Targeting the long non-coding RNA MIAT for the treatment of fibroids in an animal model.

Our previous studies indicated that there is overexpression of MIAT in fibroids and MIAT is a sponge for the miR-29 family in these tumors. The objective of the present study was to determine if the knockdown of MIAT in fibroid xenografts will increase miR-29 levels and reduce the expression of genes targeted by this miRNA such as collagen and cell cycle regulatory proteins in a mouse model for fibroids. Ovariectomized CB-17 SCID/Beige mice bearing estrogen/progesterone pellets were implanted subcutaneously in the flank with equal weight of fibroid explants which had been transduced by lentivirus for either control (empty vector) or MIAT knockdown for four weeks (n=7). Knockdown of MIAT in fibroid xenografts resulted in a 30% reduction of tumor weight and a marked increase in miR-29a, -b, and -c levels in the xenografts. There was reduced cell proliferation and expression of cell cycle regulatory genes CCND1, CDK2, and E2F1 and no significant changes in apoptosis. The xenografts with MIAT knockdown expressed lower mRNA and protein levels of FN1, COL3A1, and TGF-ß3, and total collagen protein. Targeting MIAT, which sponges the pro-fibrotic miR-29 family, is an effective therapy for fibroids by reducing cell proliferation and thereby, tumor growth and accumulation of ECM, which is a hallmark of these benign gynecologic tumors.

The Effect of Race/Ethnicity and MED12 Mutation on the Expression of Long Non-Coding RNAs in Uterine Leiomyoma and Myometrium.

The objective of this study was to elucidate the expression of long non-coding RNA (lncRNA) in leiomyomas (Lyo) and paired myometrium (Myo) and explore the impact of race and MED12 mutation. Fold change analysis (Lyo/paired Myo) indicated the expression of 63 lncRNAs was significantly altered in the mutated group but not in the non-mutated Lyo. Additionally, 65 lncRNAs exhibited an over 1.5-fold change in the Black but not the White group. Fifteen differentially expressed lncRNAs identified with next-generation sequencing underwent qRT-PCR confirmation. Compared with Myo, the expression of TPTEP1, PART1, RPS10P7, MSC-AS1, SNHG12, CA3-AS1, LINC00337, LINC00536, LINC01436, LINC01449, LINC02433, and LINC02624 was significantly higher, while the expression of ZEB2-AS1, LINC00957, and LINC01186 was significantly lower. Comparison of normal Myo with diseased Myo showed significant differences in the expression of several lncRNAs. Analysis based on race and Lyo MED12 mutation status indicated a significantly higher expression of RPS10P7, SNHG12, LINC01449, LINC02433, and LINC02624 in Lyo from Black patients. The expression of TPTEP1, PART1, RPS10P7, MSC-AS1, LINC00337, LINC00536, LINC01436, LINC01449, LINC02433, and LINC02624 was higher, while LINC01186 was significantly lower in the MED12-mutated group. These results indicate that Lyo are characterized by aberrant lncRNA expression, which is further impacted by race and Lyo MED12 mutation status.

Combination of pioglitazone, a PPARγ agonist, and synthetic surfactant B-YL prevents hyperoxia-induced lung injury in adult mice lung explants.

INTRODUCTION: Hyperoxia-induced lung injury is characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. Although a combination of aerosolized pioglitazone (PGZ) and a synthetic lung surfactant (B-YL peptide, a surfactant protein B mimic) prevents hyperoxia-induced neonatal rat lung injury, whether it is also effective in preventing hyperoxia-induced adult lung injury is unknown. METHOD: Using adult mice lung explants, we characterize the effects of 24 and 72-h (h) exposure to hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-ß signaling pathways, which are critical mediators of lung injury, 2) aberrations of lung homeostasis and injury repair pathways, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination. RESULTS: Our study reveals that hyperoxia exposure to adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates ß-catenin and LEF-1) and TGF-ß (upregulation of key TGF-ß signaling intermediates TGF-ß type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, IL-1ß, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1, and PECAM-1) markers. All of these changes were largely mitigated by the PGZ + B-YL combination. CONCLUSION: The effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo is promising to be an effective therapeutic approach for adult lung injury in vivo.

Therapeutic Effects of Long-Term Administration of Tranilast in an Animal Model for the Treatment of Fibroids.

Tranilast (N-3, 4-dimethoxycinnamoyl anthranilic acid) is an orally administered drug with antiallergic properties and approved in Japan and the Republic of Korea for the treatment of asthma and hypertrophic scars. Previous in vitro studies indicated that tranilast reduced fibroid growth through its inhibitory effects on cell proliferation and induction of apoptosis. The objective of this study was to determine the efficacy of tranilast for treatment of human-derived fibroids in a mouse model. SCID mice (ovariectomized, supplemented with estrogen and progesterone) were implanted with fibroid explants and treated for two months with tranilast (50 m/kg/daily) or the vehicle. After sacrifice, xenografts were excised and analyzed. Tranilast was well tolerated without adverse side effects. There was a 37% reduction in tumor weight along with a significant decrease in staining for Ki67, CCND1, and E2F1; a significant increase in nuclear staining for cleaved caspase 3; and reduced staining for TGF-ß3 and Masson's trichrome in the tranilast treated mice. There was a significant inhibition of mRNA and protein expression of fibronectin, COL3A1, CCND1, E2F1, and TGF-ß3 in the xenografts from the tranilast-treated mice. These promising therapeutic effects of tranilast warrant additional animal studies and human clinical trials to evaluate its efficacy for treatment of fibroids.

Differential Expression of MED12-Associated Coding RNA Transcripts in Uterine Leiomyomas.

Recent studies have demonstrated that somatic MED12 mutations in exon 2 occur at a frequency of up to 80% and have a functional role in leiomyoma pathogenesis. The objective of this study was to elucidate the expression profile of coding RNA transcripts in leiomyomas, with and without these mutations, and their paired myometrium. Next-generation RNA sequencing (NGS) was used to systematically profile the differentially expressed RNA transcripts from paired leiomyomas (n = 19). The differential analysis indicated there are 394 genes differentially and aberrantly expressed only in the mutated tumors. These genes were predominantly involved in the regulation of extracellular constituents. Of the differentially expressed genes that overlapped in the two comparison groups, the magnitude of change in gene expression was greater for many genes in tumors bearing MED12 mutations. Although the myometrium did not express MED12 mutations, there were marked differences in the transcriptome landscape of the myometrium from mutated and non-mutated specimens, with genes regulating the response to oxygen-containing compounds being most altered. In conclusion, MED12 mutations have profound effects on the expression of genes pivotal to leiomyoma pathogenesis in the tumor and the myometrium which could alter tumor characteristics and growth potential.

The Influence of Race/Ethnicity on the Transcriptomic Landscape of Uterine Fibroids.

The objective of this study was to determine if the aberrant expression of select genes could form the basis for the racial disparity in fibroid characteristics. The next-generation RNA sequencing results were analyzed as fold change [leiomyomas/paired myometrium, also known as differential expression (DF)], comparing specimens from White (n = 7) and Black (n = 12) patients. The analysis indicated that 95 genes were minimally changed in tumors from White (DF ≈ 1) but were significantly altered by more than 1.5-fold (up or down) in Black patients. Twenty-one novel genes were selected for confirmation in 69 paired fibroids by qRT-PCR. Among these 21, coding of transcripts for the differential expression of FRAT2, SOX4, TNFRSF19, ACP7, GRIP1, IRS4, PLEKHG4B, PGR, COL24A1, KRT17, MMP17, SLN, CCDC177, FUT2, MYO5B, MYOG, ZNF703, CDC25A, and CDCA7 was significantly higher, while the expression of DAB2 and CAV2 was significantly lower in tumors from Black or Hispanic patients compared with tumors from White patients. Western blot analysis revealed a greater differential expression of PGR-A and total progesterone (PGR-A and PGR-B) in tumors from Black compared with tumors from White patients. Collectively, we identified a set of genes uniquely expressed in a race/ethnicity-dependent manner, which could form the underlying mechanisms for the racial disparity in fibroids and their associated symptoms.

Perinatal exposure to nicotine alters spermatozoal DNA methylation near genes controlling nicotine action.

Perinatal smoke/nicotine exposure alters lung development and causes asthma in exposed offspring, transmitted transgenerationally. The mechanism underlying the transgenerational inheritance of perinatal smoke/nicotine-induced asthma remains unknown, but germline epigenetic modulations may play a role. Using a well-established rat model of perinatal nicotine-induced asthma, we determined the DNA methylation pattern of spermatozoa of F1 rats exposed perinatally to nicotine in F0 gestation. To identify differentially methylated regions (DMRs), reduced representation bisulfite sequencing was performed on spermatozoa of F1 litters. The top regulated gene body and promoter DMRs were tested for lung gene expression levels, and key proteins involved in lung development and repair were determined. The overall CpG methylation in F1 sperms across gene bodies, promoters, 5'-UTRs, exons, introns, and 3'-UTRs was not affected by nicotine exposure. However, the methylation levels were different between the different genomic regions. Eighty one CpG sites, 16 gene bodies, and 3 promoter regions were differentially methylated. Gene enrichment analysis of DMRs revealed pathways involved in oxidative stress, nicotine response, alveolar and brain development, and cellular signaling. Among the DMRs, Dio1 and Nmu were the most hypermethylated and hypomethylated genes, respectively. Gene expression analysis showed that the mRNA expression and DNA methylation were incongruous. Key proteins involved in lung development and repair were significantly different (FDR < 0.05) between the nicotine and placebo-treated groups. Our data show that DNA methylation is remodeled in offspring spermatozoa upon perinatal nicotine exposure. These epigenetic alterations may play a role in transgenerational inheritance of perinatal smoke/nicotine induced asthma.

Prenatal Exposure to Electronic-Cigarette Aerosols Leads to Sex-Dependent Pulmonary Extracellular-Matrix Remodeling and Myogenesis in Offspring Mice.

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

Mechanism underlying increased cardiac extracellular matrix deposition in perinatal nicotine-exposed offspring.

Although increased predisposition to cardiac fibrosis and cardiac dysfunction has been demonstrated in the perinatally nicotine-exposed heart, the underlying mechanisms remain unclear. With the use of a well-established rat model and cultured primary neonatal rat cardiac fibroblasts, the effect of perinatal nicotine exposure on offspring heart extracellular matrix deposition and the likely underlying mechanisms were investigated. Perinatal nicotine exposure resulted in increased collagen type I (COL1A1) and III (COL3A1) deposition along with a decrease in miR-29 family and an increase in long noncoding RNA myocardial infarction-associated transcript (MIAT) levels in offspring heart. Nicotine treatment of isolated primary neonatal rat cardiac fibroblasts suggested that these effects were mediated via nicotinic acetylcholine receptors including α7 and the induced collagens accumulation was reversed by a gain-of function of miR-29 family. Knockdown of MIAT resulted in increased miR-29 family and decreased COL1A1 and COL3A1 levels, suggesting nicotine-mediated MIAT induction as the underlying mechanism for nicotine-induced collagen deposition. Luciferase reporter assay and RNA immunoprecipitation studies showed an intense physical interaction between MIAT, miR-29 family, and argonaute 2, corroborating the mechanistic link between perinatal nicotine exposure and increased extracellular matrix deposition. Overall, perinatal nicotine exposure resulted in lower miR-29 family levels in offspring heart, while it elevated cardiac MIAT and collagen type I and III levels. These findings provide mechanistic basis for cardiac dysfunction in perinatal nicotine-exposed offspring and offer multiple novel potential therapeutic targets.NEW & NOTEWORTHY Using an established rat model and cultured primary neonatal cardiac fibroblasts, we show that nicotine mediated MIAT induction as the underlying mechanism for the excessive cardiac collagen deposition. These observations provide mechanistic basis for the increased predisposition to cardiac dysfunction following perinatal cigarette/nicotine exposure and offer novel potential therapeutic targets.

Role of miR-29 in mediating offspring lung phenotype in a rodent model of intrauterine growth restriction.

Considerable epidemiological and experimental evidence supports the concept that the adult chronic lung disease (CLD), is due, at least in part, to aberrations in early lung development in response to an abnormal intrauterine environment; however, the underlying molecular mechanisms remain unknown. We used a well-established rat model of maternal undernutrition (MUN) during pregnancy that results in offspring intrauterine growth restriction (IUGR) and adult CLD to test the hypothesis that in response to MUN, excess maternal glucocorticoids (GCs) program offspring lung development to a CLD phenotype by altering microRNA (miR)-29 expression, which is a key miR in regulating extracellular matrix (ECM) deposition during development and injury-repair. At postnatal day 21 and 5 mo, compared with the control offspring lung, MUN offspring lung miR-29 expression was significantly decreased in conjunction with an elevated expression of multiple downstream target ECM proteins [collagen (COL)1A1, COL3A1, COL4A5, and elastin], at both mRNA and protein levels. Importantly, MUN-induced changes in miR-29 and target gene expressions were at least partially blocked in the lungs of offspring of MUN dams treated with metyrapone, a selective GC synthesis inhibitor. Furthermore, dexamethasone treatment of cultured fetal rat lung fibroblasts significantly induced miR-29 expression along with the suppression of target ECM proteins. These data, along with the previously known role of miR-29 in regulating ECM deposition in vascular tissue in the MUN offspring, suggest miR-29 to be a common mechanistic denominator for the vascular and pulmonary phenotypes in the IUGR offspring, providing a novel potential therapeutic target.

miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids.

Maternal undernutrition increases maternal glucocorticoids (GCs) and alters microRNA expression in offspring. Given that the mechanisms of GC action on vascular development are not clear, this study examined the influence of GCs on microRNA 29c (miR-29c) and its predicted targets in primary rat aorta smooth muscle cells (RAOSMCs). Dexamethasone (Dex) and corticosterone (Cor) time-dependently increased miR-29c expression and reduced collagen type III (Col3A1), collagen type IV (Col4A5), elastin (ELN), and matrix metalloproteinase-2 (MMP2) protein in RAOSMCs. These effects were blocked by mifepristone. These genes were also targeted by miR-29c, as confirmed by a significant decrease in luciferase reporter activity of Col3A1 (34%), Col4A5 (45%), ELN (17%), and MMP2 (28%). In cells transfected with reporter plasmids, including the 3'-untranslated region of genes targeted by miR-29c, treatment with Dex or Cor also resulted in decreases in luciferase activity. Gain or loss of function of miR-29c significantly altered mRNA expression of Col3A1 (26% and 26%, respectively), Col4A5 (28% and 32%, respectively), and MMP2 (24% and 14%, respectively) but did not affect ELN. Gain or loss of function of miR-29c also significantly altered protein levels of Col3A1 (51% and 16%, respectively), Col4A5 (56% and 22%, respectively), ELN (53% and 71%, respectively), and MMP2 (28% and 53%, respectively). Coincubation of anti-miR-29c with Dex or Cor partially attenuated the effects of these steroids on protein expression of Col3A1 (25% and 24%, respectively), Col4A5 (26% and 44%, respectively), ELN (31% and 55%, respectively), and MMP2 (46% and 26%, respectively) in RAOSMCs compared with anti-miR negative controls. Our results demonstrate that GCs regulate the expression of Col3A1, Col4A5, ELN, and MMP2, at least in part, through induction of miR-29c.

In Vivo Effects of Bay 11-7082 on Fibroid Growth and Gene Expression: A Preclinical Study.

Current medical therapies for fibroids have major limitations due to their hypoestrogenic side effects. Based on our previous work showing the activation of NF-kB in fibroids, we hypothesized that inhibiting NF-kB in vivo would result in the shrinkage of tumors and reduced inflammation. Fibroid xenografts were implanted in SCID mice and treated daily with Bay 11-7082 (Bay) or vehicle for two months. Bay treatment led to a 50% reduction in tumor weight. RNAseq revealed decreased expression of genes related to cell proliferation, inflammation, extracellular matrix (ECM) composition, and growth factor expression. Validation through qRT-PCR, Western blotting, ELISA, and immunohistochemistry (IHC) confirmed these findings. Bay treatment reduced mRNA expression of cell cycle regulators (CCND1, E2F1, and CKS2), inflammatory markers (SPARC, TDO2, MYD88, TLR3, TLR6, IL6, TNFα, TNFRSF11A, and IL1ß), ECM remodelers (COL3A1, FN1, LOX, and TGFß3), growth factors (PRL, PDGFA, and VEGFC), progesterone receptor, and miR-29c and miR-200c. Collagen levels were reduced in Bay-treated xenografts. Western blotting and IHC showed decreased protein abundance in certain ECM components and inflammatory markers, but not cleaved caspase three. Ki67, CCND1, and E2F1 expression decreased with Bay treatment. This preclinical study suggests NF-kB inhibition as an effective fibroid treatment, suppressing genes involved in proliferation, inflammation, and ECM remodeling.

The immune landscape of uterine fibroids as determined by mass cytometry.

OBJECTIVE: To study the differences in immune cell profiles in uterine fibroids (Fibs) and matched myometrium (Myo). DESIGN: Observational study. SETTING: Laboratory study. PATIENT(S): The study included tissue that was collected from 10 pairs of Fib and matched Myo from women, not on hormonal medications, undergoing hysterectomy and myomectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Differences in immune cell and cytokine composition between Fib and matched Myo. RESULT(S): The mass cytometry analysis indicated that Fibs had a significantly higher number of natural killer (NK) cells, total macrophages, M2 macrophages, and conventional dendritic cells when compared with matched Myo from the same patient. In contrast, Fibs had significantly fewer CD3 and CD4 T cells when compared with Myo. The mass cytometry analysis results did not show any significant difference in the number of resting mast cells. Immunoflurorescent and immunohistochemical imaging confirmed the cytometry by time of flight results, showing a significantly higher number of NK cells, tryptase-positive mast cells indicative of mast cell activation, total macrophages, and M2 cells in Fibs and a significantly lower number of CD3 and CD4 T cells. The cytokine assay revealed significantly increased levels of human interferon α2, interleukin (IL)-1α, and platelet-derived growth factor AA and significantly lower levels of macrophage colony-stimulating factor and IL-1 receptor antagonist in Fib. CONCLUSION(S): Our results show significant differences in immune cell populations and cytokine levels between Fib and Myo. These differences could account for the increased inflammation in fib and a potential mechanism by which these tumors evade the immune system. These findings provide a foundation for further studies exploring the role of immune cells in Fib development.

Therapeutic effects of in vivo administration of an inhibitor of tryptophan 2,3-dioxygenase (680c91) for the treatment of fibroids: a preclinical study.

OBJECTIVE: Fibroids are characterized by marked overexpression of tryptophan 2,3 dioxygenase (TDO2). The objective of this study was to determine the effectiveness of in vivo administration of an inhibitor of TDO2 (680C91) on fibroid size and gene expression. DESIGN: Animal and ex vivo human study. SETTING: Academic Research Institution. SUBJECTS: Severe combined immunodeficiency mice bearing human fibroid xenografts treated with vehicle and TDO2 inhibitor. INTERVENTION: Daily intraperitoneal administration of 680C91 or vehicle for 2 months and in vitro studies with fibroid explants. MAIN OUTCOME MEASURES: Tumor weight and gene expression profile of xenografts and in vitro mechanistic experiments using fibroid explants. RESULTS: Compound 680C91 was well-tolerated with no effects on blood chemistry and body weight. Treatment of mice with 680C91 resulted in 30% reduction in the weight of fibroid xenografts after 2 months of treatment and as expected lower levels of kynurenine, the byproduct of tryptophan degradation and an endogenous ligand of aryl hydrocarbon receptor (AhR) in the xenografts. The expression of cytochrome P450 family 1 subfamily B member 1 (CYP1B1), transforming growth factor ß3 (TGF-ß3), fibronectin (FN1), cyclin-dependent kinase 2 (CDK2), E2F transcription factor 1 (E2F1), interleukin 8 (IL-8) and secreted protein acidic and cysteine rich (SPARC) mRNA were lower in the xenografts of mice treated with 680C91 compared with vehicle controls. Similarly, the protein abundance of collagen, FN1, CYP1B1, and SPARC were lower in the xenografts of 680C9- treated mice compared with vehicle controls. Immunohistochemical analysis of xenografts indicated decreased expression of collagen, Ki67 and E2F1 but no significant changes in cleaved caspase 3 expression in mice treated with 680C91. The levels of kynurenine in the xenografts showed a direct correlation with the tumor weight and FN1 levels. In vitro studies with fibroid explants showed a significant induction of CYP1B1, TGF-ß3, FN1, CDK2, E2F1, IL8, and SPARC mRNA by tryptophan, which could be blocked by cotreatment with 680C91 and the AhR antagonist CH-223191. CONCLUSION: The results indicate that correction of aberrant tryptophan catabolism in fibroids could be an effective treatment through its effect to reduce cell proliferation and extracellular matrix accumulation.

Further characterization of tryptophan metabolism and its dysregulation in fibroids.

OBJECTIVE: To determine the expression of enzymes in tryptophan (Trp) catabolism in fibroids and matched myometrium and determine the effects of race and mediator complex subunit 12 gene (MED12) mutation on their expression. DESIGN: Experimental laboratory study. SETTING: Academic research laboratory. PATIENT(S): Women of reproductive age who underwent hysterectomy while on no hormonal medications before surgery. INTERVENTION(S): Fibroids and matched myometrium were obtained from patients who underwent hysterectomy from different race or ethnic groups. MAIN OUTCOME MEASURE(S): The expression of enzymes in the Trp catabolic pathway, tryptophan transporters, and the cytochrome P450 1B1 gene (CYP1B1) in the fibroids and matched myometrium of women from different race and ethnic groups and in tumors bearing the MED12 mutation and tumors without the mutation was determined using quantitative reverse-transcription polymerase chain reaction. The levels of serotonin, kynurenic acid (KYNA), and nicotinamide adenine dinucleotide (NAD) were determined using enzyme-linked immunosorbent assay. RESULT(S): In fibroids, the expression of tryptophan hydroxylase 1 (TPH1), kynurenine amino transferase (KAT)2, large neutral amino acid transporter small subunit 2 (SLC7A8), and large neutral amino acid transporter small subunit 1 (SLC7A5) messenger RNA (mRNA) was high and that of kynureninase (KYNU) and tryptophanyl-tRNA ligase (WARS1) mRNA was low, with no changes in the expression of WARS2, kynurenine formamidase (AFMID), kynurenine 3-monooxygenase (KMO), KAT1, KAT3, and KAT4 compared with that in the matched myometrium (n = 81). The expression of CYP1B1 mRNA, a marker of the activation of the aryl hydrocarbon receptor, was higher in fibroids. Tumors bearing the MED12 mutation expressed higher levels of CYP1B1 and lower levels of WARS1, KAT1, KAT3, and KAT4 mRNAs compared with tumors without the MED12 mutation. Race or ethnicity affected the expression of KYNU, with tumors from African American and Hispanic patients expressing lower levels of KYNU mRNA compared with those from Caucasian patients. We also quantified the levels of serotonin, KYNA, and NAD, which are the end products of Trp catabolism. There were no significant differences in the levels of serotonin and KYNA, whereas the levels of NAD were lower in fibroids than in the paired myometrium. This reduction in the levels of NAD was independent of race or ethnicity. CONCLUSION(S): In addition to the expression of tryptophan 2,3-dioxygenase or indoleamine-pyrrole 2,3-dioxygenase, there was marked dysregulation in the expression of other enzymes in the Trp metabolic pathway and Trp transporters in fibroids. Both MED12 mutation status and race or ethnicity had selective effects on the expression of the components of this pathway. Additional functional studies are necessary to establish the physiologic significance of the tryptophan degradation pathway in the pathogenesis of fibroids and its potential as a target for novel therapies.

Differential Expression of Super-Enhancer-Associated Long Non-coding RNAs in Uterine Leiomyomas.

Super-enhancer-associated long non-coding RNAs (SE-lncRNAs) are a specific set of lncRNAs transcribed from super-enhancer (SE) genomic regions. Recent studies have revealed that SE-lncRNAs play essential roles in tumorigenesis through the regulation of oncogenes. The objective of this study was to elucidate the expression profile of SE-lncRNAs with concurrent assessment of associated mRNAs in leiomyomas and paired myometrium. Arraystar SE-lncRNAs arrays were used to systematically profile the differentially expressed SE-lncRNAs along with the corresponding SE-regulated protein coding genes in eight leiomyomas and paired myometrium. The analysis indicated 7680 SE-lncRNAs were expressed, of which 721 SE-lncRNAs were overexpressed, while 247 SE-lncRNAs were underexpressed by 1.5-fold or greater in leiomyoma. Thirteen novel SE-lncRNAs and their corresponding protein coding genes were selected, and their expression was confirmed in eighty-one paired leiomyoma tissues by quantitative real-time PCR. The thirteen pairs of SE-lncRNAs and their corresponding protein coding genes included RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, RP1-170O19.14/HOXA11, CASC15/PRL, EGFLAM-AS1/EGFLAM, RP11-225H22/NEURL1, RP5-1086K13.1/CD58, AC092839.3/SPTBN1, RP11-69I8.3/CTGF, TM4SF1-AS1/TM4SF1, RP11-373D23/FOSL2, RP11-399K21.11/COMTD1, and CTB-113P19.1/SPARC. Among these SE-lncRNAs, the expression of SOCS2-AS1/SOCS2, RP11-353N14.2/CBX4, RP1-170O19.14/HOXA11, and RP11-225H22/NEURL1 was significantly higher in African Americans as compared with Caucasians. The expression of RP11-353N14.2/CBX4, SOCS2-AS1/SOCS2, CASC15/PRL, and CTB-113P19.1/SPARC was significantly higher in tumors with MED12-mutation-positive as compared with MED12-mutation-negative tumors. Collectively, our results indicate that the differential expression of SE in leiomyomas is another mechanism contributing to dysregulation of protein coding genes in leiomyomas and that race and MED12 mutation can influence the expression of a select group of SE.

Human prostatic acid phosphatase, an authentic tyrosine phosphatase, dephosphorylates ErbB-2 and regulates prostate cancer cell growth.

Cellular prostatic acid phosphatase (cPAcP), an authentic tyrosine phosphatase, is proposed to function as a negative growth regulator of prostate cancer (PCa) cells in part through its dephosphorylation of ErbB-2. Nevertheless, the direct interaction between cPAcP and ErbB-2 has not been shown nor the specific dephosphorylation site of ErbB-2 by cPAcP. In this report, our data show that the phosphorylation level of ErbB-2 primarily at Tyr(1221/2) correlates with the growth rate of both LNCaP and MDA PCa2b human PCa cells. Further, cPAcP reciprocally co-immunoprecipitated with ErbB-2 in a non-permissive growth condition. Expression of wild type cPAcP, but not inactive mutant, by cDNA in cPAcP-null LNCaP C-81 cells results in decreased tyrosine phosphorylation of ErbB-2 including Tyr(1221/2). Concurrently, Tyr(317) phosphorylation of p52(Shc), proliferating cell nuclear antigen expression, and cell growth are decreased in these cells. Conversely, decreased cPAcP expression by short hairpin RNA in LNCaP C-33 cells was associated with elevated phosphorylation of ErbB-2 initially at Tyr(1221/2). Its downstream p52(Shc), ERK1/2, Akt, Src, STAT-3, and STAT-5 were activated, and cell proliferation, proliferating cell nuclear antigen, and cyclin D1 expression were increased. Stable subclones of C-33 cells by small interfering PAcP had elevated Tyr(1221/2) phosphorylation of ErbB-2 and exhibited androgen-independent growth and increased tumorigenicity in xenograft female animals. In summary, our data together indicate that in prostate epithelia, cPAcP interacts with and dephosphorylates ErbB-2 primarily at Tyr(1221/2) and hence blocks downstream signaling, leading to reduced cell growth. In PCa cells, decreased cPAcP expression is associated with androgen-independent cell proliferation and tumorigenicity as seen in advanced hormone-refractory prostate carcinomas.

Functional role of the long noncoding RNA X-inactive specific transcript in leiomyoma pathogenesis.

OBJECTIVE: To determine the expression and functional roles of a long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in leiomyoma. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Overexpression and underexpression of XIST; blockade of specific protein 1 (SP1). MAIN OUTCOME MEASURE(S): Expression of XIST in leiomyoma and its effects on microRNA 29c (miR-29c), miR-200c, and their targets. RESULT(S): Leiomyoma expressed statistically significantly more XIST as compared with matched myometrium, independent of race/ethnicity and menstrual cycle phase. By use of a three-dimensional spheroid culture system, we found reduced XIST levels in leiomyoma smooth muscle cells (LSMC) after treatment with 17ß-estradiol, progesterone, and their combination. The expression of XIST was down-regulated by treatment with the SP1-inhibitor mithramycin A and SP1 small interfering RNA. Knockdown of XIST resulted in inhibition of cell proliferation, up-regulation of miR-29c and miR-200c, and a concomitant inhibition of the target genes of these miRNAs, namely collagen type I (COL1A1), collagen type III (COL3A1), and fibronectin (FN1). By contrast, overexpression of XIST in myometrium smooth muscle cells repressed miR-29c and miR-200c, and induced COL1A1, COL3A1, and FN1 levels. By use of RNA immunoprecipitation analysis we confirmed XIST has sponge activity over miR-29c and miR-200c, which is more pronounced in leiomyoma as compared with myometrium. CONCLUSION(S): Our data demonstrate that increased expression of XIST in leiomyoma results in reduced expression of miR-29c and miR-200c with a consequent up-regulation of the genes targeted by these microRNAs including COL1A1, COL3A1, and FN1, which play key roles in extracellular matrix accumulation associated with fibroids.

Long Noncoding RNA MIAT Modulates the Extracellular Matrix Deposition in Leiomyomas by Sponging MiR-29 Family.

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-ß3 (transforming growth factor ß-3). Treatment of LSMC spheroids with TGF-ß3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-ß3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.

Tryptophan catabolism is dysregulated in leiomyomas.

OBJECTIVE: To determine the expression and functional roles of indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) in leiomyoma. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Blockade of IDO1 and TDO2. MAIN OUTCOME MEASURE(S): Expression of IDO1 and TDO2 in leiomyoma and the effects of their inhibitors on the extracellular matrix. RESULT(S): Leiomyoma expressed significantly higher levels of IDO1 and TDO2 messenger ribonucleic acid (mRNA; 60.3%, 35/58 pairs and 98.3%, 57/58 pairs, respectively) and protein (54%, 27/50 pairs and 92%, 46/50 pairs, respectively) as well as the enzyme activity marker kynurenine (78.3%, 36/46 pairs for IDO1/TDO2) compared with levels in matched myometrium. The expression of TDO2 but not IDO1 mRNA was significantly higher in fibroids from African American compared with that in Caucasian and Hispanic patients. The TDO2 but not the IDO1 protein and mRNA levels were more abundant in fibroids bearing the MED12 mutation compared with results in wild-type leiomyomas. Treatment of leiomyoma smooth muscle cell and myometrial smooth muscle cell spheroids with the TDO2 inhibitor 680C91 but not the IDO1 inhibitor epacadostat significantly repressed cell proliferation and the expression of collagen type I (COL1A1) and type III (COL3A1) in a dose-dependent manner; these effects were more pronounced in leiomyoma smooth muscle cells compared with myometrial smooth muscle cell spheroids. CONCLUSION(S): These results underscore the physiological significance of the tryptophan degradation pathway in the pathogenesis of leiomyomas and the potential utility of anti-TDO2 drugs for treatment of leiomyomas.