Let-7 Research Articles

Effect of Letrozole on hippocampal Let-7 microRNAs and their correlation with working memory and phosphorylated Tau protein in an Alzheimer's disease-like rat model

BackgroundLet-7 microRNAs (miRNAs) may contribute to neurodegeneration, including Alzheimer's disease (AD), but, they were not investigated in Streptozotocin (STZ)-induced AD. Letrozole increases the expression of Let-7 in cell lines, with conflicting evidence regarding its effects on memory. This study examined Let-7 miRNAs in STZ-induced AD, their correlation with memory and hyperphosphorylated Tau (p-Tau) and the effects of Letrozole on them.MethodsSeven groups of adult Sprague Dawley rats were used: Negative control, Letrozole, Letrozole Vehicle, STZ (with AD induced by intracerebroventricular injection of STZ in artificial cerebrospinal fluid (aCSF)), CSF Control, STZ + Letrozole (STZ-L), and CSF + Letrozole Vehicle. Alternation percentage in T-maze was used as a measure of working memory. Let-7a, b and e and p-Tau levels in the hippocampus were estimated using quantitative real-time reverse transcription–polymerase chain reaction (qRT–PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.ResultsSignificant decreases in alternation percentage and increase in p-Tau concentration were found in the STZ, Letrozole and STZ-L groups. Expression levels of all studied microRNAs were significantly elevated in the Letrozole and the STZ-L groups, with no difference between the two, suggesting that this elevation might be linked to Letrozole administration. Negative correlations were found between alternation percentage and the levels of all studied microRNAs, while positive ones were found between p-Tau concentration and the levels of studied microRNAs.ConclusionsThis study shows changes in the expression of Let-7a, b and e miRNAs in association with Letrozole administration, and correlations between the expression of the studied Let-7 miRNAs and both the status of working memory and the hippocampal p-Tau levels. These findings might support the theory suggesting that Letrozole aggravates pre-existing lesions. They also add to the possibility of Let-7’s neurotoxicity.

MicroRNA let-7 is required for hormonal regulation of metamorphosis in the silkworm, Bombyx mori

The heterochronic microRNA let-7, which was first identified in Caenorhabditis elegans, controls the timing of developmental programs, and let-7 triggers the onset of the juvenile-adult transition in bilaterians. The expression of let-7 is strongly induced during the last larval stage of C. elegans and is highly expressed in the late last instar larvae/nymphs of the fly Drosophila melanogaster and the cockroach Blattella germanica. In the silkworm Bombyx mori, the expression of let-7 remarkably increases in the corpus cardiacum-corpus allatum complex (CC-CA) at the beginning of the last larval instar and is maintained at high levels during this instar. To determine the biological function of let-7 in B. mori, we generated a let-7 knockout line and a transgenic UAS-let-7 line. The let-7 knockout larvae were developmentally arrested in the prepupal stage and became pupal-adult intermediates after apolysis. When let-7 was ubiquitously overexpressed under the transcriptional control of an Actin3-GAL4 driver, developmental timing and growth of larvae were severely impaired in the penultimate (L4) instar, and these larvae underwent precocious metamorphosis from L4. Furthermore, our results showed that reception and signaling of ecdysteroids and juvenile hormones (JHs) normally occurred in the absence of let-7, whereas the biosynthesis of ecdysone and JHs were affected by disruption and overexpression of let-7. Together, the present study demonstrates that let-7 is required for the coordination of the biosynthesis of ecdysone and JH to ensure the developmental transition during the metamorphosis of B. mori.

Effect of Basic Fibroblast Growth Factor on Expression of Let-7 MicroRNA in Proliferation of Human Dental Pulp Cells

Objective: Basic fibroblast growth factor (bFGF) plays a pivotal role in cell proliferation, differentiation and extracellular matrix turnover in various tissues. In human dental pulp cells (HDPCs), let-7 microRNA is involved in cell proliferation and differentiation. There is little information on the effect of bFGF-induced cell proliferation on let-7 microRNA in HDPCs. This study investigated the effect of bFGF on let-7g, let-7f, and let-7i microRNAs and some of the genes involved in cell proliferation including p53 and Ki67 in HDPCs.Material and Methods: HDPCs were cultured and treated with bFGF at 0, 1, and 5 ng/mL. Cell proliferation was examined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay at 24 and 48 hours. Additionally, gene expressions of let-7g, let-7f, let-7i microRNAs, and p53 and Ki67 were examined by quantitative real-time polymerase chain reaction at 24 hours. All experiments were performed in triplicate.Results: The results showed that let-7g, let-7f, and let-7i microRNAs were expressed in HDPCs. MTT assays showed that bFGF induced greater cell proliferation than the controls at 24 and 48 hours (p-value<0.050). HDPCs treated with bFGF showed a decrease in p53 expression (p-value<0.001) while Ki67 expression increased (p-value<0.001). The expression of let-7g microRNA decreased under the influence of bFGF (p-value<0.050). However, bFGF had no effect on expression of let-7f and let-7i microRNAs (p-value>0.050).Conclusion: Our preliminary study showed that exogenous bFGF could decrease let-7g microRNA expression suggesting that let-7g microRNA may be involved in bFGF-induced HDPCs proliferation.

Kap1 Regulates the Stability of Lin28A in Embryonic Stem Cells.

Lin28A is an RNA-binding protein that controls mammalian development and maintenance of the pluripotency of embryonic stem cells (ESCs) via regulating the processing of the microRNA let-7. Lin28A is highly expressed in ESCs, and ectopic expression of this protein facilitates reprogramming of somatic cells to induced pluripotent stem cells. However, the mechanisms underlying the post-translational regulation of Lin28A protein stability in ESCs remain unclear. In the present study, we identified Kap1 (KRAB-associated protein 1) as a novel Lin28A-binding protein using affinity purification and mass spectrometry. Kap1 specifically interacted with the N-terminal region of Lin28A through its coiled-coil domain. Kap1 overexpression significantly attenuated Lin28A ubiquitination and increased its stability. However, small interfering RNA-mediated knockdown of Kap1 promoted the ubiquitination of Lin28A, leading to its proteasomal degradation. Trim71, an E3 ubiquitin ligase, induced Lin28A degradation and Kap1 knockdown accelerated the Trim71-dependent degradation of Lin28A. Mutation of the lysine 177 residue of Lin28A to arginine abrogated the ubiquitination and degradation of Lin28A which were accelerated by Kap1 silencing. Moreover, Kap1 overexpression led to the accumulation of Lin28A in the cytoplasm, but not in the nucleus, and reduced the levels of let-7 subtypes. These results suggest that Kap1 plays a key role in regulation of the stability of Lin28A by modulating the Trim71-mediated ubiquitination and subsequent degradation of Lin28A, thus playing a pivotal role in the regulation of ESC self-renewal and pluripotency.

LncRNA TMPO antisense RNA 1 promotes the malignancy of cholangiocarcinoma cells by regulating let-7g-5p/ high-mobility group A1 axis

ABSTRACT Cholangiocarcinoma (CHOL) is often diagnosed at an advanced stage; therefore, exploring its key regulatory factors is important for earlier diagnosis and treatment. This study aimed to identify the mechanisms of long non-coding RNA (lncRNA) TMPO Antisense RNA 1 (TMPO-AS1), microRNA let-7 g-5p, and high-mobility group A1 (HMGA1) proteins in CHOL. Our results, through quantitative real-time PCR and Western blot detection, showed that TMPO-AS1 and HMGA1 were overexpressed while let-7 g-5p was underexpressed in CHOL. Cell function experiments in CHOL cells revealed that TMPO-AS1 knockdown inhibited cell proliferation, colony formation, and cell migration, but induced apoptosis. TMPO-AS1 knockdown also suppressed tumor growth in vivo. Together with luciferase assay and Western blotting, we found that TMPO-AS1 could sponge let-7 g-5p to promote HMGA1 expression. Moreover, HMGA1 overexpression attenuated the effect of TMPO-AS1 downregulation in CHOL cells. Overall, our findings identified the oncogenic effect of TMPO-AS1 on CHOL cells, which may put forward a novel methodology for CHOL diagnosis and therapy.

The Metabolism Reprogramming of microRNA Let-7-Mediated Glycolysis Contributes to Autophagy and Tumor Progression.

Cancer is usually a result of abnormal glucose uptake and imbalanced nutrient metabolization. The dysregulation of glucose metabolism, which controls the processes of glycolysis, gives rise to various physiological defects. Autophagy is one of the metabolic-related cellular functions and involves not only energy regeneration but also tumorigenesis. The dysregulation of autophagy impacts on the imbalance of metabolic homeostasis and leads to a variety of disorders. In particular, the microRNA (miRNA) Let-7 has been identified as related to glycolysis procedures such as tissue repair, stem cell-derived cardiomyocytes, and tumoral metastasis. In many cancers, the expression of glycolysis-related enzymes is correlated with Let-7, in which multiple enzymes are related to the regulation of the autophagy process. However, much recent research has not comprehensively investigated how Let-7 participates in glycolytic reprogramming or its links to autophagic regulations, mainly in tumor progression. Through an integrated literature review and omics-related profiling correlation, this review provides the possible linkage of the Let-7 network between glycolysis and autophagy, and its role in tumor progression.

MicroRNA let-7: A promising non-invasive biomarker for diagnosing and treating external genital endometriosis

Objective:To evaluate the possibility of using microrna let-7 and mir-9 as non-invasive biomarkers for the diagnosis and treatment of external genital endometriosis.Materials and Methods:We explored the samples of relatively healthy individuals and patients with endometriosis. All patients had undergone laparoscopic surgical treatment after clinical and laboratory examinations. We used RNA-GO to obtain total RNA from endometriosis samples excised by laparoscopic method. Next step involved reverse transcription for microRNA let-7 and mir-9. Correlation-regression analysis was performed using Mann-Whitney-Wilcoxon method. Subsequently, receiver operating characteristic analysis was conducted to determine the possibility of using let-7 microRNA for non-invasive detection of endometriosis. The results of the analysis in all groups were tested considering the normality of statistical distribution.Results:Mann-Whitney analysis showed that the difference in mir-9 mRNA between the groups with and without endometriosis, as well as between the groups with more clinically and histologically severe and mild endometriosis, was statistically insignificant. In addition, a significant difference was noted regarding let-7 microRNA between the groups with and without endometriosis, as well as between the groups with more clinically and histologically severe and mild endometriosis. Comparison with cancer antigen-125 (CA-125) showed that let-7 microRNA was a more specific test than CA-125.Conclusion:MicroRNA let-7 had the best parameters (sensitivity, specificity, and predictive value of positive and negative results) among the biomarkers studied. These biomarkers may be used for early and sometimes preclinical diagnosis of endometriosis.

Current Simultaneous Discrimination of Mismatched MicroRNAs Using Base-Flipping within the α-Hemolysin Latch.

The simultaneous discrimination of let-7 microRNAs (miRNAs) would greatly facilitate the early diagnosis and prognosis monitoring of diseases. In this work, a molecular beacon DNA probe was designed to be able to flip out its mismatched cytosine base within the α-hemolysin (α-HL) latch and generate completely separated blocking currents to identify the single-base difference. As a result, the characteristic blocking current of fully matched MB/let-7a and single-base mismatched MB/let-7f was 84.30 ± 0.92 and 87.05 ± 0.86% (confidence level P 95%), respectively. Let-7 miRNA family let-7a and let-7f were completely simultaneously discriminated, which could be attributed to the following strengths. (1) The statistic distribution of blocking current is extremely concentrated with a small relative standard deviation (RSD) of less than 1% and a narrow distribution range. (2) Complete separation is achieved with a high separation resolution of 1.54. (3) The cytosine base flipping out within the α-HL latch provides a universal labeling-free strategy to simultaneously discriminate the single-base mismatch. Overall, the target let-7f sequences were detected with a linear range from 0.001 to 10 pM in human serum samples containing 200 nM let-7a. Great potential has been demonstrated for precise detection, early diagnosis, and prognosis monitoring of diseases related to single-base difference.

LIN28B and ZBTB8B Genes Are Highly Expressed in Vitro in a CD34⁺ Cells Subpopulation of β-Thalassemia Major Patients and May be Involved in Increased HbF Production

Patients with homozygous beta thalassemia and the β⁰β⁰ genotype produce almost only fetal hemoglobin and a small proportion of hemoglobin A2. However, in the bone marrow of these patients there are descriptions of two different subpopulations of erythroblasts, one with high production of HbF (High HbF cells) and another with low production of HbF (Low HbF cells). The High F cells are probably those that survive through erythropoiesis and originate the viable red blood cells. The molecular mechanisms that are responsible for this high production of HbF are not clear. Therefore, the central aim of our work was to study, in cultures of CD34⁺ cells from β⁰β⁰ patients, differentiated to erythrocytes in vitro, the molecular characteristics of cells related to high production of HbF (High HbF), compared to those with low production of HbF (Low HbF). For this purpose, peripheral blood was collected from patients and CD34⁺ cells were identified, and further differentiated in vitro and then isolated by flow cytometry (FACS Aria). After separating the High HbF and Low HbF groups, the cells were morphologically evaluated by optical and confocal microscopy and image cytometry. Next RNA was extracted from the pool of isolated cells and the RNA sequencing assay (RNAseq) was performed, so that it was possible to analyze the transcripts that were differentially expressed between each of the groups. Finally, we validated the sequencing data by real-time PCR. The sorting was performed on the 10th day of cell culture, when the cells presented a double labeling profile for anti-transferrin (CD71) and anti-glycophorin (CD235) antibodies, the mean intensity and fluorescence (MIF) of the markers was 1058 and 1365 for CD71 in healthy control and β-thalassemia groups, respectively, and for CD235 was 430 in controls and 516 for patients. The cells were in an intermediate stage of maturation. After the isolation of sub-populations of High HbF and Low HbF cells, confocal microscopy indicated a clear difference in the intracellular HbF levels of the analyzed cells, confirming that the sorting by cytometry had occurred as desired. Imaging cytometry analysis revealed a MIF of 7.8 x 10⁵ for High HbF cells and 1.7 x 10⁵ for Low HbF cells, when we assessed the cell pools and when single cells were studied, these values ranged from 2.2 x 10⁵ for Low HbF cells to 1.9 x 10⁶ of mean intensity and fluorescence for High cells. After all data generated by RNAseq were filtered, we obtained a list of 16 enriched and differentially-expressed genes between High HbF and Low HbF cells. From these genes, it was possible to identify two that were apparently associated with the increased production of fetal hemoglobin, the genes ZBTB8B and LIN28B. ZBTB8B is a protein-coding gene with a zinc finger domain and may be involved in transcriptional regulation, it was included in our analyses since this gene is part of the same family as ZBTB7A, an important transcription factor that represses several genes involved in cell differentiation and proliferation and that has already been described as a regulator of HbF production. The LIN28B gene encodes an RNA-binding protein, which is described as a possible regulator of HbF levels during fetal development, by increasing the expression of gamma globin, culminating in the increase of intracellular levels of fetal hemoglobin through a direct action on the let-7 microRNA family and BCL11A gene.These results not only contribute to a better understanding of the mechanisms of gene regulation involved in the production of HbF, but indicate a potential new therapeutic approach to increase the production of HbF in hemoglobinopathies. DisclosuresCosta: Novartis: Consultancy.

Lin28b-Let-7-PRC1 Axis Guides Developmental Maturation of the Hematopoietic System

Polycomb group (PcG) proteins are a well-studied group of chromatin modifiers belonging to one of two distinct multi-protein complexes: Polycomb repressive complex 1 (PRC1) and PRC2. With definitive hematopoiesis, PRCs contribute to many aspects of fetal and adult blood formation. However, it is largely unknown how many of the age-specific effects of PRCs in hematopoiesis are regulated. Here, we show that the definitive hematopoietic stem and progenitor cell (HSPC) compartment is remodeled from the fetus to the neonate and into young adulthood coordinated with changes in mature blood cell output. This process is in part dependent on the PRC1 component Cbx2, which is regulated by the heterochronic Lin28b/let-7 axis.First, we quantified various population of definitive hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) using midgestation fetal liver (FL, embryonic day 14.5 (E14.5)), newborn bone marrow (BM, postnatal day 0-1), or young adult (postnatal age 6 to 8 weeks) BM. The lymphoid biased multipotent progenitor 4 (MPP4, ~0.9-fold) declined as the mice matured and aged. We also found erythroid-biased MPP2 diminished (~0.7-fold) while myeloid-biased MPP3 increased (~1.7-fold) with maturation. Using isolated long-term (LT) HSCs from these three stages, we found that E14.5 FL (~8.0-fold) and neonatal LT-HSC (~4.0-fold) showed more rapid B-cell reconstitution compared to young adult LT-HSCs upon transplantation. We found that many of these effects were regulated by Lin28b/let-7.Next, we aimed to determine the downstream mediators of Lin28/let-7's effect on HSPCs maturation. By interrogating gene regulatory subnetworks differentially active across mouse HSPC maturation and mining these subnetworks for predicted let-7 target transcripts, we found Cbx2 enriched in E14.5 FL (P=0.003) and adult HSPCs ectopically expressing LIN28B relative to wild-type adult HSPCs. In cell-based assays, we confirmed that let-7 microRNAs directly regulated CBX2 protein levels. Thus, the Lin28b/let-7 axis governs CBX2 protein levels, leading us to hypothesize that this axis exerts its wide-ranging effects on hematopoietic maturation by regulating PRC1 by controlling Cbx2 levels.As CBX2's developmental stage-specific functions have not been investigated, we generated Cbx2-/-embryos and investigated definitive FL hematopoiesis. We observed skewing of myeloerythorid progenitors to an adult-like myeloid-predominant distribution in Cbx2-/- embryos (P=0.0002), and B-cells in Cbx2-/- neonatal spleens were diminished (P=0.04). We further examined this effect using transplanted Cbx2-/- MPP4 from E14.5 FL which resulted in a decreased donor derived B-lymphoid output compared to wild-type littermates (~0.7-fold).To understand the functional role of Cbx2/PRC1 in juvenile hematopoiesis, we next investigated the role of Cbx2 in maintaining histone H2A monoubiquitinylation (H2AK119Ub) - the histone modification placed by PRC1 - in FL HSPCs. In Cbx2-/- FL HSPCs, the global distribution of H2AK119Ub localization did not change, but several specific H2AK119Ub peaks were altered. We observed differential H2AK119Ub abundance associated with a candidate enhancer within the Erg gene, suggestive of control of Erg expression by Lin28b/let-7/Cbx2. We confirmed that this enhancer activated transcription from a minimal promoter (~8-fold). Erg expression was increased in perinatal spleens of Cbx2-/- mice compared to Cbx2+/+ littermates (~4-fold). Moreover, we found that Cbx2 could repress ERG expression as well as other master HSPC transcription factors.Overall, our findings show that the Lin28b/let-7-axis controls developmental stage-specific hematopoietic output through PRC1-mediated chromatin remodeling. These findings demonstrate a key mechanism by which HSPCs alter their properties during developmental maturation with relevance to age-skewed blood disorders. DisclosuresNo relevant conflicts of interest to declare.

A Focus on Regulatory Networks Linking MicroRNAs, Transcription Factors and Target Genes in Neuroblastoma.

Simple SummaryNeuroblastoma is a tumor of the sympathetic nervous system that substantially contributes to childhood cancer mortality. Neuroblastoma originates from the neural crest cells that are subjected to genetic alterations during embryonic development. These impairments hit key genes, whose expression is activated/repressed by transcription factors and inhibited by negative regulators, named microRNAs, thereby promoting tumorigenesis. Here, we have focused on the interactions between the transcription factors MYCN and PHOX2B with their target genes ALK and LIN28B and the miRNAs let-7, miR-34 and miR-204, which should act as negative regulators of their expression. In neuroblastoma, the physiologic regulatory networks among these genes and microRNAs are disrupted, leading to a complex and aberrant oncogene expression that underlies the development of the tumor. We also looked into the role of these genetic determinants of neuroblastoma starting from their physiological role in neural crest development and ending with their pathogenic dysregulation that leads to neuroblastoma oncogenesis.Neuroblastoma (NB) is a tumor of the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. NB originates from neural crest cells (NCCs) undergoing a defective sympathetic neuronal differentiation and although the starting events leading to the development of NB remain to be fully elucidated, the master role of genetic alterations in key oncogenes has been ascertained: (1) amplification and/or over-expression of MYCN, which is strongly associated with tumor progression and invasion; (2) activating mutations, amplification and/or over-expression of ALK, which is involved in tumor initiation, angiogenesis and invasion; (3) amplification and/or over-expression of LIN28B, promoting proliferation and suppression of neuroblast differentiation; (4) mutations and/or over-expression of PHOX2B, which is involved in the regulation of NB differentiation, stemness maintenance, migration and metastasis. Moreover, altered microRNA (miRNA) expression takes part in generating pathogenetic networks, in which the regulatory loops among transcription factors, miRNAs and target genes lead to complex and aberrant oncogene expression that underlies the development of a tumor. In this review, we have focused on the circuitry linking the oncogenic transcription factors MYCN and PHOX2B with their transcriptional targets ALK and LIN28B and the tumor suppressor microRNAs let-7, miR-34 and miR-204, which should act as down-regulators of their expression. We have also looked at the physiologic role of these genetic and epigenetic determinants in NC development, as well as in terminal differentiation, with their pathogenic dysregulation leading to NB oncogenesis.

MicroRNA-mediated regulation of microRNA machinery controls cell fate decisions.

microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESCs) self-renewal (Liu et al., 2021). Here, we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of Ago2 (Liu et al., 2021), Mir182/Mir183 also repress Ago2. Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.

C. elegans LIN-28 controls temporal cell fate progression by regulating LIN-46 expression via the 5′ UTR of lin-46 mRNA

SUMMARYLin28/LIN-28 is a conserved RNA-binding protein that promotes proliferation and pluripotency and can be oncogenic in mammals. Mammalian Lin28 and C. elegans LIN-28 have been shown to inhibit biogenesis of the conserved cellular differentiation-promoting microRNA let-7 by directly binding to unprocessed let-7 transcripts. Lin28/LIN-28 also bind and regulate many mRNAs in diverse cell types. However, the determinants and consequences of LIN-28-mRNA interactions are not well understood. Here, we report that C. elegans LIN-28 represses the expression of LIN-46, a downstream protein in the heterochronic pathway. We find that lin-28 and sequences within the lin-46 5′ UTR are required to prevent LIN-46 expression at early larval stages. Moreover, we find that precocious LIN-46 expression caused by mutations in the lin-46 5′ UTR is sufficient to cause precocious heterochronic defects similar to those of lin-28(lf) animals. Thus, our findings demonstrate the biological importance of the regulation of individual target mRNAs by LIN-28.

The Long Non-Coding RNA H19 Drives the Proliferation of Diffuse Intrinsic Pontine Glioma with H3K27 Mutation.

Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and sub-cellular location of H19 lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer.

Long non-coding RNA NEAT1 facilitates the growth, migration, and invasion of ovarian cancer cells via the let-7\xa0g/MEST/ATGL axis

Background/AimGrowing evidence indicates a significant role of long non-coding RNA (lncRNA) nuclear-enriched abundant transcript 1 (NEAT1) in ovarian cancer, a frequently occurring malignant tumor in women; however, the possible effects of an interplay of NEAT1 with microRNA (miRNA or miR) let-7 g in ovarian cancer are not known. The current study aimed to investigate the role of the NEAT1/let-7 g axis in the growth, migration, and invasion of ovarian cancer cells and explore underlying mechanisms.MethodsNEAT1 expression levels were examined in clinical tissue samples and cell lines. The relationships between NEAT1, let-7 g, and MEST were then analyzed. Gain- or loss-of-function approaches were used to manipulate NEAT1 and let-7 g. The effects of NEAT1 on cell proliferation, migration, invasion, and apoptosis were evaluated. Mouse xenograft models of ovarian cancer cells were established to verify the function of NEAT1 in vivo.ResultsNEAT1 expression was elevated while let-7 g was decreased in ovarian cancer clinical tissue samples and cell lines. A negative correlation existed between NEAT1 and let-7 g, whereby NEAT1 competitively bound to let-7 g and consequently down-regulate let-7 g expression. By this mechanism, the growth, migration, and invasion of ovarian cancer cells were stimulated. In addition, let-7 g targeted mesoderm specific transcript (MEST) and inhibited its expression, leading to promotion of adipose triglyceride lipase (ATGL) expression and inhibition of ovarian cancer cell growth, migration, and invasion. However, the effect of let-7 g was abolished by overexpression of MEST. Furthermore, silencing of NEAT1 decreased the xenograft tumor growth by decreasing MEST while up-regulating let-7 g and ATGL.ConclusionsCumulatively, the findings demonstrated that NEAT1 could promote malignant phenotypes of ovarian cancer cells by regulating the let-7 g/MEST/ATGL signaling axis. Therefore, NEAT1 can be regarded as an important molecular target and biomarker for ovarian cancer.

Feedback repression of PPARα signaling by Let-7 microRNA.

SUMMARYPeroxisome proliferator-activated receptor α (PPARα) controls hepatic lipid homeostasis and is the target of lipid-lowering fibrate drugs. PPARα activation represses expression of let-7 microRNA (miRNA), but the function of let-7 in PPARα signaling and lipid metabolism is unknown. In the current study, a hepatocyte-specific let-7b/c2 knockout (let7b/c2ΔHep) mouse line is generated, and these mice are found to exhibit pronounced resistance to diet-induced obesity and fatty liver. Let-7 inhibition by hepatocyte-specific let-7 sponge expression shows similar phenotypes as let7b/c2ΔHep mice. RNA sequencing (RNA-seq) analysis reveals that hepatic PPARα signaling is repressed in let7b/c2ΔHep mice. Protein expression of the obligate PPARα heterodimer partner retinoid X receptor α (RXRα) is reduced in the livers of let7b/c2ΔHep mice. Ring finger protein 8 (Rnf8), which is a direct target of let-7, is elevated in let7b/c2ΔHep mouse liver and identified as a E3 ubiquitin ligase for RXRα. This study highlights a let-7-RNF8-RXRα regulatory axis that modulates hepatic lipid catabolism.

Overexpression of Lin28a Aggravates Psoriasis-Like Phenotype by Regulating the Proliferation and Differentiation of Keratinocytes.

PurposePsoriasis is a common and well-studied autoimmune skin disease, which is characterized by plaques. The formation of psoriasis plaques occurs through the hyperproliferation and abnormal differentiation of keratinocytes, infiltration of numerous immune cells into the dermis, increased subepidermal angiogenesis, and various autoimmune-associated cytokines and chemokines. According to previous research, Lin28 regulates the let-7 family, and let-7b is associated with psoriasis. However, the link between Lin28 and psoriasis is unclear. In this study, an association was identified between Lin28a and psoriasis progression, which promoted the pathological characteristic of psoriasis in epidermal keratinocytes.Patients and MethodsThis study aims to investigate the role of Lin28a and its underlying mechanism in psoriasis through in vivo and in vitro models, which include the Lin28a-overexpressing transgenic (TG) mice and Lin28a-overexpressing human keratinocyte (HaCaT) cell lines, respectively.ResultsIn vivo and in vitro results revealed that overexpression of Lin28a downregulated microRNA let-7 expression levels and caused hyperproliferation and abnormal differentiation in keratinocytes. In imiquimod (IMQ)-induced psoriasis-like inflammation, Lin28a overexpressing transgenic (TG) mice exhibited more severe symptoms of psoriasis.ConclusionMechanistically, Lin28a exacerbated psoriasis-like inflammation through the activation of the extracellular-signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 signaling (STAT 3) by targeting proinflammatory cytokine interleukin-6 (IL-6).

LIN28 coordinately promotes nucleolar/ribosomal functions and represses the 2C-like transcriptional program in pluripotent stem cells

LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

Novel lncRNA lncRNA001074 participates in the low salinity-induced response in the sea cucumber Apostichopus japonicus by targeting the let-7/NKAα axis.

Salinity fluctuations have severe impacts on sea cucumbers and therefore important consequences in sea cucumber farming. The responses of sea cucumbers to salinity changes are reflected in the expression profiles of multiple genes and non-coding RNAs (ncRNAs). The microRNA (let-7) which is a developmental regulator, the ion transporter gene sodium potassium ATPase gene (NKAα), and the long ncRNA lncRNA001074 were previously shown to be involved in responses to salinity changes in various marine species. To better understand the relationship between ncRNAs and target genes, the let-7/NKAα/lncRNA001074 predicted interaction was investigated in this study using luciferase reporter assays and gene knockdowns in the sea cucumber Apostichopus japonicus. The results showed that NKAα was the target gene of let-7 and NKAα expression levels were inversely correlated with let-7 expression based on the luciferase reporter assays and western blots. The let-7 abundance was negatively regulated by lncRNA001074 and NKAα both in vitro and in vivo. Knockdown of lncRNA001074 led to let-7 overexpression. These results demonstrated that lncRNA001074 binds to the 3'-UTR binding site of let-7 in a regulatory manner. Furthermore, the expression profiles of let-7, NKAα, and lncRNA001074 were analyzed in sea cucumbers after the knockdown of each of these genes. The results found that lncRNA001074 competitively bound let-7 to suppress NKAα expression under low salinity conditions. The downregulation of let-7, in conjunction with the upregulation of lncRNA001074 and NKAα, may be essential for the response to low salinity change in sea cucumbers. Therefore, the dynamic balance of the lncRNA001074, NKAα, and let-7 network might be a potential response mechanism to salinity change in sea cucumbers.

Abstract 2369: Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment

Abstract Lung cancer is the leading cause of mortality worldwide. MicroRNAs (miRNAs) are potential candidates for lung cancer therapy. However, a major limitation is the lack of an efficient delivery system to directly deliver miRNA to cancer cells while limiting exposure to healthy cells. In our previous studies on aerosolized let-7b in lung cancer prevention, let-7b showed good inhibition of B[a]P-induced lung adenoma with no side effects. In this study, we found that aerosolized let-7b decreased tumor growth in the LKR13 (KRAS mutant) syngeneic mouse model. Let-7b post-transcriptionally suppresses PD-L1 and PD-1 expression in the tumor immune microenvironment, suggesting that let-7 microRNAs may promote antitumor immunity in vivo. Single cell RNA sequencing (scRNAseq) data showed that let-7b treatment decreased the expression of PD-1 in CD8+ T cells and reduced PD-L1 expression in lung tumor cells. Let-7b treatment also significantly changed the percentages of distinct CD8+ tumor-infiltrating lymphocytes (TIL) states. The proportion of CD8+ T cells mediating anti-tumor functions (EM-like CD8+ TILs) was increased significantly by let-7b treatment compared to control. In contrast, the CD8+ T cell subpopulation that has negative effects on anti-tumor immune response, exhausted CD8+ TILs, was significantly decreased by let-7b. Flow cytometry data showed that Let-7b treatment led to the accumulation of CD8+ T cells, granzyme B+ CD8+ T cells and IFN-γ+ CD8+ T cells in tumors, and a decrease of intratumoral Granulocyte-like myeloid derived suppressor cells (G-MDSC) cells. Our results suggest that the in vivo tumor-suppressive efficacy of let-7 is mediated, at least in part, by immune-promoting effects via down-regulating PD-L1 in tumors and/or PD-1 on CD8+ T cells. Citation Format: Qi Zhang, Jing Pan, Donghai Xiong, Yian Wang, Mark S. Miller, Alberto Izzotti, Ming You. Tumor-suppressive efficacy of let-7b microRNA against lung carcinogenesis is mediated by modulating the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2369.