Comprehensive analysis of circRNA-miRNA-mRNA regulatory network and novel potential biomarkers in eutopic endometrium of adenomyosis.

Adenomyosis (ADS) is a common gynecological disorder, and its pathogenesis remains unclear. This study explores the functions of circRNAs in the eutopic endometrium of ADS and their diagnostic efficacy for ADS. High-throughput RNA sequencing was performed on 12 eutopic endometrial samples from ADS patients and 3 control endometrial samples. Additionally, circRNAs were analyzed in conjunction with clinical features. A competitive endogenous RNA network was established based on bioinformatics analysis, comprising 3 circRNAs, 1 miRNA, and 13 mRNAs. In the ADS group, the expression levels of hsa_circ_0008959 and SLC15A4 were significantly reduced, while hsa-miR-124-3p expression was increased. SLC15A4 was associated with cell proliferation and invasion. Decreased expression of hsa_circ_0008959 and SLC15A4, along with high VAS scores and elevated hsa-miR-124-3p levels, were identified as risk factors for ADS development. The combination of hsa_circ_0008959 and VAS scores demonstrated the highest diagnostic value for ADS.

miR-141-3p Regulates the Proliferation and Apoptosis of Endometrial-Myometrial Interface Smooth Muscle Cells in Adenomyosis Via JAK2/STAT3 Pathway.

Adenomyosis (ADS) is a common benign gynecological disease. Abnormal proliferation at the endometrial-myometrial interface (EMI) plays a crucial role in the occurrence and progression of ADS. miR-141-3p is associated with cell proliferation and apoptosis. However, the specific mechanism of miR-141-3p in the etiology of ADS is still unknown. In this study, we explored the effects of miR-141-3p on the proliferation and apoptosis of ADS EMI smooth muscle cells (SMCs). We collected EMI tissues for the primary culture of SMCs from 25 patients diagnosed with ADS and 20 without ADS. Real-time quantitative polymerase chain reaction and western blot were used to measure the mRNA and protein expression levels of miR-141-3p, JAK2, STAT3, phospho-JAK2, and phospho-STAT3 in ADS EMI SMCs. The cell counting kit 8 assay and flow cytometry analysis were used to evaluate the proliferation and apoptosis of EMI SMCs. The miR-141-3p mimic/inhibitor was used to increase or decrease the expression level of miR-141-3p. We added WP1066 to block the phosphorylation of JAK2/STAT3 pathway components. The miR-141-3p levels were decreased, while JAK2 and STAT3 levels were increased in ADS EMI SMCs. miR-141-3p overexpression significantly inhibited the proliferation and enhanced the apoptosis of EMI SMCs, whereas a decrease in miR-141-3p expression level was connected to the opposite results. Meanwhile, inactivated JAK2/STAT3 pathway decreased proliferation and enhanced apoptosis of EMI SMCs after WP1066 treatment. Furthermore, rescue experiments confirmed that the JAK2/STAT3 pathway was the downstream pathway of miR-141-3p and reduced the effect of miR-141-3p on the proliferation and apoptosis of EMI SMCs. These results demonstrate that miR-141-3p regulates the proliferation and apoptosis of ADS EMI SMCs by modulating the JAK2/STAT3 pathway.

RNA-seq reveals co-dysregulated circular RNAs in the adenomyosis eutopic endometrium and endometrial-myometrial interface.

BACKGROUND: Uterine adenomyosis is associated with chronic pelvic pain, abnormal uterine bleeding, and infertility. The pathogenesis of adenomyosis is still unclear. Circular RNAs (circRNAs) have been implicated in several benign diseases and malignant tumors. We aimed to explore the co-dysregulated circular RNA profile in the eutopic endometrium and endometrial-myometrial interface (EMI) of adenomyosis. METHODS: Total RNA was extracted from the eutopic endometrium and EMI of 5 patients with adenomyosis and 3 patients without adenomyosis. Next-generation sequencing was performed to identify the circRNA expression profile of the two tissue types. Bioinformatics analysis was performed to predict circRNA-binding miRNAs and miRNA-binding mRNAs and construct ceRNA networks, and functional enrichment analysis was performed to predict the biological functions of circRNAs. RESULTS: Among the adenomyosis patients, 760 circRNAs were significantly upregulated and 119 circRNAs were significantly downregulated in the EMI of adenomyosis, while 47 circRNAs were significantly upregulated and 17 circRNAs were significantly downregulated in the eutopic endometrium of adenomyosis. We identified hsa_circ_0002144 and hsa_circ_0005806 as co-upregulated and hsa_circ_0079536 and hsa_circ_0024766 as co-downregulated in the eutopic endometrium and EMI. Bioinformatics analysis was performed to construct a ceRNA network of codifferentially expressed circRNAs. The MAPK signaling pathway is the most important signaling pathway involved in the function of the ceRNA network. CONCLUSIONS: Co-dysregulated circRNAs were present in the eutopic endometrium and EMI of adenomyosis. MiRNA binding sites were observed for all of these circRNAs and found to regulate gene expression. Co-dysregulated circRNAs may induce the eutopic endometrial invagination process through the MAPK signaling pathway and promote the progression of adenomyosis.

The cannabinoid receptor CB1 affects the proliferation and apoptosis of adenomyotic human uterine smooth muscle cells of the junctional zone: a mechanism study.

BACKGROUND: The denomyotic junctional zone (JZ) plays an important role in the pathogenesis of adenomyosis. Proliferating cell nuclear antigen (PCNA) is an important nuclear marker of cell proliferation. This study aimed to evaluate the effects of the cannabinoid receptor CB1 on proliferation and apoptosis in the JZ in women with and without adenomyosis. METHODS: JZ smooth muscle cells (JZSMCs) of the adenomyosis and control groups were collected and cultivated. Immunohistochemistry and immunoblotting were used for protein localization and expression detection of CB1 and PCNA. Additionally, qRT-PCR was used to quantitatively analyse the mRNA expression of the two. AM251 and ACEA were used to regulate the function of CB1 receptors, and CCK-8 assay and flow cytometry assay were used to verify the proliferation and apoptosis of JZSMCs after regulation. RESULTS: We demonstrated that in normal JZSMCs CB1 and PCNA messenger RNA (mRNA) and protein expression was significantly higher in the proliferative phase of the menstrual cycle than in the secretory phase. CB1 and PCNA expression in JZSMCs from women with ADS was significantly higher than that in control women and did not significantly differ across the menstrual cycle. CB1 receptor antagonist AM251 inhibited the proliferation of adenomyotic JZSMCs in a dose-dependent manner. The CB1 receptor agonist ACEA significantly promoted the proliferation of adenomyotic JZSMCs. The apoptosis rate of adenomyotic JZSMCs treated with AM251 was significantly higher than that of JZSMCs from the untreated control group. The apoptosis rate was significantly decreased in the ACEA group compared with that in the untreated control group. Furthermore, AM251 suppressed the phosphorylation of AKT and Erk1/2 in adenomyotic JZSMCs. The CB1 agonist ACEA significantly promoted the phosphorylation of AKT and Erk1/2. CONCLUSIONS: Our results indicated that the levels of CB1 and PCNA were increased in patients with adenomyosis and that cyclic changes were lost. CB1 may affect uterine JZ proliferation and apoptosis in adenomyosis by enhancing AKT and MAPK/Erk signalling.

Upregulated Talin1 synergistically boosts ß-estradiol-induced proliferation and pro-angiogenesis of eutopic and ectopic endometrial stromal cells in adenomyosis.

Adenomyosis (ADS) is an estrogen-dependent gynecological disease with unspecified etiopathogenesis. Local hyperestrogenism may serve a key role in contributing to the origin of ADS. Talin1 is mostly identified to be overexpressed and involved in the progression of numerous human carcinomas through mediating cell proliferation, adhesion and motility. Whether Talin1 exerts an oncogenic role in the pathogenesis of ADS and puts an extra impact on the efficacy of estrogen, no relevant data are available yet. Here we demonstrated that the adenomyotic eutopic and ectopic endometrial stromal cells (ADS_Eu_ESC and ADS_Ec_ESC) treated with ß-estradiol (ß-E2) presented stronger proliferative and pro-angiogenetic capacities, accompanied by increased expression of PCNA, Ki67, VEGFB and ANGPTL4 proteins. Meanwhile, these promoting effects were partially abrogated by Fulvestrant (ICI 182780, an estrogen-receptor antagonist). Aberrantly upregulation of Talin1 mRNA and protein level was observed in ADS endometrial specimens and stromal cells. Through performing functional experiments in vitro, we further determined that merely overexpression of Talin1 (OV-Talin1) also enhanced ADS stromal cell proliferation and pro-angiogenesis, while the most pronounced facilitating effects were found in the co-intervention group of OV-Talin1 plus ß-E2 treatment. Results from the xenograft nude mice model showed that the hypodermic endometrial lesions from co-intervention group had the highest mean weight and volume, compared with that of individual OV-Talin1 or ß-E2 treatment. The expression levels of PCNA, Ki67, VEGFB and ANGPTL4 in the lesions were correspondingly elevated the most in the co-intervention group. Our findings unveiled that overexpressed Talin1 might cooperate withß-E2 in stimulating ADS endometrial stromal cell proliferation and neovascularization, synergistically promoting the growth and survival of ectopic lesions. These results may be beneficial to provide a new insight for clarifying the pathogenesis of ADS.

Discovery of gene module acting on ubiquitin-mediated proteolysis pathway by co-expression network analysis for endometriosis.

RESEARCH QUESTION: Is abnormal gene module expression in the eutopic endometrium related to the occurrence of endometriosis? DESIGN: Nine datasets of normal and eutopic endometrium were searched and collected through the National Center for Biotechnology Information Gene Expression Omnibus, which included genome-wide expression studies of 71 normal cases and 142 endometriosis cases. Surrogate variable analysis was used for dataset integration. The network module and hub genes were selected by weighted gene co-expression network analysis. Machine learning was used to establish a diagnostic model of endometriosis. RESULTS: A gene module that was most relevant to endometriosis was selected through weighted gene co-expression network analysis. After further analysis of this module, four hub genes that represent the function of this module were selected: SCAF11, KRAS, MDM2 and KIF3A. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the four hub genes revealed that all of them were most highly correlated with genes enriched in the ubiquitin-mediated proteolysis pathway. Moreover, in the correlation analysis between hub genes and Jab1, SCAF11 was found to be closely related to Jab1. Furthermore, hub genes were effective indicators for clinical diagnosis. The deep machine learning diagnostic model based on hub genes was highly sensitive. CONCLUSIONS: The gene module identified is highly correlated with endometriosis. The four hub genes in this module degrade p27kip1 through the ubiquitin-mediated proteolysis pathway to regulate the endometrium cell cycle and affect the development of endometriosis. The hub genes and the deep learning model based on them are valuable for clinical diagnosis.

Controversy in the management of oestrogen therapy before hysteroscopic adhesiolysis: a systematic review and meta-analysis.

The aim of this systematic review and meta-analysis was to assess the effect of oestrogen therapy as a preoperative intervention for improving clinical outcomes and fertility outcomes in women with intrauterine adhesions (IUA). A systematic search of PubMed, Embase, The Cochrane Library, clinicaltrials.gov, OVID and Chinese databases was carried out to identify relevant studies published before December 2019. Outcomes were expressed as odds ratios and 95% confidence intervals. Five cohort studies with moderate to high methodological quality were included in the meta-analysis. Preoperative oestrogen therapy was strongly associated with better clinical outcome at second-look hysteroscopy (OR 2.72; 95% CI 1.49 to 4.96; P = 0.001); whereas no significant difference was found in menstruation improvement and conception rate (OR 1.45; 95% CI, 0.95 to 2.23; P = 0.09; and OR 0.96; 95% CI 0.60 to 1.54; P = 0.87, respectively). The overall quality of the evidence ranged from moderate to very low. Preoperative oestrogen therapy may improve the short-term prognosis of IUA at second-look hysteroscopy, whereas the long-term prognosis-fertility outcome was similar to the control group. More strictly designed research studies are needed to assess the effectiveness of oestrogen administration before hysteroscopic adhesiolysis.

A retrospective validation study of sentinel lymph node mapping for high-risk endometrial cancer.

OBJECTIVES: To determine the feasibility and performance of sentinel lymph-node (SLN) mapping among women with high-risk endometrial cancer (EC). MATERIALS AND METHODS: Ninety-eight patients at high-risk EC were enrolled in this retrospective surgical trial from August 2016 to August 2018. All patients underwent intraoperative SLN biopsy, with ICG injection for laparoscopic staging; this was followed by pelvic and paraaortic lymphadenectomy (LAD). Outcomes included SLN detection rate, false-negative SLN algorithm rate, and the negative predictive value (NPV) of the SLN algorithm. The Chi-square test was used to analyze the relationship between SLN mapping and the risk factors. Then, we performed Kappa consistency check (P < 0.05 with Meaning), to estimate the consistency of SLN and lymph-node metastasis. RESULTS: Successful biopsy occurred in 94 patients (170 sides) among 98 patients (196 sides). At least 1 SLN was identified in 86.7% (170/196). Overall, the false-negative rate (FNR) was 11.8% (2/17), NPV was 97.3% (72/74), and sensitivity was 88.2% (15/17). 22/98 patients (22.4%) with high-risk EC had at least one metastatic lymph node identified. When the SLN algorithm was retrospectively applied, the FNR was 9.1% (2/22) and sensitivity was 90.9% (20/22). Considering the surgeon's experience, 68 cases of EC (except for 30 patients), the detection rate was 89.7% (122/136), NPV was 98.1% (50/51), and the FNR was 5.6% (1/18). The factor significantly affecting the detection rate of SLNs was lymphovascular space invasion (LVSI) (P = 0.016). SLN metastasis of EC was associated with depth of myometrial invasion (P = 0.034). The analysis result of SLN and the consistency of pelvic lymph-node metastasis status. As detected by Kappa coefficient was 0.939 (P < 0.001), suggests highly consistency. CONCLUSIONS: Our SLN detection rate for high-risk EC was the same as previously reported. When SLN is not detected, better after 30 patients' experience, is a reasonable alternative to complete LAD in high-risk EC. In addition, SLN shows high co-occurrence with pelvic lymph nodes. Therefore, SLN biopsy can be used to diagnose high-risk EC.

A prospective study on the predictive value of DNA methylation in cervical intraepithelial neoplasia prognosis.

PURPOSE: To study the predictive value of the DNA methylation levels of JAM3, SOX1, SLIT2, C13ORF18, and TERT in the cervical intraepithelial neoplasia prognosis. METHOD: In the present study, 139 cases were collected and followed up for 24 months. The DNA methylation levels of JAM3, SOX1, SLIT2, C13ORF18, and TERT were tested from their exfoliated cells. One-way ANOVA, receiver operating characteristic (ROC) curve analyses were conducted to analyze the data. RESULTS: The DNA methylation of the five genes was associated with prognosis of CIN. The levels of methylation increased as the progression of lesion for the prognosis. For CIN1, difference between DNA methylation of JAM3, SOX1, SLIT2, and C13ORF18 had significance statistically (P < 0.001). Sensitivity (95.2%) and specificity (93.1%) of JAM3 were the highest compared with other genes for the prognosis of CIN1. In addition, for CIN2/3, DNA methylation of JAM3, SOX1, SLIT2, TERT, and C13ORF18 had difference statistically (P < 0.001). JAM3 were also the highest in sensitivity (95.2%) and specificity (93.1%) compared with other genes for the prognosis of CIN2/3. CONCLUSIONS: Our data suggest for the first time that DNA methylation levels are associated with prognosis of CIN significantly. DNA methylation levels of some genes, especially JAM3, may serve as markers for the prediction of the CIN prognosis, including CIN1 nature prognosis and CIN2/3 after treatment.

Reproductive outcomes and risk factors of women with septate uterus after hysteroscopic metroplasty.

Background: Hysteroscopic metroplasty of the uterine septum has been the standard treatment strategy to improve reproductive outcomes, but there are still controversies about the appropriateness of metroplasty. In addition, there have been few studies of the factors related to reproductive outcomes of women after surgery. The study aimed to evaluate the reproductive outcomes and the associated risk factors that influence reproductive outcomes after hysteroscopic metroplasty of women with septate uterus and the desire to conceive. Methods: This study was an observational study. Cases were screened by searching electronic patient files, and demographic factors were collected. We conducted telephone follow-ups to collect the postoperative reproductive outcomes. The primary outcome of this study was live birth, and secondary outcomes were ongoing pregnancy, clinical pregnancy, early miscarriage, and preterm birth. Demographic variables included patients' age, body mass index (BMI), the type of septum, infertility and miscarriage history, and complications including intrauterine adhesions, endometrial polyps, endometriosis, and adenomyosis were collected to perform univariate and multivariate analyses to predict the risk factors of reproductive outcomes after surgery treatment. Results: In total, 348 women were evaluated and followed up. There were 95 cases (27.3%, 95/348) with combined infertility, 195 cases (56.0%, 195/348) with miscarriage history, and cases combined with intrauterine adhesions, endometrial polyps, endometriosis, and adenomyosis were 107 (30.7%, 107/348), 53 (15.2%, 53/348), 28 (8.0%, 28/348), and 5 (1.4%), respectively. Following surgery, the live birth rate and clinical pregnancy rate were significantly higher than prior to surgery (84.6% vs 3.7%, p= 0.000; and 78.2% vs 69.5%, p= 0.01, respectively), early miscarriage rate and preterm delivery rate were significantly lower (8.8% vs 80.6%, p= 0.000; and 7.0% vs 66.7%, p=0.000, respectively). After adjusting for body mass index, miscarriage history, and complications, multivariable logistic regression analysis revealed age ≥ 35 years and primary infertility as independent factors that affected postoperative clinical pregnancy (OR 4.025, 95% CI 2.063-7.851, p= 0.000; and OR 3.603, 95% CI 1.903-6.820, p= 0.000; respectively) and ongoing pregnancy (OR 3.420, 95% CI 1.812-6.455, p= 0.000; and OR 2.586, 95% CI 1.419-4.712, p= 0.002; respectively). Conclusions: Hysteroscopic metroplasty could lead to improved reproductive outcomes of women with septate uterus. Both age and primary infertility were independent factors for postoperative reproductive outcomes. Trial registration: Chi ECRCT20210343.

Endometrial microbiota in women with and without adenomyosis: A pilot study.

Introduction: The endometrial microbiota plays an essential role in the health of the female reproductive system. However, the interactions between the microbes in the endometrium and their effects on adenomyosis remain obscure. Materials and methods: We profile endometrial samples from 38 women with (n=21) or without (n=17) adenomyosis to characterize the composition of the microbial community and its potential function in adenomyosis using 5R 16S rRNA gene sequencing. Results: The microbiota profiles of patients with adenomyosis were different from the control group without adenomyosis. Furthermore, analysis identified Lactobacillus zeae, Burkholderia cepacia, Weissella confusa, Prevotella copri, and Citrobacter freundii as potential biomarkers for adenomyosis. In addition, Citrobacter freundii, Prevotella copri, and Burkholderia cepacia had the most significant diagnostic value for adenomyosis. PICRUSt results identified 30 differentially regulated pathways between the two groups of patients. In particular, we found that protein export, glycolysis/gluconeogenesis, alanine, aspartate, and glutamate metabolism were upregulated in adenomyosis. Our results clarify the relationship between the endometrial microbiota and adenomyosis. Discussion: The endometrial microbiota of adenomyosis exhibits a unique structure and Citrobacter freundii, Prevotella copri, and Burkholderia cepacia were identified as potential pathogenic microorganisms associated with adenomyosis. Our findings suggest that changes in the endometrial microbiota of patients with adenomyosis are of potential value for determining the occurrence, progression, early of diagnosis, and treatment oadenomyosis.

Efficacy of Freeze-Dried Amnion Grafts on Cytokines in Uterine Exudates Following Hysteroscopic Adhesiolysis of Severe Intrauterine Adhesions.

BACKGROUND: Intrauterine adhesions (IUAs) are a benign uterine disorder that results in intrauterine adhesions and scarring. This study was conducted to assess the effects of freeze-dried amnion grafts on the concentrations of adhesion-related cytokines in uterine exudates following hysteroscopic adhesiolysis of IUAs. MATERIALS AND METHODS: This study was a prospective randomized controlled trial. Thirty patients who underwent hysteroscopic adhesiolysis for severe IUAs were enrolled. They were randomly divided into a study group (with freeze-dried amnion graft treatment after surgery, N = 15) and a control group (without a graft, N = 15). After complete separation of adhesions, a Foley balloon containing a freeze-dried amnion graft was inserted into the uterine cavity in the study group, while a Foley balloon with no graft was placed in the control group. Enzyme-linked immunosorbent assays were performed to test adhesion-related cytokine concentrations in uterine exudates, including IL-1ß, TNF-α, and VEGF, at different time intervals after surgery. Second-look hysteroscopy was conducted three months after the surgery. On the basis of the American Fertility Society (AFS) scoring system, the level of adhesions was evaluated during hysteroscopy. RESULTS: The postoperative volume of the uterine exudates in the study group was significantly lower than that of the control group (P < 0.05). The adhesion-related cytokine concentrations of TNF-α, VEGF, and IL-1ß significantly increased after surgery in both groups. Their concentrations were significantly lower in the study group than in the control group (P < 0.05). Moreover, IL-1ß exhibited a long-lasting effect in the study group. AFS scores and readhesion rates were significantly lower in the study group than in the control group after hysteroscopic adhesiolysis of severe IUAs. CONCLUSION: The application of freeze-dried amnion grafts is beneficial to the postoperative recovery of patients with severe IUAs and may reduce the readhesion rate after hysteroscopic adhesiolysis by lowering adhesion-related cytokines, including TNF-α, VEGF, and IL-1ß.

Hierarchical cluster analysis in the study of the effect of cytokine expression patterns on endometrial repair and receptivity after hysteroscopic adhesiolysis.

BACKGROUND: In a previous study, we reported that amnion promotes endometrial cell growth by regulating cytokines. In this study, hierarchical cluster analysis enabled the evaluation of cytokine expression changes after amnion treatment to be explored by cluster patterns. The role of IL1B on endometrial repair and receptivity was revealed. METHODS: A total of 30 patients were recruited in this clinical trial (NCT02496052) of hysteroscopic adhesiolysis. They were randomly allocated into an amnion grafts group (amnion group) and a control group. After hysteroscopic adhesiolysis, a Foley catheter covered with a sterilized freeze-dried amnion graft was inserted into the uterine cavity of the participants in the amnion group, whereas for the control group, a Foley catheter without amnion graft was inserted. After surgery, patient follow-up was done for a year. Uterine exudates were collected every day for seven days after surgery, and analyzed by enzyme-linked immunosorbent assays. Hierarchical cluster analysis was performed to compare expression patterns of each cytokine. Single-gene gene set enrichment analysis and differentially expressed genes enrichment analysis of IL1B were performed using NCBI GEO (N=151) to evaluate its potential mechanisms and impact on endometrial receptivity. RESULTS: Compared to the control group, cytokine expression patterns of the amnion group revealed significant stratifications, which were highly correlated with the expression levels of IL1B on the sixth to seventh day after surgery, improving the pregnant rate. Wilcoxon test revealed significantly low expression levels of IL1B in the reduced endometrial receptivity group compared to the normal group. Moreover, gene set enrichment analysis showed that lysosomes, cell cycle, and calcium signaling pathways were associated with the biological processes in which IL1B plays a role. Screening and enrichment analyses of differentially expressed genes further verified the mechanisms of action of IL1B on endometrial repair and receptivity recovery. CONCLUSIONS: Amnion promotes endometrial repair and receptivity by altering the expression levels and patterns of IL1B. Furthermore, by affecting lysosomal, cell cycle, and calcium signaling pathways, IL1B may be one of the factors involved in endometrial repair and receptivity recovery.

Talin1 Induces Epithelial-Mesenchymal Transition to Facilitate Endometrial Cell Migration and Invasion in Adenomyosis Under the Regulation of microRNA-145-5p.

Adenomyosis (ADS) is a commonly encountered benign gynecological disorder. Epithelial-mesenchymal transition (EMT) may serve a pivotal role in the pathogenesis of ADS. Talin1 has been identified to be implicated in multiple human carcinomas, probably through inducing EMT process. However, available data on the precise molecular mechanism of Talin1 in the pathogenesis of ADS remain extremely scanty. In the present study, we aim to investigate the clinical roles of Talin1 and its effects on uterine endometrial cell migration, invasion, and EMT in ADS. Relative mRNA expression of Talin1, microRNA-145-5p (miR-145-5p), and EMT-related markers was determined by qRT-PCR. Immunohistochemistry and immunofluorescence were performed to examine the distribution of Talin1 in ADS endometrium. Protein levels of Talin1, EMT-related markers, and wnt/ß-catenin pathway were measured by western blot. Wound healing assay and transwell assay were utilized for evaluating cell migration and invasion respectively. Dual-luciferase reporter assay was performed to verify the relationship between Talin1 and miR-145-5p. We found Talin1 was markedly overexpressed in ADS endometrial tissue and cells, whereas miR-145-5p was downregulated. Elevated Talin1 mRNA level might be closely related to some clinicopathological features of ADS. Through functional experiments, we demonstrated that overexpression of Talin1 induced EMT and enhanced migration and invasion ability of ADS eutopic and ectopic endometrial epithelial cells (ADS_Eu_EEC and ADS_Ec_EEC) in vitro through activating the canonical wnt/ß-catenin pathway. From a mechanistic perspective, Talin1 was inversely regulated by miR-145-5p as a direct target. Our findings unveiled that under the regulation of miR-145-5p, Talin1 might promote endometrial cell migration and invasion through inducing EMT, presenting a novel insight for elucidating the pathogenesis of ADS.

Elevated Circular RNA PVT1 Promotes Eutopic Endometrial Cell Proliferation and Invasion of Adenomyosis via miR-145/Talin1 Axis.

Several theories on the origin of adenomyosis (ADS) have been proposed, of which the most widely accepted is the fundamental pathogenic role of uterine eutopic endometrium. Emerging evidence suggests that circular RNAs participate in the multiple tumorgenesis. The vital importance of circular RNA PVT1 (circPVT1) in the pathological progress like malignancies has been well documented. Nevertheless, its underlying correlation with ADS remains elusive yet. The purpose of this study was to investigate the expression pattern, regulatory effect, and internal mechanism of circPVT1 in ADS. qRT-PCR was performed to detect the relative mRNA expression of circPVT1, miR-145, and Talin1 in ADS endometrial tissue and cells. The protein level of Talin1 was measured by Western blot and immunochemistry. Immunofluorescence was used to identify the primary endometrial epithelial and stromal cells. circPVT1 knockdown in vitro was achieved by transfecting with specific lentivirus vector CCK-8, and colony formation assays were utilized to assess cell proliferation; meanwhile, the transwell assay was employed for evaluating cell invasion ability. By conducting bioinformatics, dual-luciferase reporter assay, or RNA immunoprecipitation (RIP) experiment, the interaction between miR-145 and circPVT1 or Talin1 was verified. Rescue experiments further determined the regulatory effect of circPVT1/miR-145/Talin1 axis. We found both circPVT1 and Talin1 were markedly upregulated in ADS endometrial tissue and cells, whereas miR-145 was decreased. Elevated expression of circPVT1 was closely related to the severity of dysmenorrhea, menorrhagia, and uterine enlargement of patients with ADS. Knockdown of circPVT1 inhibited adenomyotic epithelial and stromal cell proliferation and invasion. Further mechanistic experiments revealed that circPVT1 negatively regulated miR-145 through serving as a molecular sponge. And the facilitating effect of circPVT1 was partially reversed by miR-145. Talin1 was demonstrated to be a down target of miR-145 and indirectly affected by circPVT1. Our findings unveiled that enhanced circPVT1 may be involved in the pathogenesis of ADS via stimulating endometrial cell proliferation and invasion. The establishment of circPVT1/miR-145/Talin1 pathway might present a novel therapeutic insight for ADS.

Exploration of Potential Diagnostic Value of Protein Content in Serum Small Extracellular Vesicles for Early-Stage Epithelial Ovarian Carcinoma.

Epithelial ovarian carcinoma (EOC) is one of the most common gynecologic malignancies with a high mortality rate. Serum biomarkers and imaging approaches are insufficient in identifying EOC patients at an early stage. This study is to set up a combination of proteins from serum small extracellular vesicles (sEVs) for the diagnosis of early-stage EOC and to determine its performance. A biomarker for early-stage ovarian cancer (BESOC) cohort was used as a Chinese multi-center population-based biomarker study and registered as a Chinese Clinical Trial ChiCTR2000040136. The sEV protein levels of CA125, HE4, and C5a were measured in 299 subjects. Logistic regression was exploited to calculate the odds ratio and to create the sEV protein model for the predicted probability and subsequently receiver-operating characteristic (ROC) analysis. The combined sEV marker panel of CA125, HE4, and C5a as a sEV model obtained an area under curve (AUC) of 0.912, which was greater than the serum model (0.809), by ROC analysis to identify EOC patients from the whole cohort. With the cutoff of 0.370, the sensitivity and specificity of the sEV model were 0.80 and 0.89, which were much better performance than the serum markers (sensitivity: 0.55~0.66; specificity: 0.59~0.68) and the risk of ovarian malignancy algorithm (ROMA) index approved by the U.S. Food and Drug Administration (sensitivity: 0.65; specificity: 0.61), to identify EOC patients from patients with benign ovarian diseases or other controls. The sEV levels of CA125 significantly differed among early-stage and late-stage EOC (p < 0.001). Moreover, the AUC of ROC to identify early-stage EOC patients was 0.888. Further investigation revealed that the sEV levels of these 3 proteins significantly decreased after cytoreductive surgery (CA125, p = 0.008; HE4, p = 0.025; C5a, p = 0.044). In summary, our study showed that CA125, HE4, and C5a levels in serum sEVs can identify EOC patients at the early stage, elucidating the possibility of using a sEV model for the diagnosis of early-stage EOC.