Let-7 Research Articles

Let-7 MicroRNAs Regulate Microglial Function and Suppress Glioma Growth through Toll-Like Receptor 7

Microglia express Toll-like receptors (TLRs) that sense pathogen- and host-derived factors, including single-stranded RNA. In the brain, let-7 microRNA (miRNA) family members are abundantly expressed, and some have recently been shown to serve as TLR7 ligands. We investigated whether let-7 miRNA family members differentially control microglia biology in health and disease. We found that a subset of let-7 miRNA family members function as signaling molecules to induce microglial release of inflammatory cytokines, modulate antigen presentation, and attenuate cell migration in a TLR7-dependent manner. The capability of the let-7 miRNAs to control microglial function is sequence specific, mapping to a let-7 UUGU motif. In human and murine glioblastoma/glioma, let-7 miRNAs are differentially expressed and reduce murine GL261 glioma growth in the same sequence-specific fashion through microglial TLR7. Taken together, these data establish let-7 miRNAs as key TLR7 signaling activators that serve to regulate the diverse functions of microglia in health and glioma.

Discovery of Small-Molecule Modulators of Protein-RNA Interactions by Fluorescence Intensity-Based Binding Assay.

Protein-RNA interactions mediate various cellular processes, the dysregulation of which has been associated with a list of diseases. Thus, novel experimental tools for monitoring protein-RNA interactions are highly desirable to identify new chemical modulators of these therapeutic targets. In this study, we constructed simple fluorescence intensity-based protein-RNA binding assays by testing multiple environment-sensitive organic fluorophores. We selected the oncogenic interaction between Lin28 and the let-7 microRNA and the important immunomodulatory Roquin-Tnf CDE interaction as representative targets. We adapted this assay to high-throughput screening for the identification of pyrazolyl thiazolidinedione-type molecules as potent small-molecule inhibitors of protein-microRNA interactions. We clearly showed the structure-activity relationships of this new class of Lin28-let-7 interaction inhibitors, and confirmed that cellular mature let-7 microRNAs and their target genes could be modulated upon treatment with the pyrazolyl thiazolidinedione-type inhibitor. We expect that our simple and adaptable screening approach can be applied for the development of various assay systems aimed at the identification of bioactive small molecules targeting protein-RNA interactions.

Abstract TMIM-065: MICRO-RNA LET-7 REGULATION AND FUNCTION IN OVARIAN CANCER AND EARLY EMBRYONIC DEVELOPMENT

Abstract INTRODUCTION: Epithelial ovarian cancer (EOC) is the fifth deadliest malignancy in women in the United States, with a high death-to-incidence ratio and five-year survival of 30%. Aggressiveness and recurrence of EOC has been attributed to cancer stem cells (CSC) within tumors, which contribute to chemoresistance and relapse. Deregulation of miRNAs has been linked to cancer initiation, progression, and the stem cell state. We focus on let-7 miRNA, which is repressed in many cancers and is associated with decreased survival. Let-7 is essential for cell differentiation, and its repression is required for reprogramming. Decreased let-7 level in cancer is associated with stemness. OBJECTIVE: Purpose for our research is to understand let-7 expression and function in EOC cell-lines and patients-derived samples in order to understand mechanisms of its abnormal regulation and develop novel treatments. MATERIALS AND METHODS: Patient-derived samples are used along with EOC cell-lines. Over-expression of Let-7 is achieved by mimic transfection and confirmed via RT-qPCR. Level of miRNA and pluripotency markers mRNA is detected via RT-qPCR, and protein expression confirmed by Western blot. Functional assays to analyze cancer phenotype include wound healing, invasion and spheroid formation assays. Embryoid body (EB) formation is used to mimic murine early embryonic development and guided toward primitive streak via cytokines. Let-7 expression is analyzed via RT-qPCR. RESULTS: We demonstrate that patient-derived EOC samples have decreased Let-7 levels and increased expression of pluripotency markers, therefore, we hypothesize that up-regulation of let-7 in EOC will decrease aggressiveness and increase chemosensitivity. We have demonstrated that up-regulation of let-7 via mimic transfection reduced cancer stem cell properties demonstrated by reduction of pluripotency markers and spheroid formation. Let-7 also decreased in vitro migration and invasion. Our data show that let-7 suppresses stem cell-like phenotype of EOC, indicating that it may be a treatment option in conjunction with conventional chemotherapies. In order to understand deregulation of let-7 in cancer, understanding its normal regulation and function during development is essential. By using mESCs we demonstrate that let-7 is dynamically expressed, contradicting conventional belief that let-7 levels slowly increase upon differentiation. Previously published data demonstrating that Hmga2, a let-7 target, peaks and is required upon exit from pluripotency supports our results. CONCLUSION: In conclusion, let-7 is repressed in EOC, and its over-expression demonstrates tumor suppressive functions via decreasing stemness, migration, invasion, and spheroid formation. During development let-7 is dynamically expressed, demonstrating complex regulation. Citation Format: Evgeny A Chirshev, Anthony Nguyen, Hojo Nozomi, Hill Alyse, Juli Unternaehrer. MICRO-RNA LET-7 REGULATION AND FUNCTION IN OVARIAN CANCER AND EARLY EMBRYONIC DEVELOPMENT [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-065.

Let-7 regulates cell cycle dynamics in the developing cerebralcortex and retina

In the neural progenitors of the developing central nervous system(CNS), cell proliferation is tightly controlled and coordinated with cell fatedecisions. Progenitors divide rapidly during early development and their cell cyclelengthens progressively as development advances to eventually give rise to a tissueof the correct size and cellular composition. However, our understanding of themolecules linking cell cycle progression to developmental time is incomplete. Here,we show that the microRNA (miRNA) let-7 accumulates in neural progenitors over timethroughout the developing CNS. Intriguingly, we find that the level and activity oflet-7 oscillate as neural progenitors progress through the cell cycle by in situ hybridization and fluorescent miRNA sensoranalyses. We also show that let-7 mediates cell cycle dynamics: increasing the levelof let-7 promotes cell cycle exit and lengthens the S/G2 phase of the cell cycle,while let-7 knock down shortens the cell cycle in neural progenitors. Together, ourfindings suggest that let-7 may link cell proliferation to developmental time andregulate the progressive cell cycle lengthening that occurs duringdevelopment.

Detection of Several Homologous MicroRNAs by a Single Smart Probe System Consisting of Linear Nucleic Acid Blockers.

We report a universal smart probe (SP) that is capable of detecting several homologous let-7 microRNAs (miRNAs). While the SP is complementary to let-7a, and therefore, strongly binds to this target, due to sequence homology, the SP also has equal propensity to non-specifically hybridize with let-7b and let-7c, which are homologous to let-7a. The fluorescence signal of the SP was switched off in the absence of any homologous member target, but the signal was switched on when any of the three homologous members was present. With the assistance of nucleic acid blockers (NABs), this SP system can discriminate between homologous miRNAs. We show that the SP can discriminate between let-7a and the other two sequences by using linear NABs (LNABs) to block non-specific interactions between the SP and these sequences. We also found that LNABs used do not cross-react with the let-7a target due to the low LNABs:SP molar ratio of 6:1 used. Overall, this SP represents a universal probe for the recognition of a homologous miRNA family. The assay is sensitive, providing a detection limit of 6 fmol. The approach is simple, fast, usable at room temperature, and represents a general platform for the in vitro detection of homologous microRNAs by a single fluorescent hairpin probe.

Matrine suppression of self-renewal was dependent on regulation of LIN28A/Let-7 pathway in breast cancer stem cells.

Matrine, a natural product extracted from the root of Sophora flavescens Ait, was the main chemical ingredient of compounds of Kushen injection, which has been widely used for its remarkable anticancer effects for years. The underlying mechanisms for Matrine regulations of human breast cancer stem cells (BrCSCs) are barely known. LIN28, a well-characterized suppressor of Let-7 microRNA biogenesis, playing vital roles in regulations of stem cells' renewal and tumorigenesis. Here we show that the compounds of Kushen injection derived Matrine could suppress the BrCSCs differentiation and self-renewal through downregulating the expression of Lin28A, resulting in the inactivation of Wnt pathway through a Let-7b-dependent way. In opposite to Matrine, Cisplatin treatment increases the ability of tumorsphere formation and the expression of BrCSCs markers, which was partially blocked by either Let-7b overexpression or CCND1 inhibition. Furthermore, Matrine sensitized BrCSCs to cisplatin's suppression of cancer expansion in vitro and in vivo. Our study uncovers the role of the LIN28A/Let-7 in BrCSCs renewal, and more importantly, elucidated a novel mechanism by which Matrine induces breast cancer involution.

New regulatory mechanism of tumor suppressor microRNA let-7s (comprehensive approach for tumor suppressors in vitro)

Abstract Purpose Cancer suppressor microRNA let-7 families have a unique control mechanism that interacts with Lin28, while Lin28-independent expression of let-7 families has been reported. We attempted to identify the new let-7 regulatory genes and analyze their functions on 1123 genes for which RNA binding is predicted by GeneOntology. Methods We tried to identify the new let-7 regulatory genes from the gene expression library by using a luciferase reporter in which the complementary sequence of let-7a was inserted into 3’UTR. Suppression of the candidate genes and preparation of its mutants were carried out and the effect on the expression and maturation/processing of let-7 families was evaluated by using qPCR, Northern blotting, and CLIP-PCR. The selectivity of microRNAs was analyzed using TaqMan Array of 377 microRNAs. In addition, the effect of target gene on cell proliferation was evaluated by MTT assay and subcutaneous implantation model in mouse. Expression and survival rate of candidate genes were investigated using TCGA database. Results In addition to Lin28, that is a known let-7 regulator, several unknown genes were identified by this screening. One of them selectively reduced the expression level of primary let-7, increased that of mature let-7. The affinity of primary let-7 and DGCR-8 in the nucleus was increased. The post-transcriptional regulation of microRNAs were suspected. Furthermore, this regulation had a strong selectivity to let-7 families. This gene suppressed cell growth and its suppression is improved by inhibition of let7s. In TCGA database, this gene was involved in survival in some carcinomas. Conclusion We identified a new gene that controls let-7 families. It is strongly suggested that this gene regulates the maturation process of the let-7 family after transcription and is potentially the candidate of tumor suppressor gene.

LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

Abnormal placental development is one of the main etiological factors for intrauterine growth restriction (IUGR). Here, we show that LIN28A and LIN28B are significantly lower and lethal-7 (let-7) microRNAs (miRNAs) significantly higher in term human IUGR vs. normal placentas. We hypothesize that let-7 miRNAs regulate genes with known importance for human placental development [high-mobility group AT-hook 1 (HMGA1), transcriptional regulator Myc-like (c-myc), vascular endothelial growth factor A (VEGF-A), and Wnt family member 1 (WNT1)] by targeting the AT-rich interacting domain (ARID)-3B complex. ACH-3P cells with LIN28A and LIN28B knockout (DKOs) significantly increased let-7 miRNAs, leading to significantly decreased ARID3A, ARID3B, and lysine demethylase 4C (KDM4C). Similarly, Sw.71 cells overexpressing LIN28A and LIN28B (DKIs) significantly decreased let-7 miRNAs, leading to significantly increased ARID3A, ARID3B, and KDM4C. In ACH-3P cells, ARID3A, ARID3B, and KDM4C make a triprotein complex [triprotein complex comprising ARID3A, ARID3B, and KDM4C (ARID3B-complex)] that binds the promoter regions of HMGA1, c-MYC, VEGF-A, and WNT1. ARID3B knockout in ACH-3P cells disrupted the ARID3B-complex, leading to a significant decrease in HMGA1, c-MYC, VEGF-A, and WNT1. DKOs had a significant reduction, whereas DKIs had a significant increase in HMGA1, c-MYC, VEGF-A, and WNT1, potentially due to regulation by the ARID3B-complex. This is the first study showing regulation of let-7 targets in immortalized human trophoblast cells by the ARID3B-complex.-Ali, A., Anthony, R. V., Bouma, G. J., Winger, Q. A. LIN28-let-7 axis regulates genes in immortalized human trophoblast cells by targeting the ARID3B-complex.

Upregulation of Insulin-Like Growth Factor-1 Receptor (IGF-1R) Reverses the Inhibitory Effect of Let-7g-5p on Migration and Invasion of Nasopharyngeal Carcinoma.

BackgroundLet-7 microRNAs (miRNAs) have the effects of inhibiting tumor growth and metastasis, however, the research in nasopharyngeal carcinoma (NPC) is limited. This study focused on the effects of Let-7 on NPC migration and invasion and the mechanism of action.Material/MethodsPlasmid transfection was used to upregulate the expression levels of Let-7g-5p and insulin-like growth factor-1 receptor (IGF-1R). Cell counting kit-8 (CCK-8) assay was applied to test the cell viability. Scratch assay and Transwell assay were performed to detect the migration and invasion abilities. Bioinformatics prediction and luciferase reporter assay were used to determine and verify the downstream target genes for Let-7g-5p. Protein and mRNA were detected by western blot and real-time quantitative polymerase chain reaction (RT-qPCR), respectively.ResultsLet-7g-5p was under-expressed in human NPC cells. Overexpression of Let-7g-5p could inhibit cell viability and inhibit the migration and invasion of SUNE1 cells. The dual-luciferase reporter assay showed that IGF-1R was a direct target gene of Let-7g-5p, which was directly regulated IGF-1R expression by 3′UTR. Let-7g-5p overexpression could inhibit the expression of IGF-1R gene, and upregulation of IGF-1R gene expression reversed the inhibitory effect of Let-7g-5p on cell viability and epithelial-mesenchymal transition processes.ConclusionsLet-7g-5p is lowly expressed in NPC and it was the first to discover that IGF-1R was a target gene of let-7g-5p in NPC. Upregulation of IGF-1R reversed the inhibitory effect of Let-7g-5p on epithelial-mesenchymal transition.

MicroRNA let-7-TGFBR3 signalling regulates cardiomyocyte apoptosis after infarction

BackgroundMyocardial infarction (MI) is a life-threatening disease, often leading to heart failure. Defining therapeutic targets at an early time point is important to prevent heart failure. MethodsMicroRNA screening was performed at early time points after MI using paired samples isolated from the infarcted and remote myocardium of pigs. We also examined the microRNA expression in plasma of MI patients and pigs. For mechanistic studies, AAV9-mediated microRNA knockdown and overexpression were administrated in mice undergoing MI. FindingsMicroRNAs let-7a and let-7f were significantly downregulated in the infarct area within 24 h post-MI in pigs. We also observed a reduction of let-7a and let-7f in plasma of MI patients and pigs. Inhibition of let-7 exacerbated cardiomyocyte apoptosis, induced a cardiac hypertrophic phenotype, and resulted in worsened left ventricular ejection fraction. In contrast, ectopic let-7 overexpression significantly reduced those phenotypes and improved heart function. We then identified TGFBR3 as a target of let-7, and found that induction of Tgfbr3 in cardiomyocytes caused apoptosis, likely through p38 MAPK activation. Finally, we showed that the plasma TGFBR3 level was elevated after MI in plasma of MI patients and pigs. InterpretationTogether, we conclude that the let-7-Tgfbr3-p38 MAPK signalling plays an important role in cardiomyocyte apoptosis after MI. Furthermore, microRNA let-7 and Tgfbr3 may serve as therapeutic targets and biomarkers for myocardial damage. FundMinistry of Science and Technology, National Health Research Institutes, Academia Sinica Program for Translational Innovation of Biopharmaceutical Development-Technology Supporting Platform Axis, Thematic Research Program and the Summit Research Program, Taiwan.

Modeling the Dynamics of Let-7-Coupled Gene Regulatory Networks Linking Cell Proliferation to Malignant Transformation.

Let-7 microRNA controls the expression of proteins that belong to two distinct gene regulatory networks, namely, a cyclin-dependent kinase (Cdk) network driving the cell cycle and a cell transformation network that can undergo an epigenetic switch between a non-transformed and a malignant transformed cell state. Using mathematical modeling and transcriptomic data analysis, we here investigate how Let-7 controls the Cdk-dependent cell cycle network, and how it couples the latter with the transformation network. We also assess the consequence of this coupling on cancer progression. Our analysis shows that the switch from a quiescent to a proliferative state depends on the relative levels of Let-7 and several cell cycle activators. Numerical simulations further indicate that the Let-7-coupled cell cycle and transformation networks mutually control each other, and our model identifies key players for this mutual control. Transcriptomic data analysis from The Cancer Genome Atlas (TCGA) suggests that the two networks are activated in cancer, in particular in gastrointestinal cancers, and that the activation levels vary significantly among patients affected by a same cancer type. Our mathematical model, when applied to a heterogeneous cell population, suggests that heterogeneity among tumors may in part result from stochastic switches between a non-transformed cell state with low proliferative capability and a transformed cell state with high proliferative property. The model further predicts that Let-7 may reduce tumor heterogeneity by decreasing the occurrence of stochastic switches toward a transformed, proliferative cell state. In conclusion, we identified the key components responsible for the qualitative dynamics of two networks interconnected by Let-7. The two networks are heterogeneously activated in several cancers, thereby stressing the need to consider patient’s specific characteristics to optimize therapeutic strategies.

Abstract 3660: A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation

Abstract BACKGROUND/OBJECTIVES: MYC family proteins are implicated in many human cancers, but their therapeutic targeting has proven challenging. MYCN and MYC amplification in childhood neuroblastoma (NB) and medulloblastoma (MB) are associated with aggressive disease and high mortality, underscoring a dire need for novel therapies. Let-7 microRNAs (miRNAs) inhibit tumor progression and regulate metabolism by degrading several mRNAs, including MYC. Let-7 miRNAs are therefore frequently repressed in cancer, including MYC-driven NB and MB. We previously reported that the mRNA translation elongation regulator eukaryotic Elongation Factor-2 Kinase (eEF2K) is a pivotal mediator of cancer cells adaptation to nutrient deprivation (ND). Publicly available transcriptomic database analyses indicate that eEF2K expression significantly correlate with MYCN and MYC expression in multiple tumor cohorts. Our preliminary data also indicate that the eEF2K 3’ untranslated region (UTR) harbors a potential binding site for let-7 miRNAs. In addition, eEF2K mRNA and let-7 miRNA expression negatively correlates in NB and MB, suggesting a potential regulation of the former by the latter. We therefore hypothesized that let-7 down-regulation induces eEF2K expression, thereby supporting MYC-driven NB and MB adaptation to ND and tumor progression. METHODS: Immunohistochemistry for eEF2K substrate (p-eEF2) was performed on NB and MB tissue microarrays to link results with MYC expression and outcome. Effects of eEF2K pharmacological and genetic inhibition on NB and MB cell survival were evaluated in vitro by MTT assay and PI staining. The ability of let-7 to degrade eEF2K mRNA was assessed by let-7 miRNAs transfection into MB cells, followed by RT-PCR and Western Blotting for eEF2K. Binding of let-7 to the eEF2K 3’UTR was validated by luciferase reporter assay. Finally, NB xenograft mouse models were used to confirm in vitro observations. RESULTS: High eEF2K activity is linked to MYC over-expression and reduced survival in NB and MB (p<0.05). Pharmacological and genetic inhibition of eEF2K significantly reduces survival of MYC/MYCN-amplified NB and MB cell lines under ND. Let-7 miRNAs transfection decreases eEF2K mRNA and protein levels (by ~40-50%), and down-regulation of luciferase activity by let-7 miRNAs is impaired upon mutation of the let-7 binding site on the eEF2K 3’UTR. Knockdown of eEF2K determines a twofold growth decrease of MYCN-amplified NB xenografts when mice are kept under caloric restriction diet. CONCLUSIONS: Let-7 miRNAs degrade eEF2K mRNA by binding to its 3'UTR, indicating that let-7 repression in MYC-driven NB and MB is partially responsible for eEF2K increased levels and activity. Moreover, the let-7-eEF2K axis represents a critical mechanism for MYC-driven NB and MB adaptation to ND, constituting a promising therapeutic target. Citation Format: Alberto Delaidelli, Gian Luca Negri, Brian Cho, Simran Sidhu, Stefan Pfister, Michael Taylor, Gabriel Leprivier, Marcel Kool, Poul Soresnsen. A link between miRNAs and mRNA translation elongation: The let7-eEF2K axis in MYC-driven pediatric tumors adaptation to nutrient deprivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3660.

INCREASED EXPRESSION OF THE NERVE GROWTH FACTOR (NGF) IN PULMONARY ARTERY IS ACCOMPANIED BY CHANGES OF PARTICULAR MICRORNAS LEVELS IN MONOCROTALINE-INDUCED PULMONARY HYPERTENSION

Objective: NGF is a member of the neurotrophin family, whose role has been described in the development and persistence of pulmonary hypertension (PH). Let-7 family is a group of microRNAs that targets NGF and downregulates its expression. On the other hand, microRNA miR-21, has been shown to support NGF signalling, and its expression is increased after NGF stimulation in neuronal cells. Furthermore, miR-21 was reported to contribute to proliferation and migration of lung vascular smooth muscle cells and to decrease BMPR-II expression. In this study, we examined the role of these microRNAs in regulation of NGF expression in pulmonary artery and lungs and, consequently, in the development of experimental monocrotaline-induced PH. Design and method: Male Wistar rats were injected either with monocrotaline (s.c. 60 mg/kg, MCT group) or with vehicle (CON group). Four weeks after MCT treatment, vital functions and right ventricular pressure of rats were measured. Expression of let-7 family, miR-21 and NGF was determined by qRT-PCR in rat pulmonary artery and whole lungs. Results: After MCT administration, we observed decreased oxygen saturation, increased right ventricular pressure and right ventricular hypertrophy (P < 0.05 vs CON). NGF gene expression was significantly increased in pulmonary artery (+106%, P < 0.05 vs CON). In the let-7 microRNA family, expression of two members, namely let-7 g and let-7i was significantly decreased (−31% and −34% respectively, P < 0.05 vs CON), whereas expression of miR-21 was significantly increased (+37%, P < 0.05 vs CON). In lung tissue, we did not note any change in NGF gene expression, together with no significant changes in expression of let-7 family. However, there was a significant increase in miR-21 expression (+92%, P < 0.05 vs CON). Conclusions: The decreased expression of let-7 g and let-7i microRNAs is in line with the increase in NGF expression in pulmonary artery. Additionally, increased miR-21 expression could further enhance NGF signalling. Therefore, we conclude that the let-7 – NGF – miR-21 axis could be a significant contributor to pulmonary vascular pathophysiology in MCT-induced PH.

RETRACTED: Modulation of TNFα Activity by the microRNA Let-7 Coordinates Zebrafish Heart Regeneration

Summary The adult zebrafish is capable of regenerating heart muscle, resolving collagen tissue, and fully restoring heart function throughout its life. In this study, we show that the highly upregulated, epicardium-enriched microRNA let-7i functions in wound closure and cardiomyocyte proliferation. RNA sequencing experiments identified upregulated expression of members of the tumor necrosis factor (TNF) signaling pathway in the absence of let-7. Importantly, co-suppression of TNF and let-7 activity rescued epicardium migration and cardiomyocyte proliferation defects induced by depletion of let-7 alone. Sensitizing animals to low levels of TNF activity before injury culminated in repressed cardiomyocyte proliferation and wound closure defects, suggesting that levels of inflammation at the onset of injury are critical for heart regeneration. Our studies indicate that injury-induced reduction in TNF signaling by let-7 in the epicardium creates a pro-regenerative environment for cardiomyocyte proliferation during adult heart regeneration.

Long Noncoding RNA H19 Contributes to Cholangiocyte Proliferation and Cholestatic Liver Fibrosis in Biliary Atresia.

Biliary atresia (BA) is a neonatal liver disease featuring cholestasis and severe liver fibrosis (LF). Despite advances in the development of surgical treatment, lacking an early diagnostic marker and intervention of LF invariably leads to death from end-stage liver disease in the early years of life. We previously reported that knockout of sphingosine 1-phosphate receptor 2 (S1PR2) protected mice from bile duct ligation (BDL)-induced cholangiocyte proliferation and LF. Our recent studies further showed that both hepatic and serum exosomal long noncoding RNA H19 (lncRNAH19) levels are correlated with cholestatic injury in multidrug resistance 2 knockout (Mdr2-/- ) mice. However, the role of lncRNAH19 in BA progression remains unclear. Here, we show that both hepatic and serum exosomal H19 levels are positively correlated with severity of fibrotic liver injuries in BA patients. H19 deficiency protects mice from BDL-induced cholangiocyte proliferation and LF by inhibiting bile-acid-induced expression and activation of S1PR2 and sphingosine kinase 2 (SphK2). Furthermore, H19acts as a molecular sponge for members of the microRNA let-7 family, which results in up-regulation of high-mobility group AT-hook 2 (HMGA2), a known target of let-7 and enhancement of biliary proliferation. Conclusion: These results indicate that H19 plays a critical role in cholangiocyte proliferation and cholestatic liver injury in BA by regulating the S1PR2/SphK2 and let-7/HMGA2 axis. Serum exosomal H19 may represent a noninvasive diagnostic biomarker and potential therapeutic target for BA.

C/EBP\u03b2-LIP induces cancer-type metabolic reprogramming by regulating the let-7/LIN28B circuit in mice

The transcription factors LAP1, LAP2 and LIP are derived from the Cebpb-mRNA through the use of alternative start codons. High LIP expression has been associated with human cancer and increased cancer incidence in mice. However, how LIP contributes to cellular transformation is poorly understood. Here we present that LIP induces aerobic glycolysis and mitochondrial respiration reminiscent of cancer metabolism. We show that LIP-induced metabolic programming is dependent on the RNA-binding protein LIN28B, a translational regulator of glycolytic and mitochondrial enzymes with known oncogenic function. LIP activates LIN28B through repression of the let-7 microRNA family that targets the Lin28b-mRNA. Transgenic mice overexpressing LIP have reduced levels of let-7 and increased LIN28B expression, which is associated with metabolic reprogramming as shown in primary bone marrow cells, and with hyperplasia in the skin. This study establishes LIP as an inducer of cancer-type metabolic reprogramming and as a regulator of the let-7/LIN28B regulatory circuit.

Dual mechanisms of posttranscriptional regulation of Tet2 by Let-7 microRNA in macrophages

Tet methylcytosine dioxygenase 2 (Tet2) is an epigenetic regulator that removes methyl groups from deoxycytosine residues in DNA. Tet2-deficient murine macrophages show increased lipopolysaccharide (LPS)-induced and spontaneous inflammation at least partially because Tet2 acts to restrain interleukin (IL)-1β and IL-6 expression in induced cells. MicroRNAs have emerged as critical regulatory noncoding RNAs that tune immune cell responses to physiological perturbations and play roles in pathological conditions in macrophages. To determine if a microRNA played any role in Tet2 activity, we examined the interrelationship of Tet2 action and the let-7 microRNA family, utilizing several let-7 microRNA engineered murine models. We first showed that Tet2, but not Tet3, is a direct target of the let-7a-1/let-7d/let-7f-1 (let-7adf) microRNAs in macrophages. We found that overexpression or deletion of the let-7adf gene cluster causes altered IL-6 induction both in tissue culture cells induced by LPS treatment in vitro as well as in a Salmonella infection mouse model in vivo. Mechanistically, let-7adf promotes IL-6 by directly repressing Tet2 levels and indirectly by enhancing a Tet2 suppressor, the key TCA cycle metabolite, succinate. We found that Let-7adf promotes succinate accumulation by regulating the Lin28a/Sdha axis. We thereby identify two pathways of let-7 control of Tet2 and, in turn, of the key inflammatory cytokine, IL-6, thus characterizing a regulatory pathway in which a microRNA acts as a feedback inhibitor of inflammatory processes.

Put the Pedal to the METTL1: Adding Internal m7G Increases mRNA Translation Efficiency and Augments miRNA Processing.

Complementary papers by Zhang, Liu, and colleagues (Zhang etal., 2019) and Pandolfini, Barbieri, and colleagues (Pandolfini etal., 2019) develop new sequencing techniques that reveal that METTL1 N7-methylates internal guanosines in mRNAs and miRNAs to increase translation efficiency and miRNA processing, respectively.

A Regulator of Metabolic Reprogramming: MicroRNA Let-7

Let-7, a gene firstly known to control the timing of Caenorhabditis elegans larval development does not code for a protein but instead produces small non-coding RNAs, microRNAs. Higher animals have multiple isoforms of mature let-7 microRNAs. Mature let-7 family members share the same “seed sequence” and distinct from each other slightly by ‘non-seed’ sequence region. Let-7 has emerged as a central regulator of systemic energy homeostasis and it displays remarkable plasticity in metabolic responses to nutrients availability and physiological activities. In this review, we discuss recent studies highlighting post-transcriptional mechanisms that govern metabolic reprogramming in distinct cells by let-7. We focus on the participation of the let-7 clusters in immune cells, and suggest that tissue-specific regulation of the let-7 clusters by engineered mouse models might impact metabolic homeostasis and will be required to elucidate their physiological and pathological roles in the in vivo disease models.

The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR–LIN28–LET7 signaling

Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions.