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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.Experimental laboratory study.Academic research laboratory.Women of reproductive age who underwent hysterectomy while on no hormonal medications before surgery.Fibroids and matched myometrium were obtained from patients who underwent hysterectomy from different race or ethnic groups.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.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.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.

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.

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-β3 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-β3 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.

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.

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.

Introduction: Acute lung injury and acute respiratory distress syndrome are characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. We previously reported that a combination of aerosolized pioglitazone (PGZ) and synthetic lung surfactant B-YL peptide as surfactant protein B mimic prevents hyperoxia-induced neonatal rat lung injury, but this has not been tested in adult lung injury. Method: We characterize effects of hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of hyperoxia-induced lung injury, 2) aberrations of lung homeostasis and injury repair following 24 or 72-hour exposure of adult mice lung explants to hyperoxia, 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 of adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, MCP-1, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1 and PECAM-1) markers. All of these changes were largely prevented by PGZ+B-YL combination. Conclusions: The effectiveness of the PGZ+B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo sets the stage to test this promising therapeutic approach for adult lung injury in vivo.

Nicotine inhalation via electronic cigarettes (e‐cigs) is an emerging concern. However, little is known about the acute toxicity in the lungs following inhalation of nicotine‐containing e‐cig aerosols. We hypothesized that acute exposure to aerosolized nicotine causes lung toxicity by eliciting inflammatory and dysregulated repair responses. Adult C57BL/6J mice were exposed 2 hours daily for 3 days to e‐cig aerosols containing propylene glycol (PG) with or without nicotine. Acute exposure to nicotine‐containing e‐cig aerosols induced inflammatory cell influx (neutrophils and CD8a+ T lymphocytes), and release of pro‐inflammatory cytokines in bronchoalveolar lavage fluid in a sex‐dependent manner. Inhalation of e‐cig aerosol containing PG alone significantly augmented the lung levels of various homeostasis/repair mediators (PPARγ, ADRP, ACTA2, CTNNB1, LEF1, β‐catenin, E‐cadherin, and MMP2) in a sex‐dependent manner when compared to air controls. These findings were accompanied by an increase in protein abundance and altered gene expression of lipogenic markers (PPARγ, ADRP) and myogenic markers (fibronectin, α‐smooth muscle actin and β‐catenin), suggesting a dysregulated repair response in mouse lungs. Furthermore, exposure to nicotine‐containing e‐cig aerosols or PG alone differentially affected the release of pro‐inflammatory cytokines in healthy and COPD human 3D EpiAirway tissues. Overall, acute exposure to nicotine‐containing e‐cig aerosols was sufficient to elicit a pro‐inflammatory response and altered mRNA and protein levels of myogenic, lipogenic, and extracellular matrix markers in mouse lung in a sex‐dependent manner. Thus, acute exposure to inhaled nicotine via e‐cig leads to dysregulated repair and inflammatory responses, which may lead to airway remodeling in the lungs.

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-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.

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.

Objective To determine the mechanism by which tranilast induces miR-200c expression in leiomyoma smooth muscle cells (LSMCs). Design Experimental study. Setting Academic research laboratory. Patient(s) Women undergoing hysterectomy for leiomyoma. Intervention(s) Blockade of RelA/p65. Main Outcome Measure(s) Effects of tranilast and blockade of RelA/p65 on miR-200c expression. Result(s) Tranilast, an inflammation inhibitor, dose-dependently induced miR-200c in LSMCs and myometrium smooth muscle cells (MSMCs), with a more profound effect in LSMCs than in MSMCs. The treatment of LSMCs with Bay 117082, an inhibitor of IκB phosphorylation, further enhanced miR-200c induction by tranilast. The knockdown of RelA/p65 by small interfering RNA also induced miR-200c expression in LSMCs. Although tranilast had no effect on total RelA/p65 protein levels in LSMCs, it significantly induced RelA/p65 phosphorylation at S536 while reducing its activity as well as its nuclear translocation. ChIP assay indicated that tranilast reduces the binding ability of RelA/p65 to miR-200c promoter, resulting in miR-200c induction. Tranilast also inhibited interleukin-8 (IL8) expression in LSMCs. The induction of miR-200c by tranilast partially mediates the inhibitory effect of tranilast on the expression of IL8 and cyclin-dependent kinase 2 in LSMCs. Conclusion(s) Induction of miR-200c by tranilast in LSMCs is mediated through a transcriptional mechanism involving inhibition of the nuclear factor κB signaling pathway. These results highlight the significance of inflammation in the pathogenesis of leiomyoma and the potential utility of antiinflammatory drugs for treatment of leiomyomas.

The objective of this study was to identify the expression profile of long noncoding RNAs (lncRNAs) with concurrent assessment of microRNA (miRNA) and messenger RNA (mRNA) profiles in leiomyomas and paired myometrium using next-generation RNA sequencing and assembly of RNA transcripts. Total RNA was isolated from leiomyoma and paired myometrium (N = 8) and samples from 3 pairs were subjected to RNA sequencing. Normalized assembly of over 48 000 lncRNAs resulted in identification of 45 936 lncRNAs. Of these lncRNAs, 22 148 representing overlapping, intergenic, intronic, and antisense subtypes were expressed in all paired tissues, with 5941 (2813 up- and 3128 downregulated at ≥1.5 fold) differentially expressed in leiomyomas. Concurrent RNA sequencing revealed the expression of 2588 miRNAs and 21 814 mRNAs, of which 392 miRNAs and 16 559 mRNAs were expressed in all paired tissues. Of these transcripts, 56 and 92 miRNAs and 2030 and 1825 mRNAs were up- or downregulated at ≥1.5 fold, respectively, in leiomyoma as compared to myometrium. Using quantitative reverse transcription-polymerase chain reaction (QRT-PCR), we confirmed the expression of hepatocellular carcinoma upregulated (HULC), lnc-maternally expressed 3 (MEG3), long intergenic ncRNA 890 (LINC00890), TSIX, long intergenic ncRNA 473 (LINC00473), lnc-KLF9-1, and lnc-POTEM-3 (lncRNA-ATB) in leiomyoma and matched myometrium (N = 8). Collectively, the results presented here provide a comprehensive expression profile of lncRNAs in leiomyomas with concurrent integrated expression of miRNAs and mRNAs and implicate potential regulatory functions of lncRNAs through interactions with specific miRNAs and mRNAs which are known to be critical in the pathogenesis of leiomyoma.

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, XIST levels were induced in leiomyoma smooth muscle cells (LSMC) after treatment with 17β-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.

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.

The objective of this study was to determine whether miR-93, miR-29c, and miR-200c, which we previously reported to be downregulated in leiomyomas, target cell cycle regulatory proteins that influence cell proliferation. Based on TargetScan algorithm 3 cell cycle regulatory proteins namely, E2F transcription factor 1 (E2F1), Cyclin D1 (CCND1) and CDK2 which were predicted to be targets of these miRNAs were further analyzed. In 30 hysterectomy specimens, we found the expression of E2F1 and CCND1 messenger RNA (mRNA) was increased in leiomyoma as compared to matched myometrium, with no significant changes in CDK2 mRNA levels. There was a significant increase in the abundance of all 3 proteins in leiomyoma in comparison with matched myometrium. Using luciferase reporter assay, we demonstrated E2F1 and CCND1 are targets of miR-93 and CDK2 is a target of miR-29c and miR-200c. We confirmed these findings through transfection studies in which transfection of primary leiomyoma cells with miR-93 resulted in a significant decrease in the expression of E2F1 and CCND1 mRNA and protein levels, whereas knockdown of miR-93 had the opposite effect. Similarly, overexpression of miR-29c and miR-200c in leiomyoma cells inhibited the expression of CDK2 protein and mRNA, whereas knockdown of this microRNAs (miRNA) had the opposite effect. Transfection of miR-29c, miR-200c, and miR-93 in primary leiomyoma cells resulted in a time-dependent inhibition of cell proliferation and cell motility. These results collectively indicate that the 3 miRNAs known to be downregulated in fibroid tumors are critical in regulation of cell proliferation because of their effects on 3 key cell cycle regulatory proteins, which are overexpressed in uterine leiomyomas.

The objective of this study was to determine the underlying mechanism by which glucocorticoids (GCs) induce of miR-29c expression in vascular smooth muscle cells.QRT-PCR was used for miR-29c detection. Protein levels were determined by western blotting. Knockdown of SP1, DNMT1 and DNMT3A was achieved through transfection with their specific respective siRNAs. The effect of GCs on SP1 activity was determined by luciferase reporter assay and the methylation status in miR-29c promoter was detected by methylation specific PCR. CHIP assay was used to determine the binding ability of SP1 and glucocorticoid receptor (GR) in miR-29c promoter.Treatment of RASMC with SP1 siRNA or SP1 inhibitor, mithramycin A, as well as DNMT1 and DNMT3A siRNAs and an inhibitor of DNMTs, Decitabine, resulted in increased expression of miR-29c (P0.05). Treatment RASMC with dexamethasone (DEX, 0.1μM) for 24h reduced the expression of SP1 and phosphorylated SP1 at threonine 453 protein levels, repressed SP1 activity, and inhibited the expression of DNMT1 and DNMT3A proteins (P0.05). Treatment with mifepristone, a GR antagonist, blocked the inhibitory effect of DEX on DNMT1 and DNMT3A protein expression. DEX also suppressed SP1 binding ability in miR-29c promoter and inhibited methylation of miR-29c promoter (P0.05). Treatment of RASMC with DEX (0.1μM) significantly (P0.05) increased the binding of GR to the miR-29c promoter.The stimulatory effect of GCs on miR-29c expression is mediated by these three mechanisms: transcriptionally regulated by SP1, and epigenetically through a methylation-dependent process and GR.

Tranilast (N-3,4-dimethoxycinnamoyl anthranilic acid) is an antiallergic agent with inhibitory effects on cell proliferation and extracellular matrix production. Here we assess the effect of tranilast on the expression of miR-29c and genes functionally involved in cell proliferation, fibrosis, and epigenetic regulation in isolated leiomyoma smooth muscle cells (LSMC). Tranilast significantly inhibited the rate of LSMC proliferation, which was associated with downregulation of cell cycle progression genes cyclin D1 (CCND1) and cyclin-dependent kinase 2 (CDK2) expression at messenger RNA and protein levels ( P < .05). Tranilast also suppressed the expression of collagen type I (COL1), collagen type III alpha 1 chain (COL3A1), the profibrotic cytokine, transforming growth factor β-3 (TGF-β3), DNA (cytosine-5)-methyltransferase 1 (DNMT1), and enhancer of zeste homolog 2 (EZH2), which regulate epigenetic status of gene promoters ( P < .05). Tranilast also significantly induced the expression of cellular and secreted miR-29c through downregulation of methylation status of miR-29c promoter ( P < .05). In addition, tranilast suppressed the activity of luciferase reporter containing 3'UTR of COL3A1 and CDK2, which are downstream targets of miR-29c ( P < .05). Knockdown of miR-29c expression attenuated the inhibitory effects of tranilast on COL3A1 and CDK2 protein expression ( P < .05). Collectively, these findings suggest that tranilast could have therapeutic potential as an inhibitory agent for leiomyoma growth and its associated symptoms.

MicroRNA-200c (miR-200c) through repression of specific target genes has been associated with cellular transition, tumorigenesis, and tissue fibrosis. We explored the expression and functional aspects of miR-200c in genesis of leiomyomas (LYO), benign uterine tumors with fibrotic characteristic. Using LYO and matched myometrium (MYO;n=76) from untreated and from patients exposed to hormonal therapies (GNRH agonist (GNRHa), Depo-Provera, and oral contraceptives), we found that miR-200c was expressed at significantly lower levels (PZEB1/ZEB2mRNAs and proteins, with concurrent increase in E-cadherin (CDH1) and reduction in vimentin expression, phenotypic alteration, and inhibition of MSMC and LSMC proliferations. We further validatedTIMP2, FBLN5, andVEGFAas direct targets of miR-200c through interaction with their respective 3′ UTRs, and other genes as determined by microarray analysis. At tissue levels, LYO expressed lower levels ofTIMP2andFBLN5mRNAs but increased protein expressions, which to some extent altered due to hormonal exposure. Given the regulatory functions ofZEBs, VEGFA, FBLN5, andTIMP2on cellular activities that promote cellular transition, angiogenesis, and matrix remodeling, we concluded that altered expression of miR-200c may have a significant impact on the outcome of LYO growth, maintenance of their mesenchymal and fibrotic characteristics, and possibly their associated symptoms.

miR-93/106b and their host gene minichromosome maintenance complex component 7 (MCM7) reside at chr7q22, a region frequently rearranged in leiomyomas. We explored the expression of miR-93/106b in leiomyoma and paired myometrium (n = 63) from untreated and patients exposed to hormonal therapies (GnRH agonist, Depo-Provera, and oral contraceptives) from African-Americans and Caucasians and their regulatory functions in isolated paired (n = 15) leiomyoma and myometrial smooth muscle cells and the leiomyosarcoma cell line. At tissue level leiomyomas expressed significantly lower levels of miR-93 and elevated MCM7 as compared with myometrium with limited racial influence or hormonal exposure on their expression. Assessing the regulatory function of miR-93/106b through doxycycline-inducible lentiviral transduction in a microarray analysis, tissue factor (F3) and IL8 were identified as their possible targets. At the tissue level, leiomyomas expressed a significantly lower level of F3 and an elevated IL-8 level, which exhibited an inverse relationship with miR-93 but with limited racial or hormonal influences. The gain of function of miR-93/106b in leiomyoma smooth muscle cells, myometrial smooth muscle cells, and the leiomyosarcoma cell line dose dependently repressed F3 and IL8 through direct interactions with their respective 3′-untranslated region and indirectly through F3 repression inhibited IL8, CTGF, and PAI-1 expression, confirmed by using small interfering RNA silencing or factor Vlla (FVIIa) activation of F3, as well as reducing the rate of proliferation, while increasing caspase-3/7 activity. We concluded that differential expression of miR-93/106b and their direct and/or indirect regulatory functions on F3, IL8, CTGF, and PAI-1 expression, with key roles in inflammation and tissue turnover may be of significance in the outcome of leiomyoma growth and associated symptoms.

Transforming growth factor-beta (TGF-beta), Smads, and the cyclin-dependent kinase (cdk) inhibitor p21(WAF1) are important in the pathogenesis of diabetic tubular hypertrophy. Phosphoinositide 3 kinase (PI3K)/Akt kinase activity is increased in diabetic glomerular hypertrophy. Thus, we studied the role of PI3K in high glucose (30 mM)-induced p21(WAF1), Smad2/3, and cell cycle-dependent hypertrophy in LLC-PK1 cells. We found that high glucose time-dependently (1-48 h) increased PI3K/Akt kinase activity. LY294002 (a PI3K inhibitor) attenuated high glucose-induced cell cycle-dependent (G(0)/G(1) phase) hypertrophy at 72 h while attenuating high glucose-induced p21(WAF1) gene transcription and protein expression at 36-48 h. LY294002 also attenuated high glucose-induced binding of p21(WAF1) to the cyclin E/cdk2 complex, whereas attenuating high glucose-induced TGF-beta bioactivity, Smad2/3 phosphorylation, and Smad2/3 DNA-binding activity at 36-48 h. We concluded that PI3K is required for high glucose-induced cell cycle-dependent hypertrophy, p21(WAF1) transcription and expression, p21(WAF1) binding to the cyclin E/cdk2 complex, TGF-beta bioactivity, and Smad2/3 activity in LLC-PK1 cells.

Objective To determine the expression of miR-29c and its target genes in leiomyoma and the role of NF-κB, specific protein 1 (SP1), and DNA methylation in its regulation. Design Experimental study. Setting Academic research laboratory. Patient(s) Women undergoing hysterectomy for leiomyoma. Intervention(s) Over- and underexpression of miR-29c; blockade of transcription factors. Main Outcome Measure(s) MiR-29c and its target gene levels in leiomyoma and the effects of blockade of transcription factors on miR-29c expression. Result(s) Leiomyoma as compared with myometrium expressed significantly lower levels of miR-29c, with an inverse relationship with expression of its targets, COL3A1 and DNMT3A. Gain of function of miR-29c inhibited the expression of COL3A1 and DNMT3A at protein and mRNA levels, secreted COL3A1, and rate of cell proliferation. Loss of function of miR-29c had the opposite effect. E 2 , P, and their combination inhibited miR-29c in leiomyoma smooth muscle cells (LSMC). Phosphorylated NF-κB (p65) and SP1 protein expression were significantly increased in leiomyoma. SiRNA knockdown of SP1 and DNMT3A or their specific inhibitors significantly increased the expression of miR-29c, accompanied by the inhibition of cellular and secreted COL3A1 in siRNA-treated cells. Knockdown of p65 also induced miR-29c expression but had no effect on COL3A1 expression. Conclusion(s) MiR-29c expression is suppressed in leiomyoma, resulting in an increase in expression of its targets COL3A1 and DNMT3A. The suppression of miR-29c in LSMC is primarily mediated by SP1, NF-κB signaling, and epigenetic modification. Collectively, these results indicate a significant role for miR-29c in leiomyoma pathogenesis.

The purpose of this study was to examine the hypothesis that excess maternal glucocorticoids in response to maternal undernutrition programs the expression of extracellular matrix (ECM) components potentially by miR-29c. We measured the expression of mRNA (qRT-PCR) and protein (Western blot) for collagen 3A1, collagen 4A5 and matrix metalloproteinase 2 (MMP2) in offspring carotid arteries from three groups of dams: 50% food-restricted in latter half of gestation [maternal undernutrition (MUN)], MUN dams who received metyrapone (MET) (500 mg/ml ) in drinking water from day 10 of gestation to term, and control dams fed anad libitumdiet. The expression of miR-29c was significantly decreased at 3 weeks, 3 months and 9 months in MUN carotid arteries, and these decreases in expression were partially blocked by treatment of dams with MET. The expression pattern of ECM genes that are targets of miR-29c correlated with miR-29c expression. Expression of mRNA was increased for elastin (ELN) and MMP2 mRNA in 3-week MUN carotids; in 9-month carotids there were also significant increases in expression of Col3A1 and Col4A5. These changes in mRNA expression of ECM genes at 3 weeks and 9 months were blocked by MET treatment. Similarly, the expression of ELN and MMP2 proteins at 3 weeks were increased in MUN carotids, and by 9 months there were also increases in expression of Col3A1 and Col4A5, which were blocked by MET in MUN carotids. Overall, the results demonstrate a close correlation between expression of miR-29c and the ECM proteins that are its targets thus supporting our central hypothesis.

The effects of polymeric IgA1 (pIgA1) and monomeric IgA1 (mIgA1) from patients with IgA nephropathy (IgAN) on the renin-angiotensin system (RAS) and TGF-beta synthesis were examined in cultured human mesangial cells (HMC). Both pIgA1 and mIgA1 induced renin gene expression in HMC, in a dose-dependent manner. Similar findings were observed for TGF-beta gene and protein expression. The values measured in HMC incubated with pIgA1 were significantly higher than those in HMC incubated with equivalent amounts of mIgA1. When similar experiments were performed with the addition of either captopril or losartan, there was a significant increase in the renin gene expression by HMC, whereas the synthesis of TGF-beta was markedly reduced. The TGF-beta signal transduction pathways in HMC were studied by measuring the receptor-regulated Smad proteins (Smad 2 and 3) and common-partner Smad proteins (Smad 4). pIgA1 from patients with IgAN upregulated Smad activity in HMC, and the activity observed in HMC that had been preincubated with pIgA1 was readily suppressed with optimal concentrations of captopril or losartan. The effects of pIgA1 on the RAS were further examined in HMC incubated with IgA isolated from 30 patients with IgAN, 30 healthy subjects, and disease control subjects with other diseases. pIgA1 induction of angiotensin II or TGF-beta synthesis in HMC was significantly greater with preparations from patients with IgAN, compared with healthy or disease control subjects. The findings support a pathogenetic role of pIgA1 in IgAN through upregulation of the RAS and TGF-beta, leading to chronic renal failure with renal fibrosis.

Uterine leiomyosarcoma is a relatively rare malignancy with high mortality due to metastasis and chemoresistance. Leiomyosarcomas share similar morphological characteristics with leiomyomas which are considered to have the potential of transformation into leiomyosarcoma. Accumulated evidence suggests that microRNAs acting as regulators of gene expression at the posttranscriptional level play key roles in diverse biological processes including cellular transformation and tumorigenesis. We hypothesized that miR-200c, whose expression is altered in leiomyomas, equally plays a key role in pathogenesis of leiomyosarcoma. Using SK-LMS-1 leiomyosarcoma cell line as an in vitro model here, we found that the level of expression of miR-200c was significantly lower as compared to isolated leiomyoma smooth muscle cells. Overexpression (gain-of-function) of miR-200c in SK-LMS-1 through direct interaction with 3'-untranslated region of IKBKB, IL8, CDK2, and CCNE2, respectively, resulted in suppression of their expression as determined by quantitative polymerase chain reaction and Western blot analysis. Additionally, gain-of-function of miR-200c through inhibition of IKBKB expression resulted in decreased p65 transcriptional activity in IL8 promoter. Gain-of-function of miR-200c also increased SK-LMS-1 caspase 3/7 activity and inhibited their proliferation and migration. In summary, the results suggest that a progressive decline in miR-200c expression which alters transcriptional regulation of specific target genes that control nuclear factor-κB signaling pathway, inflammation, cell cycle, and migration, in part may promote development and progression of leiomyosarcomas, including their transformation from leiomyomas.

To test the hypothesis that inhibition of offspring angiogenesis by maternal undernutrition (MUN) is mediated by maternal glucocorticoids, 3 groups of dams were studied: controls received ad libitum food; MUN dams were food restricted by 50% from day 10 of gestation; and metyrapone (MET) dams were food restricted and treated with 0.5 mg/mL of MET, a glucocorticoid synthesis inhibitor. The MUN reduced birth weights, reduced vascular endothelial growth factor (VEGF) abundance in P1 aortas, reduced VEGF and VEGF-R2 abundances in P1 mesenteric arterioles, reduced arteriolar endothelial nitric oxide synthase abundance, reduced microvessel density in the anterior tibialis, reduced endothelial cell branching in culture, reduced arteriolar immunoreactivity for proliferating cell nuclear antigen (PCNA), increased active caspase 3 in P1 mesenteric arterioles, and decreased matrix metalloproteinase (MMP)-2 and MMP-9 abundances in lysates of P1 aortas. All of these effects were prevented by treatment with metyrapone. Collectively, these findings suggest that reduced angiogenesis in MUN offspring involves direct inhibitory effects of maternal glucorticoid on fetal VEGF and its receptors.

PURPOSE The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes. METHODS Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3' untranslated region (UTR) of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative real-time PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration. RESULTS At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at -14.33. Pre-miR-133b targeted the 3' UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor β1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFβ1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF. CONCLUSIONS Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation.

A number of microRNAs (miRNAs), including miR-200 family, are aberrantly expressed in endometriosis and endometrial cancer. Here we assessed the expression and functional aspects of miR-200c in endometrial tissues (N = 52) from normal endometrial biopsies (N = 15), endometrial tissues including those exposed to hormonal therapies (N = 20), and grade I-III endometrial cancer (N = 17). miR-200c expression was elevated in normal endometrial biopsies from mid- and late-luteal phase, and in endometrial tumors as compared to endometrial tissues from peri- and postmenopausal period (P < .05) and its pattern of temporal expression displayed an inverse relationship with the expression of ZEBs. The expression of E-cadherin (CDH1) varied, but expressed at low levels, specifically in endometrial tissues and endometrial tumors. The endometrial expression of ZEBs and CDH1 in patients who were exposed to Depo-Provera and gonadotropin-releasing hormone agonist GnRHa displayed a trend toward lower expression as compared to proliferative phase; however, treatment of Ishikawa cells with 17β-estradiol, progesterone, and medroxy progesterone acetate had modest effects on the expression of miR-200c and ZEBs without affecting CDH1 expression. Gain of function of miR-200c in Ishikawa cells repressed ZEBs, as well as VEGFA, FLT1, IKKβ, and KLF9 expression at transcriptional and translational levels through direct interaction with their respective 3′untranslated regions and increased the rate of their proliferation. These results indicated that endometrial miR-200c expression undergoes dynamic changes during transition from normal into cancerous states; possibly influenced by hormonal milieu and by targeting the expression of specific genes with key regulatory functions in cellular transformation, inflammation, and angiogenesis may influence these events during normal and disease progression.

Evidence suggests that a number of microRNA (miRNA) are aberrantly expressed in endometrial disorders with potential posttranscriptional regulation of their specific target genes, including ovarian steroid receptors.Our objective was to assess the endometrial expression of miR-98 and miR-181a and their respective target genes, progesterone (P4) receptor membrane component 1 (PGRMC1) and P4 receptor (PGR).We evaluated tissue expression and in vitro regulation at an academic university medical center in endometrial biopsies and endometrial tissues from follicular and luteal phases with and without exposure to hormonal therapies and grade I-III endometrial cancer (n = 52).INTERVENTIONS included endometrial biopsies and in vitro transfection.We evaluated expression and function of miR-98 and miR-181a.Aberrant expression of miR-98 and miR-181a is associated with endometrial transition from normal into cancerous states, which to some extent is influenced by hormonal milieu, and exhibited an inverse relationship with PGMRC1 and PGR expression, respectively. Treatments of Ishikawa cells with 17β-estradiol, P4, or medroxyprogesterone acetate had limited effects on miR-98, miR-181a, and PGRMC1 expression, whereas 17β-estradiol treatment increased PGR expression. In Ishikawa cells, gain of function of miR-98 repressed PGRMC1 and CYP19A1, and miR-181a repressed PGR, DDX3X, and TIMP3 at mRNA and protein levels through direct interactions with their respective 3'-untranslated regions and CCNE1 through miR-181a-induced DDX3X repression, with miR-98 reducing the rate of cell proliferation as compared with controls.miR-98 and miR-181a through their regulatory functions on PGRMC1, PGR, CYP19A1, TIMP3, and DDX3X expression may influence a wide range of endometrial cellular activities during normal menstrual cycle and transition into disease states, including endometrial cancer.

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.

The cyclin-dependent kinase inhibitor p21(WAF1) is required for diabetic glomerular hypertrophy. High glucose-induced hypertrophy in proximal tubule cells is dependent on transforming growth factor-beta (TGF-beta). Many of the TGF-beta-induced effects are dependent on Smad2/3. Thus, the molecular mechanisms of high glucose-induced p21(WAF1) and hypertrophy were studied in high glucose-cultured proximal tubule-like LLC-PK(1) cells. We found that high glucose (30 mM) induced hypertrophy at 72 h. High glucose also increased the expression of p21(WAF1) protein and p21(WAF1) mRNA transcription and abundance at 48 h. The DNA element in the 5' regulatory region of p21(WAF1) gene essential for high glucose-induced p21(WAF1) gene transcription was identified as Sp1 by a series of the 5' regulatory region of p21(WAF1) gene deletion mutants. Moreover, high glucose activated Smad2/3 while increasing the Sp1 DNA-binding activity. High glucose also increased the Sp1-dependent transcriptional activity of p21(WAF1) gene. High glucose-induced hypertrophy was attenuated by p21(WAF1) short interfering RNA and Smad3 dominant-negative plasmid transfection. We concluded that high glucose induced hypertrophy via Sp1-Smad2/3-dependent activation of p21(WAF1) gene transcription in LLC-PK(1) cells.

High glucose inhibits endothelial cell proliferation. Thus, we studied cyclin-dependent kinase inhibitor p15(INK4b) in high glucose-induced effects in human umbilical endothelial cells at 24h. High glucose decreased cell proliferation while arresting cells in G(0)/G(1) phase of the cell cycle. High glucose increased phospho-extracellular signal regulated kinase (ERK)1/2, p15(INK4b) protein and mRNA expression. High glucose-inhibited cell proliferation was attenuated by antisense p15(INK4b) oligonucleotide. Moreover, PD98059 attenuated high glucose-induced p15(INK4b) protein expression. High glucose increased transforming growth factor-beta (TGF-beta) gene transcriptional activity and mRNA expression. However, neither SB431542 (type I TGF-beta receptor blocker) nor TGF-beta1 antibody affected high glucose-induced p15(INK4b) protein expression. Additionally, N-acetylcysteine (an antioxidant) attenuated high glucose-induced growth arrest and p15(INK4b) protein expression. Thus, high glucose-induced growth arrest is dependent on p15(INK4b) and oxidative stress in endothelial cells. Moreover, high glucose-induced p15(INK4b) protein expression is dependent on ERK1/2 and oxidative stress.

Objective To determine the effects of maternal undernutrition (MUN) on the reproductive axis of aging offspring. Design Animal (rat) study. Setting Research laboratory. Animal(s) Female Sprague-Dawley rats. Intervention(s) Food restriction during the second half of pregnancy in rats. Main Outcome Measure(s) Circulating gonadotropins, antimullerian hormone (AMH), ovarian morphology, estrous cyclicity, and gene expression studies in the hypothalamus and ovary in 1-day-old (P1) and aging adult offspring. Result(s) Offspring of MUN dams had low birth weight (LBW) and by adult age developed obesity. In addition, 80% of adult LBW offspring had disruption of estrous cycle by 8 months of age, with the majority of animals in persistent estrous. Ovarian morphology was consistent with acyclicity, with ovaries exhibiting large cystic structures and reduced corpora lutea. There was an elevation in circulating T, increased ovarian expression of enzymes involved in androgen synthesis, an increase in plasma LH/FSH levels, a reduction in E 2 levels, and no changes in AMH in adult LBW offspring compared with in control offspring. Hypothalamic expression of leptin receptor (ObRb), estrogen receptor-α (ER-α), and GnRH protein was altered in an age-dependent manner with increased ObRb and ER-α expression in P1 LBW hypothalami and a reversal of this expression pattern in adult LBW hypothalami. Conclusion(s) Our data indicate that the maternal nutritional environment programs the reproductive potential of the offspring through alteration of the hypothalamic-pituitary-gonadal axis. The premature reproductive senescence in LBW offspring could be secondary to the development of obesity and hyperleptinemia in these animals in adult life.