NF-κB-Inducing Kinase Provokes Insulin Resistance in Skeletal Muscle of Obese Mice.

Skeletal muscle is crucial for preserving glucose homeostasis. Insulin resistance and abnormalities in glucose metabolism result from a range of pathogenic factors attacking skeletal muscle in obese individuals. To relieve insulin resistance and restore glucose homeostasis, blocking the cell signaling pathways induced by those pathogenic factors seems an attractive strategy. It has been discovered that insulin sensitivity in obese people is inversely linked with the activity of NF-κB inducing kinase (NIK) in skeletal muscle. In order to evaluate NIK's pathological consequences, mechanism of action, and therapeutic values, an obese mouse model reproduced by feeding a high-fat diet was treated with a NIK inhibitor, B022. C2C12 myoblasts overexpressing NIK were utilized to assess insulin signaling and glucose uptake. B022 thus prevented high-fat diet-induced NIK activation and insulin desensitization in skeletal muscle. The insulin signaling in C2C12 myoblasts was compromised by the upregulation of NIK brought on by oxidative stress, lipid deposition, inflammation, or adenoviral vector. This inhibition of insulin action is mostly due to an inhibitory serine phosphorylation of IRS1 caused by ERK, JNK, and PKC that were activated by NIK. In summary, NIK integrates signals from several pathogenic factors to impair insulin signaling by igniting a number of IRS1-inhibiting kinases, and it also has significant therapeutic potential for treating insulin resistance.

Discovery of novel, potent, and orally bioavailable HDACs inhibitors with LSD1 inhibitory activity for the treatment of solid tumors.

Histone deacetylases (HDACs) and lysine-specific demethylase 1 (LSD1) are attractive targets for epigenetic cancer therapy. There is an intimate interplay between the two enzymes. HDACs inhibitors have shown synergistic anticancer effects in combination with LSD1 inhibitors in several types of cancer. Herein, we describe the discovery of compound 5e, a highly potent HDACs inhibitor (HDAC1/2/6/8; IC50 = 2.07/4.71/2.40/107 nM) with anti-LSD1 potency (IC50 = 1.34 µM). Compound 5e exhibited marked antiproliferative activity in several cancer cell lines. 5e effectively induced mitochondrial apoptosis with G2/M phase arrest, inhibiting cell migration and invasion in MGC-803 and HCT-116 cancer cells. It also showed good liver microsomal stability and acceptable pharmacokinetic parameters in SD rats. More importantly, orally administered compound 5e demonstrated higher in vivo antitumor efficacy than SAHA in the MGC-803 (TGI = 71.5%) and HCT-116 (TGI = 57.6%) xenograft tumor models accompanied by good tolerability. This study provides a novel lead compound with dual inhibitory activity against HDACs and LSD1 to further develop epigenetic drugs for solid tumor therapy. Further optimization is needed to improve the LSD1 activity to achieve dual inhibitors with balanced potency on LSD1 and HDACs.

A tRNA-derived fragment of ginseng protects heart against ischemia/reperfusion injury via targeting the lncRNA MIAT/VEGFA pathway.

Traditional Chinese medicines (TCMs) have been widely used for treating ischemic heart disease (IHD), and secondary metabolites are generally regarded as their pharmacologically active components. However, the effects of nucleic acids in TCMs remain unclear. We reported for the first time that a 22-mer double-strand RNA consisting of HC83 (a tRNA-derived fragment [tRF] from the 3' end of tRNAGln(UUG) of ginseng) and its complementary sequence significantly promoted H9c2 cell survival after hypoxia/reoxygenation (H/R) in vitro. HC83_mimic could also significantly improve cardiac function by maintaining both cytoskeleton integrity and mitochondrial function of cardiomyocytes. Further in vivo investigations revealed that HC83_mimic is more potent than metoprolol by >500-fold against myocardial ischemia/reperfusion (MI/R) injury. In-depth studies revealed that HC83 directly downregulated a lncRNA known as myocardial infarction-associated transcript (MIAT) that led to a subsequent upregulation of VEGFA expression. These findings provided the first evidence that TCM-derived tRFs can exert miRNA-like functions in mammalian systems, therefore supporting the idea that TCM-derived tRFs are promising RNA drug candidates shown to have extraordinarily potent effects. In summary, this study provides a novel strategy not only for discovering pharmacologically active tRFs from TCMs but also for efficiently exploring new therapeutic targets for various diseases.

Full-Range Profiling of tRNA Modifications Using LC-MS/MS at Single-Base Resolution through a Site-Specific Cleavage Strategy.

Transfer RNAs (tRNAs) are the most heavily modified RNA species. Liquid chromatography coupled with mass spectrometry (LC-MS/MS) is a powerful tool for characterizing tRNA modifications, which involves pretreating tRNAs with base-specific ribonucleases to produce smaller oligonucleotides amenable to MS. However, the quality and quantity of products from base-specific digestions are severely impacted by the base composition of tRNAs. This often leads to a loss of sequence information. Here, we report a method for the full-range profiling of tRNA modifications at single-base resolution by combining site-specific RNase H digestion with the LC-MS/MS and RNA-seq techniques. The key steps were designed to generate high-quality products of optimal lengths and ionization properties. A linear correlation between collision energies and the m/z of oligonucleotides significantly improved the information content of collision-induced dissociation (CID) spectra. False positives were eliminated by up to 95% using novel inclusion criteria for collecting a census of modifications. This method is illustrated by the mapping of mouse mitochondrial tRNAHis(GUG) and tRNAVal(UAC), which were hitherto not investigated. The identities and locations of the five species of modifications on these tRNAs were fully characterized. This approach is universally applicable to any tRNA species and provides an experimentally realizable pathway to the de novo sequencing of post-transcriptionally modified tRNAs with high sequence coverage.

LC-MS/MS Profiling of Post-Transcriptional Modifications in Ginseng tRNA Purified by a Polysaccharase-Aided Extraction Method.

Transfer RNAs (tRNAs) are the most heavily modified RNA species in life entities. Post-transcriptional modifications severely impact the structure and function of tRNAs. To date, hundreds of modifications have been identified in tRNAs, mainly from microorganisms and animals. However, tRNAs in plant roots or tubers that have been widely used for food and medical purpose for centuries are rarely studied because isolation of RNA from plants still remains a challenge. In this paper, a polysaccharase-aided RNA isolation (PARI) method for extraction of high-quality RNA from plants containing large quantities of polysaccharides is developed. This method presents a new strategy of "digesting" polysaccharides that is completely different from the conventional method of "dissolving" the contaminants. By using this method, RNA of high integrity and purity were successfully extracted from ginseng roots because polysaccharide contaminations were removed efficiently with α-amylase digestion. Ginseng tRNAs were first sequenced by NGS and a total of 41 iso acceptors were identified. ChloroplastictRNAGly(GCC) in ginseng root was purified and four modified nucleosides, including m7G, D, T, and Ψ, were identified by LC-MS/MS. The results also revealed that the m7G occurs at a novel position 18, which may be related to the deformation of D-loop. PARI is the first enzyme-assisted technique for RNA isolation from plants, which could fundamentally solve the problem of polysaccharide contaminations. By using the PARI method, more individual tRNAs could be isolated easily from polysaccharide-rich plant tissues, which would have a positive impact on the feasibility of research on structure and function of tRNA in plants.