Hepatic Snai1 and Snai2 promote liver regeneration and suppress liver fibrosis in mice.

Liver injury stimulates hepatocyte replication and hepatic stellate cell (HSC) activation, thereby driving liver regeneration. Aberrant HSC activation induces liver fibrosis. However, mechanisms underlying liver regeneration and fibrosis remain poorly understood. Here, we identify hepatic Snai1 and Snai2 as important transcriptional regulators for liver regeneration and fibrosis. Partial hepatectomy or CCl4 treatment increases occupancies of Snai1 and Snai2 on cyclin A2 and D1 promoters in the liver. Snai1 and Snai2 in turn increase promoter H3K27 acetylation and cyclin A2/D1 expressions. Hepatocyte-specific deletion of both Snai1 and Snai2, but not one alone, suppresses liver cyclin A2/D1 expression and regenerative hepatocyte proliferation after hepatectomy or CCl4 treatments but augments CCl4-stimulated HSC activation and liver fibrosis. Conversely, Snai2 overexpression in the liver enhances hepatocyte replication and suppresses liver fibrosis after CCl4 treatment. These results suggest that hepatic Snai1 and Snai2 directly promote, via histone modifications, reparative hepatocyte replication and indirectly inhibit liver fibrosis.

Minimally Invasive Delivery of Percutaneous Ablation Agent via Magnetic Colloidal Hydrogel Injection for Treatment of Hepatocellular Carcinoma.

Percutaneous thermotherapy, a minimally invasive operational procedure, is employed in the ablation of deep tumor lesions by means of target-delivering heat. Conventional thermal ablation methods, such as radiofrequency or microwave ablation, to a certain extent, are subjected to extended ablation time as well as biosafety risks of unwanted overheating. Given its effectiveness and safety, percutaneous thermotherapy gains a fresh perspective, thanks to magnetic hyperthermia. In this respect, an injectable- and magnetic-hydrogel-construct-based thermal ablation agent is likely to be a candidate for the aforementioned clinical translation. Adopting a simple and environment-friendly strategy, a magnetic colloidal hydrogel injection is introduced by a binary system comprising super-paramagnetic Fe3O4 nanoparticles and gelatin nanoparticles. The colloidal hydrogel constructs, unlike conventional bulk hydrogel, can be easily extruded through a percutaneous needle and then self-heal in a reversible manner owing to the unique electrostatic cross-linking. The introduction of magnetic building blocks is exhibited with a rapid magnetothermal response to an alternating magnetic field. Such hydrogel injection is capable of generating heat without limitation of deep penetration. The materials achieve outstanding therapeutic results in mouse and rabbit models. These findings constitute a new class of locoregional interventional thermal therapies with minimal collateral damages.

Inflammatory Fibroblast-Like Synoviocyte-Derived Exosomes Aggravate Osteoarthritis via Enhancing Macrophage Glycolysis.

The severity of osteoarthritis (OA) and cartilage degeneration is highly associated with synovial inflammation. Although recent investigations have revealed a dysregulated crosstalk between fibroblast-like synoviocytes (FLSs) and macrophages in the pathogenesis of synovitis, limited knowledge is available regarding the involvement of exosomes. Here, increased exosome secretion is observed in FLSs from OA patients. Notably, internalization of inflammatory FLS-derived exosomes (inf-exo) can enhance the M1 polarization of macrophages, which further induces an OA-like phenotype in co-cultured chondrocytes. Intra-articular injection of inf-exo induces synovitis and exacerbates OA progression in murine models. In addition, it is demonstrated that inf-exo stimulation triggers the activation of glycolysis. Inhibition of glycolysis using 2-DG successfully attenuates excessive M1 polarization triggered by inf-exo. Mechanistically, HIF1A is identified as the determinant transcription factor, inhibition of which, both pharmacologically or genetically, relieves macrophage inflammation triggered by inf-exo-induced hyperglycolysis. Furthermore, in vivo administration of an HIF1A inhibitor alleviates experimental OA. The results provide novel insights into the involvement of FLS-derived exosomes in OA pathogenesis, suggesting that inf-exo-induced macrophage dysfunction represents an attractive target for OA therapy.

WTAP-mediated m6A modification of FRZB triggers the inflammatory response via the Wnt signaling pathway in osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis. However, the exact pathogenesis remains unclear. Emerging evidence shows that N6-methyladenosine (m6A) modification may have an important role in OA pathogenesis. This study aimed to investigate the role of m6A writers and the underlying mechanisms in osteoarthritic cartilage. Among m6A methyltransferases, Wilms tumor 1-associated protein (WTAP) expression most significantly differed in clinical osteoarthritic cartilage. WTAP regulated extracellular matrix (ECM) degradation, inflammation and antioxidation in human chondrocytes. Mechanistically, the m6A modification and relative downstream targets in osteoarthritic cartilage were assessed by methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing, which indicated that the expression of frizzled-related protein (FRZB), a secreted Wnt antagonist, was abnormally decreased and accompanied by high m6A modification in osteoarthritic cartilage. In vitro dysregulated WTAP had positive effects on ß-catenin expression by targeting FRZB, which finally contributed to the cartilage injury phenotype in chondrocytes. Intra-articular injection of adeno-associated virus-WTAP alleviated OA progression in a mouse model, while this protective effect could be reversed by the application of a Wnt/ß-catenin activator. In summary, this study revealed that WTAP-dependent RNA m6A modification contributed to Wnt/ß-catenin pathway activation and OA progression through post-transcriptional regulation of FRZB mRNA, thus providing a potentially effective therapeutic strategy for OA treatment.

Doxorubicin-Loaded Microalgal Delivery System for Combined Chemotherapy and Enhanced Photodynamic Therapy of Osteosarcoma.

Osteosarcoma (OS) is considered the most frequent type of primary malignant bone tumor. Currently, radiotherapy, photodynamic (PDT), and other therapies for osteosarcoma are limited by tumor hypoxia and single efficacy and serve side-effects. Herein, we reported a microalgal drug delivery system (SpiD), doxorubicin (DOX)-loaded Spirulina platensis (Spi) for OS therapy. The specific surface of Spirulina platensis allowed for effective loading of DOX via surface channels and electrostatic interactions. Under 650 nm laser irradiation, SpiD enabled high oxygen production by photosynthesis and enhanced reactive oxygen species (ROS) generation via chlorophyll-assisted photosensitization, synergistically killing tumor cells with the released DOX. Combined chemotherapy and enhanced PDT mediated by SpiD exerted synergic antitumor effects and resulted in potent therapeutic efficacy in orthotopic osteosarcoma mice. Furthermore, SpiD could reduce the side-effects of chemotherapy, showing excellent blood and tissue safety. Taken together, this microalgal drug delivery system provided a natural, efficient, safe, and inexpensive strategy for OS treatment.

miR-30e-5p regulates leukemia stem cell self-renewal through the Cyb561/ROS signaling pathway.

Leukemia stem cells (LSC) represent a crucial and rare subset of cells present in acute myeloid leukemia (AML); they play a pivotal role in the initiation, maintenance, and relapse of this disease. Targeting LSC holds great promise for preventing AML relapse and improving long-term outcomes. However the precise molecular mechanisms governing LSC self-renewal are still poorly understood. Here, we present compelling evidence that the expression of miR-30e-5p, a potential tumor-suppressive microRNA, is significantly lower in AML samples than in healthy bone marrow samples. Forced expression of miR- 30e effectively inhibits leukemogenesis, impairs LSC self-renewal, and delays leukemia progression. Mechanistically, Cyb561 acts as a direct target of miR-30e-5p in LSC, and its deficiency restricts the self-renewal of LSC by activating reactive oxygen series signaling and markedly prolongs recipients' survival. Moreover, genetic or pharmacological overexpression of miR-30e-5p or knockdown of Cyb561 suppresses the growth of human AML cells. In conclusion, our findings establish the crucial role of the miR-30e-5p/Cyb561/ROS axis in finely regulating LSC self-renewal, highlighting Cyb561 as a potential therapeutic target for LSC-directed therapies.

Silver Nanowire Reinforced Conductive and Injectable Colloidal Gel for Effective Wound Healing via Electrical Stimulation.

The remarkable biocapacity, injectability, and adaptability of colloidal gels have led to their widespread usage in tissue engineering as irregular defect implants. However, multifunctionalities including electroconductivity and antibacterial property are highly required for colloidal gels. In addition, the inherently weak mechanical property of physically crosslinked colloidal gels limits their application. Herein, we present Ag nanowires (Ag NWs)-reinforced colloidal gels composed of biocompatible gelatin nanoparticles and polydopamine-modified Ag NWs through the controlled electrostatic assembly, which are injectable and conductive. One-dimensional Ag NWs can significantly improve the mechanical and electrical properties of the colloidal gel while maintaining its inherent excellent injectability. Owing to the network of Ag NWs, the storage modulus and conductivity of the optimized Ag NW colloidal gel are 7.5 and 13 times higher, respectively, than those of the colloidal gel made up of polydopamine-modified Ag nanoparticles with equivalent Ag concentration. Furthermore, this Ag NW colloidal gel can adapt to sharp wounds on skin, which accelerates the healing of a MRSA-infected wound via electrical stimulation. This article is protected by copyright. All rights reserved.

Long noncoding RNA TUG1 promotes malignant progression of osteosarcoma by enhancing ZBTB7C expression.

BACKGROUND: Dysregulation of long non-coding RNAs (lncRNAs) is an important component of tumorigenesis. Aberrant expression of lncRNA taurine upregulated gene 1 (lncTUG1) has been reported in various tumors; however, its precise role and key targets critically involved in osteosarcoma (OS) progression remains unclear. METHODS: The expression profiles of lncRNAs and its regulated miRNAs related to OS progression were assessed by bioinformatics analysis and confirmed by qRT-PCR of OS cells. The miRNA targets were identified by transcriptome sequencing and verified by luciferase reporter and RNA pull-down assays. Several in vivo and in vitro approaches, including CCK8 assay, western blot, qRT-PCR, lentiviral transduction and OS cell xenograft mouse model were established to validate the effects of lncTUG1 regulation of miRNA and the downstream target genes on OS cell growth, apoptosis and progression. RESULTS: We found that lncTUG1 and miR-26a-5p were inversely up or down-regulated in OS cells, and siRNA-mediated lncTUG1 knockdown reversed the miR-26a-5p down-regulation and suppressed proliferation and enhanced apoptosis of OS cells. Further, we identified that an oncoprotein ZBTB7C was also upregulated in OS cells that were subjected to lncTUG1/miR-26a-5p regulation. More importantly, ZBTB7C knockdown reduced the ZBTB7C upregulation and ZBTB7C overexpression diminished the anti-OS effects of lncTUG1 knockdown in the OS xenograft model. CONCLUSIONS: Our data suggest that lncTUG1 acts as a miR-26a-5p sponge and promotes OS progression via up-regulating ZBTB7C, and targeting lncTUG1 might be an effective strategy to treat OS.

ZBTB7C m6A modification incurred by METTL3 aberration promotes osteosarcoma progression.

Aberrant N6-methyladenosine (m6A) modification of mRNAs contributes significantly to the epigenetic tumorigenesis, however, its precise role and the key targets in osteosarcoma (OS) are not defined. Here we reported that selective METTL3 (methyltransferase like 3) elevation and the consequential increase of m6A modification causally affect OS progression. The fast-growing OS cells displayed preferential upregulation of METTL3 and increased m6A modification. Conversely, m6A inhibition by 3-deazaadenosine, siRNA-mediated METTL3 knockdown or a METTL3-selective inhibitor STM2457 effectively inhibits OS cell growth and induced OS cell apoptosis. Further investigation revealed that an oncogenic protein ZBTB7C was likely a critical m6A target that mediated the oncogenic effects. ZBTB7C mRNA contains a typical m6A motif of high confidence and its mRNA and protein were enriched with increased m6A modification in OS samples/cells. In an OS xenograft model, STM2457 or siRNA-mediated METTL3 knockdown effectively lowed ZBTB7C abundance. More importantly, the anti-OS effects of STM2457 were significantly reduced when ZBTB7C was overexpressed by lentivirus. Together, our results demonstrate that the METTL3 aberration and the resultant ZBTB7C m6A modification form an important epigenetic regulatory loop that promotes OS progression, and targeting the METTL3/ZBTB7C axis may provide novel insights into the potential strategies for OS therapy.

LepRb+ cell-specific deletion of Slug mitigates obesity and nonalcoholic fatty liver disease in mice.

Leptin exerts its biological actions by activating the long-form leptin receptor (LepRb). LepRb signaling impairment and leptin resistance are believed to cause obesity. The transcription factor Slug - also known as Snai2 - recruits epigenetic modifiers and regulates gene expression by an epigenetic mechanism; however, its epigenetic action has not been explored in leptin resistance. Here, we uncover a proobesity function of neuronal Slug. Hypothalamic Slug was upregulated in obese mice. LepRb+ cell-specific Slug-knockout (SlugΔLepRb) mice were resistant to diet-induced obesity, type 2 diabetes, and liver steatosis and experienced decreased food intake and increased fat thermogenesis. Leptin stimulated hypothalamic Stat3 phosphorylation and weight loss to a markedly higher level in SlugΔLepRb than in Slugfl/fl mice, even before their body weight divergence. Conversely, hypothalamic LepRb+ neuron-specific overexpression of Slug, mediated by AAV-hSyn-DIO-Slug transduction, induced leptin resistance, obesity, and metabolic disorders in mice on a chow diet. At the genomic level, Slug bound to and repressed the LepRb promoter, thereby inhibiting LepRb transcription. Consistently, Slug deficiency decreased methylation of LepRb promoter H3K27, a repressive epigenetic mark, and increased LepRb mRNA levels in the hypothalamus. Collectively, these results unravel what we believe to be a previously unrecognized hypothalamic neuronal Slug/epigenetic reprogramming/leptin resistance axis that promotes energy imbalance, obesity, and metabolic disease.

Injectable magnetic montmorillonite colloidal gel for the postoperative treatment of hepatocellular carcinoma.

Bioactive materials have been extensively developed for the adjuvant therapy of cancer. However, few materials can meet the requirements for the postoperative resection of hepatocellular carcinoma (HCC) due to massive bleeding and high recurrence. In particular, combination therapy for HCC has been highly recommended in clinical practice, including surgical resection, interventional therapy, ablation therapy and chemotherapy. Herein, an injectable magnetic colloidal gel (MCG) was developed by controllable electrostatic attraction between clinically available magnetic montmorillonites and amphoteric gelatin nanoparticles. The optimized MCG exhibited an effective magnetic heating effect, remarkable rheological properties, and high gel network stability, realizing the synergistic treatment of postoperative HCC by stimuli-responsive drug delivery, hemostasis and magnetic hyperthermia. Furthermore, a minimal invasive MCG-induced interventional magnetic hyperthermia therapy (MHT) under ultrasound guidance was realized on hepatic tumor rabbits, providing an alternative therapeutics to treat the postoperative recurrence. Overall, MCG is a clinically available injectable formulation for adjuvant therapy after HCC surgical resection.

Hepatic Slug epigenetically promotes liver lipogenesis, fatty liver disease, and type 2 diabetes.

De novo lipogenesis is tightly regulated by insulin and nutritional signals to maintain metabolic homeostasis. Excessive lipogenesis induces lipotoxicity, leading to nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Genetic lipogenic programs have been extensively investigated, but epigenetic regulation of lipogenesis is poorly understood. Here, we identified Slug as an important epigenetic regulator of lipogenesis. Hepatic Slug levels were markedly upregulated in mice by either feeding or insulin treatment. In primary hepatocytes, insulin stimulation increased Slug expression, stability, and interactions with epigenetic enzyme lysine-specific demethylase-1 (Lsd1). Slug bound to the fatty acid synthase (Fasn) promoter where Slug-associated Lsd1 catalyzed H3K9 demethylation, thereby stimulating Fasn expression and lipogenesis. Ablation of Slug blunted insulin-stimulated lipogenesis. Conversely, overexpression of Slug, but not a Lsd1 binding-defective Slug mutant, stimulated Fasn expression and lipogenesis. Lsd1 inhibitor treatment also blocked Slug-stimulated lipogenesis. Remarkably, hepatocyte-specific deletion of Slug inhibited the hepatic lipogenic program and protected against obesity-associated NAFLD, insulin resistance, and glucose intolerance in mice. Conversely, liver-restricted overexpression of Slug, but not the Lsd1 binding-defective Slug mutant, had the opposite effects. These results unveil an insulin/Slug/Lsd1/H3K9 demethylation lipogenic pathway that promotes NAFLD and type 2 diabetes.

A Cdh1-FoxM1-Apc axis controls muscle development and regeneration.

Forkhead box M1 (FoxM1) transcriptional factor has a principal role in regulating cell proliferation, self-renewal, and tumorigenesis. However, whether FoxM1 regulates endogenous muscle development and regeneration remains unclear. Here we found that loss of FoxM1 in muscle satellite cells (SCs) resulted in muscle atrophy and defective muscle regeneration. FoxM1 functioned as a direct transcription activator of adenomatous polyposis coli (Apc), preventing hyperactivation of wnt/ß-catenin signaling during muscle regeneration. FoxM1 overexpression in SCs promoted myogenesis but impaired muscle regeneration as a result of spontaneous activation and exhaustion of SCs by transcriptional regulation of Cyclin B1 (Ccnb1). The E3 ubiquitin ligase Cdh1 (also termed Fzr1) was required for FoxM1 ubiquitylation and subsequent degradation. Loss of Cdh1 promoted quiescent SCs to enter into the cell cycle and the SC pool was depleted by serial muscle injuries. Haploinsufficiency of FoxM1 ameliorated muscle regeneration of Cdh1 knock-out mice. These data demonstrate that the Cdh1-FoxM1-Apc axis functions as a key regulator of muscle development and regeneration.

Inhibition of Slug effectively targets leukemia stem cells via the Slc13a3/ROS signaling pathway.

Leukemia stem cells (LSCs) are the rare populations of acute myeloid leukemia (AML) cells that are able to initiate, maintain, and propagate AML. Targeting LSCs is a promising approach for preventing AML relapse and improving long-term outcomes. While Slug, a zinc-finger transcription repressor, negatively regulates the self-renewal of normal hematopoietic stem cells, its functions in AML are still unknown. We report here that Slug promotes leukemogenesis and its loss impairs LSC self-renewal and delays leukemia progression. Mechanistically, Slc13a3, a direct target of Slug in LSCs, restricts the self-renewal of LSCs and markedly prolongs recipient survival. Genetic or pharmacological inhibition of SLUG or forced expression of Slc13a3 suppresses the growth of human AML cells. In conclusion, our studies demonstrate that Slug differentially regulates self-renewal of LSCs and normal HSCs, and both Slug and Slc13a3 are potential therapeutic targets of LSCs.

The transcription factor Slug represses p16Ink4a and regulates murine muscle stem cell aging.

Activation of the p16Ink4a-associated senescence pathway during aging breaks muscle homeostasis and causes degenerative muscle disease by irreversibly dampening satellite cell (SC) self-renewal capacity. Here, we report that the zinc-finger transcription factor Slug is highly expressed in quiescent SCs of mice and functions as a direct transcriptional repressor of p16Ink4a. Loss of Slug promotes derepression of p16Ink4a in SCs and accelerates the entry of SCs into a fully senescent state upon damage-induced stress. p16Ink4a depletion partially rescues defects in Slug-deficient SCs. Furthermore, reduced Slug expression is accompanied by p16Ink4a accumulation in aged SCs. Slug overexpression ameliorates aged muscle regeneration by enhancing SC self-renewal through active repression of p16Ink4a transcription. Our results identify a cell-autonomous mechanism underlying functional defects of SCs at advanced age. As p16Ink4a dysregulation is the chief cause for regenerative defects of human geriatric SCs, these findings highlight Slug as a potential therapeutic target for aging-associated degenerative muscle disease.

Application of TALE-Based Approach for Dissecting Functional MicroRNA-302/367 in Cellular Reprogramming.

MicroRNAs are small 18-24 nt single-stranded noncoding RNA molecules involved in many biological processes, including stemness maintenance and cellular reprogramming. Current methods used in loss-of-function studies of microRNAs have several limitations. Here, we describe a new approach for dissecting miR-302/367 functions by transcription activator-like effectors (TALEs), which are natural effector proteins secreted by Xanthomonas and Ralstonia bacteria. Knockdown of the miR-302/367 cluster uses the Kruppel-associated box repressor domain fused with specific TALEs designed to bind the miR-302/367 cluster promoter. Knockout of the miR-302/367 cluster uses two pairs of TALE nucleases (TALENs) to delete the miR-302/367 cluster in human primary cells. Together, both TALE-based transcriptional repressor and TALENs are two promising approaches for loss-of-function studies of microRNA cluster in human primary cells.

CRISPR/Cas9-Mediated Genome Editing Corrects Dystrophin Mutation in Skeletal Muscle Stem Cells in a Mouse Model of Muscle Dystrophy.

Muscle stem cells (MuSCs) hold great therapeutic potential for muscle genetic disorders, such as Duchenne muscular dystrophy (DMD). The CRISP/Cas9-based genome editing is a promising technology for correcting genetic alterations in mutant genes. In this study, we used fibrin-gel culture system to selectively expand MuSCs from crude skeletal muscle cells of mdx mice, a mouse model of DMD. By CRISP/Cas9-based genome editing, we corrected the dystrophin mutation in expanded MuSCs and restored the skeletal muscle dystrophin expression upon transplantation in mdx mice. Our studies established a reliable and feasible platform for gene correction in MuSCs by genome editing, thus greatly advancing tissue stem cell-based therapies for DMD and other muscle disorders.

Selective Expansion of Skeletal Muscle Stem Cells from Bulk Muscle Cells in Soft Three-Dimensional Fibrin Gel.

Muscle stem cells (MuSCs) exhibit robust myogenic potential in vivo, thus providing a promising curative treatment for muscle disorders. Ex vivo expansion of adult MuSCs is highly desired to achieve a therapeutic cell dose because of their scarcity in limited muscle biopsies. Sorting of pure MuSCs is generally required for all the current culture systems. Here we developed a soft three-dimensional (3D) salmon fibrin gel culture system that can selectively expand mouse MuSCs from bulk skeletal muscle preparations without cell sorting and faithfully maintain their regenerative capacity in culture. Our study established a novel platform for convenient ex vivo expansion of MuSCs, thus greatly advancing stem cell-based therapies for various muscle disorders. Stem Cells Translational Medicine 2017;6:1412-1423.

Zfp281 Coordinates Opposing Functions of Tet1 and Tet2 in Pluripotent States.

Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, we performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. We identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. Our study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states.