Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.
Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.
Uterine leiomyoma is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires maintenance and proliferation of a stem cell population. Dysregulated DNA methylation has been reported in leiomyoma, but its role in leiomyoma stem cell regulation remains unclear. Here, we FACS sorted cells from human leiomyoma tissues into three populations: stem-cell like cells (LSC, 5%), intermediate cells (LIC, 7%), and differentiated cells (LDC, 88%) and analyzed the transcriptome and epigenetic landscape of leiomyoma cells at different differentiation stages. LSC harbored a unique methylome, with 8862 differentially methylated regions compared to LIC and 9444 compared to LDC, most of which were hypermethylated. Consistent with global hypermethylation, transcript levels of TET1 and TET3 methylcytosine dioxygenases were lower in LSC. Integrative analyses revealed an inverse relationship between methylation and gene expression changes during LSC differentiation. In LSC, hypermethylation suppressed genes important for myometrium- and leiomyoma-associated functions, including muscle contraction and hormone action, to maintain stemness. The hypomethylating drug, 5'-Aza stimulated LSC differentiation, depleting the stem cell population and inhibiting tumor initiation. Our data suggest that DNA methylation maintains the pool of LSC, which is critical for the regeneration of leiomyoma tumors.
Cellular senecence is an important biologic endpoint. Naturally occuring (aging) senescence is common in uterine leiomyoma (ULM). AKT is one of major pathways in promoting ULM growth and survival. Inactivation of AKT by MK2206 in ULM resulted in stress-induced senescence in vitro. Study of the senescent phenotypes and molecular changes in ULM may greatly facilitate the understanding of the tumor biology and potential clinical therapy for this common disease associated with high morbidity. To study senescence in a model system that closely resembles primary ULM in vivo, we applied an ex vivo model of three-dimensional (3D) spheroid culture system which maintained the molecular and cellular characteristics of primary ULM and matched myometrium as seen in vivo. Gene expression profiling done on ULM induced to undergo replication (passaging) or stress-induced (MK2206) senescence revealed that ROS and hypoxic-related genes were upregulated in the two types of senescences. Overexpression of two selected genes, WIPI1 and SLITKR4, induced cellular senescence in ULM spheroids. Additionally, administration of ABT263 (a BH3 mimetic) effectively reduced the senescent cells induced in ULM spheroids. This study identified novel genes associated with senescence in ULM and demonstrated a BH3 mimetic to act as a senolytic to remove senescent cells.
ObjectiveThe aim of this study was to systematically review the available clinical trials examining male infertility after inguinal hernias were repaired using mesh procedures.MethodsThe Cochrane Library, PubMed, Embase, Web of Science, and Chinese Biomedical Medicine Database were investigated. The Jada score was used to evaluate the quality of the studies, “Oxford Centre for Evidence-based Medicine-Levels of Evidence” was used to assess the level of the trials, and descriptive analysis was used to evaluate the studies.ResultsTwenty nine related trials with a total of 36,552 patients were investigated, including seven randomized controlled trials (RCTs) with 616 patients and 10 clinical trials (1230 patients) with mesh or non-mesh repairs. The Jada score showed that there were six high quality RCTs and one low quality RCT. Levels of evidence determined from the Oxford Centre for Evidence-based Medicine further demonstrated that those six high quality RCTs also had high levels of evidence. It was found that serum testosterone, LH, and FSH levels declined in the laparoscopic group compared to the open group; however, the testicular volume only slightly increased without statistical significance. Testicular and sexual functions remained unchanged after both laparoscopic transabdominal preperitoneal hernia repair (TAPP) and totally extra-peritoneal repair (TEP). We also compared the different meshes used post-surgeries. VyproII/Timesh lightweight mesh had a diminished effect on sperm motility compared to Marlex heavyweight mesh after a one-year follow-up, but there was no effect after 3 years. Additionally, various open hernia repair procedures (Lichtenstein, mesh plug method, posterior pre-peritoneal mesh repair, and anterior tension-free repair) did not cause infertility.ConclusionsThis systematic review suggests that hernia repair with mesh either in an open or a laparoscopic procedure has no significant effect on male fertility.
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