Abbreviations: a-SMA, smooth muscle a-actin; Ad-shCtrl, adenoviral vectors expressing the scrambled shRNA; Ad-shMALAT1, adenoviral vectors expressing shRNA against MALAT1. EdU, 5-Ethyny-2 0 -deoxyuridine; Ago2, Argonaute-2; BDL, bile duct ligation; CCl 4 , carbon tetrachloride; ceRNAs, competing endogenous RNAs; Col1a1, collagen type I, a 1; DMEM, Dulbecco's modified Eagle's medium; HSCs, hepatic stellate cells; IgG, immunoglobulin G; IHC, immunohistochemical; lncRNAs, long noncoding RNAs; MALAT1, Metastasis-associated lung adenocarcinoma transcript 1; miR-101, microRNA-101; miR-NC, miR-101b negative control; miRNAs, microRNAs; ncRNAs, non-coding RNAs; qRT-PCR, quantitative real-time PCR; Rac1, RAS-related C3 botulinum substrate 1; siCtrl, scrambled siRNA; siRNAs, small interfering RNAs.Emerging evidence shows that Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a pivotal role in cell proliferation, migration, and invasion in tumors. However, the biological role and underlying mechanism of MALAT1 in liver fibrosis remains undefined. In this study, up-regulation of MALAT1 was observed in fibrotic liver tissues and in activated hepatic stellate cells (HSCs). In addition, depletion of MALAT1 inhibited the activation of HSCs in vitro and attenuated collagen deposits in vivo. Our results demonstrated that MALAT1 expression is negatively correlated with microRNA-101b (miR-101b) expression. Furthermore, there was a negative feedback loop between the levels of MALAT1 and miR-101b. Luciferase reporter assay indicated that MALAT1 and RAS-related C3 botulinum substrate 1 (Rac1) are targets of miR-101b. We uncovered that MALAT1 regulates Rac1 expression through miR-101b as a competing endogenous RNA (ceRNA), thereby influencing the proliferation, cell cycle and activation of primary HSCs. Collectively, The ceRNA regulatory network may prompt a better understanding of liver fibrogenesis and contribute to a novel therapeutic strategy for liver fibrosis.

Doxorubicin is a potent anticancer drug used to treat a variety of cancer types. However, its use is limited by doxorubicin-induced cardiotoxicity (DIC). A missense variant in the RARG gene (S427L; rs2229774) has been implicated in susceptibility to DIC in a genome wide association study. The goal of this study was to investigate the functional role of this RARG variant in Dic. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) from patients treated with doxorubicin. iPSC-CMs from individuals who experienced DIC (cases) showed significantly greater sensitivity to doxorubicin compared to iPSC-CMs from doxorubicin-treated individuals who did not develop DIC (controls) in cell viability and optical mapping experiments. Using CRISPR/Cas9, we generated isogenic cell lines that differed only at the RARG locus. Genetic correction of RARG-S427L to wild type resulted in reduced doxorubicin-induced double stranded DnA breaks, reactive oxygen species production, and cell death. conversely, introduction of RARG-S427L increased susceptibility to doxorubicin. Finally, genetic disruption of the RARG gene resulted in protection from cell death due to doxorubicin treatment. Our findings suggest that the presence of RARG-S427L increases sensitivity to DIC, establishing a direct, causal role for this variant in Dic. Doxorubicin is an anthracycline chemotherapeutic drug used in the treatment of solid organ and hematological malignancies for both adult and pediatric patients 1. Despite its effectiveness as an anti-cancer agent, the use of doxorubicin is limited by cardiotoxicity which affects up to one quarter of patients 2. Doxorubicin-induced cardiotoxicity (DIC) leads to cardiomyopathy with systolic dysfunction, arrhythmia, congestive heart failure, and in some cases death 1,3. Despite extensive investigation, the mechanisms of DIC remain incompletely understood, and we lack the ability to predict this adverse drug reaction in individual patients. Genetic association studies have identified several genetic variants that are associated with DIC 4. However, lack of replication of these findings and the absence of functional validation has precluded their implementation as clinical tests. Among the variants with the strongest genetic evidence is a non-synonymous coding variant (rs2229774, S427L) in the retinoic acid receptor gamma gene, RARG, which was identified in a genome-wide association study (GWAS) of DIC (odds ratio = 4.7, p = 5.9 10 −8) 5. However, the directionality of this effect is controversial 6 , and genetic association studies cannot prove a causal relationship between a genetic locus and the phenotype of interest. As a consequence, direct functional assessment of the role of this variant in DIC is essential.

Endomembrane specialization allows functional compartmentalization but imposes physical constraints to information flow within the cell. However, the evolution of an endomembrane system was associated with the emergence of contact sites facilitating communication between membrane-bound organelles. Contact sites between the endoplasmic reticulum (ER) and mitochondria are highly conserved in terms of their morphological features but show surprising molecular diversity within and across eukaryote species. ER-mitochondria contact sites are thought to regulate key processes in oncogenesis but their molecular composition remains poorly characterized in mammalian cells. In this study, we investigate the localization of pannexin 2 (Panx2), a membrane channel protein showing tumor-suppressing properties in cancer cells. Using a combination of subcellular fractionation, particle tracking in live-cell, and immunogold electron microscopy, we show that Panx2 localizes at ER-mitochondria contact sites in mammalian cells and sensitizes cells to apoptotic stimuli.

Mast cells are important immune cells that have significant roles in mediating allergy and asthma. Therefore, studying the molecular mechanisms regulating these and other processes in mast cells is important to elucidate. Methods such as lipofection, transduction, and electroporation are often employed to dissect these mechanisms by disrupting gene expression in mast cell lines. However, as with other leukocytes, human mast cells (HMCs) are often refractory to the delivery of plasmids by lipofection. In this study, we investigated the utility of lipid nanoparticles (LNPs) containing the ionizable cationic lipids 1,2-dioleoyloxy-3-dimethylaminopropane, 1,2-dioleyloxy-3-dimethylaminopropane, or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane for the delivery of plasmid DNA into HMC lines. Herein, we demonstrate for the first time the use of LNPs to achieve significant and reproducible levels of plasmid DNA transfection in HMC-1.2 and laboratory of allergic diseases 2 (LAD2) cells. These levels reached 53.2% and 16.0% in HMC-1.2 and LAD2 cells, respectively; and outperformed Lipofectamine 3000 in both cases. Moreover, cell viability in the transfected cells remained above 65% for all LNP conditions tested. Together, these observations illustrate the efficacy of this technique for mast cell researchers and further support the use of LNPs for nucleic acid delivery into leukocytes.