Cytosolic microRNA-inducible nuclear translocation of Cas9 protein for disease-specific genome modification.

MicroRNA-dependent mRNA decay plays an important role in gene silencing by facilitating posttranscriptional and translational repression. Inspired by this intrinsic nature of microRNA-mediated mRNA cleavage, here, we describe a microRNA-targeting mRNA as a switch platform called mRNA bridge mimetics to regulate the translocation of proteins. We applied the mRNA bridge mimetics platform to Cas9 protein to confer it the ability to translocate into the nucleus via cleavage of the nuclear export signal. This system performed programmed gene editing in vitro and in vivo. Combinatorial treatment with cisplatin and miR-21-EZH2 axis-targeting CRISPR Self Check-In improved sensitivity to chemotherapeutic drugs in vivo. Using the endogenous microRNA-mediated mRNA decay mechanism, our platform is able to remodel a cell's natural biology to allow the entry of precise drugs into the nucleus, devoid of non-specific translocation. The mRNA bridge mimetics strategy is promising for applications in which the reaction must be controlled via intracellular stimuli and modulates Cas9 proteins to ensure safe genome modification in diseased conditions.

Artificial intelligence-driven new drug discovery targeting serine/threonine kinase 33 for cancer treatment.

BACKGROUND: Artificial intelligence (AI) is capable of integrating a large amount of related information to predict therapeutic relationships such as disease treatment with known drugs, gene expression, and drug-target binding. AI has gained increasing attention as a promising tool for next-generation drug development. METHODS: An AI method was used for drug repurposing and target identification for cancer. Among 8 survived candidates after background checking, N-(1-propyl-1H-1,3-benzodiazol-2-yl)-3-(pyrrolidine-1-sulfonyl) benzamide (Z29077885) was newly selected as an new anti-cancer drug, and the anti-cancer efficacy of Z29077885 was confirmed using cell viability, western blot, cell cycle, apoptosis assay in MDA-MB 231 and A549 in vitro. Then, anti-tumor efficacy of Z29077885 was validated in an in vivo A549 xenograft in BALB/c nude mice. RESULTS: First, we discovered an antiviral agent, Z29077885, as a new anticancer drug candidate using the AI deep learning method. Next, we demonstrated that Z29077885 inhibits Serine/threonine kinase 33 (STK33) enzymatic function in vitro and showed the anticancer efficacy in various cancer cells. Then, we found enhanced apoptosis via S-phase cell cycle arrest as the mechanism underlying the anticancer efficacy of Z29077885 in both lung and breast cancer cells. Finally, we confirmed the anti-tumor efficacy of Z29077885 in an in vivo A549 xenograft. CONCLUSIONS: In this study, we used an AI-driven screening strategy to find a novel anticancer medication targeting STK33 that triggers cancer cell apoptosis and cell cycle arrest at the s phase. It will pave a way to efficiently discover new anticancer drugs.

Exosomal miRNA-19a and miRNA-614 Induced by Air Pollutants Promote Proinflammatory M1 Macrophage Polarization via Regulation of RORα Expression in Human Respiratory Mucosal Microenvironment.

Air pollution exposure leads to various inflammatory diseases in the human respiratory system. Chronic rhinosinusitis is an inflammatory disease caused by viruses, bacteria, or air pollutants. However, the underlying molecular mechanisms through which air particulate matter (PM) causes inflammation and disease remain unclear. In this article, we report that the induction of exosomal microRNAs (miRNAs) from human nasal epithelial cells upon airborne PM exposure promotes proinflammatory M1 macrophage polarization via downregulated RORα expression. Exposure of human nasal epithelial cells to PM results in inflammation-related miRNA expression, and more miRNA is secreted through exosomes delivered to macrophages. Among these, miRNA-19a and miRNA-614 directly bind to the 3'-untranslated region of RORα mRNA and downregulate RORα expression, which leads to inflammation due to inflammatory cytokine upregulation and induces macrophages to a proinflammatory M1-like state. Finally, we showed enhanced expression of miRNA-19a and miRNA-614 but reduced RORα expression in a chronic rhinosinusitis patient tissue compared with the normal. Altogether, our results suggest that PM-induced exosomal miRNAs might play a crucial role in the proinflammatory mucosal microenvironment and macrophage polarization through the regulation of RORα expression.

Identification of XAF1-MT2A mutual antagonism as a molecular switch in cell-fate decisions under stressful conditions.

XIAP-associated factor 1 (XAF1) is a tumor suppressor that is commonly inactivated in multiple human neoplasms. However, the molecular mechanism underlying its proapoptotic function remains largely undefined. Here, we report that XAF1 induction by heavy metals triggers an apoptotic switch of stress response by destabilizing metallothionein 2A (MT2A). XAF1 directly interacts with MT2A and facilitates its lysosomal degradation, resulting in the elevation of the free intercellular zinc level and subsequent activation of p53 and inactivation of XIAP. Intriguingly, XAF1 is activated as a unique transcription target of metal-regulatory transcription factor-1 (MTF-1) in signaling apoptosis, and its protein is destabilized via the lysosomal pathway by MTF-1-induced MT2A under cytostatic stress conditions, indicating the presence of mutual antagonism between XAF1 and MT2A. The antagonistic interplay between XAF1 and MT2A acts as a key molecular switch in MTF-1-mediated cell-fate decisions and also plays an important role in cell response to various apoptotic and survival factors. Wild-type (WT) XAF1 but not MT2A binding-deficient mutant XAF1 increases the free intracellular zinc level and accelerates WT folding of p53 and degradation of XIAP. Consistently, XAF1 evokes a more drastic apoptotic effect in p53+/+ versus isogenic p53-/- cells. Clinically, expression levels of XAF1 and MT2A are inversely correlated in primary colon tumors and multiple cancer cell lines. XAF1-depleted xenograft tumors display an increased growth rate and a decreased apoptotic response to cytotoxic heavy metals with strong MT2A expression. Collectively, this study uncovers an important role for XAF1-MT2A antagonism as a linchpin to govern cell fate under various stressful conditions including heavy metal exposure.

Boiling Histotripsy-induced Partial Mechanical Ablation Modulates Tumour Microenvironment by Promoting Immunogenic Cell Death of Cancers.

Boiling histotripsy is a promising non-invasive High-Intensity Focused Ultrasound (HIFU) technique that employs HIFU mechanical effects to fractionate solid tumours without causing any significant thermal damage. It has been suggested that boiling histotripsy may induce a strong immune response due to the absence of denatured antigenic protein at the HIFU focus. The underlying immunological mechanisms of this technique are, however, poorly understood. In this study, we demonstrated the feasibility of using boiling histotripsy to mechanically fractionate human breast adenocarcinoma cells (MDA-MB-231) and the potential immunological effects induced by boiling histotripsy, for the first time. Our results showed that mechanical stresses produced by boiling histotripsy promote immunogenic cell death of cancer cells via TNF-induced necrosis signaling pathway. This immunogenic cell death significantly increases secretions of damage-associated molecular patterns (CRT, HSP70, HMGB-1), pro-inflammatory cytokines (IFN-γ, IL-1α, IL-1ß, IL-18) and chemokines (IL-8) which are related to M1 macrophage activation. Furthermore, the levels of these signaling proteins increase with the degree of mechanical damage induced by boiling histotripsy. Together, the results presented can suggest that boiling histotripsy could be a potential therapeutic approach for not only mechanically destroying solid tumours (e.g., breast cancer) but also promoting immunogenic cell death via TNF-induced necrosis to trigger antitumour immunity.