Uterine Leiomyoma Tissues Research Articles

Serum-Resistant Ternary DNA Polyplexes for Suicide Gene Therapy of Uterine Leiomyoma.

Uterine leiomyoma (UL) is a prevalent benign tumor in women that frequently gives rise to a multitude of reproductive complications. The use of suicide gene therapy has been proposed as a highly promising method for treating UL. To achieve successful gene therapy, it is essential to develop carriers that can efficiently transport nucleic acids into targeted cells and tissues. The instability of polyplexes in blood and other biological fluids is a crucial factor to consider when using non-viral carriers. In this study, we present serum-resistant and cRGD-modified DNA complexes for targeted delivery genes to UL cells. Ternary polyplexes were formed by incorporating cystine-cross-linked polyglutamic acid modified with histidine residues. We employed two techniques in the production of cross-linked polyanionic coating: matrix polymerization and oxidative polycondensation. In this study, we investigated the physicochemical properties of ternary DNA complexes, including the size and zeta-potential of the nanoparticles. Additionally, we evaluated cellular uptake, toxicity levels, transfection efficiency and specificity in vitro. The study involved introducing the HSV-TK gene into primary UL cells as a form of suicide gene therapy modeling. We have effectively employed ternary peptide-based complexes for gene delivery into the UL organtypic model. By implementing in situ suicide gene therapy, the increase in apoptosis genes expression was detected, providing conclusive evidence of apoptosis occurring in the transfected UL tissues. The results of the study strongly suggest that the developed ternary polyplexes show potential as a valuable tool in the implementation of suicide gene therapy for UL.

Search for key genes, key signaling pathways, and immune cell infiltration in uterine fibroids by bioinformatics analysis.

Uterine fibroids grow in the myometrium and are benign tumors. The etiology and molecular mechanism are not fully understood. Here, we hope to study the potential pathogenesis of uterine fibroids by bioinformatics. Our aim is to search for the key genes, signaling pathways and immune infiltration about the development of uterine fibroids. The GSE593 expression profile was downloaded from the Gene Expression Omnibus database, which contains 10 samples, including 5 uterine fibroids samples and 5 normal controls. Bioinformatics methods were used to find differentially expressed genes (DEGs) in tissues and further analyze the DEGs. R (version 4.2.1) software was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathway enrichment analysis of DEGs in uterine leiomyoma tissues and normal control. STRING database was used to generate protein-protein interaction (PPI) networks of key genes. Then, CIBERSORT was used to assess the infiltration of immune cells in uterine fibroids. A total of 834 DEGs were identified, of which 465 were up-regulated and 369 were down-regulated. GO andKEGG pathway analysis showed that the DEGs were mainly concentrated in extracellular matrix and cytokine related signaling pathways. We identified 30 key genes in DEGs from the PPI network. There were some differences in infiltration immunity between the 2 tissues. This study indicated that screening key genes, signaling pathways and immune infiltration by comprehensive bioinformatics analysis is helpful to understand the molecular mechanism of uterine fibroids and provide new insights into understanding the molecular mechanism.

Pivotal Role of Ubiquitin Carboxyl-Terminal Hydrolase L1 (UCHL1) in Uterine Leiomyoma.

Uterine leiomyomas are smooth-muscle tumors originating in the myometrium and are the most common pelvic tumors in women of reproductive age. Symptomatic tumors may result in abnormal uterine bleeding, bladder dysfunction, pelvic discomfort, and reproductive issues, such as infertility and miscarriage. There are currently few non-invasive treatments for leiomyoma, but there are no practical early intervention or preventive methods. In this study, human uterine leiomyoma and myometrial tissues were used to detect the protein and mRNA expression levels of UCHL1. To explore the effects of UCHL1 knockdown and inhibition in leiomyoma and myometrial cells, we determined the mRNA expressions of COL1A1 and COL3A1. Collagen gel contraction and wound-healing assays were performed on myometrial and leiomyoma cells. We found that UCHL1 expression was considerably higher in uterine leiomyomas than in the myometrium. COL1A1 and COL3A1 expression levels were downregulated after inhibition of UCHL1 in human leiomyoma cells. Furthermore, the elimination of UCHL1 significantly decreased the migration and contractility of leiomyoma cells. In conclusion, these results indicate that UCHL1 is involved in the growth of leiomyoma in humans. For the treatment of uterine leiomyoma, targeting UCHL1 activity may be a unique and possible therapeutic strategy.

Hsa_circ_0056686, derived from cancer-associated fibroblasts, promotes cell proliferation and suppresses apoptosis in uterine leiomyoma through inhibiting endoplasmic reticulum stress.

Abnormal expression of circular RNAs (circRNAs) in cancer-associated fibroblasts (CAFs) is involved in the tumor-promoting ability of CAFs. Hsa_ circ_ 0056686 has been reported to affect leiomyoma size. The purpose of this study is to investigate the regulatory role of hsa_circ_0056686 in CAFs on uterine leiomyoma (ULM). The primary CAFs and corresponding normal fibroblasts (NFs) were isolated from the tumor zones of ULM tissues and adjacent, respectively. Hsa_circ_0056686 level was higher in CAFs than NFs, and also higher in ULM tissues than in adjacent tissues. CAFs-CM significantly increased the proliferation and migration and inhibited apoptosis of ULM cells, as confirmed by CCK-8, transwell, and flow cytometry assays. Moreover, conditioned medium (CM) from CAFs transfected with hsa_circ_0056686 shRNA (CAFssh-circ_0056686-CM) abolished CAFs-mediated proliferation, migration and apoptosis of ULM cells. CAFs-CM suppressed the expression of endoplasmic reticulum stress (ERS) marker proteins and induced the expression of extracellular matrix (ECM) marker proteins, thus suppressing ERS and increasing ECM accumulation, which could be declined by CAFssh-circ_0056686-CM. Meanwhile, knockdown of hsa_circ_0056686 reversed the inhibitory effects of CAFs-CM on brefeldin A-induced cell apoptosis. Luciferase gene reporter and RNA pull-down assays indicated that miR-515-5p directly bound with hsa_circ_0056686. MiR-515-5p overexpression restored the hsa_circ_0056686-shRNA-mediated malignant biological behaviors of ULM cells. Hsa_circ_0056686 contributed to tumor-promoting effects of CAFs in ULM, manifested by promoting ULM cell proliferation and migration and reducing ERS-induced apoptosis through sponging miR-515-5p.

MiR-335-5p Inhibits Progression of Uterine Leiomyoma by Targeting ARGLU1.

Studies have demonstrated that miR-335-5p exhibits an essential role in the progress of multiple tumors, including thyroid cancer, pancreatic cancer, and non-small-cell lung cancer. However, the possible expression, the detailed role, and the underlying mechanisms of miR-335-5p in uterine leiomyoma (UL) still remained unclear. Therefore, the present study was designed to investigate the mechanism and function of miR-335-5p in UL. In our study, microRNA-335-5p (miR-335-5p) is significantly downregulated in UL tissues and UL cell lines, especially in HCC1688 and SK-UT-1 cells. Functionally, overexpression of miR-335-5p notably inhibits the viability of UL cell lines by CCK-8 assay. Besides, upregulation of miR-335-5p inhibits proliferation of UL cell lines by colony formation assay and decreases the protein levels of PCNA and Ki-67 detected by western blot assay. In addition, overexpression of miR-335-5p induces UL cell cycle arrest at G1 phase. Upregulation of miR-335-5p decreases the levels of Cyclin A1, Cyclin B1, and Cyclin D2 and upregulates the expression of p27 protein. Additionally, upregulation of miR-335-5p promotes the apoptosis of UL cell lines, increases the protein levels of Bax, Cleaved caspase-3, and Cleaved caspase-9, and decreases the protein expression of Bcl-2. Moreover, Arginine and Glutamate-Rich protein 1 (ARGLU1) is predicted as a target of miR-335-5p by ENCORI and miRDB and confirmed by dual-luciferase reporter assay. ARGLU1 is negatively associated with miR-335-5p. Furthermore, overexpression of ARGLU1 partly restores the effects of miR-335-5p mimic on the viability, proliferation, cell cycle, and apoptosis of UL cell lines. To conclude, miR-335-5p may play a repressive role in UL by targeting ARGLU1 and serve as a potential therapeutic target for the treatment of UL.

The Potential of Transforming Growth Factor-beta Inhibitor and Vascular Endothelial Growth Factor Inhibitor as Therapeutic Agents for Uterine Leiomyoma.

Background: Uterine leiomyoma is the most common benign tumor in women of reproductive age, and it can cause infertility. The growth of uterine leiomyoma is mediated by various steroids and growth factors. The purpose of this study was to evaluate the expression of various growth factors in uterine leiomyoma. Additionally, comparing the effects of existing medication and specific growth factor inhibitors on leiomyoma and the normal myometrium, we aimed to see the potential of transforming growth factor-beta (TGF-β) inhibitors and vascular endothelial growth factor (VEGF) inhibitors as therapeutic drugs for uterine leiomyoma.Methods: This in vitro study included uterine leiomyoma samples from 12 patients who underwent hysterectomy by laparoscopy or laparotomy at Seoul St. Mary's Hospital between May 2016 and March 2018. Normal myometrium and uterine leiomyoma tissue were obtained from each patient and the expression of growth factors was compared using immunohistochemical staining. After the primary culture of normal myometrial and leiomyoma cells, cell viability was evaluated following treatment with 100 nM ulipristal acetate (UPA) and mifepristone for 48 h. Western blot analysis was performed to determine the protein expression of each growth factor. Cell viability was determined following treatment with a 10-µM TGF-β inhibitor (LY364947) and a 5-µM VEGF inhibitor (axitinib) for 24 h in cultured normal myometrium and leiomyoma cells.Results: Immunohistochemical staining revealed the significantly higher intensity of TGF-β and VEGF in the leiomyoma tissue than in the normal myometrium (P < 0.05). Mifepristone treatment decreased VEGF expression by 62% in the leiomyoma cells (P < 0.05). According to the cell counting kit-8 (CCK-8) assay, cell viability was decreased after UPA, mifepristone, TGF-β1 inhibitor, and VEGF inhibitor treatments in the normal myometrium and leiomyoma tissue. The effects of the TGF-β1 inhibitor significantly differed between normal myometrium and leiomyoma tissue, with a greater decrease in cell survival in the leiomyoma tissue (P < 0.05). Post-hoc analysis showed that the TGF-β1 and VEGF inhibitors had a greater inhibitory effect on leiomyoma tissue compared with that of UPA.Conclusion: TGF-β and VEGF inhibitors significantly decreased the viability of uterine leiomyoma cells, showing stronger effects than the conventional drug, UPA. TGF-β1 inhibitors affect both leiomyoma tissue and the normal uterus; thus, targeted local treatment rather than systemic treatment should be considered.

Peptide ancestry informative markers in uterine neoplasms from women of European, African, and Asian ancestry

SummaryCharacterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step to connect patient ancestry with disease pathogenesis. Nonsynonymous single-nucleotide polymorphisms encoding missense substitutions within tryptic peptides exhibiting high allele frequencies in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs), were prioritized from 1000 genomes. In silico analysis identified that as few as 20 pAIMs can determine ancestry proportions similarly to >260K SNPs (R2 = 0.99). Multiplexed proteomic analysis of >100 human endometrial cancer cell lines and uterine leiomyoma tissues combined resulted in the quantitation of 62 pAIMs that correlate with patient race and genotype-confirmed ancestry. Candidates include a D451E substitution in GC vitamin D-binding protein previously associated with altered vitamin D levels in African and European populations. pAIMs will support generalized proteoancestry assessment as well as efforts investigating the impact of ancestry on the human proteome and how this relates to the pathogenesis of uterine neoplasms.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid: a therapeutic approach to treat human uterine leiomyoma

To evaluate the effect of inhibition of histone deacetylases (HDACs) by suberoylanilide hydroxamic acid (SAHA) treatment of human uterine leiomyoma primary (HULP) cells invitro on cell proliferation, cell cycle, extracellular matrix (ECM) formation, and transforming growth factor β3 (TGF-β3) signaling. Prospective study comparing uterine leiomyoma (UL) vs. adjacent myometrium (MM) tissue and cells with or without SAHA treatment. Hospital and university laboratories. Women with UL without any hormone treatment. Myomectomy or hysterectomy surgery in women for leiomyoma disease. HDAC activity was assessed by enzyme-linked immunosorbent assay, and gene expression was assessed by quantitative real-time polymerase chain reaction. Effects of SAHA on HULP cells were analyzed by CellTiter(Promega, Madison, Wisconsin), Western blot, and quantitative real-time polymerase chain reaction. The expression of HDAC genes (HDAC1, fold change [FC] = 1.65; HDAC3, FC = 2.08; HDAC6, FC = 2.42) and activity (0.56 vs. 0.10 optical density [OD]/h/mg) was significantly increased in UL vs. MM tissue. SAHA decreased HDAC activity in HULP cells but not in MM cells. Cell viability significantly decreased in HULP cells (81.68% at 5 μM SAHA, 73.46% at 10 μM SAHA), but not in MM cells. Proliferating cell nuclear antigen expression was significantly inhibited in SAHA-treated HULP cells (5 μM SAHA, FC = 0.556; 10 μM SAHA, FC = 0.622). Cell cycle markers, including C-MYC (5 μM SAHA, FC = 0.828) and CCND1 (5 μM SAHA, FC = 0.583; 10 μM SAHA, FC = 0.482), were significantly down-regulated after SAHA treatment. SAHA significantly inhibited ECM protein expression, including FIBRONECTIN (5 μM SAHA, FC = 0.815; 10 μM SAHA, FC = 0.673) and COLLAGEN I (5 μM SAHA, FC = 0.599; 10 μM SAHA, FC = 0.635), in HULP cells. TGFβ3 and MMP9 gene expression was also significantly down-regulated by 10 μM SAHA (TGFβ3, FC = 0.596; MMP9, FC = 0.677). SAHA treatment inhibits cell proliferation, cell cycle, ECM formation, and TGF-β3 signaling in HULP cells, suggesting that histone deacetylation may be useful for treatment of UL.

Uterine leiomyomas in hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome can be identified through distinct clinical characteristics and typical morphology.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) constitute a tumor susceptibility syndrome caused by germline mutations in the fumarate hydratase (FH) gene. The most common features are leiomyomas of the uterus and the skin. The syndrome includes a predisposition to early-onset, aggressive renal cell cancer. It is important to identify women with HLRCC among other uterine leiomyoma patients in order to direct them to genetic counseling and to identify other affected family members. We conducted a nationwide historical study to identify typical clinical characteristics, uterine leiomyoma morphology, and immunohistochemistry for diagnosing HLRCC. The study included 20 women with a known FH germline mutation and 77 women with sporadic uterine leiomyomas. The patient records of all women were reviewed to obtain clinical details regarding their leiomyomas. Uterine leiomyoma tissue specimens from 43 HLRCC-related leiomyomas and 42 sporadic leiomyomas were collected and prepared for histology analysis. A morphologic description was performed on hematoxylin & eosin-stained tissue slides, and immunohistochemical analysis was carried out for CD34, Bcl-2, and p53 stainings. The women with HLRCC were diagnosed with uterine leiomyomas at a young age compared with the sporadic leiomyoma group (mean 33.8years vs. 45.4years, P<0.0001), and their leiomyomas occurred as multiples compared with the sporadic leiomyoma group (more than four tumors 88.9% vs. 30.8%, P<0.0001). Congruently, these women underwent surgical treatment at younger age compared with the sporadic leiomyoma group (mean 37.3years vs. 48.3years, P<0.0001). HLRCC leiomyomas had denser microvasculature highlighted by CD34 immunostaining when compared with the sporadic leiomyoma group (112.6mean count/high-power field, SD 20.8 vs. 37.4 mean count/high-power field, SD 21.0 P<0.0001) and stronger anti-apoptotic protein Bcl-2 immunostaining when compared with the sporadic leiomyoma group (weak 4.7%, moderate 44.2%, strong 51.2% vs. 26.2%, 52.4%, 21.4%, respectively, P=0.003). No differences were observed in p53 staining. Women with HLRCC may be identified through the distinct clinical characteristics: symptomatic and numerous leioymyomas at young age, and morphologic features of FH-mutant leiomyomas, aided by Bcl-2 and CD34 immunohistochemistry. Further, distinguishing individuals with a germline FH mutation enables proper genetic counseling and regular renal monitoring.

5-aza-2\u2032-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/\u03b2-catenin pathway in human uterine leiomyomas

BackgroundUterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2′-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option.MethodsProspective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR.ResultsDNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 μM (85.3%). Treatment with 10 μM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/β-catenin pathway final targets, including WISP1 protein expression (10 μM, FC = 0.699), c-MYC gene expression (2 μM, FC = 0.745 and 10 μM, FC = 0.728), and MMP7 gene expression (5 μM, FC = 0.520 and 10 μM, FC = 0.577).Conclusions5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/β-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

Evaluation of vitamin D receptor expression in uterine leiomyoma and nonneoplastic myometrial tissue: a cross\u2010sectional controlled study

BackgroundUterine myomas are the most frequent benign solid pelvic tumors in women of reproductive age. At present, uterine myomas are the most common indication for hysterectomy because of the morbidity they cause, including intense bleeding, compression of adjacent organs, pain, and infertility. Some studies show that vitamin D receptor (VDR) expression is correlated with the etiology of uterine leiomyomas. This study aimed to assess the expression of VDR in uterine leiomyoma and nonneoplastic myometrial tissue.Methods A controlled cross-sectional study involving 40 women who underwent abdominal hysterectomy the Department of Gynecology of the Getúlio Vargas Hospital of Federal University of Piauí, Brazil, was performed to compare the immunohistochemical expression of VDR in samples of uterine leiomyoma tissue with adjacent nonneoplastic myometrial tissue. The mean percetages of stained nuclei in the two groups was compared by Student’s t teste, with significance established at p < 0.05.ResultsThe percentage of cells with nuclei stained by anti-VDR in the myometrial and leiomyoma tissue was 79.52 % (± 4.32) and 60.22 % (± 7.24), respectively (p < 0.0001).ConclusionsThe mean percentage of nuclei expressing VDR was significantly lower in the uterine leiomyoma than in nonneoplastic myometrial tissue.

Proteogenomic landscape of uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer patients

Pathogenic mutations in fumarate hydratase (FH) drive hereditary leiomyomatosis and renal cell cancer (HLRCC) and increase the risk of developing uterine leiomyomas (ULMs). An integrated proteogenomic analysis of ULMs from HLRCC (n = 16; FH-mutation confirmed) and non-syndromic (NS) patients (n = 12) identified a significantly higher protein:transcript correlation in HLRCC (R = 0.35) vs. NS ULMs (R = 0.242, MWU p = 0.0015). Co-altered proteins and transcripts (228) included antioxidant response element (ARE) target genes, such as thioredoxin reductase 1 (TXNRD1), and correlated with activation of NRF2-mediated oxidative stress response signaling in HLRCC ULMs. We confirm 185 transcripts previously described as altered between HLRCC and NS ULMs, 51 co-altered at the protein level and several elevated in HLRCC ULMs are involved in regulating cellular metabolism and glycolysis signaling. Furthermore, 367 S-(2-succino)cysteine peptides were identified in HLRCC ULMs, of which sixty were significantly elevated in HLRCC vs. NS ULMs (LogFC = 1.86, MWU p < 0.0001). These results confirm and define novel proteogenomic alterations in uterine leiomyoma tissues collected from HLRCC patients and underscore conserved molecular alterations correlating with inactivation of the FH tumor suppressor gene.

Differential regulatory effect of progesterone on the proliferation and apoptosis of uterine leiomyoma tissue explants and primary leiomyoma cell cultures.

ObjectiveThe growth of uterine leiomyomas is regulated by progesterone, although the underlying molecular mechanisms are not fully understood.MethodsPrimary leiomyoma cells were isolated by standard method from 16 samples of uterine leiomyoma tissue. Uterine leiomyoma explants and primary leiomyoma cell cultures were exposed to progesterone in concentrations of 0.01 µg/ml, 0.1 µg/ml and 1 µg/ml for 24 h. Cell apoptosis was assessed with Annexin V assays performed in cell cultures by flow cytometry. The expression of PR-A, PR-B, Ki67, Akt, ERK, PTEN and PPARγ mRNAs was estimated in cultured leiomyoma cells and tissue explants by real time RT-PCR.ResultsTreatment with progesterone promoted viability and proliferation of cultured leiomyoma cells in a dose-dependent manner. Low and high doses of progesterone decreased early apoptosis of leiomyoma cells. High concentrations of progesterone increased the number of living cells in Annexin V assays. High doses of progesterone increased the expression of Ki67 mRNA, while low doses increased the expression of PR-A mRNA in cultured leiomyoma cells and tissue explants. In cell cultures, low doses of progesterone increased the expression of PR-B mRNA and the expression of PTEN and PPARγ mRNAs in a dose-dependent manner. Exposure of leiomyoma tissue explants to progesterone led to increased expression of PR-B and ERK mRNAs in a dose-dependent manner.ConclusionsThe effects of progesterone on the apoptosis and proliferation of leiomyoma cells was dose-dependent and different in cell cultures and leiomyoma explants, possibly as a result of impacts derived from the tumor microenvironment.

Bioinformatic analysis identifies potential key genes in the pathogenesis of uterine leiomyoma

Objective: The present study aimed to screen hub genes for pathology of uterine leiomyoma. Methods: The microarray data of GSE31699, containing 16 uterine leiomyoma tissue samples and 16 matched normal myometrium samples, were downloaded from the Gene Expression Omnibus database (GEO). The “limma” R language package was used to identify differently-expressed genes (DEGs) between uterine leiomyoma and myometrium. Gene Ontology (GO) and pathway enrichment analyses were performed by using clusterprofiler, the DEGs were mostly enriched in post-synapse assembly, response to glucocorticoid, extracellular matrix receptor interaction and coagulation cascades. Subsequently, a protein-protein interaction (PPI) network of DEGs was constructed by Search Tool for the Retrieval of Interacting Genes Database (STRING) and visualized by utilizing Cytoscape software. We screened hub clusters of PPI network by the plug-in Molecular Complex Detection (MCODE) in Cytoscape, then clusterprofiler was also utilized to analyze functions and pathways enrichment of the genes in the hub clusters. Furthermore, we employed the “WGCNA” package in R to conduct co-expression network for all genes in GSE31699. Ultimately, we selected the overlapped genes in hub clusters of DEGs’ PPI network and WGCNA. Results: Five genes (COL5A2, ALDH1A1, GNG11, EFEMP1, ANXA1) were finally validated in other GEO datasets (GSE64763, GSE764, GSE593) and Oncomine database. Gene set enrichment analysis (GSEA) was also performed for the hub genes. The expression of COL5A2 was significantly higher in uterine leiomyoma compared with that in myometrium, while the expression of the other hub genes was significantly lower in uterine leiomyoma. Conclusion: The results indicated that COL5A2, ALDH1A1, GNG11, EFEMP1 and ANXA1 may be the key pathological genes in uterine leiomyoma.

CYP24A1 expression analysis in uterine leiomyoma regarding MED12 mutation profile

Uterine leiomyoma (ULM) is the most common gynecological tumor. Recent studies have revealed the role of hypovitaminosis D as a major risk factor in thedisease development. CYP24A, a mitochondrial enzyme that catalyzes the degradation of 1,25(OH)2D3, is reported to be over-expressed in several human cancers. In this study, we aimed to investigate the expression level of CYP24A1 in leiomyoma samples compared with the adjacent tissues regarding the MED12 mutation profile. In the present study, 61 ULMs and adjacent tissue samples were collected from 51 women undergoing hysterectomy and myomectomy. The samples were Sanger sequenced for MED12 mutation, and the expression level of CYP24A1 was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The results demonstrated that CYP24A1 gene was ectopically expressed in 18% of uterine leiomyoma tissues, although this expression was independent of the MED12 mutation profile. The findings of the present study support current evidence that dysregulation of vitamin D signaling and metabolic pathways may be involved in at least some subtypes of ULMs.

Circular RNA expression profiling identifies novel biomarkers in uterine leiomyoma

BackgroundCircular RNAs (circRNAs) have recently been identified in the development and progression of multiple human diseases. However, the significance of circRNAs in uterine leiomyoma (ULM) remains to be elucidated. Here, we aim to explore the expression profile of circRNAs in ULM and the potential of cicRNAs to be used as biomarkers or therapeutic targets.MethodsGlobal circRNA expression Profiles for ULM was performed by microarray in ULM tissue and matched adjacent normal myometrium counterpart. Bioinformatics analysis, qRT-PCR validation, and receiver operating characteristic (ROC) diagnostic accuracy was applied for differentially expressed circRNAs. Cell proliferation and spheroid formation assay were performed to assess the functional role of candidate circRNA.Results579 up- and 625 down-regulated circRNAs were identified between ULMs and adjacent normal myometrium tissues. Bioinformatics analysis suggested that most differentially expressed circRNAs participate in pathways were related to pathological processes of ULM. The qRT-PCR validation results for 6 circRNAs (hsa_circ_0083920, hsa_circ_0056686, hsa_circ_0062558, hsa_circ_0020376, hsa_circ_0043597, hsa_circ_0026353, and circ_0017248) matched the microarray results. ROC analysis showed that hsa_circ_0083920, hsa_circ_0056686, hsa_circ_0062558, hsa_circ_0020376, and hsa_circ_0043597 could accurately distinguish the ULM samples from the myometrium samples. Additionally, hsa_circ_0056686 was validated to be upregulated in ULM and was associated with the leiomyoma size (P = 0.0446). Reduction of endogenous hsa_circ_0056686 expression significantly suppressed leiomyoma cell proliferation and spheroid formation capacity.ConclusionsThis study provides an integrated analysis of circRNAs in ULM, and gives new insight into the complex epigenetic mechanisms of ULM. Aberrantly expressed circRNAs may contribute to the pathogenesis of ULM and hsa_circ_0056686 might be a potential therapeutic target.

Deoxyelephantopin, a novel naturally occurring phytochemical impairs growth, induces G2/M arrest, ROS-mediated apoptosis and modulates lncRNA expression against uterine leiomyoma

Deoxyelephantopin (DOE), a phytochemical, extracted and purified from Elephantopus scaber, has been shown to exhibit antitumor activities. Objective of the present study was to investigate anti-tumor and apoptosis-inducing properties of DOE against uterine leiomyoma (UL) and to explore their molecular mechanisms. Primary cell cultures from fresh UL tissue were established and maintained up to 12 passages. The cells exhibited continuous proliferation with 24 -h doubling time until 12 passages and was then subjected to molecular characterization. The growth inhibitory effect of DOE on UL cells was confirmed by colony formation, cellular senescence, AO/PI and DAPI staining. Fluorescent-activated cell sorting (FACS) assay for apoptosis and cell cycle arrest analysis revealed that DOE significantly (p < 0.05) inhibited the UL cell proliferation via cell cycle arrest at G2/M phase and induced apoptosis via ROS production by lowering mitochondrial membrane potential. Subsequently, the DOE induced ROS was markedly attenuated by co-treatment of N-Acetyl-Cysteine (NAC). Our quantitative RT-PCR and western blot results showed up-regulation of Bax, Caspase-3 and down-regulation of Bcl2, P53, αSMA, COL4A2, VEGF, PCNA, Cyclin B1 and oncogenic lncRNAs (H19, HOTAIR, BANCR and ROR) in DOE treated UL cells which further strengthen our findings. In conclusion, DOE inhibits growth of UL cells via cell cycle arrest at G2/M phase, induces ROS-dependent caspase-3-mediated mitochondrial intrinsic apoptotic pathway and down-regulation of oncogenic lncRNA in UL cells. Our findings suggest that DOE deserves for further systematic investigation in the uterine leiomyoma animal model as a novel apoptosis inducer for potential applications in the prevention or treatment of uterine leiomyoma.

The role of apoptosis in the complex pathogenesis of the most common obstetrics and gynaecology diseases.

We aimed to assess the etiological role of apoptotic genes Bcl-2 and Bax in the background of major obstetric and gynaecological diseases. Placental tissue samples were collected from 101 pregnancies with intrauterine growth restriction and 104 pregnancies with premature birth with 140 controll samples from term, eutrophic newborns. In addition, gene expression assessment of the genes Bax and Bcl-2 was performed in 101 uterine leiomyoma tissue samples at our disposal with 110 control cases. Gene expression levels were assessed by PCR method. The expression of the Bcl-2 gene was decreased in placental samples with intrauterine growth restriction. Significant overexpression of the proapoptotic Bax gene was detected in samples from premature infants. Antiapoptotic Bcl-2 gene expression was found to be significantly increased in fibroid tissues. Apoptosis plays a crucial role in the development of the most common OB/GYN conditions. Decrease in the placental expression of the antiapoptotic gene Bcl-2 may upset the balance of programmed cell death.

NAV3, a Tumor Suppressor Gene, Is Decreased in Uterine Leiomyoma Tissue and Cells.

NAV 3 is a tumor suppressor of unknown function in leiomyomas. The objective of this study is to assess NAV3 expression and its potential role in human uterine leiomyomas. NAV3 protein expression was examined in patient leiomyoma and patient-matched myometrial tissue samples by Western blot and immunohistochemistry. NAV3 mRNA and protein expression was assessed in leuprolide acetate- and cetrorelix-treated cell line leiomyoma samples. RNAseq analysis of placebo-treated leiomyoma compared with myometrium demonstrated the presence of transcripts encoding for several neuronal proteins. For NAV3, RNA sequence analysis demonstrated decreased expression in leiomyoma as compared with myometrium (0.86 ± 0.03 fold). Presence of NAV3 mRNA was also decreased in leiomyoma surgical samples (0.43 fold ± 0.05, p = 0.026) compared with patient-matched myometrium. Confirmatory qRT-PCR results on immortalized leiomyoma and myometrial cell lines similarly demonstrated a decrease in expression of NAV3 in leiomyomas (0.28 ± 0.02, p = 0.00075). Immunohistochemical analysis demonstrated a significant decrease in NAV 3 protein in leiomyomas (H-score 154.7 ± 6.2) as compared with myometrium (H-score; 312.5 ± 14.7, p < 0.0001). Leuprolide acetate-treated leiomyoma cells demonstrated an increase in NAV 3 mRNA expression (1.53 ± 0.13, p < 0.0001). Similarly, Western blot analysis on leuprolide-treated leiomyoma cells showed a non-significant increase in NAV 3 protein expression (1.26 ± 0.09, p = 0.063). NAV 3, a tumor suppressor in numerous cancers, is decreased in leiomyoma cells and tissue compared with myometrium, and increased by GnRH analog treatment, suggesting that NAV3 may mediate steroid hormone-independent leiomyoma regulation by GnRH analogs.

Ectopic expression of CYP24A1 circular RNA hsa_circ_0060927 in uterine leiomyomas.

BackgroundAs a novel class of non‐coding RNAs, the role of circular RNAs (circRNAs) in tumor biogenesis and progression has been proved in a number of human tumors; however, up to now, the relation between circRNAs and uterine leiomyomas (ULM) remains unclear.MethodsIn this study, we have estimated the expression level of CYP24A1 hsa_circ_0060927 in uterine leiomyoma and adjacent tissues considering the mediator complex subunit 12 gene (MED12) mutation profile by quantitative real‐time polymerase chain reaction (qRT‐PCRs).ResultsUsing Sanger sequencing method, somatic mutations in the MED12 exon 2 were detected in 14 (35.90%) ULM samples, including 10 (71.43%) missense mutations and 4 (28.57%) in‐frame deletions. Our results revealed that hsa_circ_0060927 was ectopically expressed in 33.33% of ULM tissues; although, this expression was independent of the MED12 mutation profile in the ULM samples.ConclusionsPresent results provide primary evidence for the role of circular RNAs in the leiomyoma development; however, further studies are essential to confirm the importance of these molecules as potential biomarkers for diagnosis and/or prognosis in ULM.