LncRNA BANCR Promotes Endometrial Stromal Cell Proliferation and Invasion in Endometriosis via the miR-15a-5p/TRIM59 Axis

Long non-coding RNA (LncRNA) emerges as a regulator in various diseases, including endometriosis (EM). This study aims to uncover the role of long non-coding RNA BRAF-activated non-protein coding RNA (lncRNA BANCR)-mediated competing endogenous RNA mechanism in endometrial stromal cell (ESC) proliferation and invasion in EM by regulating miR-15a-5p/TRIM59. ESCs were isolated from eutopic and ectopic endometrial tissues, followed by the determination of Cytokeratin 19 and Vimentin expressions in cells. Then, expressions of lncRNA BANCR, microRNA (miR)-15a-5p, and tripartite motif-containing 59 (TRIM59) in tissues and cells were determined by real-time quantitative polymerase chain reaction or Western blot assay, and cell proliferation and invasion were evaluated by cell counting kit-8 and transwell assays. After that, the subcellular localization of lncRNA BANCR and binding of miR-15a-5p to lncRNA BANCR or TRIM59 were analyzed. LncRNA BANCR was upregulated in ectopic endometrial tissues and ectopic ESCs (Ect-ESCs). Silencing lncRNA BANCR suppressed Ect-ESC proliferation and invasion. LncRNA BANCR inhibited miR-15a-5p to promote TRIM59 expression. miR-15a-5p downregulation or TRIM59 overexpression both reversed the effects of silencing lncRNA BANCR on Ect-ESC proliferation and invasion. In summary, our findings suggested that lncRNA BANCR facilitated Ect-ESC proliferation and invasion by inhibiting miR-15a-5p and promoting TRIM59.


Introduction
Endometriosis (EM) is termed a complex gynecological disorder, diagnosed by the presence of endometrial-like tissue, glands, and stroma outside the uterine cavity [1]. EM will affect about 10% of the female population and not only can it result in heavy social and economic burdens, including, pain, infertility, depression and anxiety, health care costs, and indirect productivity loss, but data are accumulating that malignant transformation is an important consideration [2,3]. EM is a benign disease similar to malignancy in some perspectives, including estrogen-stimulated proliferation, recurrence, and tendency to metastasis [4]. Pathologi-cally, endometrial stromal cells (ESCs), the major resident cells in human endometrium, are switched to invasive and proliferative phenotypes under the stimulation of cytokines and chemokines, thus exacerbating EM progression [5,6]. Most current therapies focus on alleviation of pain symptoms and prevention of recurrence but lack the curable intent [7]. Therefore, it is prudent to explore novel molecules sensitive to EM proliferation and invasion, in an effort to improve the prognosis of EM patients.
Long non-coding RNA (LncRNA), the well-studied RNA molecules that regulate multiple cellular and biological processes, are documented to participate in hormone response, behaviors of ESCs, autophagy, and immune disorder in the context of EM, thus affecting EM progression [8]. LncRNA BRAF-activated non-protein coding RNA (BANCR), a wellestablished oncogenic lncRNA in human cancers, is involved in cell proliferation, migration, invasion, apoptosis, and epithelial to mesenchymal transition [9,10]. More importantly, lncRNA BANCR inhibitor is potent to reduce the volume of eutopic endometrium in animal models by suppressing the production of angiogenic factors [11]. However, the expression pattern and role of lncRNA BANCR in ectopic ESCs (Ect-ESCs) remain indistinct.
Taking the aforementioned associations into consideration, we raised a hypothesis that lncRNA BANCR mediates a ceRNA network with miR-15a-5p/TRIM59 to affect Ect-ESC proliferation and invasion. In this manner, the present study sought to investigate the molecular mechanism of lncRNA BANCR in EM, hoping to provide a novel theoretical reference for EM treatment.

Methods and Materials
2.1. Acquisition of Clinical Samples. A total number of 20 EM patients (28-40 years old) who received tissue resection in The Affiliated Hospital of Inner Mongolia Medical University were included in this study for the collection of eutopic and ectopic endometrial tissues. The procedures of sample collection were ratified by the medical ethics committee of The Affiliated Hospital of Inner Mongolia Medical University and conformed to the Declaration of Helsinki. The written informed content was signed by each patient. The collected samples were stored in liquid nitrogen and kept at -80°C before the subsequent uses.
Inclusion criteria of EM patients were as follows [27]: (1) at reproductive age (19-45 years old); (2) at the proliferative phase of the menstrual cycle; (3) EM was confirmed by lap-aroscopic surgery and postoperative histological examination. Exclusion criteria of EM patients were as follows: (1) with any history of malignancy; (2) with autoimmune or metabolic disorders; (3) taking any hormonal medications or dietary supplements within the last three months before the surgery; (4) pregnancy or lactation.
2.2. Isolation of Primary Human Endometrial Stromal Cells (hESCs). hESCs were obtained from eutopic and ectopic endometrial tissues of EM patients in The Affiliated Hospital of Inner Mongolia Medical University. The procedures of cell isolation were as follows: firstly, endometrial tissues were shredded, detached with 4% collagenase (60 min), and centrifuged (500 × g, 5 min); secondly, the cell suspensions were centrifuged (3000 × g, 10 min); thirdly, cell precipitates were resuspended in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 mg/L streptomycin (Solarbio, Beijing, China). Then, isolated ESCs were placed in an incubator and kept at 37°C, and the medium was renewed after the first 24 h of cell culture and then replaced every 2-3 days.
2.7. Transwell Assay. The invasion potential of Ect-ESCs was analyzed using transwell assay. Simply put, the apical transwell chamber was pre-coated with Matrigel (BD Biosciences, San Jose, CA, USA), followed by cell seeding (4 × 10 4 cells/ 0.1 mL). Then, the basolateral transwell chamber was supplemented with DMEM supplemented with 10% FBS. After 24 h of cell culture, cells were stained with crystal violet. Non-invasive cells were wiped out, and the number of invasive cells was counted under a microscope.

Real-Time-Quantitative Polymerase Chain Reaction
(RT-qPCR). After extraction of the total RNA content in cells and tissues using the TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), the extracted RNA was reverse-transcribed into the complementary DNA using RT-PCR assay kits (K1621, Thermo Fisher Scientific, Waltham, MA, USA). The quantification of gene expression was achieved by real-time PCR. The relative amount of gene expression was quantified according to the 2 -ΔΔCt method, with GAPDH and U6 as endogenous references [28]. The primers used in RT-qPCR are shown in Table 1.
2.9. Western Blot Assay. The total protein samples were extracted from tissues or cells using radioimmunoprecipitation assay buffer. According to the instructions of the producer, the protein concentration was determined using the bicinchoninic acid protein assay kit (ab102536, Abcam, Cambridge, UK). An equal amount of protein (30 μg) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. After blockade with Tris-HCl buffered saline containing 0.1% Tween-20 and 5% skim milk, membranes were incubated with primary antibodies against TRIM59 (ab166793, 1 : 500, Abcam) and GAPDH (ab181602, 1 : 10000, Abcam) overnight and then with secondary antibody against IgG (ab6721, 1 : 2000, Abcam) for 1 h. Membranes were visualized using the enhanced chemiluminescence-PLUS (Amersham Biosciences, Sweden). Eventually, the grayscale was quantified using Image-Pro Plus (Media Cybernetics, San Diego, CA, USA).
2.11. Subcellular Fractionation Assay. Following the manufacturer's protocol, the nucleus and cytoplasm were separated using PARIS kits (AM1921, Thermo Fisher Scientific). In brief, Ect-ESCs were collected, lysed with the preparation buffer, and then centrifuged. The fractionation of the supernatant and nuclear pellet was achieved using the cell disruption buffer, and the nuclear pellet was preserved for RNA extraction and analysis. Lastly, lncRNA BANCR was analyzed by RT-qPCR, with U6 (the control of nucleus) and GAPDH (the control of cytoplasm) as internal references.
2.12. Dual-Luciferase Assay. The synthesized BANCR fragments containing the binding sites to miR-15a-5p and BANCR fragments containing the mutant binding sites to miR-15a-5p, as well as TRIM59 fragments containing the binding sites to miR-15a-5p and TRIM59 fragments containing the mutant binding sites to miR-15a-5p were inserted into pmirGLO-reporter plasmids. The above-constructed luciferase reporter plasmids (BANCR-WT or BANCR-MUT, TRIM59-WT or TRIM59-MUT) were co-transfected into Ect-ESCs with mimics-NC or miR-15a-5p mimics (miRmimics). After 48 h, cells were harvested and lysed, and the luciferase activity was quantified using the luciferase assay kits (16186, Thermo Fisher Scientific). All plasmids were supplied by GenePharma.
2.13. Statistical Analysis. Data analysis and graphing were conducted with the help of SPSS21.0 software (IBM Corp, Armonk, NY, USA) and GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). Measurement data were formalized as mean ± standard deviation (SD). First, data were subjected to normality and homogeneity of variance tests, which revealed that data conformed to normal distribution and homogeneity of variance. Then, pairwise comparisons were analyzed using the t test, and multi-group comparisons were analyzed using one-way or two-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test. The P values were attained by two-sided tests, and a value of P < 0:01 was indicative of extremely statistical significance.

TRIM59 Overexpression Reverses the Effects of Silencing lncRNA BANCR on Inhibiting Ect-ESC Proliferation and
Invasion. To evaluate the role of TRIM59 in Ect-ESC proliferation and invasion, Ect-ESCs were transfected with oe-TRIM59 to upregulate TRIM59 expression (P < 0:05, Figure 5(a)-5(b)), followed by combined treatment with si-BANCR#1 which had better knockdown effectiveness. Our subsequent experiments revealed that TRIM59 overexpression enhanced the proliferation potential of Ect-ESCs (P < 0:05, Figure 5(c)-5(d)), as well as the invasion potential of Ect-ESCs (P < 0:05, Figure 5(e)). Altogether, the preceding data suggested that TRIM59 overexpression reversed the effects of silencing lncRNA BANCR on inhibiting Ect-ESC proliferation and invasion.

Discussion
EM is a gynecological disorder with malignant potential and remains a challenge in the clinic due to the lack of therapies with curable efficacy [7]. ECSs are identified as the major resident cells in the endometrium and alterations in their behaviors, such as proliferation, invasion, migration, and epithelial-mesenchymal transition, are associated with the pathogenesis of EM [33,36,37]. In addition, lncRNAs are involved in gene regulation in EM and the alteration of lncRNA expression level affects EM progression [38]. Apart from lncRNAs, miRNAs and circRNAs also play crucial roles in the regulation of EM progression [39][40][41]. In the present study, our findings supported that lncRNA BANCR promotes Ect-ESC proliferation and invasion via the miR-15a-5p/TRIM59 axis. LncRNA BANCR is a putative biomarker of cell malignancy [9,10]. A pioneering study by Zhu and colleagues has uncovered that knockdown of lncRNA BANCR restrains angiogenesis in ectopic endometrial tissues by targeting the extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway [11]. In this study, augmented lncRNA BANCR levels were evident in ectopic endometrial tissues of EM patients. Then, ESCs were isolated from eutopic and ectopic endometrial tissues and it was found that lncRNA BANCR was prominently expressed in Ect-ESCs. To further evaluate the functionality of lncRNA BANCR in Ect-ESC proliferation and invasion, lncRNA BANCR was silenced in Ect-ESCs using si-BANCR, upon which the proliferation and invasion potentials of Ect-ESCs were declined. It might be correlated with the effects of lncRNA BANCR on , and (e) were analyzed using one-way ANOVA, and data in figure (c) were analyzed using two-way ANOVA, followed by Tukey's multiple comparison test. ** P < 0:01. International Journal of Genomics promoting the expression levels of ESCs mobility-related proteins, such as matrix metalloproteinase 1 and 2 [42][43][44]. Therefore, it is plausible to suggest that silencing lncRNA BANCR suppresses Ect-ESC proliferation and invasion. LncRNAs further function as the sponges of miRNAs to play performance in human diseases [45,46]. miRNAs can serve as desirable biomarkers for EM diagnosis and treatment due to their stability in body fluids, specific and distinct signatures relative to clinical controls, and biological relevance to EM pathogenesis [47]. To further analyze the downstream mechanism of lncRNA BANCR, the lncLocator database and subcellular fractionation assay validated the cytoplasmic localization of lncRNA BANCR in Ect-ESCs, suggesting the possibility of lncRNA BANCR as a ceRNA in Ect-ESCs. A prior microarray analysis disclosed the downregulation of miR-15a-5p in endometriotic tissues and its association with angiogenesis-related proteins [48]. In addition, miR-15a-5p was previously demonstrated to repress proliferation, migration, and invasion of Ect-ESCs and mobility and angiogenesis of endometrial mesenchymal stem cells [18,20], further highlighting the participation of miR-15a-5p in EM pathogenesis. Accordingly, the dualluciferase assay verified the binding relationship between lncRNA BANCR and miR-15a-5p, and decreased miR-15a-5p expression levels were found in ectopic endometrial tissues and Ect-ESCs and elevated in response to silencing lncRNA BANCR, suggesting that lncRNA BANCR negatively regulated miR-15a-5p in EM. Subsequently, miR-15a-5p was downregulated in Ect-ESCs in the si-BANCR group using the miR-15a-5p inhibitor, upon which the proliferation and invasion potentials of Ect-ESCs were enhanced. In accordance, miR-15a-5p was capable of improving pro-apoptosis B-cell lymphoma 2-associated X protein (Bcl2) and decreasing anti-apoptosis B-cell lymphoma 2 (Bax) wherein the ratio of Bax/Bcl2 is associated with apoptosis of ESCs [49,50]. Collectively, the above findings and shreds of evidence initially demonstrated that lncRNA BANCR promotes Ect-ESC proliferation and invasion by inhibiting miR-15a-5p.
Furthermore, the subsequent bioinformatic data directed our attention to TRIM59. As indicated by a previous study, TRIM59 functioned as a positive regulator of Ect-ESC invasion [26]. The binding relationship between miR-15a-5p and TRIM59 was testified by the dual-luciferase assay. Meanwhile, increased TRIM59 expression levels were found in ectopic endometrial tissues and Ect-ESCs, downregulated by silencing lncRNA BANCR and upregulated by inhibition of miR-15a-5p, indicative of a positive correlation between lncRNA BANCR and TRIM59 and a negative correlation between miR-15a-5p and TRIM59. To confirm the effects of TRIM59 on Ect-ESC proliferation and invasion, TRIM59 was overexpressed in Ect-ESCs in the si-BANCR group using oe-TRIM59, upon which Ect-ESC proliferation and invasion were accelerated. Likewise, TRIM59 acts as a booster of proliferation and invasion of endometriosis-associated cancer, such as ovarian, breast, and cervical cancers [51][52][53]. Altogether, the above findings initially supported that lncRNA BANCR promotes Ect-ESC proliferation and invasion by inducing TRIM59 upregulation.

Conclusion
To conclude, our study was the first of its kind that unveiled the promotive role of the lncRNA BANCR-mediated ceRNA network in Ect-ESC proliferation and invasion and provided a novel theoretical reference for the clinical study of lncRNA BANCR in EM. However, whether other downstream miR-NAs of lncRNA BANCR and downstream target genes of miR-15a-5p are involved in functions of Ect-ESCs remains unknown. In the next step, experimentation will be designed to investigate other downstream mechanisms of lncRNA BANCR and miR-15a-5p in Ect-ESCs and the upstream mechanism of lncRNA BANCR in Ect-ESCs.

Data Availability
The data that support this study are available from the corresponding author upon reasonable request.

Ethical Approval
The procedures of sample collection were ratified by the medical ethics committee of The Affiliated Hospital of Inner Mongolia Medical University and conformed to the Declaration of Helsinki.

Consent
The written informed content was signed by each patient.

Conflicts of Interest
The authors have no relevant financial or non-financial interests to disclose.

Supplementary Materials
Table S1: The downstream miRNAs of lncRNA BANCR were predicted on the RNA22 database. Table S2: The target genes of miR-15a-5p were predicted on the StarBase database. Table S3: The target genes of miR-15a-5p were predicted on the miRWalk database. (Supplementary Materials)