Let-7 suppresses liver fibrosis by inhibiting hepatocyte apoptosis and TGF-ß production.

OBJECTIVE: FAS-mediated apoptosis of hepatocytes and aberrant TGF-ß signaling are major drivers of liver fibrosis. Decreased miRNA let-7 expression in the livers of patients and animals with fibrosis suggests a mechanistic link of let-7 to hepatic fibrogenesis. METHODS: Using transient transfection we tested the effects of let-7 overexpression and TET3 siRNA knockdown on FAS and TGF-ß1 expression and FAS-mediated apoptosis in human and mouse primary hepatocytes. We assessed the therapeutic activity of let-7 miRNA delivered via adeno-associated viral vectors in mouse models of carbon tetrachloride (CCl4)-induced and bile duct ligation (BDL)-induced liver fibrosis. RESULTS: Let-7 decreased TGF-ß1 production from hepatocytes through a negative feedback loop involving TET3. On the other hand, let-7 post-transcriptionally inhibits FAS expression, thereby suppressing hepatocyte apoptosis. Hepatic-specific delivery of let-7 miRNA mitigated liver fibrosis in both CCl4 and BDL mouse models. CONCLUSIONS: Let-7 is a crucial node in the signaling networks that govern liver fibrosis progression. Let-7 and/or its derivatives may be used as therapeutic agents for liver fibrosis.

H19/TET1 axis promotes TGF-ß signaling linked to endothelial-to-mesenchymal transition.

While emerging evidence suggests the link between endothelial activation of TGF-ß signaling, induction of endothelial-to-mesenchymal transition (EndMT), and cardiovascular disease (CVD), the molecular underpinning of this connection remains enigmatic. Here, we report aberrant expression of H19 lncRNA and TET1 in endothelial cells (ECs) of human atherosclerotic coronary arteries. Using primary human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAoECs) we show that TNF-α, a known risk factor for endothelial dysfunction and CVD, induces H19 expression which in turn activates TGF-ß signaling and EndMT via a TET1-dependent epigenetic mechanism. We also show that H19 regulates TET1 expression at the posttranscriptional level. Further, we provide evidence that this H19/TET1-mediated regulation of TGF-ß signaling and EndMT occurs in mouse pulmonary microvascular ECs in vivo under hyperglycemic conditions. We propose that endothelial activation of the H19/TET1 axis may play an important role in EndMT and perhaps CVD.

H19 lncRNA identified as a master regulator of genes that drive uterine leiomyomas.

Uterine leiomyomas or fibroids (UFs) are benign tumors characterized by hyperplastic smooth muscle cells and excessive deposition of extracellular matrix (ECM). Afflicting ~80% of women, and symptomatic in 25%, UFs bring tremendous suffering and are an economic burden worldwide; they cause severe pain and bleeding, and are the leading cause of hysterectomy. Yet, UFs are severely understudied with few effective treatment options available; those that are available frequently have significant side effects such as menopausal symptoms. Recently, integrated genome-scale studies have revealed mutations and fibroid subtype-specific expression changes in key driver genes, with MED12 and HMGA2 together contributing to nearly 90% of all UFs, but their regulation of expression is poorly characterized. Here we report that the expression of H19 long noncoding RNA (lncRNA) is aberrantly increased in UFs. Using cell culture and genome-wide transcriptome and methylation profiling analyses, we demonstrate that H19 promotes expression of MED12, HMGA2, and key ECM-remodeling genes via multiple mechanisms including a new class of epigenetic modification by TET3. Our results mark the first example of an evolutionarily conserved lncRNA in pathogenesis of UFs and regulation of TET expression. Given the link between a H19 single-nucleotide polymorphism (SNP) and increased risk and tumor size of UFs, and the existence of multiple fibroid subtypes driven by key pathway genes regulated by H19, we propose a unifying mechanism for pathogenesis of uterine fibroids mediated by H19 and identify a pathway for future exploration of novel target therapies for uterine leiomyomas.

H19 lncRNA alters methylation and expression of Hnf4α in the liver of metformin-exposed fetuses.

Metformin is the most widely used anti-diabetic medication worldwide. However, human and animal studies suggest that prenatal metformin exposure may increase the risk of metabolic disorders in adult offspring, yet the underpinning mechanism remains unclear. Here we report that metformin-exposed mouse fetuses exhibit elevated expression of the H19 long noncoding RNA, which induces hypomethylation and increased expression of hepatocyte nuclear factor 4α (HNF4α). As a transcription factor essential for morphological and functional differentiation of hepatocytes, HNF4α also has an indispensable role in the regulation of expression of gluconeogenic genes. Consistently, H19 overexpression in a human liver cell line leads to decreased methylation and increased expression of Hnf4α, with concomitant activation of the gluconeogenic program. Mechanistically, we show that the methylation change of Hnf4α is induced by H19-mediated regulation of S-adenosylhomocysteine hydrolase. We also provide evidence that altered H19 expression is a direct effect of metformin in the fetal liver. Our results suggest that metformin from the mother can directly act upon the fetal liver to modify Hnf4α expression, a key factor for both liver development and function, and that perturbation of this H19/Hnf4α-mediated pathway may contribute to the fetal origin of adult metabolic abnormalities.

H19 lncRNA alters DNA methylation genome wide by regulating S-adenosylhomocysteine hydrolase.

DNA methylation is essential for mammalian development and physiology. Here we report that the developmentally regulated H19 lncRNA binds to and inhibits S-adenosylhomocysteine hydrolase (SAHH), the only mammalian enzyme capable of hydrolysing S-adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases that methylate diverse cellular components, including DNA, RNA, proteins, lipids and neurotransmitters. We show that H19 knockdown activates SAHH, leading to increased DNMT3B-mediated methylation of an lncRNA-encoding gene Nctc1 within the Igf2-H19-Nctc1 locus. Genome-wide methylation profiling reveals methylation changes at numerous gene loci consistent with SAHH modulation by H19. Our results uncover an unanticipated regulatory circuit involving broad epigenetic alterations by a single abundantly expressed lncRNA that may underlie gene methylation dynamics of development and diseases and suggest that this mode of regulation may extend to other cellular components.

The H19/let-7 double-negative feedback loop contributes to glucose metabolism in muscle cells.

The H19 lncRNA has been implicated in development and growth control and is associated with human genetic disorders and cancer. Acting as a molecular sponge, H19 inhibits microRNA (miRNA) let-7. Here we report that H19 is significantly decreased in muscle of human subjects with type-2 diabetes and insulin resistant rodents. This decrease leads to increased bioavailability of let-7, causing diminished expression of let-7 targets, which is recapitulated in vitro where H19 depletion results in impaired insulin signaling and decreased glucose uptake. Furthermore, acute hyperinsulinemia downregulates H19, a phenomenon that occurs through PI3K/AKT-dependent phosphorylation of the miRNA processing factor KSRP, which promotes biogenesis of let-7 and its mediated H19 destabilization. Our results reveal a previously undescribed double-negative feedback loop between sponge lncRNA and target miRNA that contributes to glucose regulation in muscle cells.

PreImplantation factor promotes neuroprotection by targeting microRNA let-7.

Dysfunction and loss of neurons are the major characteristics of CNS disorders that include stroke, multiple sclerosis, and Alzheimer's disease. Activation of the Toll-like receptor 7 by extracellular microRNA let-7, a highly expressed microRNA in the CNS, induces neuronal cell death. Let-7 released from injured neurons and immune cells acts on neighboring cells, exacerbating CNS damage. Here we show that a synthetic peptide analogous to the mammalian PreImplantation factor (PIF) secreted by developing embryos and which is present in the maternal circulation during pregnancy inhibits the biogenesis of let-7 in both neuronal and immune cells of the mouse. The synthetic peptide, sPIF, destabilizes KH-type splicing regulatory protein (KSRP), a key microRNA-processing protein, in a Toll-like receptor 4 (TLR4)-dependent manner, leading to decreased production of let-7. Furthermore, s.c. administration of sPIF into neonatal rats following hypoxic-ischemic brain injury robustly rescued cortical volume and number of neurons and decreased the detrimental glial response, as is consistent with diminished levels of KSRP and let-7 in sPIF-treated brains. Our results reveal a previously unexpected mechanism of action of PIF and underscore the potential clinical utility of sPIF in treating hypoxic-ischemic brain damage. The newly identified PIF/TLR4/KSRP/let-7 regulatory axis also may operate during embryo implantation and development.

Is cryoballoon ablation preferable to radiofrequency ablation for treatment of atrial fibrillation by pulmonary vein isolation? A meta-analysis.

OBJECTIVE: Currently radiofrequency and cryoballoon ablations are the two standard ablation systems used for catheter ablation of atrial fibrillation; however, there is no universal consensus on which ablation is the optimal choice. We therefore sought to undertake a meta-analysis with special emphases on comparing the efficacy and safety between cryoballoon and radiofrequency ablations by synthesizing published clinical trials. METHODS AND RESULTS: Articles were identified by searching the MEDLINE and EMBASE databases before September 2013, by reviewing the bibliographies of eligible reports, and by consulting with experts in this field. Data were extracted independently and in duplicate. There were respectively 469 and 635 patients referred for cryoballoon and radiofrequency ablations from 14 qualified clinical trials. Overall analyses indicated that cryoballoon ablation significantly reduced fluoroscopic time and total procedure time by a weighted mean of 14.13 (95% confidence interval [95% CI]: 2.82 to 25.45; P = 0.014) minutes and 29.65 (95% CI: 8.54 to 50.77; P = 0.006) minutes compared with radiofrequency ablation, respectively, whereas ablation time in cryoballoon ablation was nonsignificantly elongated by a weighted mean of 11.66 (95% CI: -10.71 to 34.04; P = 0.307) minutes. Patients referred for cryoballoon ablation had a high yet nonsignificant success rate of catheter ablation compared with cryoballoon ablation (odds ratio; 95% CI; P: 1.34; 0.53 to 3.36; 0.538), and cryoballoon ablation was also found to be associated with the relatively low risk of having recurrent atrial fibrillation (0.75; 0.3 to 1.88; 0.538) and major complications (0.46; 0.11 to 1.83; 0.269). There was strong evidence of heterogeneity and low probability of publication bias. CONCLUSION: Our findings demonstrate greater improvement in fluoroscopic time and total procedure duration for atrial fibrillation patients referred for cryoballoon ablation than those for radiofrequency ablation.

The imprinted H19 lncRNA antagonizes let-7 microRNAs.

Abundantly expressed in fetal tissues and adult muscle, the developmentally regulated H19 long noncoding RNA (lncRNA) has been implicated in human genetic disorders and cancer. However, how H19 acts to regulate gene function has remained enigmatic, despite the recent implication of its encoded miR-675 in limiting placental growth. We noted that vertebrate H19 harbors both canonical and noncanonical binding sites for the let-7 family of microRNAs, which plays important roles in development, cancer, and metabolism. Using H19 knockdown and overexpression, combined with in vivo crosslinking and genome-wide transcriptome analysis, we demonstrate that H19 modulates let-7 availability by acting as a molecular sponge. The physiological significance of this interaction is highlighted in cultures in which H19 depletion causes precocious muscle differentiation, a phenotype recapitulated by let-7 overexpression. Our results reveal an unexpected mode of action of H19 and identify this lncRNA as an important regulator of the major let-7 family of microRNAs.

Decidual cell regulation of natural killer cell-recruiting chemokines: implications for the pathogenesis and prediction of preeclampsia.

First trimester human decidua is composed of decidual cells, CD56(bright)CD16(-) decidual natural killer (dNK) cells, and macrophages. Decidual cells incubated with NK cell-derived IFN-γ and either macrophage-derived TNF-α or IL-1ß synergistically enhanced mRNA and protein expression of IP-10 and I-TAC. Both chemokines recruit CXCR3-expressing NK cells. This synergy required IFN-γ receptor 1 and 2 mediation via JAK/STAT and NFκB signaling pathways. However, synergy was not observed on neutrophil, monocyte, and NK cell-recruiting chemokines. Immunostaining of first trimester decidua localized IP-10, I-TAC, IFN-γR1, and -R2 to vimentin-positive decidual cells versus cytokeratin-positive interstitial trophoblasts. Flow cytometry identified high CXCR3 levels on dNK cells and minority peripheral CD56(bright)CD16(-) pNK cells and intermediate CXCR3 levels on the majority of CD56(dim)CD16(+) pNK cells. Incubation of pNK cells with either IP-10 or I-TAC elicited concentration-dependent enhanced CXCR3 levels and migration of both pNK cell subsets that peaked at 10 ng/mL, whereas each chemokine at a concentration of 50 ng/mL inhibited CXCR3 expression and pNK cell migration. Deciduae from women with preeclampsia, a leading cause of maternal and fetal morbidity and mortality, displayed significantly lower dNK cell numbers and higher IP-10 and I-TAC levels versus gestational age-matched controls. Significantly elevated IP-10 levels in first trimester sera from women eventually developing preeclampsia compared with controls, identifying IP-10 as a novel, robust early predictor of preeclampsia.

TNF, acting through inducibly expressed TNFR2, drives activation and cell cycle entry of c-Kit+ cardiac stem cells in ischemic heart disease.

TNF, signaling through TNFR2, has been implicated in tissue repair, a process that in the heart may be mediated by activated resident cardiac stem cells (CSCs). The objective of our study is to determine whether ligation of TNFR2 can induce activation of resident CSCs in the setting of ischemic cardiac injury. We show that in human cardiac tissue affected by ischemia heart disease (IHD), TNFR2 is expressed on intrinsic CSCs, identified as c-kit(+)/CD45(-)/VEGFR2(-) interstitial round cells, which are activated as determined by entry to cell cycle and expression of Lin-28. Wild-type mouse heart organ cultures subjected to hypoxic conditions both increase cardiac TNF expression and show induced TNFR2 and Lin-28 expression in c-kit(+) CSCs that have entered cell cycle. These CSC responses are enhanced by exogenous TNF. TNFR2(-/-) mouse heart organ cultures subjected to hypoxia increase cardiac TNF but fail to induce CSC activation. Similarly, c-kit(+) CSCs isolated from mouse hearts exposed to hypoxia or TNF show induction of Lin-28, TNFR2, cell cycle entry, and cardiogenic marker, α-sarcomeric actin (α-SA), responses more pronounced by hypoxia in combination with TNF. Knockdown of Lin-28 by siRNA results in reduced levels of TNFR2 expression, cell cycle entry, and diminished expression of α-SA. We conclude that hypoxia-induced c-kit(+) CSC activation is mediated by TNF/TNFR2/Lin-28 signaling. These observations suggest that TNFR2 signaling in resident c-kit(+) CSCs induces cardiac repair, findings which provide further understanding of the unanticipated harmful effects of TNF blockade in human IHD.

Lin28 regulates BMP4 and functions with Oct4 to affect ovarian tumor microenvironment.

Emerging evidence suggests that the tumor microenvironment plays a critical role in regulating cancer stem cells (CSCs) and tumor progression through both autocrine and paracrine signaling. Elevated production of bone morphogenetic proteins (BMPs) from human ovarian cancer cells and stroma has been shown to increase CSC proliferation and tumor growth. Here, we report that Lin28, a stem cell factor, binds to BMP4 mRNA in epithelial ovarian carcinoma cells, thereby promoting BMP4 expression at the post-transcriptional level. As co-expression of Lin28 and Oct4 (another stem cell factor) has been implicated in ovarian cancer CSCs, we also determined that high levels of Lin28 are associated with an unfavorable prognosis when co-expressed with high levels of Oct4. Together, these findings uncover a new level of regulation of BMP4 expression and imply a novel Lin28/Oct4/BMP4-mediated mechanism of regulating ovarian tumor cell growth, thus holding potential for the development of new strategies for the diagnosis and treatment of ovarian cancer.

Does Lin28 Antagonize miRNA-Mediated Repression by Displacing miRISC from Target mRNAs?

Lin28 is a developmentally regulated RNA-binding protein that plays important roles in diverse physiological and pathological processes including oncogenesis and brain synaptic function. These pleiotropic roles of Lin28 are mechanistically linked both to its ability to directly stimulate translation of genes involved primarily in cell growth and metabolism and to its ability to block biogenesis of a subset of miRNAs including the let-7 family of miRNAs. In the case of direct stimulation of gene expression, Lin28 binds to targeted mRNAs through recognition of Lin28-responsive elements (LREs) within mRNAs and recruits RNA helicase A (RHA) to promote translation. RHA belongs to the DEAD-box protein family of RNA helicases, which generally catalyze ATP-dependent unwinding of RNA duplexes or remodeling of ribonucleoprotein complexes (RNPs). Since any given mRNA can potentially be inhibited by miRNAs bearing complementary sequences, we hypothesize that binding of Lin28 to LREs not only nucleates the binding of multiple Lin28 molecules to the same mRNA, but also leads to remodeling of RNPs through recruitment of RHA and causes release of inhibitory miRNA-induced silencing complexes bound to the mRNA. This mode of action may contribute to Lin28-mediated stimulation of translation in both tumor and neuronal cells.

Drosha mediates destabilization of Lin28 mRNA targets.

Lin28 plays important roles in development, stem cell maintenance, oncogenesis and metabolism. As an RNA-binding protein, it blocks the biogenesis primarily of let-7 family miRNAs and also promotes translation of a cohort of mRNAs involved in cell growth, metabolism and pluripotency, likely through recognition of distinct sequence and structural motifs within mRNAs. Here, we show that one such motif, shared by multiple Lin28-responsive elements (LREs) present in Lin28 mRNA targets also participates in a Drosha-dependent regulation and may contribute to destabilization of its cognate mRNAs. We further show that the same mutations in the LREs known to abolish Lin28 binding and stimulation of translation also abrogate Drosha-dependent mRNA destabilization, and that this effect is independent of miRNAs, uncovering a previously unsuspected coupling between Drosha-dependent destabilization and Lin28-mediated regulation. Thus, Lin28-dependent stimulation of translation of target mRNAs may, in part, serve to compensate for their intrinsic instability, thereby ensuring optimal levels of expression of genes critical for cell viability, metabolism and pluripotency.

Lin28 regulates HER2 and promotes malignancy through multiple mechanisms.

The RNA binding protein Lin28 and its paralog Lin28B are associated with advanced human malignancies. Blocking the biogenesis of let-7 miRNA, a tumor suppressor, by Lin28/Lin28B has been thought to underlie their roles in cancer. Here we report that the mRNA for the human epidermal growth factor receptor 2 (HER2), a HER-family receptor tyrosine kinase known to play a critical role in cell proliferation and survival and also a major therapeutic target in breast cancer, is among several targets of Lin28 regulation. We show that Lin28 stimulates HER2 expression at the posttranscriptional level, and that enforced Lin28 expression promotes cancer cell growth via multiple mechanisms. Consistent with its pleiotropic role in regulating gene expression, Lin28 overexpression in primary breast tumors is a powerful predictor of poor prognosis, representing the first report on the impact of Lin28 expression on clinical outcome in human cancer. While revealing another layer of regulation of HER2 expression in addition to gene amplification, our studies also suggest novel mechanistic insights linking Lin28 expression to disease outcome and imply that targeting multiple pathways is a common mechanistic theme of Lin28-mediated regulation in cancer.

A mirror of two faces: Lin28 as a master regulator of both miRNA and mRNA.

Lin28 is an evolutionarily conserved RNA-binding protein that plays important roles in development, pluripotency, tumorigenesis, and metabolism. Emerging evidence suggests that the pleiotropic roles of Lin28 in the diverse physiological and pathological processes are mechanistically linked to its ability to modulate not only the biogenesis of miRNAs, particularly the let-7 family miRNAs, but also the translation of mRNAs important for cell growth and metabolism. Let-7 negatively regulates the translation of oncogenes, cell cycle regulators, and metabolic pathway components. Lin28 relieves this repression by blocking the production of mature let-7. Lin28 binds to the terminal loops of let-7 precursors, leading to inhibition of processing and the induction of uridylation and precursor degradation. Lin28 also is a direct translational regulator: it selectively binds to a cohort of mRNAs and stimulates their translation. Recent advances in our understanding of Lin28-mediated mechanisms of posttranscriptional regulation of gene expression reveal important roles of this protein in the fields of development, stem cells, metabolic diseases, and cancer.

Determinants of mRNA recognition and translation regulation by Lin28.

Lin28 is critical for stem cell maintenance and is also associated with advanced human malignancies. Our recent genome-wide studies mark Lin28 as a master post-transcriptional regulator of a subset of messenger RNAs important for cell growth and metabolism. However, the molecular basis underpinning the selective mRNA target regulation is unclear. Here, we provide evidence that Lin28 recognizes a unique motif in multiple target mRNAs, characterized by a small but critical 'A' bulge flanked by two G:C base pairs embedded in a complex secondary structure. This motif mediates Lin28-dependent stimulation of translation. As Lin28 is also known to inhibit the biogenesis of a cohort of miRNAs including let-7, we propose that Lin28 binding to different RNA types (precursor miRNAs versus mRNAs) may facilitate recruitment of different co-factors, leading to distinct regulatory outcomes. Our findings uncover a putative yet unexpected motif that may constitute a mechanistic base for the multitude of functions regulated by Lin28 in both stem cells and cancer cells.

Evidence that Lin28 stimulates translation by recruiting RNA helicase A to polysomes.

The stem cell protein Lin28 functions to inhibit the biogenesis of a group of miRNAs but also stimulates the expression of a subset of mRNAs at the post-transcriptional level, the underlying mechanism of which is not yet understood. Here we report the characterization of the molecular interplay between Lin28 and RNA helicase A (RHA) known to play an important role in remodeling ribonucleoprotein particles during translation. We show that reducing Lin28 expression results in decreased RHA association with polysomes while increasing Lin28 expression leads to elevated RHA association. Further, the carboxyl terminus of Lin28 is necessary for interaction with both the amino and carboxyl termini of RHA. Importantly, a carboxyl terminal deletion mutant of Lin28 that retains RNA-binding activity fails to interact with RHA and exhibits dominant-negative effects on Lin28-dependent stimulation of translation. Taken together, these results lead us to suggest that Lin28 may stimulate translation by actively recruiting RHA to polysomes.

Lin28-mediated post-transcriptional regulation of Oct4 expression in human embryonic stem cells.

Lin28 acts as a repressor of microRNA processing and as a post-transcriptional regulatory factor for a subset of mRNAs. Here we report that in human embryonic stem cells Lin28 facilitates the expression of the pivotal pluripotency factor Oct4 at the post-transcriptional level. We provide evidence that Lin28 binds Oct4 mRNA directly through high affinity sites within its coding region and that an interaction between Lin28 and RNA helicase A (RHA) may play a part in the observed regulation. We further demonstrate that decreasing RHA levels impairs Lin28-dependent stimulation of translation in a reporter system. Taken together with previous studies showing that RHA is required for efficient translation of a specific class of mRNAs, these findings suggest a novel mechanism by which Lin28 may affect target mRNA expression and represent the first evidence of post-transcriptional regulation of Oct4 expression by Lin28 in human embryonic stem cells.

RNA processing in the polyoma virus life cycle.

Not only is gene regulation in polyoma interesting, but it has also proven to be highly informative and illustrative of a number of novel concepts in gene regulation. Of special interest and importance are the mechanisms by which this virus switches from the expression of early gene products to late gene products after the onset of viral DNA replication. This switch is mediated at least in part by changes in transcription elongation and polyadenylation in the late region, and by the formation and editing of dsRNA in the nucleus. In this review we will summarize the regulation of RNA synthesis and processing during polyoma infection, and will point out in particular those aspects that have been most novel.