Leiomyoma Tissue Research Articles

Activation of protein kinase B by WNT4 as a regulator of uterine leiomyoma stem cell function

To investigate the functional interaction between the Wnt/β-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). Laboratory study. Research laboratory. Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. None. Gene expression, protein phosphorylation, and cell proliferation. Cells from human leiomyoma tissues were sorted by fluorescence-activated cell sorting (FACS) into three populations: LSC, intermediate cells (LIC), and differentiated cells (LDC) with the function of the Wnt/β-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/β-catenin signaling pathway components were differentially expressed in each leiomyoma cell population. WNT4 was distinctly overexpressed in LIC, and its receptor FZD6 was primarily expressed in LSC. WNT4 stimulated Akt phosphorylation, activated β-catenin, and increased primary leiomyoma cell proliferation. These stimulatory effects were abolished by cotreatment with the Akt inhibitor, MK-2206. WNT4 up-regulated the expression of pro-proliferative genes, c-Myc and cyclin D1, specifically in LSC; this was also abrogated by Akt inhibition. Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent β-catenin activation, representing a key step toward a better understanding of LSC regulation and potentially novel therapeutic targets.

Targeting DNA Methylation Depletes Uterine Leiomyoma Stem Cell-enriched Population by Stimulating Their Differentiation.

Uterine leiomyoma (LM) is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires the maintenance and proliferation of a stem cell population. Dysregulated deoxyribonucleic acid (DNA) methylation has been reported in LM, but its role in LM stem cell regulation remains unclear. Here, we fluorescence-activated cell sorting (FACS)-sorted cells from human LM tissues into 3 populations: LM stem cell–like cells (LSC, 5%), LM intermediate cells (LIC, 7%), and differentiated LM cells (LDC, 88%), and we analyzed the transcriptome and epigenetic landscape of LM cells at different differentiation stages. Leiomyoma stem cell–like cells 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 the genes important for myometrium- and LM-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 LM tumors.

TRIM9 overexpression promotes uterine leiomyoma cell proliferation and inhibits cell apoptosis via NF-κB signaling pathway

AimsUterine leiomyoma (UM) is the most common benign gynecological tumor and the leading indication for hysterectomy. Our study explored the roles of TRIM9 in leiomyoma formation and investigated the underlying molecular mechanisms. Material and methodsThe relationship between TRIM9 expression and fibroids formation was deciphered from the GEO database after bioinformatics analysis and identified by qPCR in human leiomyoma tissues. Both TRIM9 mRNA and protein expression were further detected in primary cultured uterine leiomyoma cells (UMC). The tumorigenesis potentials of TRIM9 in cell proliferation, cell cycle, cell apoptosis; cyclin D1, survivin and cleaved-caspase 3 protein expressions in primary UMC with TRIM9 overexpression (UMC-oeTRIM9); and uterine smooth muscle cells (SMC) with TRIM9 knockdown (SMC-siTRIM9) were evaluated in vitro. NF-κB p65 and its phosphorylation were further examined by western blotting, and rescue experiments on cell proliferation, cell cycle and cell apoptosis were conducted. Key findingsTRIM9 showed higher expression in UM tissue and UMC compared with normal myometrium. The overexpression of TRIM9 in UMC notably promoted UM growth via enhancement of cell proliferation, reduction of cell apoptosis, and regulation of cyclin D1, survivin, cleaved-caspase 3, and nuclear NF-κB expression, which were reversed in SMC-siTRIM9 and PDTC (an NF-κB inhibitor) intervention in UMC-oeTRIM9. SignificanceTo our knowledge, this was the first study demonstrating the roles of TRIM9 in cell growth progression of UM development. TRIM9 may be a potential therapeutic target for UM, by promoting leiomyoma cell proliferation and reducing cell apoptosis via activation of the NF-κB pathway.

G protein-coupled estrogen receptor 1 expression in normal myometrium, leiomyoma, and adenomyosis tissues of premenopausal women

To verify the different expression of G protein-coupled estrogen receptor 1 (GPER1) among normal uterine, leiomyoma, and adenomyosis tissues. Normal uterine, leiomyoma, and adenomyosis tissue samples were obtained from women aged 35–52 years from a tertiary university hospital. The tissue samples were subjected to immunohistochemical, Western blot, and reverse-transcription polymerase chain reaction (RT-PCR) analyses of GPER1. GPER1 protein expression was confirmed in the tissues by immunohistochemical and Western blot analyses and compared with GPER1 mRNA levels using RT-PCR. GPER1 was detected in the tissue samples of leiomyoma and adenomyosis, which are estrogen-dependent diseases. GPER1 expression was similar between normal uterine and leiomyoma tissues but was reduced in adenomyosis tissue. The level of phosphorylated extracellular signal-regulated kinases 1/2 was lower and higher in leiomyoma and adenomyosis tissues, respectively, than in normal tissue, but the differences among the groups were not statistically significant. Our immunohistochemical, Western blot, and RT-PCR results suggest that GPER1 expression is involved in cell proliferation in leiomyoma and in cell invasion and migration in adenomyosis. Functional studies of GPER1 involving larger sample sizes should be performed to confirm the adenomyosis and leiomyoma disease mechanisms and eventually to develop new therapeutic interventions for these diseases.

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.

Altered chromatin landscape and enhancer engagement underlie transcriptional dysregulation in MED12 mutant uterine leiomyomas

Uterine leiomyomas (fibroids) are a major source of gynecologic morbidity in reproductive age women and are characterized by the excessive deposition of a disorganized extracellular matrix, resulting in rigid benign tumors. Although down regulation of the transcription factor AP-1 is highly prevalent in leiomyomas, the functional consequence of AP-1 loss on gene transcription in uterine fibroids remains poorly understood. Using high-resolution ChIP-sequencing, promoter capture Hi-C, and RNA-sequencing of matched normal and leiomyoma tissues, here we show that modified enhancer architecture is a major driver of transcriptional dysregulation in MED12 mutant uterine leiomyomas. Furthermore, modifications in enhancer architecture are driven by the depletion of AP-1 occupancy on chromatin. Silencing of AP-1 subunits in primary myometrium cells leads to transcriptional dysregulation of extracellular matrix associated genes and partly recapitulates transcriptional and epigenetic changes observed in leiomyomas. These findings establish AP-1 driven aberrant enhancer regulation as an important mechanism of leiomyoma disease pathogenesis.

Uterine intravenous leiomyomatosis with an isolated large metastasis to the right atrium: a case report

BackgroundAn intravenous leiomyomatosis is a special type of uterine leiomyoma characterized by the formation of benign leiomyomatous tissue within the vascular wall. Although histologically benign, intracardiac metastasis can lead to circulatory failure, and death, if untreated. Herein, we report on a case of a uterine intravenous leiomyomatosis with an isolated large adherent metastasis in the right atrium of the heart.Case PresentationA 52-year-old Japanese woman sought medical attention at our hospital for lower abdominal pain. A 27-cm uterine mass was detected on clinical imaging, with a 78 × 47-mm mass in the right atrium detected on preoperative echocardiography. Intracardiac mass resection and tricuspid annuloplasty were performed as the first-stage surgery. The pedicle of the tumor was adherent to the wall of the atrium. On histological examination, the tumor was found to consist of spindle-shaped cells with eosinophilic cytoplasm, without atypia, but with a myxoid change, and rich microvascularization of the pedicle. Total abdominal hysterectomy was performed as the second-stage surgery, with confirmation of the diagnosis as uterine intravenous leiomyomatosis with an isolated metastasic lesion to the right atrium. There has been no evidence of tumor recurrence in the 15 months since surgery.ConclusionWe report a unique case in which a large right atrial leiomyoma was identified following a uterine intravenous leiomyomatosis. Our case exemplifies that intravenous leiomyomatosis metastatic tumors have the potential to grow via their vascularization.

Molecular Bio-Imaging Probe for Non-Invasive Differentiation Between Human Leiomyoma Versus Leiomyosarcoma.

Leiomyosarcoma is the most frequent subtype of the deadly uterine sarcoma and shares many common clinical grounds with leiomyoma, which is in turn the most common solid benign uterine neoplasm. With the recent progress in minimally invasive techniques for managing leiomyomas, accurate preoperative diagnosis of uterine masses has become the most important selection criterion for the safest therapeutic option. Therefore, different imaging modalities would be playing a key role in management of uterine masses. Testing for a sarcoma-specific promoter that expresses its downstream reporter gene only in leiomyosarcoma and not in leiomyoma or healthy uterine tissue. Adenoviral vectors were utilized both in vitro and in vivo to test the specificity of the promoters. Quantitative studies of downstream gene expression of these promoters was carried out both in vitro and in vivo. Our data indicated that human leiomyosarcoma cells highly expressed the reporter gene downstream to survivin promoter (Ad-SUR-LUC) when compared with benign leiomyoma or normal cells (p value of 0.05). Our study suggested that survivin is the unique promoter capable of distinguishing between the deadly sarcoma and the benign counterparts.

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.

Steroid hormones and hormone antagonists regulate the neural marker neurotrimin in uterine leiomyoma

To characterize the role of steroid hormone and antihormone exposure on neurotrimin (NTM) expression in human leiomyoma and myometrial tissue and cells. Laboratory study of placebo and ulipristal acetate (UPA)-treated patient tissue. Invitro assessment of immortalized myometrial and leiomyoma cell lines after hormone and antihormone exposure. Academic research center. Not applicable. Exposure of leiomyoma cell lines to 17β-E2, medroxyprogesterone acetate (MPA), UPA, and fulvestrant. Messenger RNA expression quantified with the use of RNASeq analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels quantified by means of Western blot analysis. Immunohistochemistry (IHC) on placebo- and UPA-treated patient uterine tissue specimens. Expression of NTM in human uterine leiomyoma specimens according to RNASeq was increased compared with myometrium (5.22 ± 0.57-fold), which was confirmed with the use of qRT-PCR (1.95 ± 0.05). Furthermore, NTM protein was elevated in leiomyoma tissue compared with matched myometrium (2.799 ± 0.575). IHC revealed increased staining intensity in leiomyoma surgical specimens compared with matched myometrium of placebo patients. Western blot analysis in immortalized leiomyoma cell lines demonstrated an up-regulation of NTM protein expression (2.4 ± 0.04). Treatment of leiomyoma cell lines with 17β-E2 yielded a 1.98 ± 0.11-fold increase in NTM protein expression; however, treatment with fulvestrant showed no significant change compared with control. Leiomyoma cell lines demonstrated a 1.91 ± 0.97-fold increase in NTM protein expression after progesterone treatment. RNASeq analysis demonstrated a reduced expression in patient leiomyoma after UPA treatment (0.75 ± 0.14). Treatment of leiomyoma cells with UPA demonstrated a reduced total NTM protein amount (0.54 ± 0.31) in patients, which was confirmed with the use of IHC (UPA10 147.2 ± 9.40, UPA20 182.8 ± 8.98). Invitro studies with UPA treatment revealed a concentration-dependent effect that supported these findings. NTM, a neural cell adhesion molecule, is increased in leiomyoma compared with myometrium in patient tissue and invitro models after estrogen and progesterone treatment. Down-regulation of expression occurs after UPA treatment, but not after fulvestrant exposure. NCT00290251.

From 2646 to 15: differentially regulated microRNAs between progenitors from normal myometrium and leiomyoma

Uterine leiomyomas (fibroids) are smooth muscle neoplasms of the myometrial layer of the uterus and are the most common benign tumors in women. Although their etiology is still unclear, progenitor cells seem to be implicated. To identify the dysregulated pathways involved in leiomyoma onset by microRNA profiling of progenitor cells isolated from normal myometrium and leiomyoma tissue. Pairs of normal myometrium and uterine fibroid specimens were collected from 12 myomectomy patients. Myometrial progenitor cells and leiomyoma progenitor cells were isolated and characterized for stemness. After total RNA extraction and profiling of their 2646 microRNAs, DIANA-miRPath analysis was applied to find any dysregulated pathways. Only 30 microRNAs showed a significant differential regulation between myometrial progenitor cells and leiomyoma progenitor cells. Removal of those that had values close to the cut-off or that were not consistent among triplicates left 15 microRNAs, of which 7 were downregulated and 8 were upregulated in leiomyoma progenitor cells compared to myometrial progenitor cells. According to DIANA-miRPath analysis, the 7 downregulated microRNAs (hsa-miR-146b-5p; hsa-miR-335-3p; hsa-miR-335-5p; hsa-miR-135b-5p; hsa-miR-10a-3p; hsa-miR-10a-5p; hsa-miR-200a-3p) are all related to 3 pathways, "ECM-receptor interaction" (33 targeted genes), "Adherens junction" (33 targeted genes), and "Hippo signaling" (69 targeted genes), whereas the 8 upregulated miRNAs (hsa-miR-146a-5p; hsa-miR-576-3p; hsa-miR-122-5p; hsa-miR-1246; hsa-miR-595; hsa-miR-658; hsa-miR-4284; hsa-miR-924) are related to 4 pathways, "PI3K-Akt signaling pathway" (71 targeted genes), "Pathways in Cancer" (80 targeted genes), "Cell Cycle" (37 targeted genes), and "Regulation of actin cytoskeleton" (41 targeted genes). The findings that only 15 of 2646 microRNAs are differentially regulated in normal myometrium and leiomyoma and that they are involved in 7 dysregulated pathways provides interesting insights into the development of uterine fibroids, and lends support to the hypothesis that leiomyoma onset is the result of alterations affecting progenitor cells.

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.

256P - Lnc-AL445665.1-4 may be involved in the development of multiple uterine leiomyoma through interacting with miR-146b-5p

BackgroundThe clinical behaviors and cytogenetics of solitary uterine leiomyomas (SUL) and multiple uterine leiomyomas (MUL) vary. Our previous study demonstrated that miR-146b-5p played important roles in the development of SUL and MUL. Long non-coding RNAs (lncRNAs) can participate in the pathogenesis of several diseases by regulating microRNAs; however, their roles in regulating miR-146b-5b and in the pathology of leiomyomas are unclear. MethodsPair-matched uterine leiomyoma and adjacent normal myometrium tissue samples were collected from 37 patients with leiomyomas, including 15 with SUL and 22 with MUL. Six paired samples were used for lncRNAs microarray analysis. Targeted lncRNAs were selected by bioinformatics analysis, and were verified by qRT-PCR and a dual-luciferase reporter assay.Growth curve analysis and qRT-PCR were used to evaluate the effect of silencing the lncRNA lnc-AL445665.1-4 on cell proliferation and miR-146b-5p expression, respectively. ResultsFifty-five of the selected lncRNAs were predicted to target miR-146b-5p. Four lncRNAs were selected after Venn diagram analysis showing common dysregulation in the three groups. Lnc-AL445665.1-4 was selected for further exploration. qRT-PCR showed that lnc-AL445665.1-4 expression was significantly up-regulated in MUL compared with SUL in an additional 12 and 19 paired SUL-normal and MUL-normal samples, respectively. The dual-luciferase reporter assaydemonstrated the presence of binding sites on lnc-AL445665.1 for miR-146b-5p. Silencing lnc-AL445665.1-4 not only inhibited cell proliferation but also negatively regulated the expression of miR-146b-5p. ConclusionsOur results suggest that lnc-AL445665.1-4 may be involved in the development of MUL by interacting with miR-146b-5p. Lnc-AL445665.1-4 could be a novel target for genetic therapy or serve as a biomarker for predicting the recurrence of MUL in patients that have undergone myomectomy. Legal entity responsible for the studyZhengyu Li and E. Yang. FundingHas not received any funding.All authors have declared no conflicts of interest.

Parasitic Pelvic and Infradiaphragmatic Leiomyomatosis Following Laparoscopic Myomectomy

Purpose: Uterine leiomyomas are the most common indication for gynecological surgery. The use of power morcellators has facilitated myomectomy, but appears to be associated with leiomyoma seeding, leading to parasitic leiomyomatosis. This is a case report of a patient with parasitic leiomyomatosis following laparoscopic myomectomy, which was detected incidentally during elective gallbladder surgery. Case Report: A 42-year-old female was treated for symptomatic cholelithiasis in July 2018 by elective laparoscopic cholecystectomy. Three years earlier, the patient had undergone laparoscopic myomectomy using a power morcellator for the removal of a subserosal uterine fibroid 21 cm in diameter. During laparoscopy, a firm nodule, roughly 2 cm in diameter was observed on the right infradiaphragmatic surface, and two smaller, similar pelvic nodules. The infradiaphragmatic nodule was excised and histopathological examination identified it as leiomyoma tissue, confirming the case as parasitic leiomyomatosis. Discussion: Parasitic leiomyomatosis is a rare entity first described in 1909. Reports of its incidence have increased since the advent of laparoscopic surgery for the treatment of uterine fibroids and there appears to be an association with the use of power morcellators for fragmentation of large myomas, which may promote seeding. Parasitic myoma cells display increased expression of estrogen and progesterone receptors, and increased angiogenetic capacities compared with their uterine counterparts. Further studies are required to ascertain whether power morcellators play a key role in myoma dissemination, and to evaluate possible therapeutic solutions.

Identification of PDGFRα+ cells in uterine fibroids - link between angiogenesis and uterine telocytes.

IntroductionTelocytes (TCs), also called interstitial Cajal-like cells (ICLC), CD34+ cells or PDGFRα+ cells (platelet-derived growth factor receptor α positive cells), a new type of cell of mesenchymal origin, were described over one decade ago. The unique nature of these cells still deserves attention from the scientific community. Telocytes make homo- and heterocellular contact with myocytes, immunocytes and nerves, have their own immunohistochemical and secretome profiles and thus might regulate local regenerative processes including angiogenesis and fibrosis. The aim of our study was to observe the missing link between angiogenesis and telocytes in leiomyoma, the most common benign tumors affecting women of reproductive age.Material and methodsWe observed uterine tissue samples from leiomyoma, adjacent myometrium and unchanged tissue from patients with leiomyoma and control subjects using routine histology, histochemistry, immunofluorescence (CD117, CD31, CD34, PDGFRα, tryptase, sFlt-1) and image analysis methods.ResultsThe decline of the telocyte density in the foci of fibroids correlated with poor vascularization inside the leiomyoma. Moreover, the expression of sFlt-1 (anti-angiogenic-related factor) significantly increased inside a fibroid. In leiomyoma the decrease of telocyte and blood micro-vessel density was accompanied by prevalence of collagen deposits, unlike the unchanged myometrium.ConclusionsOur results demonstrate TCs in human uterine fibroids and highlight their possible involvement in the pathogenesis of myometrial pathology in the context of angiogenesis.

Lnc-AL445665.1\u20134 may be involved in the development of multiple uterine leiomyoma through interacting with miR-146b-5p

BackgroundThe clinical behaviors and cytogenetics of solitary uterine leiomyomas (SUL) and multiple uterine leiomyomas (MUL) vary, which greatly affects the choice of treatments for reproductive-aged patients with leiomyomas. Our previous study demonstrated that a series of microRNAs, including miR-146b-5p, are dysregulated and play important roles in the development of SUL and MUL. Long non-coding RNAs (lncRNAs) can participate in the pathogenesis of several diseases by regulating the expression of microRNAs; however, their roles in regulating miR-146b-5b and in the pathology of leiomyomas are unclear.MethodsPair-matched uterine leiomyoma and adjacent normal myometrium tissue samples were collected from 37 patients with leiomyomas, including 15 with SUL and 22 with MUL. Six paired samples (three SUL and three MUL samples) were used for lncRNAs microarray analysis. Targeted lncRNAs were selected by bioinformatics analysis, and were verified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and a dual-luciferase reporter assay. Growth curve analysis and qRT-PCR were used to evaluate the effect of silencing the lncRNA lnc-AL445665.1–4 on cell proliferation and miR-146b-5p expression, respectively.ResultsThere were 245 up-regulated and 243 down-regulated lncRNAs in SUL, and 119 up-regulated and 447 down-regulated lncRNAs in MUL. Fifty-five of the selected lncRNAs were predicted to target miR-146b-5p, which is up-regulated in SUL and down-regulated in MUL. Four lncRNAs were selected after Venn diagram analysis showing common dysregulation in the three groups. Lnc-AL445665.1–4 was selected for further exploration. qRT-PCR showed that lnc-AL445665.1–4 expression was significantly up-regulated in MUL compared with SUL in an additional 12 and 19 paired SUL-normal and MUL-normal samples, respectively. The dual-luciferase reporter assay demonstrated the presence of binding sites on lnc-AL445665.1 for miR-146b-5p. Silencing lnc-AL445665.1–4 not only inhibited cell proliferation but also negatively regulated the expression of miR-146b-5p.ConclusionsOur results suggest that lnc-AL445665.1–4 may be involved in the development of MUL by interacting with miR-146b-5p. Further investigation of the roles of lncRNAs and miRNAs may help to optimize the clinical management of leiomyoma patients. Lnc-AL445665.1–4 could be a novel target for genetic therapy or serve as a biomarker for predicting the recurrence of MUL in patients that have undergone myomectomy.

DRUG SCREENING OF ORAL CARCINOMA CELL LINES USING PLASTIC, MOUSE OR HUMAN TUMOR DERIVED MATRICES

Objectives Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Traditionally, cancer cell lines cultured in 2D are used to predict the efficacy of new anti-cancer compounds. However, this method has low predicting value for efficacy since more than 80% of the cancer drugs, which have promising effect in pre-clinical studies, fail in Phase II clinical trials. Our group has developed human matrix based product, Myogel, which is extracted from leiomyoma tissue. Our hypothesis is that Myogel represents better the in vivo condition compared with the 2D plastic wells, or even wells coated with mouse derived Matrigel®. We selected 12 OSCC cell lines and 19 anti-cancer compounds, targeting mTOR and epidermal growth factor receptor (EGFR) signalling pathways. The High Throughput Drug Screening method with five different conditions were used: cells in 2D plastic wells; on top and within Matrigel® or Myogel. Additionally, the morphology of OSCC cells and EGRF location were studied using immunofluorescence staining and confocal microscope. Findings Cancer cells on top and within Myogel were less responsive to EGFR inhibitors compared to cells cultured in 2D plastic or Matrigel®. However, in case of mTOR inhibitors, similar efficacy of the drugs in all conditions was seen. The morphology of the carcinoma cells differed depending on the matrix. Within Matrigel, the cells formed isolated round-shaped organoids, whereas the cells within Myogel were stellate-shaped. Immunofluorescent staining revealed that in 2D and Matrigel, EGFR was located primarily on the cell membranes, while in Myogel, the staining was mainly in the cytoplasm. Conclusions Carcinoma cells showed different behaviours and responses to anti-cancer compounds depending on the testing conditions. Comparison between clinical data and our in vitroresults are still needed to reveal the most reliable condition for cancer drug testing.

Calcifying fibrous tumor and inflammatory myofibroblastic tumor are epigenetically related: A comparative genome-wide methylation study

Based on histological findings, calcifying fibrous tumor (CFT) may be a late (burned out) stage of inflammatory myofibroblastic tumor (IMT). This concept, however, has not been proven by molecular means.Five CFTs were analyzed for IMT-related rearrangements in ALK, ROS1 and RET using fluorescence in situ hybridization (FISH). Additionally, genome-wide methylation patterns were investigated and compared with IMT (n = 7), leiomyoma (n = 7), angioleiomyoma (n = 9), myopericytoma (n = 7) and reactive soft tissue lesions (n = 10) using unsupervised hierarchical cluster analysis and t distributed stochastic neighbor embedding.CFT patients, 4 females and 1 male, had a median age of 20 years ranging from 7 to 43 years. Two patients were younger than 18 years old. The tumors originated in the abdomen (n = 4) and axilla (n = 1). Histologically, all lesions were (multi) nodular and hypocellular consisting of bland looking (myo)fibroblasts embedded in a collagenous matrix with calcifications.FISH analysis brought up negative results for ALK, RET and ROS1 rearrangements. However, genome-wide methylation analysis revealed overlapping methylation patterns of CFT and IMT forming a distinct homogeneous methylation cluster with exception of one case clustering with myopericytoma/angioleiomyoma.In conclusion, DNA methylation profiling supports the concept that CFT and IMT represent both ends of a spectrum of one entity with CFT being the burn out stage of IMT.

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay.

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Nuclear TFE3 expression is a diagnostic marker for Desmoid-type fibromatosis

BackgroundDesmoid-type fibromatosis (DTF) is a lesion characterized by clonal proliferation of myofibroblasts, which exhibits an infiltrative growth pattern. It is necessary for them to be distinguished from other fibroblastic and myofibroblastic lesions as well as spindle cell tumors. Altered Wnt signaling can act as a defining characteristic of DTF, with nuclear β-catenin serving as a diagnostic marker for. Transcription factor E3 (TFE3) has been linked to Wnt pathway activation and regulation, and may add value to the diagnosis of DTF. The present study, therefore, sought to assess whether TFE3 is a specific diagnostic marker for DTF.MethodsNuclear TFE3 and β-catenin staining was performed on a wide range of tumor types such as DTF (n = 46), nodular fasciitis (n = 14), neurofibroma (n = 5), dermatofibrosarcoma protuberans (n = 5), gastrointestinal stromal tumor (n = 10), sclerosing epithelioid fibrosarcoma (n = 2), synovial sarcoma (n = 5), leiomyoma (n = 3) and cutaneous scar tissue (n = 4) using an immunohistochemical approach. In addition, the clinicopathological features and localization of these tumors were summarized. FISH assay was carried out to examine Xp11.2 translocations/TFE3 gene fusions. Statistical difference between immunohistochemical expression of TFE3 and β-catenin was analyzed.ResultsThe expression of nuclear TFE3 protein was found in 43 (93.5%) DTF tissue samples, ranging from moderate to intense expression levels. The distribution rates of TFE3 positivity in nodular fasciitis, gastrointestinal stromal tumor, leiomyoma and scar tissue samples were 42.9, 40, 25 and 33%, respectively. All studied samples of neurofibroma, synovial sarcoma, sclerosing epithelioid fibrosarcoma and dermatofibrosarcoma protuberans were negative for TFE3.ConclusionsThis study reveal that TFE3 has a potential to serve as a diagnostic marker capable of assisting in the differential diagnosis of DTF and other spindle cell lesions.