Delivery of Lipid Nanoparticles with ROS Probes for Improved Visualization of Hepatocellular Carcinoma.

Reactive oxygen species (ROS) are highly reactive products of the cell metabolism derived from oxygen molecules, and their abundant level is observed in many diseases, particularly tumors, such as hepatocellular carcinoma (HCC). In vivo imaging of ROS is a necessary tool in preclinical research to evaluate the efficacy of drugs with antioxidant activity and for diagnosis and monitoring of diseases. However, most known sensors cannot be used for in vivo experiments due to low stability in the blood and rapid elimination from the body. In this work, we focused on the development of an effective delivery system of fluorescent probes for intravital ROS visualization using the HCC model. We have synthesized various lipid nanoparticles (LNPs) loaded with ROS-inducible hydrocyanine pro-fluorescent dye or plasmid DNA (pDNA) with genetically encoded protein sensors of hydrogen peroxide (HyPer7). LNP with an average diameter of 110 ± 12 nm, characterized by increased stability and pDNA loading efficiency (64 ± 7%), demonstrated preferable accumulation in the liver compared to 170 nm LNPs. We evaluated cytotoxicity and demonstrated the efficacy of hydrocyanine-5 and HyPer7 formulated in LNP for ROS visualization in mouse hepatocytes (AML12 cells) and in the mouse xenograft model of HCC. Our results demonstrate that obtained LNP could be a valuable tool in preclinical research for visualization ROS in liver diseases.

Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

Ratiometric i-Motif-Based Sensor for Precise Long-Term Monitoring of pH Micro Alterations in the Nucleoplasm and Interchromatin Granules.

DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau1/2 <1 s for 7.2 → 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.

Intravital electrochemical nanosensor as a tool for the measurement of reactive oxygen/nitrogen species in liver diseases.

Reactive oxygen/nitrogen species (ROS/RNS) are formed during normal cellular metabolism and contribute to its regulation, while many pathological processes are associated with ROS/RNS imbalances. Modern methods for measuring ROS/RNS are mainly based on the use of inducible fluorescent dyes and protein-based sensors, which have several disadvantages for in vivo use. Intravital electrochemical nanosensors can be used to quantify ROS/RNS with high sensitivity without exogenous tracers and allow dynamic ROS/RNS measurements in vivo. Here, we developed a method for quantifying total ROS/RNS levels in the liver and evaluated our setup in live mice using three common models of liver disease associated with ROS activation: acute liver injury with CCl4, partial hepatectomy (HE), and induced hepatocellular carcinoma (HCC). We have demonstrated using intravital electrochemical detection that any exposure to the peritoneum in vivo leads to an increase in total ROS/RNS levels, from a slight increase to an explosion, depending on the procedure. Analysis of the total ROS/RNS level in a partial hepatectomy model revealed oxidative stress, both in mice 24 h after HE and in sham-operated mice. We quantified dose-dependent ROS/RNS production in CCl4-induced injury with underlying neutrophil infiltration and cell death. We expect that in vivo electrochemical measurements of reactive oxygen/nitrogen species in the liver may become a routine approach that provides valuable data in research and preclinical studies.

Murine Falcor/LL35 lncRNA Contributes to Glucose and Lipid Metabolism In Vitro and In Vivo.

Glucose and lipid metabolism are crucial functional systems in eukaryotes. A large number of experimental studies both in animal models and humans have shown that long non-coding RNAs (lncRNAs) play an important role in glucose and lipid metabolism. Previously, human lncRNA DEANR1/linc00261 was described as a tumor suppressor that regulates a variety of biological processes such as cell proliferation, apoptosis, glucose metabolism and tumorigenesis. Here we report that murine lncRNA Falcor/LL35, a proposed functional analog of human DEANR1/linc00261, is predominantly expressed in murine normal hepatocytes and downregulated in HCC and after partial hepatectomy. The application of high-throughput approaches such as RNA-seq, LC-MS proteomics, lipidomics and metabolomics analysis allowed changes to be found in the transcriptome, proteome, lipidome and metabolome of hepatocytes after LL35 depletion. We revealed that LL35 is involved in the regulation of glycolysis and lipid biosynthesis in vitro and in vivo. Moreover, LL35 affects Notch and NF-κB signaling pathways in normal hepatocytes. All observed changes result in the decrease in the proliferation and migration of hepatocytes. We demonstrated similar phenotype changes between murine LL35 and human linc00261 depletion in vitro and in vivo that opens the opportunity to translate results for LL35 from a liver murine model to possible functions of human lncRNA linc00261.

Effect of Surface Modification of Multifunctional Nanocomposite Drug Delivery Carriers with DARPin on Their Biodistribution In Vitro and In Vivo.

We present a targeted drug delivery system for therapy and diagnostics that is based on a combination of contrasting, cytotoxic, and cancer-cell-targeting properties of multifunctional carriers. The system uses multilayered polymer microcapsules loaded with magnetite and doxorubicin. Loading of magnetite nanoparticles into the polymer shell by freezing-induced loading (FIL) allowed the loading efficiency to be increased 5-fold, compared with the widely used layer-by-layer (LBL) assembly. FIL also improved the photoacoustic signal and particle mobility in a magnetic field gradient, a result unachievable by the LBL alone. For targeted delivery of the carriers to cancer cells, the carrier surface was modified with a designed ankyrin repeat protein (DARPin) directed toward the epithelial cell adhesion molecule (EpCAM). Flow cytometry measurements showed that the DARPin-coated capsules specifically interacted with the surface of EpCAM-overexpressing human cancer cells such as MCF7. In vivo and ex vivo biodistribution studies in FvB mice showed that the carrier surface modification with DARPin changed the biodistribution of the capsules toward epithelial cells. In particular, the capsules accumulated substantially in the lungs─a result that can be effectively used in targeted lung cancer therapy. The results of this work may aid in the further development of the "magic bullet" concept and may bring the quality of personalized medicine to another level.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA-protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2'-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA-protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA-protein interactions because of high selectivity of cysteine trapping.

Reactive oxygen species-responsive RNA interference.

Due to oxidative instability, arylboronic acids are not compatible with the solid-phase synthesis of nucleic acids. We solved this problem and, based on these findings, developed siRNA prodrugs activated in the presence of reactive oxygen species (ROS) in vivo. These prodrugs can be used for specific targeting of ROS-rich cancer cells.

Modulation of RNA Splicing by Oligonucleotides: Mechanisms of Action and Therapeutic Implications.

Dysregulation of RNA splicing causes many diseases and disorders. Several therapeutic approaches have been developed to correct aberrant alternative splicing events for the treatment of cancers and hereditary diseases, including gene therapy and redirecting splicing, using small molecules or splice switching oligonucleotides (SSO). Significant advances in the chemistry and pharmacology of nucleic acid have led to the development of clinically approved SSO drugs for the treatment of spinal muscular dystrophy and Duchenne muscular dystrophy (DMD). In this review, we discuss the mechanisms of SSO action with emphasis on "less common" approaches to modulate alternative splicing, including bipartite and bifunctional SSO, oligonucleotide decoys for splice factors and SSO-mediated mRNA degradation via AS-NMD and NGD pathways. We briefly discuss the current progress and future perspectives of SSO therapy for rare and ultrarare diseases.

Antitumor efficacy of multi-target in situ vaccinations with CpG oligodeoxynucleotides, anti-OX40, anti-PD1 antibodies, and aptamers.

To overcome immune tolerance to cancer, the immune system needs to be exposed to a multi-target action intervention. Here, we investigated the activating effect of CpG oligodeoxynucleotides (ODNs), mesyl phosphoramidate CpG ODNs, anti-OX40 antibodies, and OX40 RNA aptamers on major populations of immunocompetent cells ex vivo. Comparative analysis of the antitumor effects of in situ vaccination with CpG ODNs and anti-OX40 antibodies, as well as several other combinations, such as mesyl phosphoramidate CpG ODNs and OX40 RNA aptamers, was conducted. Antibodies against programmed death 1 (PD1) checkpoint inhibitors or their corresponding PD1 DNA aptamers were also added to vaccination regimens for analytical purposes. Four scenarios were considered: a weakly immunogenic Krebs-2 carcinoma grafted in CBA mice; a moderately immunogenic Lewis carcinoma grafted in C57Black/6 mice; and an immunogenic A20 B cell lymphoma or an Ehrlich carcinoma grafted in BALB/c mice. Adding anti-PD1 antibodies (CpG+αOX40+αPD1) to in situ vaccinations boosts the antitumor effect. When to be used instead of antibodies, aptamers also possess antitumor activity, although this effect was less pronounced. The strongest effect across all the tumors was observed in highly immunogenic A20 B cell lymphoma and Ehrlich carcinoma.

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads.

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.

Level of Murine DDX3 RNA Helicase Determines Phenotype Changes of Hepatocytes In Vitro and In Vivo.

DDX3 RNA helicase is intensively studied as a therapeutic target due to participation in the replication of some viruses and involvement in cancer progression. Here we used transcriptome analysis to estimate the primary response of hepatocytes to different levels of RNAi-mediated knockdown of DDX3 RNA helicase both in vitro and in vivo. We found that a strong reduction of DDX3 protein (>85%) led to similar changes in vitro and in vivo-deregulation of the cell cycle and Wnt and cadherin pathways. Also, we observed the appearance of dead hepatocytes in the healthy liver and a decrease of cell viability in vitro after prolonged treatment. However, more modest downregulation of the DDX3 protein (60-65%) showed discordant results in vitro and in vivo-similar changes in vitro as in the case of strong knockdown and a different phenotype in vivo. These results demonstrate that the level of DDX3 protein can dramatically influence the cell phenotype in vivo and the decrease of DDX3, for more than 85% leads to cell death in normal tissues, which should be taken into account during the drug development of DDX3 inhibitors.

Magnetic Nanoparticles as a Tool for Remote DNA Manipulations at a Single-Molecule Level.

Remote control of cells and single molecules by magnetic nanoparticles in nonheating external magnetic fields is a perspective approach for many applications such as cancer treatment and enzyme activity regulation. However, the possibility and mechanisms of direct effects of small individual magnetic nanoparticles on such processes in magneto-mechanical experiments still remain unclear. In this work, we have shown remote-controlled mechanical dissociation of short DNA duplexes (18-60 bp) under the influence of nonheating low-frequency alternating magnetic fields using individual 11 nm magnetic nanoparticles. The developed technique allows (1) simultaneous manipulation of millions of individual DNA molecules and (2) evaluation of energies of intermolecular interactions in short DNA duplexes or in other molecules. Finally, we have shown that DNA duplexes dissociation is mediated by mechanical stress and produced by the movement of magnetic nanoparticles in magnetic fields, but not by local overheating. The presented technique opens a new avenue for high-precision manipulation of DNA and generation of biosensors for quantification of energies of intermolecular interaction.

Identification of a long non-coding RNA regulator of liver carcinoma cell survival.

Genomic studies have significantly improved our understanding of hepatocellular carcinoma (HCC) biology and have led to the discovery of multiple protein-coding genes driving hepatocarcinogenesis. In addition, these studies have identified thousands of new non-coding transcripts deregulated in HCC. We hypothesize that some of these transcripts may be involved in disease progression. Long non-coding RNAs are a large class of non-coding transcripts which participate in the regulation of virtually all cellular functions. However, a majority of lncRNAs remain dramatically understudied. Here, we applied a pooled shRNA-based screen to identify lncRNAs essential for HCC cell survival. We validated our screening results using RNAi, CRISPRi, and antisense oligonucleotides. We found a lncRNA, termed ASTILCS, that is critical for HCC cell growth and is overexpressed in tumors from HCC patients. We demonstrated that HCC cell death upon ASTILCS knockdown is associated with apoptosis induction and downregulation of a neighboring gene, protein tyrosine kinase 2 (PTK2), a crucial protein for HCC cell survival. Taken together, our study describes a new, non-coding RNA regulator of HCC.

Multifunctional nanostructured drug delivery carriers for cancer therapy: Multimodal imaging and ultrasound-induced drug release.

Development of multimodal systems for therapy and diagnosis of neoplastic diseases is an unmet need in oncology. The possibility of simultaneous diagnostics, monitoring, and therapy of various diseases allows expanding the applicability of modern systems for drug delivery. We have developed hybrid particles based on biocompatible polymers containing magnetic nanoparticles (MNPs), photoacoustic (MNPs), fluorescent (Cy5 or Cy7 dyes), and therapeutic components (doxorubicin). To achieve high loading efficiency of MNP and Dox to nanostructured carriers, we utilized a novel freezing-induced loading technique. To reduce the systemic toxicity of antitumor drugs and increase their therapeutic efficacy, we can use targeted delivery followed by the remote control of drug release using high intensity-focused ultrasound (HIFU). Loading of MNPs allowed performing magnetic targeting of the carriers and enhanced optoacoustic signal after controlled destruction of the shell and release of therapeutics as well as MRI imaging. The raster scanning optoacoustic mesoscopy (PA, RSOM), MRI, and fluorescent tomography (FT) confirmed the ultrasound-induced release of doxorubicin from capsules: in vitro (in tubes and pieces of meat) and in vivo (after delivery to the liver). Disruption of capsules results in a significant increase of doxorubicin and Cy7 fluorescence initially quenched by magnetite nanoparticles that can be used for real-time monitoring of drug release in vivo. In addition, we explicitly studied cytotoxicity, intracellular localization, and biodistribution of these particles. Elaborated drug delivery carriers have a good perspective for simultaneous imaging and focal therapy of different cancer types, including liver cancer.

Panel of potential lncRNA biomarkers can distinguish various types of liver malignant and benign tumors.

PURPOSE: Liver cancers are among the deadliest malignancies due to a limited efficacy of early diagnostics, the lack of appropriate biomarkers and insufficient discrimination of different types of tumors by classic and molecular methods. In this study, we searched for novel long non-coding RNA (lncRNA) as well as validated several known candidates suitable as probable biomarkers for primary liver tumors of various etiology. METHODS: We described a novel lncRNA HELIS (aka "HEalthy LIver Specific") and estimated its expression by RT-qPCR in 82 paired tissue samples from patients with hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), combined HCC-CCA, pediatric hepatoblastoma (HBL) and non-malignant hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH). Additionally, we examined expression of cancer-associated lncRNAs HULC, MALAT1, UCA1, CYTOR, LINC01093 and H19, which were previously studied mainly in HCC. RESULTS: We demonstrated that down-regulation of HELIS strongly correlates with carcinogenesis; whereas in tumors with non-hepatocyte origin (HBL, CCA) or in a number of poorly differentiated HCC, this lncRNA is not expressed. We showed that recently discovered LINC01093 is dramatically down-regulated in all malignant liver cancers; while in benign tumors LINC01093 expression is just twice decreased in comparison to adjacent samples. CONCLUSION: Our study revealed that among all measured biomarkers only down-regulated HELIS and LINC01093, up-regulated CYTOR and dysregulated HULC are perspective for differential diagnostics of liver cancers; whereas others demonstrated discordant results and cannot be considered as potential universal biomarkers for this purpose.

Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors.

BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

Genomic DNA i-motifs as fast sensors responsive to near-physiological pH microchanges.

We report the design of robust sensors for measuring intracellular pH, based on the native DNA i-motifs (iMs) found in neurodegeneration- or carcinogenesis-related genes. Those iMs appear to be genomic regulatory elements and might modulate transcription in response to pH stimuli. Given their intrinsic sensitivity to minor pH changes within the physiological range, such noncanonical DNA structures can be used as sensor core elements without additional modules other than fluorescent labels or quenchers. We focused on several iMs that exhibited fast folding/unfolding kinetics. Using stopped-flow techniques and FRET-melting/annealing assays, we confirmed that the rates of temperature-driven iM-ssDNA transitions correlate with the rates of the pH-driven transitions. Thus, we propose FRET-based hysteresis analysis as an express method for selecting sensors with desired kinetic characteristics. For the leading fast-response sensor, we optimized the labelling scheme and performed intracellular calibration. Unlike the commonly used small-molecule pH indicators, that sensor was transferred efficiently to cell nuclei. Considering its favourable kinetic characteristics, the sensor can be used for monitoring proton dynamics in the nucleus. These results argue that the 'genome-inspired' design is a productive approach to the development of biocompatible molecular tools.

Upregulation of Mcl-1S Causes Cell-Cycle Perturbations and DNA Damage Accumulation.

As an important regulator of apoptosis, Mcl-1 protein, a member of the Bcl-2 family, represents an attractive target for cancer treatment. The recent development of novel small molecule compounds has allowed Mcl-1-inhibitory therapy to proceed to clinical trials in cancer treatment. However, the possible adverse effects of either direct inhibition of Mcl-1 or upregulation of Mcl-1S, proapoptotic isoform resulting from alternative splicing of Mcl-1, remain unclear. Here, we investigated changes in Mcl-1S levels during cell cycle and the cell cycle-related functions of Mcl-1 isoforms to address the above-mentioned concerns. It was shown that an anti-mitotic agent monastrol caused accumulation of Mcl-1S mRNA, although without increasing the protein level. In contrast, both mRNA and protein levels of Mcl-1S accrued during the premitotic stages of the normal cell cycle progression. Importantly, Mcl-1S was observed in the nuclear compartment and an overexpression of Mcl-1S, as well as knockdown of Mcl-1, accelerated the progression of cells into mitosis and resulted in DNA damage accumulation. Surprisingly, a small molecule inhibitor of Mcl-1, BH3-mimetic S63845, did not affect the cell cycle progression or the amount of DNA damage. In general, upregulated Mcl-1S protein or genetically inhibited Mcl-1L were associated with the cell cycle perturbations and DNA damage accumulation in normal and cancer cells. At the same time, BH3-mimetic to Mcl-1 did not affect the cell cycle progression, suggesting that direct inhibition of Mcl-1 is devoid of cell-cycle related undesired effects.

Murine Long Noncoding RNA Morrbid Contributes in the Regulation of NRAS Splicing in Hepatocytes In Vitro.

The coupling of alternative splicing with the nonsense-mediated decay (NMD) pathway maintains quality control of the transcriptome in eukaryotes by eliminating transcripts with premature termination codons (PTC) and fine-tunes gene expression. Long noncoding RNA (lncRNA) can regulate multiple cellular processes, including alternative splicing. Previously, murine Morrbid (myeloid RNA repressor of Bcl2l11 induced death) lncRNA was described as a locus-specific controller of the lifespan of short-living myeloid cells via transcription regulation of the apoptosis-related Bcl2l11 protein. Here, we report that murine Morrbid lncRNA in hepatocytes participates in the regulation of proto-oncogene NRAS (neuroblastoma RAS viral oncogene homolog) splicing, including the formation of the isoform with PTC. We observed a significant increase of the NRAS isoform with PTC in hepatocytes with depleted Morrbid lncRNA. We demonstrated that the NRAS isoform with PTC is degraded via the NMD pathway. This transcript is presented almost only in the nucleus and has a half-life ~four times lower than other NRAS transcripts. Additionally, in UPF1 knockdown hepatocytes (the key NMD factor), we observed a significant increase of the NRAS isoform with PTC. By a modified capture hybridization (CHART) analysis of the protein targets, we uncovered interactions of Morrbid lncRNA with the SFPQ (splicing factor proline and glutamine rich)-NONO (non-POU domain-containing octamer-binding protein) splicing complex. Finally, we propose the regulation mechanism of NRAS splicing in murine hepatocytes by alternative splicing coupled with the NMD pathway with the input of Morrbid lncRNA.