimental procedures of mice were approved by the Institutional Animal Care and Use Committee at Fudan University.

Quantitative real-time PCR (QPCR)

Total RNA was extracted from cells or homogenized tissues using TRIzol reagent (T9108, Takara) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using PrimeScript™ RT Master Mix (RR036, Takara) and then amplified using SYRB Green PCR Master Mix (RR820, Takara) with ABI PRISM 7900 Sequence Detection System (Applied Biosystems, Waltham, MassachusettsMA, USA). β-Actin (Actb) was used as an internal control to normalize the relative changes in gene expression using the 2−△△Ct method. Human primer sequences for QPCR: Rev-erbα, forward 5′-TGGACTCCAACAACAACACAG -3′ and reverse 5′-GATGGTGGGAAGTAGGTGGG-3′; Rev-erbβ, forward 5′- TCATGCTTGCGAAGGCTGTAA-3′ and reverse 5′-CGCTTAGGAATACGACCAAACC-3′; Bmal1, forward 5′-CATTAAGAGGTGCCACCAATCC-3′ and reverse 5′-TCATTCTGGCTGTAGTTGAGGA-3′; Clock, forward 5′-TGCGAGGAACAATAGACCCAA-3′ and reverse 5′-ATGGCCTATGTGTGCGTTGTA-3′; IGFBP1, forward 5′-CGAAGGCTCTCCATGTCACCA-3′ and reverse 5′-TGTCTCCTGTGCCTTGGCTAAAC-3′; PGR, forward 5′-TGTATTTGTGCGTGTGGGTG-3′ and reverse 5′-TACAGCCCATTCCCAGGAAG-3′; C/EBPβ, forward 5′-CTTCAGCCCGTACCTGGAG -3′ and reverse 5′-GGAGAGGAAGTCGTGGTGC-3′. Mouse primer sequences for QPCR: Rev-erbα, forward 5′-TACATTGGCTCTAGTGGCTCC-3′ and reverse 5′-CAGTAGGTGATGGTGGGAAGTA-3′; Rev-erbβ: forward 5′- TGAACGCAGGAGGTGTGATTG-3′ and reverse 5′-GAGGACTGGAAGCTATTCTCAG-3′; Bmal1: forward 5′-GGCGTCGGGACAAAATGAAC-3′ and reverse 5′-TCTTCCCTCGGTCACATCCT-3′; Dtprp: forward 5′-AAGAATGCCCTTCAGCGAGC-3′ and reverse 5′-AGCTGGTGGGTTTGTGACAT-3′; Wnt4: forward 5′-AGACGTGCGAGAAACTCAAAG-3′ and reverse 5′-GGAACTGGTATTGGCACTCCT-3′; Bmp2: forward 5′-GGGACCCGCTGTCTTCTAGT-3′ and reverse 5′-TCAACTCAAATTCGCTGAGGAC-3′, IL-6, forward 5′- ATCCAGTTGCCTTCTTGGGACTGA-3′ and reverse 5′-TAAGCCTCCGACTTGTGAAGTGGT-3′; PGR, forward 5′-CTCCGGGACCGAACAGAGT-3′ and reverse 5′-ACAACAACCCTTTGGTAGCAG-3′.

Human samples

Human endometrial tissues during secretory phase were collected from women with regular menstrual cycles who did not have underlying endometrial abnormalities and did not receive exogenous steroidal hormones therapy for three months preceding biopsy collection. Human decidual tissues (gestational age: 6–12 weeks) were obtained from healthy pregnancies who were aged between 22 and 40 and artificially terminated for non-medical reasons or miscarriages who were diagnosed as unexplained abortion excluding chromosomal defects, genetic abnormalities, infection, endocrine and other factors. All participants were required to complete the questionnaire of patients pittsburgh sleep quality index (PSQI). Participants with PSQI ≤ 5 were considered to have normal sleep, Participants with PSQI > 5 were considered to have sleep disturbance. Written informed consent was obtained from all participants. All performances were approved by Human Research Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University.

Cell culture and treatment

Human endometrial tissues were digested with 1.0 mg/mL collagenase IV (C5138, Sigma-Aldrich) to obtain hESCs and they were cultured in complete medium (Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F12 supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin) as described previously [27]. Human decidual stromal cells (hDSCs) were separated from decidual tissues after digestion with 1.0 mg/mL collagenase IV (C5138, Sigma-Aldrich) and 150 U/mL DNase I in DMEM/F12 and density gradient centrifugation with percoll, as described previously [28].

Mouse endometrial stromal cells (mESCs) were isolated from mouse uteruses during diestrous phase followed by prior studies [29, 30]. Briefly, mouse uteruses were cut into 2–3 mm pieces and digested with 6 mg/ml dispase II (17105041, Gibco) and 25 mg/ml trypsin (T8150, Solarbio) for 1 h at 4 °C on a shaker, 1 h at room temperature without shaking, and 30 min at 37 °C without shaking, after which tissues were washed twice with hank's balanced salt solution. The remaining tissues were digested with 0.5 mg/ml collagenase at 37 °C for 30 min, and then filtered through 70 μm filter to obtain stromal cells. The stromal cells were cultured in complete medium for 1 h, and then the mixed complete medium was replaced with fresh complete medium.

For si-RNA transfection, h/mESCs were dealt with Rev-erbα/PGR/C/EBPβ-specific siRNA (Si-RNA for hESCs: si-Rev-erbα: CATGTCCTATGAACATGTA; si-PGR: GCACCTGATCTAATACTAA; si–C/EBPβ: CCATGGAAGTGGCCAACTT. Si-RNA for mESCs: si-Rev-erbα: GTACAAACGGTGTCTGAAA; si-PGR: CCATGTAAAGAGCACCATA; si–C/EBPβ: GAGCGACGAGTACAAGATG) for 20 h using transfection reagent (L3000015, Invitrogen) according to the manufacturer’s instructions. For in vitro decidualization, hESCs were treated with 1 mM MPA and 0.2 mg/mL cAMP (T1418, Topscience, Shanghai, China) in complete medium for 48 h; mESCs were treated with 10 nM estradiol (E2) (T1048, Topscience, Shanghai, China) and 1 μM progesterone (P4) (T0478, Topscience, Shanghai, China) in complete medium for 72 h. For IL-6 treatment, h/mESCs were dealt with IL-6 (200-06-5, PeproTech; 216–16, PeproTech) with indicated concentrations for 4 h before in vitro decidual treatment. For antibody neutralizing or inhibitor tests, h/mESCs were treated with 2.5 μg/mL anti-IL-6 (501125, biolegend; 504513, Biolegend) or 100 mg /mL Tocilizumab (IL-6R inhibitor) (HY-P9917, MedChemExpress) for 4 h before si-RNA transfection.

Western blot

Western blot was performed as described previously [28]. The primary antibodies were as follows: anti-IGFBP1 (ab180948, Abcam), anti-Rev-erbα (sc-393215, Santa Cruze), anti-β-Actin (ab179467, Abcam), anti-β-Tubulin (ab179513, Abcam), anti-PR (human, 8757, Cell Signaling Technology), anti-C/EBPβ (ab32358, Abcam); anti-IL-6 (human, ab233706, Abcam), anti-IL-6R (human, ab222101, Abcam), anti-PR (mouse, ab133526, Abcam), anti-IL-6 (mouse, ab229381, Abcam), anti-IL-6R (mouse, ab300581, Abcam), anti-Wnt4 (sc-376279, Santa Cruze). β-Tubulin and β-Actin were used as internal standards.

RNA-Seq

Total RNA was extracted from hESCs treated with si-RNA transfection and in vitro decidualization using TRIzol reagent according to the manufacturer’s instructions. mRNA was enriched from total RNA and then constructed a cDNA library, which was sequenced on the BGISEQ-500 sequencing platform (BGI-shenzhen Technology Co., Ltd).

Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR)

HESCs were fixed and cross-linked with 1% formaldehyde for 10 min at room temperature. And then they were sonicated into fragments of 200–700 bp after terminated cross-linking with 125 mM glycine. Sonicated products were divided into two groups, one group was used as the input control. Another group was incubated with antibodies (anti-Rev-erbα, 13418, Cell Signaling Technology; anti-IgG, ab172730, Abcam) overnight at 4 ℃, and then incubated with protein A/G immunomagnetic beads to obtain protein-DNA complex. After DNA was purified, qPCR was used to identify the enriched genes. Primers were as follows: IL-6, forward 5′-TGCACTTTTCCCCCTAGTTG-3′ and reverse 5′-TCATGGGAAAATCCCACATT -3′; IL-6R, forward 5′-GAGGGCAGAGGCACTTACTG-3′ and reverse 5′-AGTTGCCCAACTCTTCCAGA-3′; Negative, forward 5′-TGTGTGGAGCCAACAGTCTC-3′ and reverse 5′-CAGAAAAGCCCAGATGGAAA-3′.

Immunofluorescence and hematoxylin–eosin (HE) staining

Paraffin-embedded section of decidual tissues were dewaxed using dimethylbenzene and rehydrated in ethanol at different concentrations (100%, 95%, 90%, 80%, 70% and 50%). For immunofluorescence, the sections were blocked with 10% donkey serum after antigen retrieval using citrate sodium solution, and then they were incubated with primary antibodies (anti- Rev-erbα (sc-393215, Santa Cruze); anti-Vimentin (ab92547, Abcam), anti-Wnt4 (sc-376279, Santa Cruze)) overnight at 4 ℃. The sections were incubated with secondary antibodies for 2 h at room temperature after washed three times with tris-buffered saline (TBS) (10 min each), followed by 4′,6-diamidino-2-phenylindole (DAPI) staining. Mean gray value was calculated using ImageJ software. Relative mean gray value = mean gray value of cells /the mean value of mean gray value of cells from human/mouse with normal sleep. For hematoxylin–eosin (HE) staining, the sections were stained with hematoxylin solution for 5 min, and then washed with ultrafiltration water for 5 s. Next, the sections were stained with eosin solution for 3 min and dehydrated in ethanol at different concentrations (50%, 70%, 80%, 90%, 95% and 100%) and dimethylbenzene in turn. The slides were sealed with mounting medium and taken pictures using a fluorescence microscope.

Statistical analysis

GraphPad Prism version 7 was used to analyze the statistical difference. A Student’s tail t-test was performed to determine the statistical significance of differences between two groups. P < 0.05 was considered as statistically significant difference. Data were showed as mean ± standard error of the mean (SEM).

留言 (0)

沒有登入
gif