Introduction
Adenomyosis (ADS) is a commonly encountered benign gynecological disorder, predominantly occurring in women of reproductive age. It is typically characterized as the aberrant displacement of eutopic uterine endometrial glands and stroma, deeply and haphazardly involved into myometrium [
1]. The concomitant myometrial hyperplasia and hypertrophy may be associated with a series of subinfertility. However, the only radical treatment strategy is still hysterectomy, by which fertility preservation is compromised. In particular, the pathogenesis of ADS remains uncertain as yet, therefore, individualized therapy and targeted agents are still difficult to achieve.
According to the invagination and EMID (endometrial-myometrial interface disruption) theory [
2,
3], ADS may actually derives from the excessive invasion of altered endometrial basalis into myometrium, after passing through the disrupted EMI. Despite the mechanism that triggers the progressive invasion of endometrium has not been fully elucidated, accumulating evidence supported that there were biochemical and functional abnormalities related to the endometrial cells. It has been postulated that sustained proliferation and survival of eutopic or ectopic endometrial cells may, along with enhanced migratory prosperities, permit the deeper invasion and down-growth of ectopic lesions [
4,
5]. Meanwhile, angiogenesis is considered to be an essential component during the development of ADS, as the implantation of ectopic endometrium requires a blood supply to maintain its growth and survival [
6].
ADS is a recognized estrogen-dependent disease. Thus far, multiple observations have implied that elevated local E
2 concentration and ER overexpression might be central and crucial to the pathogenesis of ADS. Based on the available data, the local hyperestrogen milieu may result in increased endometrial cell proliferation, enhanced pro-angiogenetic prosperity and induction of epithelial-mesenchymal transition (EMT), thereby promoting the implantation and invasion of ectopic endometrium into myometrium, eventually contributing to the onset of ADS [
4,
7,
8]. However, whether the effects of active estradiol (β-E
2) on adenomyotic endometrium could be altered by certain pathogenic genes, the direct evidence is still limited.
Talin1, a ubiquitous intracellular cytoskeletal protein containing 2541 amino acids, is a key regulator of integrin activation [
9]. It has been well identified that Talin1 is closely connected with the progression of multiple human cancers through mediating cell proliferation, migration and invasion [
10]. Previously, we have demonstrated that aberrantly overexpressed Talin1 might induce EMT to facilitate ADS endometrial epithelial cell migration and invasion via activating wnt/β-catenin pathway [
11]. However, whether Talin1 serves an extra role in β-E
2-induced proliferation and angiogenesis of adenomyotic endometrium, and then affects the invasive implantation of ectopic lesions, it remains undiscovered yet.
Therefore, the main purpose of the study was to investigate whether Talin1 participates in the development of ADS by directly influencing the regulatory effects of β-E2 on proliferation and pro-angiogenesis of endometrium in vitro and in vivo.
Materials and methods
Sample collection
All tissue samples were collected with informed consent. Our study was in accordance with the requirements of the Medical Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University (IEC-C-29-V02-FJ1). The eutopic and corresponding ectopic uterine endometrium were obtained during hysterectomy from 28 women diagnosed with ADS, which were utilized for subsequent primary culture of endometrial stromal cells. Meanwhile, normal endometrial tissues were collected from 22 women undergoing hysterectomies for benign ovarian tumors or cervical intraepithelial neoplasia II-III, without histological evidence of ADS. All participants were premenopausal with regular menstrual cycles and at proliferative phase during the procedure. Any signs or symptoms of pathologic changes in endometrium, a history of hormone therapy within 3 months, or concomitant with endometriosis were not included in the study [
12]. All endometrial specimens were obtained from February 2019 to January 2020.
Cell culture
As we reported previously [
13], the primary adenomyotic eutopic and ectopic endometrial stromal cells (ADS_Eu_ESC and ADS_Ec_ESC,
n = 7 respectively) as well as the normal uterine endometrial stromal cells as control (Ctrl_ESC,
n = 5) were isolated from the corresponding endometrium and cultured in vitro. Briefly, after being rinsed with PBS 2 ~ 3 times to remove impurities and blood cells, the separated endometrial specimen was then minced into pieces less than 2mm
3. Subsequently, 0.02% type I collagenase (Sigma, USA) mixed with 0.005% deoxyribonuclease (Invitrogen, USA) was added to aptly digest the tissue debris for 45 ~ 60 min at 37 °C. To accelerate the digestion process, a gentle shaking every 5 ~ 10 min was helpful. Afterwards, DMEM/f12 (Hyclone, USA) containing 12.5% fetal bovine serum (FBS, BD, USA) was utilized for stopping the digestion. Filtered through the 100 μm cell strainer, the cell suspension free from mucosa and remnants was obtained. After successive centrifugation at room temperature (720 rpm, 3 min) and filtration through the 40 μm cell strainer, the filtrate was then centrifuged twice (1200 rpm, 3 min). Finally, the primary uterine endometrial stromal cells were isolated and seeded in culture dishes. Cells were cultured with DMEM/f12 medium containing 12.5% FBS and 1% penicillin/streptomycin (Gibco, USA). When reaching 80% confluence, the primary cells were trypsinzed and passaged. We selected the cells at (Passage 3 to Passage 6) P
3-P
6 for subsequent experiments. (Supplementary Figure S
1).
The estrogen receptor positive (ER+) Ishikawa cells (Human Asia endometrial adenocarcinoma cell line) and HUVECs (human umbilical vein endometrial cells) were purchased from China Infrastructure of Cell Line Resource and cultivated in DMEM/f12 medium with 10% FBS. All cells were incubated at 37 °C and 5% CO2 in a humidified atmosphere.
Drug treatment
To remove the confounding effects of endogenous steroids, ADS_Eu_ESC, ADS_Ec_ESC and IshikawaER+ cells were cultured in phenol red-free DMEM/f12 medium for 48 h before drug treatment. Subsequently, cells were incubated in fresh medium (as control), β-E2 (10nM, Sigma, USA), Fulvestrant (10 nM, ICI 182780, a selective ER antagonist, MedChem Express, USA) or β-E2 plus Fulvestrant for 24 h.
Gene regulation
The lentivirus vectors containing Talin1 overexpression plasmid pSGLV (OV-Talin1) and its corresponding negative control (OV-NC) were constructed by Gene Chem (Shanghai, China). Meanwhile, the short hairpin RNA (ShRNA) against Talin1 (Sh-Talin1) and its negative control ShRNA (Sh-NC) were also synthesized and generated by Gene Chem. The ADS_Eu_ESC, ADS_Ec_ESC and IshikawaER+ cells were firstly seeded into 6-well plates at a density of 2х105 cells/well. When reaching 50 ~ 60% cell confluence, the OV-Talin1 or OV-NC vector was transfected into cells using a lipofectime 3000 (Invitrogen, USA) according to the manufacturer’s instructions. Meanwhile, the cells were subjected to lentivirual transduction with 5 μg/ml polybrene for 24 h, and the medium was then changed. A qRT-PCR or western blot assay was performed for further detecting the transfection efficiency. Especially, the ADS_Eu_ESC and ADS_Ec_ESC cells transfected with OV-Talin1 continued to receive 10 nM β-E2 treatment for 24 h, as mentioned above, after removal of the endogenous steroid hormones in the phenol red-free medium.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
The RNA isoPlus (Takara, BioInc, Japan) was used for the total RNA extraction from endometrial tissue samples and cells. For the reverse transcription of Talin1, the PrimeScript RT Reagent Kit (Takara) was utilized to synthesize the cDNA. The subsequent quantitative PCR traction was performed following the protocol of a SYBR Green PCR Kit (Takara) through an ABI 7500 system (Applied Biosystems, Grand Island, USA). The thermocycling conditions were as follows: 95 °C for 5 s and 60 °C for 30 s. β-actin was selected as the reference gene. The experiment was independently repeated for 3 times and the results were analyzed with 2-ΔΔCT method. The following primers specific to Talin1 were used for quantitative real-time PCR: sense primer, 5′-CTATATGCCACACCCGCCTC-3′ and antisense primer, 5′-CCCAGGATTCCACGGGACTA-3′. The primers for internal control β-actin were as follows: forward, 5′-GCCGTGGTGGTGAAGCTGT-3′ and reverse, 5′-ACCCACACTGTGCCCATCTA-3′. All the primers in the study were generated by Sangon Biotech (Shanghai, China).
Western blot
As previously described [
14], total protein from each sample was extracted with RIPA lysis buffer (Sigma, St, Lousis) containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF, Solarbio). A phosphorylase inhibitor and a protease inhibitor cocktail (1:100, Solarbio) were added to prevent degrading of proteins in the extracts. tThe protein concentration was quantified using a BCA Protein Assay Kit (Beyotime, China). Afterwards, the equal boiled protein samples (30 μg) were loaded on 8 ~ 12% sodium dodecyl sulfatepolyacrylamide gels (Solarbio Life Sciences), electrophoretically separated, and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, Massachusetts), which had been wetted in 100% methanol for 15 s previously.. Then the membranes were blocked in 5% skim milk containing Tris-buffered Saline Tween (TBST, Solarbio, China) at room temperature for 1 h to reduce nonspecific bindings. Subsequently, the membranes were incubated with the primary antibodies (1:500 ~ 1:1000 dilutions, Cell Signaling Technology, USA) overnight at 4 °C with gentle agitation. Getting washed 2 ~ 3 times with TBST, the secondary antibodies (1:2000 dilution) were added for incubation for 1 h. Finally, the immunoreactive bands were detected with Chemiluminescent HRP Substrate (Merck Millipore) and quantified through a Image Lab software (Bio-Rad, Hercules, USA) .
Cell viability assay
After receiving the drug treatment and/or gene regulation as described above, the ADS_Eu_ESC and ADS_Ec_ESC cells were seeded in 96-well plates (4 × 103 cells /well) and cultured for 96 h. Then 10 μl/well cell-counting kit-8 (CCK-8, Dojindo, Japan) reagent was added at indicated time points (24 h, 48 h, 72 h, 96 h) and the corresponding cells were incubated for another 1 h at 37 °C. The absorbance of each well at 450 nm was measured with a microplate reader (Bio-Rad, USA) and cell viability was evaluated.
The ADS_Eu_ESC and ADS_Ec_ESC cells were seeded in 6-well plates at a density of 2 × 103 cells /well. Thereafter, cells were incubated for 14 days to allow colony formation, during which the medium was refreshed every 2 ~ 3 days. Then 1 ml/well 4% paraformaldehyde and 0.1% crystal violet were used to fix and stain the cells, respectively. Eventually, the number of visible colonies was counted after full decolorization.
Capillary tube and network formation assay
The pro-angiogenetic activity of ADS_Eu_ESC and ADS_Ec_ESC was evaluated by a capillary tube and network formation assay. Firstly, 24-well ice-cold plates were coated with 250 μl/well Matrigel (BD, Bioscience, USA, 1:4 dilution in serum-free DMEM/f12) and incubated for 30 min at 37 °C. Meanwhile, the ADS_Eu_ESC and ADS_Ec_ESC cells with different treatment were collected, centrifuged and filtered to obtain the corresponding conditioned medium. Then HUVECs (2 × 103, P2-P4) in 250 μl conditioned medium were overlaid on the Matrigel. After incubation at 37 °C for 6 h, 4 μM Calcein Acetoxymethyl Ester (Calcein AM) was added for cell staining followed by incubation for 30 min. Finally, after replacement with fresh medium, the number of new capillary formation was observed and counted under a fluorescence microscope.
Xengraft model establishment and treatment
A total of 33 BALB/c female nude mice (4 week old) were purchased from Beijing Vital River Laboratory Animal Technology. Co., Ltd. (Beijing, China). The guidelines for animal care were approved by the committee on Animal Study of Beijing Obstetrics and Gynecology Hospital, Capital Medical University. The mice were raised under specific pathogen-free (SPF) conditions. All of them got bilaterally ovariectomized and left untreated for 2 weeks.
The Ishikawa
ER+ cells transfected with OV-Talin1, OV-NC, treated with β-E
2 or β-E
2 + OV-Talin1 were made into single cell suspension (2 × 10
7/ml) with a mixture of 200 μl PBS and 50 μl Matrigel. Subsequently, the cell suspension was delivered by subcutaneous injection into the right axilla lesions of nude mice. The length (L), width (W) and height (H) of the nodule lesions were measured every week and the lesion volumes were calculated according to the following formula [
15]: V = π/6 (L x W x H). On the 84th day after cell inoculation, the mice were euthanized. Then the exfoliated lesion nodules were weighted and stored for subsequent experiment.
Statistical analysis
Experiments were performed triplicately or more for statistical significance. The results were analyzed using SPSS 23.0 and Graphpad Prism software. The measurement data were expressed as mean ± standard deviation. Continuous variables in two groups were analyzed by independent sample t-test. One-way analysis of variance (ANOVA) was used for comparison of multiple groups. The difference was considered statistically significant at P < 0.05.
Discussion
ADS is a commonly encountered estrogen-dependent disorder affecting approximately 20% of women of reproductive age and shows an increased incidence among women with infertility [
2,
14,
16]. Despite the recent advantages of diagnostic tools, a shared definition and classification as well as the awareness of the condition are still lacking. Currently, there was no international guideline to follow regarding the completely effective management on ADS, except for hysterectomy [
17,
18]. Although the precise etiology and pathogenesis of ADS remain to be further elucidated, several theories or hypotheses addressing the progress of the disease have been put forward [
4], including the enhanced invasion and invagination of endometrium into myometrium; metaplasia or differentiation of stem cells; endometrial-myometrial interface disruption (EMID); induction of aberrant local hormones and some genetic or epigenetic modifications.
According to one of the most accepted theories, ADS may result from the invagination of basalis endometrium into the myometrium after crossing an altered or interrupted EMT, a highly specialized hormone-responsive structure, eventually establishing ectopic lesions [
19]. Emerging evidence have demonstrated that enhanced endometrial proliferation, more active cell migration and invasion through the EMT phenotype, as well as increased neovascularization were much more common in eutopic and ectopic endometrium of ADS [
20‐
23]. These alterations from endometrial cells have been postulated to be extremely beneficial to endometrium invading into deeper myometrium and maintaining the subsequent growth and survival of adenomyotic ectopic lesions. Notably, during the course of endometrial invagination and implantation, steroid hormones are likely to serve a central role. Particularly, the local supraphysiological estrogen levels may be a preliminary status contributing to the origin of ADS, since it has been manifested that high β-E
2 initiated and facilitated the microtrauma of EMI as a positive feedback [
2,
4]. As described in earlier reports, elevated β-E
2 induced a shift of epithelial to mesenchymal markers and fostered endometrial cell migration and migration in ADS [
8,
24,
25]. Furthermore, our previous study also demonstrated that β-E
2 could cause hyperproliferation of adenomyotic smooth muscle cells (SMCs) in EMI through activating ER-enhanced RoA-Rock signaling pathway [
26,
27]. Also, hyperestrogen has been found to be involved in overexpression of annexin A2 in adenomyotic endometrium, which mediated the angiogenetic process via β-catenin/T-cell factor signaling [
28]. Although a growing body of evidence recently linked the pathogenesis of ADS to a remarkable disorder of estrogen metabolism, the molecular mechanisms of this disease still remain largely unelucidated.
In the present study, we further demonstrated that a suitable dose of β-E
2 exhibited a significant promoting effect on adenomyotic endometrial stromal cell proliferation and pro-angiogenesis. According to reports from Herndon etal. and Guo etal., the molecular mechanism underlying decreased apoptosis and increased proliferation likely derive from excessive E
2 in adenomyotic endometrium [
29,
30]. Huang etal. Also revealed β-E
2-induced angiogenesis could contribute to ADS by activating the slug/VEGF axis in endometrial epithelial cells [
31]. On the premise of not contradicting the previous results, however, our research mainly featured the use of primary isolated and cultured endometrial stromal cells of human ADS instead of merely endometriod adenocarcinoma cell lines, which means better fitting the cellular biological model of ADS. At the same time, we intervened with β-E
2 in both eutopic and ectopic endometrial stromal cells, which may provide a more powerful supplementary basis for verifying the role of local hyperestrogenism in different positions and stages during the development of ADS. Concomitant treatment with an ER antagonist (Fulvestrant, ICI 182780), which not only abolished the stimulative effects of β-E
2 on cell proliferation and pro-angiogenesis from the perspective of functional experiment, but also abrogated the expression of markers including PCNA, Ki67, VEGFB and ANGPTL4, further supported an ER-dependent mechanism in ADS. Indeed, these observations may account for elevated β-E
2-mediated overproliferation and hyperangiogenesis in adenomyotic endometrium. However, whether the key links in β-E
2-guided ADS can be affected by other factors synchronously, the relevant evidence is still less sufficient.
Talin1, a ubiquitous macromolecular (270-KDa) protein highly enriched at the cell-matrix attachment sites, mostly functions as the key regulator of integrin activation, which is encoded by TLN1 [
32]. Since a crucial final step in activating integrin is binding of the N-terminal head domain of Talin1 to the β-integrin cytoplasmic domain, Fadi etal. Recently demonstrated that Talin1-dependent integrin activation could regulate VE-cadherin localization and endothelial cell barrier function, which was critical for vascular sprouting development and stability [
33]. Furthermore, an important property of integrin is the modulation of affinity for extracellular ligands, a process termed integrin activation or ″inside-out integrin signaling″. So far, abundant robust evidence has confirmed that Talin1 can bind and activate integrin through modulating its affinity. Once activated, the integrin initiates the activation of FAK, thereby mediating numerous processes concerning cell proliferation, adhesion and mobility [
34]. As previous studies implicated, Talin1 was mostly identified to be overexpressed and involved in the progression of multiple human cancers, during which the tumor cell invasion or metastasis was stimulated [
35]. On the contrary, Somcyeh etal. Revealed that cytoplasmic expression of Talin1 was associated with advanced pathological features in colorectal cancer, based on the observations that a negative correlation between Talin1 protein level and advanced TNM stage (
P = 0.028) as well as worse disease specific survival (
P = 0.011) [
36]. As regards the effects of Talin1 in gynecological diseases, it has been reported that Talin1 dysregulation in uterine endometrium of patients with missed abortion would negatively alter the endometrial epithelial cell adhesive capacity during the early stage of pregnancy, thus impeding implantation [
37,
38]. Besides, as per available literature, Talin1 was detected to be upregulated in the eutopic and ectopic endometrial glands of ADS by Jiang etal [
39],. which was consistent with our previous study. As a partial improvement of Jiang′s research on the specific role and internal mechanism of Talin1, we have previously proved that abnormally overexpressed Talin1 induced EMT in ADS endometrial cells via triggering wnt/β-catenin pathway [
11]. Collectively, we have preminarily confirmed that Talin1 could promote the infiltration of adenomyotic endometrium into myometrium. However, whether Talin1 could alter the subsequent proliferation and pro-angiogenesis of endometrial cells acquiring a stromal phenotype to maintain the ectopic implantation and survival after EMT, especially whether Talin1 influences the efficacy of β-E
2, is still disputed. Therefore, we designed the present study for further investigation.
As expected, our study unveiled that a trend of gradually increasing expression of Talin1 protein from normal uterine endometrium to ADS ectopic endometrium, eutopic endometrium, the corresponding Ctrl_ESC, and primary ADS_Ec_ESC as well as ADS_Eu_ESC cells. More importantly, we provided novel data to present that Talin1 overexpression (OV-Talin1) can serve a positive role in facilitating adenomyotic endometrial stromal cell proliferation and pro-angiogenesis. Histopathologically, ADS is a benign disease, but published work has confirmed that its endometrial cells, especially the stromal cells, are more active in proliferation, migration, invasion and angiogenesis, which are similar to the biological characteristics of tumor cells [
4,
19]. Considering the aberrant enrichment of Talin1 has been identified in several tumors and there is strong evidence linking it to oncogenic progress, our findings suggested that Talin1 might also play some distinct roles in the development of ADS. In addition, Pulous etal.demonstrated that Talin1-dependent integrin activation was required for endothelial proliferation and postnatal angiogenesis [
33], which may account for the enhanced pro-angiogenesis of endometrial stromal cells transfected with Talin1 overexpression vector in our study. Furthermore, accumulating studies have reported that upregulated Talin1 stimulates overproliferation of glioblastoma multiform cells, ovarian carcinoma cells and HCC cells through triggering FAK signaling [
40‐
42]. Interestingly, results from our previous study indicated that FAK could regulate endometrial stromal cell proliferation, migration and invasion in ADS [
43]. Thus, given to the current study, it can be speculated that FAK pathway may also play an important role in Talin1 stimulated proliferation and neovascularization in ADS stromal cells, although more mechanism experiments are needed to conduct.
Based on validating the promoting effects of Talin1 overexpression or β-E2 treatment on ADS endometrial stromal cell proliferation and pro-angiogenesis respectively, the synergistic effects of the two were first linked and verified both in vitro and in vivo. Specifically, the ADS_Eu_ESC and ADS_Ec_ESC cells, treated with OV-Talin1 or β-E2, acquiring originally higher proliferative and pro-angiogenetic capabilities, presented even more enhanced abilities after co-treated with β-E2 plus OV-Talin1. Of note, the xenograft nude mice model was established through inoculation of IshikawaER+ cells intervened with β-E2 or Talin1 overexpression, which further supported the addictive effects of Talin1 on β-E2 induced growth and neovascularization of the ectopic endometrial lesions. To our best knowledge, our research has supplemented more data to the factors that alter the efficacy of β-E2in the pathogenesis of ADS. Especially for the first time, it has revealed that accompanying up-regulation of Talin1 could positively amply the effects of β-E2, and the combination of the two might make it easier for inducing ADS. Our findings may provide a novel therapeutic insight for ADS, for instance, through simultaneous blocking on the effects of β-E2 and Talin1.
This study also raised several points that warrant further exploration and improvement. Firstly, concerning the potential molecular mechanism of Talin1 cooperating with β-E2, more experimental research is needed. In addition to improving the proliferation and angiogenesis of eutopic and ectopic endometrial stromal cells, it is well worth speculating and verifying whether Talin1 and β-E2 serve a joint role during other related pathogenesis of ADS. Furthermore, based on the results from the present study, we are still unable to fully determine the regulatory relationship between Talin1 and β-E2. For example, whether β-E2 was more likely to be dominant and whether β-E2 also regulated the expression and efficacy of Talin1 are under exploration. We should also admit that some figures from the western blot assay were not of very good quality, therefore the densitometric analysis and the final results probably got affected to some extent. In this regard, we need to expand the sample size as much as possible and re-conduct the experiment in our future research. Another limitation in our study is that the model was established through subcutaneous injection of Ishikawa cells, which might fail to completely reshape the clinicopathological process of human ADS, although this method is simpler and has been reported previously.
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