Endometriosis (EM) denotes the occurrence, growth, and infiltration of glandular and mesenchymal endometrial tissue outside the endometrium and is often characterized by repeated bleeding or nodule formation [
The endometrial cell line CRL-7566 (ATCC) was purchased from the American Type Culture Collection, Manassas, Virginia, USA. Antibodies reactive against LC3-II, P62, actin, and ATG5 were purchased from Cell Signaling Technology (Danvers, USA). Antibodies against fascin-1 were from Abcam (Cambridge, USA). Antibodies against FLAG-tagged proteins were from Sigma-Aldrich, USA. The plasmid encoding FLAG-fascin-1 was purchased from Addgene (Cambridge, USA), and the ATG5 siRNA interference fragment was synthesized by Genepharma (Shanghai, China).
Rapamycin, 3-MA, and MTT were purchased from Sigma-Aldrich, USA. Lipofectamine 2000 was from Invitrogen (San Diego, USA). All other reagents were from Shanghai Shengwu Huaxue Company, China.
The CRL-7566 cells were cultured in high-glucose DMEM complete medium (Invitrogen, San Diego, USA) with the addition of 5% fetal bovine serum (FBS; Invitrogen, San Diego, USA), in an atmosphere comprising 5% CO2 at 37°C. Plasmid transfection and siRNA interference were conducted using Lipofectamine 2000 (Invitrogen, San Diego, USA), according to the manufacturer’s instructions.
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The endometrial CRL-7566 cells were seeded at approx. 1×105 cells/well into the wells of a 24-well plate with sterilized round glass coverslips and cultured in DMEM with 10% FBS at 37% in an atmosphere comprising 5% CO2. After the culture medium was removed, the cells were fixed with 4% paraformaldehyde/30% sucrose/PBS for 15min at 25°C, washed with PBS for 5min two times, permeabilized with 0.1% Triton X-100/PBS for 10min at 25°C, washed with PBS for 10min two times, blocked with 5% BSA in PBS for 30min at 25°C, incubated with the primary antibody diluted as 1:1000 in 5% BSA/PBS at room temperature for 1h, washed with PBS for 5min five times, blocked with 5% BSA/PBS for 20min at 25°C, incubated with the secondary antibody diluted as 1:10000 in 5% BSA/PBS at room temperature for 1h, washed with PBS for 5min seven times, stained for 2min with 1
The cells were lysed using 2×SDS sample buffer for 10min at 25°C and ultrasonicated for 5s three times. The BCA protein assay kit was used to determine the protein concentration, using a sample amount of 20
A 6-cm diameter culture dish was seeded with 1×105 cells; 200
After transfected cells were trypsinized routinely, they were resuspended in complete medium and agitated to yield a single-cell suspension. The resulting suspension revealed a single-cell ratio exceeding 95% upon microscopic inspection. Subsequently, the cells were counted on a hemocytometry glass slide, and the cell concentration was adjusted with complete medium to yield a defined dilution ratio. Using a dilution that corresponds to 500 cells per Petri dish, 10-cm diameter dishes were seeded with 12mL of the cell suspension and swayed to thoroughly mix the cells. The thus seeded culture dishes were incubated at 37°C in an atmosphere comprising 5% CO2 for 14 days, with medium changes every three days. The medium was supplemented with 200
The cells were cultured to 90% confluency in six-well plates; then a thin scratch (wound) was made in the central area using a 10-ml pipette tip. Detached and damaged cells were carefully removed with PBS and the medium was replaced with serum-free medium. Wound closure was observed by light microscopy and images were captured after 24h.
Pathological samples and normal endometrial tissue from 20 cases of EM were collected according to the ethical requirements of Hunan Province People’s Hospital, Changsha, China, and signed informed consent statements were collected from all study participants. Detailed clinical manifestations, including pathological indices, status of metastasis, recurrence, and follow-up, were collected from all patients. Normal eutopic endometrial tissues were obtained from premenopausal women undergoing hysterectomy for uterine leiomyoma. Ectopic endometriotic tissues were obtained from ovarian endometriotic cysts. A total of 20 patients had histological and laparoscopic evidence of advanced stage endometriosis (stage III or IV) and had regular menstrual cycles. Study participant had not received any hormonal therapy during the previous 3 months. All tissues were classified into five categories according to day of menstrual cycle as early proliferative (days 4-7), mid proliferative (days 8-10), late proliferative (days 11-14), early secretory (days 15-18), mid secretory (days 19-22), or late secretory (days 23-28) phases. Menstrual cycle day was established based on the patient’s menstrual history. The average age of participants was 38.2+4.6 years for eutopic endometrial tissues and 29.3+6.6 years for ectopic endometrial tissues. Of the 20 eutopic and 20 ectopic endometrial samples, four cases were each in early proliferative, late proliferative, early secretory, mid-secretory, and late secretory phases.
After the tissue was homogenized using 0.1% Triton X-100 in PBS, an equal volume of 2×SDS sample buffer was added to each sample, lysed for 10min at 25°C, ultrasonicated for 5s three times, and used in the same way as described in the Immunoblotting.
The data were analyzed using GraphPad Prism 5 software. Analysis of variance (ANOVA) and Tukey’s test (for within-group two-by-two comparisons) or Dunnett’s multiple comparison test (for comparisons of each group with the control group) was used. Differences with
We used two different methods to interfere with the autophagy level of endometrial CRL-7566 cells-treatment with the autophagy inhibitor 3-MA and transfection with an RNA interference fragment targeting the ATG5 gene in order to repress autophagy. The results of immunoblotting have shown that the expression level of ATG5 was obviously reduced upon RNA interference (Figure
Interference with the autophagy level of endometrial CRL-7566 cells. (a) Immunofluorescence showing the influence of each of the interference methods on the abundance of LC3-positive puncta in endometrial CRL-7566 cells. (b) Immunoblot- determination of the expression level of ATG5 after transfection with an RNAi fragment targeting the corresponding gene. Actin was used as internal control. (c) Statistical analysis of the results obtained from (a).
We used two different methods to interfere with the autophagy level of endometrial CRL-7566 cells—treatment with the autophagy inhibitor 3-MA and transfection with an RNA interference fragment targeting the ATG5 gene in order to repress autophagy.
The results of cell proliferation and clonogenic assays showed that the autophagy inhibitor 3-MA and transfection with the RNAi fragment targeting ATG5 were both able to significantly promote the cells’ proliferation and clonogenicity, while rapamycin was able to suppress the proliferation and clonogenicity of CRL-7566 cells. Moreover, the differences were all statistically significant (Figure
The effect of interference with the autophagy level of endometrial CRL-7566 cells on cell proliferation and clonogenicity. (a) The cell proliferation assay showed that each of the interference methods had an effect on the proliferation of endometrial CRL-7566 cells. (b) The clonogenic assay showed that each of the interference methods had an effect on the clonogenicity of endometrial CRL-7566 cells.
In order to simplify the observation of the cells’ filopodia, we overexpressed the tagged filopodial marker protein FLAG-fascin 1, enabling the visualization of filopodia through immunostaining. As can be seen from Figure
The effects of treatment with the autophagy activator rapamycin on the formation of filopodia and cellular invasiveness. The tagged filopodial marker protein FLAG-fascin was overexpressed to visualize the filopodia (red) and DAPI staining was used to visualize the nuclei (gray). (a) Control treatment (buffer). (b) Rapamycin treatment. (c) Results of the statistical analysis of the relative length of filopodia. The results were normalized to the control group, which was set to 1. (d) Scratch assay analysis of the cells’ invasiveness. The results were normalized to the control group, which was set to 100%.
In a further step aimed at investigating the mechanism by which the autophagy activator influences the cells’ invasiveness, we measured the influence of rapamycin on the invasiveness of cells overexpressing fascin-1. The results showed that extrinsic expression of fascin-1 did not change the level of autophagy but was nevertheless able to counteract the suppression of cellular invasiveness induced by rapamycin treatment (Figure
The effect of fascin-1 overexpression on the reduction of cellular invasiveness by rapamycin. (a) Immunoblotting to determine the expression of fascin-1 and LC3-II. Actin was used as internal control. (b) Statistical analysis of the effect of fascin-1 overexpression on the change of cellular invasiveness brought upon by rapamycin treatment.
We collected pathological samples and normal control tissues from 20 cases of EM and used antibodies against LC3-II and p62 to determine their respective protein levels. LC3-II is an autophagic marker protein, and its reduced levels can indicate a reduced rate of autophagy. By contrast, p62 is a substrate of the autophagic-lysosomal degradation pathway, and an increase of its levels is considered an indicator of suppressed autophagy. Immunohistochemistry and immunostaining result showed the protein levels of p62 in EM tissues are higher than in the controls (Figure
The levels of the autophagy marker LC3-II and the autophagic substrate p62 in endometriotic tissue samples and corresponding normal tissue controls. (a) Immunohistochemistry results showing the protein levels of p62 in EM tissues and controls. (b) Immunoblotting results showing the protein levels of LC3-II in EM tissues and controls. Actin was used as internal control. (c) Statistical analysis of the protein levels of LC3-II and p62 in EM tissues and controls.
A large number of recent studies have shown that autophagy plays a significant role in the emergence and development of neoplasms. In fact, changes of autophagic activity have been observed in a large number of tumors, and the expression levels of the autophagy-related protein LC3 differ among different tumors, including a smaller number of reports in brain tumors and lung, cervical, and ovarian cancer [
Our research shows that the level of autophagy in EM tissues is lowered. Furthermore, different methods of interfering in vitro with the autophagy levels of endometrial CRL-7566 cells demonstrated the important influence autophagy has on the cells’ growth. This in turn corroborates the function of autophagy in the proliferation and invasiveness of endometriotic cells. Taken together, this study opens the prospect of elucidating the mechanisms leading to the development of EM, as well as offering new ideas and a theoretical basis for its prevention and treatment.
The authors declare no competing financial interests.
Xiaomei Luo, Shizhang Wang, and Wei Cheng performed the experiments. Zhihong Chen and Jieqiong Tan conceived and designed the experiments. Zhihong Chen and Jieqiong Tan wrote the paper.
This work was supported by grants from the Science and Technology Planning Project of Hunan Province (2015JC3116, 2018JJ2225), the Science and Technology Foundation of Health and Family Planning Commission of Hunan Province (B2015-93), and the National Natural Science Foundation of China (31500832).