Tsai-Der Chuang

Maternal undernutrition induces premature reproductive senescence in adult female rat offspring.

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, antimüllerian 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 E2 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.

Excess Maternal Glucocorticoids in Response to In Utero Undernutrition Inhibit Offspring Angiogenesis

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.

The regulatory function of miR-200c on inflammatory and cell-cycle associated genes in SK-LMS-1, a leiomyosarcoma cell line.

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.

Mechanisms underlying aberrant expression of miR-29c in uterine leiomyoma

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. E2, 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.

Tranilast Inhibits Genes Functionally Involved in Cell Proliferation, Fibrosis, and Epigenetic Regulation and Epigenetically Induces miR-29c Expression in Leiomyoma Cells.

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.

Long-term effects of maternal undernutrition on offspring carotid artery remodeling: role of miR-29c

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 an ad libitum diet. 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.

Expression Profiling of lncRNAs, miRNAs, and mRNAs and Their Differential Expression in Leiomyoma Using Next-Generation RNA Sequencing:

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.

Next-generation sequencing reveals differentially expressed small noncoding RNAs in uterine leiomyoma

OBJECTIVE To determine the expression profile of small noncoding RNAs (sncRNAs) in leiomyoma, which has not been investigated to date. DESIGN Laboratory-based investigation. SETTING Academic center. PATIENT(S) Women undergoing hysterectomy for benign indications. INTERVENTION(S) Next-generation sequencing and screening of an sncRNA database with confirmatory analysis by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). MAIN OUTCOME MEASURE(S) Expression profile of sncRNAs in leiomyoma and matched myometrium. RESULT(S) Screening our previously determined RNA sequencing data with the sncRNA database resulted in identification of 15 small nuclear (sn) RNAs, 284 small nucleolar (sno) RNAs, 98 Piwi-interacting (pi) RNAs, 152 transfer (t) RNAs, and 45 ribosomal (r) RNAs, of which 15 snoRNAs, 24 piRNAs, 7 tRNAs, and 6 rRNAs were differentially expressed at a 1.5-fold change cutoff in leiomyoma compared with myometrium. We selected 5 snoRNAs, 4 piRNAs, 1 tRNA, and 1 rRNA that were differentially expressed and confirmed their expression in paired tissues (n = 20) from both phases of the menstrual cycle with the use of qRT-PCR. The results indicated up-regulation of the snoRNAs (SNORD30, SNORD27, SNORA16A, SNORD46, and SNORD56) and down-regulation of the piRNAs (piR-1311, piR-16677, piR-20365, piR-4153), tRNA (TRG-GCC5-1), and rRNA (RNA5SP202) expression in leiomyoma compared with myometrium (P<.05). The pattern of expression of these sncRNAs was similar to RNA sequencing analysis, with no menstrual cycle-dependent differences detected except for SNORD30. Because Argonaute 2 (AGO2) is required for sncRNA-mediated gene silencing, we determined its expression and found greater abundance in leiomyoma. CONCLUSION(S) Our results provide the first evidence for the differential expression of additional classes of sncRNAs and AGO2 in leiomyoma, implicating their roles as a gene regulatory mechanism.

Glucocorticoids regulate MiR-29c levels in vascular smooth muscle cells through transcriptional and epigenetic mechanisms.

Aims: 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. Main methods: 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. Key findings: 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 (P < 0.05). Treatment RASMC with dexamethasone (DEX, 0.1 &mgr;M) for 24 h 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 (P < 0.05). 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 (P < 0.05). Treatment of RASMC with DEX (0.1 &mgr;M) significantly (P < 0.05) increased the binding of GR to the miR‐29c promoter. Significance: 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.

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.