2. 1 Clinical samples
This study was approved and supervised by the Medical Ethics Committee of Xiangya Hospital, Central South University. Written informed consent was obtained from all subjects for participation. Twenty patients with ovarian endometriomas at stage III–IV who provided both eutopic endometrium(EU) and ectopic endometrium(EC) and twenty women without endometriosis as normal control were recruited during the proliferative phase. All subjects were at the age of 20–45 years old, with regular menstrual cycles, who received steroid hormone treatment at least 3 months before specimen collection.
2.2 Fluorescent in situ hybridization
The EU and EC tissues were embedded in paraffin and sliced. Slices were dewaxed in xylene, dehydrated in anhydrous ethanol, and then treated with protease K (20ug/ml). Subsequently,3% methanol-H2O2 was added to block endogenous peroxidase. After prehybridization, the hybridization solution with a digoxigenin(DIG)-labeled probe was added and hybridized overnight at 42℃. Sections were then incubated with mouse anti-digoxigenin labelled peroxidase (anti-DIG-HRP, Jackson, USA) at 37°C for 50 minutes. After washing for 3 times, fresh prepared FITC-TSA chromogenic reagent (Servicebio, China) was added reaction was in dark for 5 minutes at room temperature reaction, and then the sections were incubated with DAPI for 5 minutes. Finally, the slices were sealed and observed under Nikon Laser Scanning Confocal Microscope (Nikon, Japan).
2.3 Cell culture and identification
The endometrial tissue was rinsed with Hanks solution, and cut into pieces in F12/DMEM culture medium, followed by collagenase digestion for 60 min and DNase I digestion for 30 min. After centrifugation, the cells were suspended in F12/DMEM with 10% fetal bovine serum, filtered through a cell strainer (70µm) to collect endometrial stromal cells. The endometrial stromal cells were inoculated in F12/DMEM with 10% fetal bovine serum in an incubator with 5% CO2 at 37 ℃. The isolated endometrial stromal cells were identified by staining with vimentin using immunofluorescent techniques.
2.4 Cell transfection
Primary eutopic and normal endometrial stromal cells were treated at the third passage, with the cell density at 1 × 104 ml. The lentivirus vectors (Genepharma, China) targeting to the junction region of hsa_circ_0000673 were constructed to knockdown hsa_circ_0000673 expression in endometrial stroma cells. All the lentivirus vectors were transfected into eutopic and normal endometrial stromal cells with transfection enhancers, and the sequences were as follows: LV-1, 5’-GTGGTTCTTGCAGATTATCTC-3’; LV-2, 5’- CTTGCAGATTATCTCCCTCCA-3’; LV- negative control (NC), 5’-TTCTCCGAACGTGTCACGT-3’. The transfection efficiency was confirmed undfer fluorescence microscopy after 24 hours and cells were harvested for further function experiments after 48 hours. Has-miR-616-3p mimic or inhibitor (Guangzhou RiboBio, China) was transfected using Lipofectamine 2000 regents (Invitrogen, USA) according to the manufacturer’s protocol. All experiments were done three times with triplicates in each assay.
2.5 CCK-8 assay
The CCK8 assay was carried out via CCK8 assay as the manufacturer's protocol (NCM Biotech, China). Briefly, cells were seeded into 96-well plates and divided into four groups with four duplicate wells in each group. After cell attachment, the lentivirus vectors were added and the plates were incubated at 37℃ for 24 hours. 10 µ L CCK-8 regent was added into each well and incubated at 37℃ for 2hours. The cell proliferation was determined by testing the absorbance value at 450 nm.
2.6 Colony formation assay
Cells were paved in six-well plates with 1000 cells per well. LV-circ_0000673 or miR-616-3p mimic/inhibitor was transfected and the cells were cultured for 2 weeks. After washing, the cells were successively treated by 4% paraformaldehyde and 0.1% crystal violet. Then, the colonies in each well were calculated under microscope.
2.7 Wound healing assay
Wound healing assay was performed to assess the migration capability of endometrial stroma cells after knockdown of circ_000673. Briefly, endometrial stromal cells were seeded in six-well plates, and LV-circ_0000673 or miR-616-3p mimic/inhibitor was transfected. Then the monolayer cells were scraped with a micropipette tip to make a straight wound, and cultured with serum-free medium for 24h. The gap width at 0 and 24h was recorded under microscopy.
The cells were collected 72 hours after transfection, and total RNA was extracted using Trizol regent. The concentration and purity of RNA were measured by NanoVue Plus spectrophotometer (Healthcare Bio-ScienceAB, Uppsala, Sweden). The primers were listed as follows: hsa_circ_0000673, 5’-ATCTGTAAACCTTCTGTCCAAGA-3’(forward) and 5’-TCAAAACTGCTCAGAAGGCG-3’(reverse); PTEN, 5’-ACTATTCCCAGTCAGAGGCG-3’ (forward) and 5’-TCACCTTTAGCTGGCAGACC-3’ (reverse); PI3K, 5’-GCACCTGAATAGGCAAGTC-3’ (forward) and 5’-TCGCACCACCTCAATAAGT-3’ (reverse); AKT, 5’-GTGGAGGACCAGATGATGC-3’ (forward) and 5’-TGCCCCTGCTATGTGTAAG-3’ (reverse); GAPDH, 5’-TGCACCACCAACTGCTTAGC-3(forward) and 5’-GGCATGGACTGTGGTCATGAG-3’(reverse). miR-616-3p stem loop reverse transcription primers and U6 control were designed and synthesized by Guangzhou Ruibo Biological Co., Ltd. The reverse transcriptase kit (TaKaRa, China), was used for reverse transcriptional reaction. Gene expression was performed with SYBR Green qPCR mix (Bio-Rad, USA) using Applied Biosystems 7900 Real-Time PCR system. Relative gene expression was analyzed using the 2−ΔΔCt method.
2.9 Western blot assay
Endometrial cells were lysed in RIPA lysis buffer to extract total protein. 50 µ g total protein was taken for 10% SDS-PAGE electrophoresis, transferred onto a 0.45µm PVDF membrane and blocked with 5% nonfat milk successively. Primary antibodies of PTEN (1:1000), PI3K (1:1000), pAKT (1:2000), and GAPDH (1:1000) were added and incubated overnight at 4 ℃. Then after washing for three times, the secondary antibodies were added incubated at room temperature for one hour. At the end, enhanced chemiluminescence reagent (CST, USA) was added and the reactivity was determined by an enhanced chemiluminescence detection system (Amersham, Pittsburg, PA).
2.10 Dual luciferase assay
Dual luciferase assay was performed to assess the bonding between circ_0000673, PTEN and miR-616-3p. The wild-type (WT) of circ_0000673 and PTEN 3’UTR were inserted into pMIR-reporter plasmids (OBio Technology Corp, China) and the mutant (MUT) type was designed by mutating the binding site of the seed sequence. The WT and MUT luciferase reporter plasmids were respectively co-transfected with miR-616-3p mimics/negative control into 293T cells. Then, the relative luciferase activity was measured by luciferase reporter kits (Promega, Madison, WI, USA).
2.11 Statistical analysis
Data were expressed as mean ± standard deviation (SD). Two-tailed student’s t test was performed for pairwise comparisons, and the one-way Variance (ANOVA) Analysis was used for multiple comparisons. All experiments were performed in triplicate and P value < 0.05 was set as statistical significance. The statistical analysis was conducted by the SPSS 25.0 and GraphPad Prism 9.0.