Specific Gene Silencing Research Articles

PssRNAit: A Web Server for Designing Effective and Specific Plant siRNAs with Genome-Wide Off-Target Assessment.

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit The pssRNAit server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.

The pharyngeal nervous system orchestrates feeding behavior in planarians.

Planarians exhibit traits of cephalization but are unique among bilaterians in that they ingest food by means of goal-directed movements of a trunk-positioned pharynx, following protrusion of the pharynx out of the body, raising the question of how planarians control such a complex set of body movements for achieving robust feeding. Here, we use the freshwater planarian Dugesia japonica to show that an isolated pharynx amputated from the planarian body self-directedly executes its entire sequence of feeding functions: food sensing, approach, decisions about ingestion, and intake. Gene-specific silencing experiments by RNA interference demonstrated that the pharyngeal nervous system (PhNS) is required not only for feeding functions of the pharynx itself but also for food-localization movements of individual animals, presumably via communication with the brain. These findings reveal an unexpected central role of the PhNS in the linkage between unique morphological phenotypes and feeding behavior in planarians.

Deciphering the internalization mechanism of WRAP:siRNA nanoparticles

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases and, indeed, the first therapeutic siRNA was approved by the FDA in 2018. As an alternative to the traditional delivery systems for nucleic acids, peptide-based nanoparticles (PBNs) have been applied successfully for siRNA delivery. Recently, we have developed amphipathic cell-penetrating peptides (CPPs), called WRAP allowing a rapid and efficient siRNA delivery into several cell lines at low doses (20 to 50 nM).In this study, using a highly specific gene silencing system, we aimed to elucidate the cellular uptake mechanism of WRAP:siRNA nanoparticles by combining biophysical, biological, confocal and electron microscopy approaches. We demonstrated that WRAP:siRNA complexes remain fully active in the presence of chemical inhibitors of different endosomal pathways suggesting a direct cell membrane translocation mechanism. Leakage studies on lipid vesicles indicated membrane destabilization properties of the nanoparticles and this was supported by the measurement of WRAP:siRNA internalization in dynamin triple-KO cells. However, we also observed some evidences for an endocytosis-dependent cellular internalization. Indeed, nanoparticles co-localized with transferrin, siRNA silencing was inhibited by the scavenger receptor A inhibitor Poly I and nanoparticles encapsulated in vesicles were observed by electron microscopy in U87 cells.In conclusion, we demonstrate here that the efficiency of WRAP:siRNA nanoparticles is mainly based on the use of multiple internalization mechanisms including direct translocation as well as endocytosis-dependent pathways.

TRNA 2'-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila.

2′-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

Specific Gene Silencing in Leptospira biflexa by RNA-Guided Catalytically Inactive Cas9 (dCas9).

Easy, practical, and affordable gene silencing techniques are constantly progressing, and genetic tools such as TALEs, RNAi, and CRISPR/Cas9 have emerged as new techniques for understanding the basic biology and virulence mechanisms of pathogenic organisms, including bacteria. Here, we describe one-step targeted gene silencing in Leptospira biflexa by using plasmids expressing catalytically inactive Streptococcus pyogenes Cas9 (dCas9) and a single-guide RNA (sgRNA) capable of pairing to the coding strand of a desired gene.

Targeted and direct intracellular delivery of native DNAzymes enables highly specific gene silencing.

DNAzymes exhibit high potential as gene silencing agents for therapeutic applications. Such purposes, however, are significantly challenged by the targeted and successful delivery of unmodified DNAzymes into cells with minimal side effects. Here, we set out to formulate and demonstrate a new stimuli-responsive and constrained aptamer/DNAzyme (Apt/Dz) catenane nanostructure for highly specific gene silencing. The rational design of the Apt/Dz catenane nanostructure with the respective integration of the aptamer sequence and the completely closed catenane format enables both the targeted capability and significantly improved nuclease resistance, facilitating the stable and targeted delivery of unmodified Dz into cancer cells. Moreover, the Dz enzymatic activity in the constrained structure can only be conditionally regulated by the specific intracellular mRNA sequences to silence the target gene with highly reduced side effects. Results show that the Apt/Dz catenane nanostructure can effectively inhibit the expression of the target gene and the proliferation of cancer cells with high specificity.

Glucan-Encapsulated siRNA Particles (GeRPs) for Specific Gene Silencing in Kupffer Cells in Mouse Liver.

Here, we describe a protocol to prepare and administer glucan-encapsulated RNAi particles (GeRPs), for specific delivery of siRNA and subsequent gene silencing in Kupffer cells (KCs) in mice. This technology is based on baker's yeast and allows gene manipulation in macrophages in a tissue-specific manner depending on the route of administration and the model that is used. GeRP administered by intravenous injection in mice are delivered to KCs. Therefore, using the GeRP technology to silence genes provides a unique method to study the function of factors expressed by KCs in the regulation of liver function.

Identification of miRNA precursors in the phloem of Cucurbita maxima.

Plant development and responses to environmental cues largely depend on mobile signals including microRNAs (miRNAs) required for post-transcriptional silencing of specific genes. Short-range cell-to-cell transport of miRNA in developing tissues and organs is involved in transferring positional information essential for determining cell fate. Among other RNA species, miRNAs are found in the phloem sap. Long-distance transport of miRNA via the phloem takes a part in regulation of physiological responses to changing environmental conditions. As shown for regulation of inorganic phosphorus and sulfate homeostasis, mature miRNAs rather than miRNAs precursors are transported in the phloem as signaling molecules. Here, a bioinformatics analysis of transcriptomic data for Cucurbita maxima phloem exudate RNAs was carried out to elucidate whether miRNA precursors could also be present in the phloem. We demonstrated that the phloem transcriptome contained a subset of C. maxima pri-miRNAs that differed from a subset of pri-miRNA sequences abundant in a leaf transcriptome. Differential accumulation of pri-miRNA was confirmed by PCR analysis of C. maxima phloem exudate and leaf RNA samples. Therefore, the presented data indicate that a number of C. maxima pri-miRNAs are selectively recruited to the phloem translocation pathway. This conclusion was validated by inter-species grafting experiments, in which C. maxima pri-miR319a was found to be transported across the graft union via the phloem, confirming the presence of pri-miR319a in sieve elements and showing that phloem miRNA precursors could play a role in long-distance signaling in plants.

Hydrophobically Modified let-7b miRNA Enhances Biodistribution to NSCLC and Downregulates HMGA2 In Vivo

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules.

Targeting WNT10B-Driven Signalling through Induction of FZD6 By Porcupine Inhibition in T Cell Acute Lymphoblastic Leukemia

Background Wnt/Fzd signaling is known to play a pervasive influence in hematopoietic stem cell maintenance, T-cell development in the thymus and function as well as an important role in T-cell acute lymphoblastic leukemia (T-ALL) establishment. We have previously described a recurrent rearrangement involving the WNT10Blocus (WNT10BR) expressing a transcript variant (WNT10BIVS1) in acute myeloid leukemia. To determine the occurrence of this rearrangement in T-ALL we analyzed retrospectively an italian cohort of patients (n=20) and detected the WNT10BRrearrangement with a high prevalence (14/20). We also confirmed the relevance of these findings to human disease, detecting the molecular circuit triggered by the WNT10B over-expression using the MOLT-4 T-ALL cell model.In this report, we examined the expression of components of the Wnt signaling cascade mediated by WNT10B and the effects of specific gene silencing by short hairpin RNA (shRNA) and exposure to the potent PORCN inhibitor (LGK974), or the TGFbRI inhibitor (A83-01) on the WNT10B-mediated Wnt signaling activation. Methods We used the T-ALL model MOLT-4 cell line to assess the WNT10B/FZD signaling axis driven by WNT10BR. In order to identify interaction between WNT10B and FZD receptors we performed in situ proximity ligation assay (PLA) a method used to visualize protein-protein interactions.MOLT4 cells were infected with WNT10B/WNT10BIVS1-shRNA silencing lentiviral vectors versus empty vector control and treated with increased concentration of LGK974 or A83-01, subsequently the effects of pharmacological inhibition on the WNT10B/FZD interactions and on Wnt effector proteins were evaluated by PLA and expression analyses. Cell proliferation and cell death were measured by EdU assay and Annexin-V/Propidium Iodide (PI) analyses. Results We found that WNT10BRdrives Wnt signaling activity in T-ALL through interaction of WNT10B with FZD6 receptor. The effects of WNT10B/FZD6 interaction on Wnt-mediated signal in MOLT-4 were interfered by short hairpin RNAs (shRNAs)-mediated gene silencing and by small molecules-mediated disruption of Wnt-dependent signaling. We performed WNT10BIVS1knockdown or pharmacological inhibition of WNT10B release by the porcupine (PORCN) inhibitor LGK974 and these in turn progressively down-modulate WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. Finally, we induced interference to the WNT10B/FZD6 protein complex formation by exposure to the TGFbRI inhibitor A83-01 via inhibiting FZD6 expression, confirming its role in the WNT10B-mediated signaling activation. Conclusion Our study describes the molecular circuit of WNT10BR-mediated activation and highlight a strategy for a major improvement in T-ALL treatment.By altering FZD6-WNT10B complex formation, may provide the basis for therapeutic strategies to eradicate leukemic stem cells in patients selectively deployed depending on the underlying genetics of disease. Disclosures No relevant conflicts of interest to declare.

Engineered Artificial MicroRNA Precursors Facilitate Cloning and Gene Silencing in Arabidopsis and Rice.

Plant genome sequences are presently deciphered at a staggering speed, due to the rapid advancement of high-throughput sequencing technologies. However, functional genomics significantly lag behind due to technical obstacles related to functional redundancy and mutant lethality. Artificial microRNA (amiRNA) technology is a specific, reversible, and multiplex gene silencing tool that has been frequently used in generating constitutive or conditional mutants for gene functional interrogation. The routine approach to construct amiRNA precursors involves multiple polymerase chain reactions (PCRs) that can increase both time and labor expenses, as well as the chance to introduce sequence errors. Here, we report a simplified method to clone and express amiRNAs in Arabidopsis and rice based on the engineered Arabidopsis miR319a or rice miR528 precursor, which harbor restriction sites to facilitate one-step cloning of a single PCR product. Stem-loop reverse-transcriptase quantitative PCR (RT-qPCR) and functional assays validated that amiRNAs can be accurately processed from these modified precursors and work efficiently in plant protoplasts. In addition, Arabidopsis transgenic plants overexpressing the modified miR319a precursor or its derived amiRNA could exhibit strong gene silencing phenotypes, as expected. The simplified amiRNA cloning strategy will be broadly useful for functional genomic studies in Arabidopsis and rice, and maybe other dicotyledon and monocotyledon species as well.

Therapeutic potential of functionalized siRNA nanoparticles on regression of liver cancer in experimental mice

Short interfering RNA (siRNA) possesses special ability of silencing specific gene. To increase siRNA stability, transportation and its uptake by tumor cells, effective delivery to the appropriate target cells is a major challenge of siRNA-based therapy. In the present study, an effective, safe and biocompatible survivin siRNA encapsulated, GalNAc decorated PEGylated PLGA nanoconjugates (NCs) viz., GalNAc@PEG@siRNA-PLGA were engineered and their synergistic antitumor efficacy was evaluated for targeted delivery in HCC bearing experimental mice. GalNAc@PEG@siRNA-PLGA NCs were characterized for size, bioavailability, toxicity and biocompatibility. Their antitumor potential was evaluated considering gene silencing, apoptosis, histopathology and survival of treated mice. Exceptional accumulation of hepatocytes, reduction in survivin expression and prominent regression in tumor size confirmed the ASGPR-mediated uptake of ligand-anchored NCs and silencing of survivin gene in a targeted manner. Increased DNA fragmentation and potential modulation of caspase-3, Bax and Bcl-2 factors specified the induction of apoptosis that helped in significant inhibition of HCC progression. The potential synchronous and tumor selective delivery of versatile NCs indicated the effective payloads towards the target site, increased apoptosis in cancer cells and improved survival of treated animals.

Expression and Localization Profiles of Rhoptry Proteins in Plasmodium berghei Sporozoites.

In the Plasmodium lifecycle two infectious stages of parasites, merozoites, and sporozoites, efficiently infect mammalian host cells, erythrocytes, and hepatocytes, respectively. The apical structure of merozoites and sporozoites contains rhoptry and microneme secretory organelles, which are conserved with other infective forms of apicomplexan parasites. During merozoite invasion of erythrocytes, some rhoptry proteins are secreted to form a tight junction between the parasite and target cell, while others are discharged to maintain subsequent infection inside the parasitophorous vacuole. It has been questioned whether the invasion mechanisms mediated by rhoptry proteins are also involved in sporozoite invasion of two distinct target cells, mosquito salivary glands and mammalian hepatocytes. Recently we demonstrated that rhoptry neck protein 2 (RON2), which is crucial for tight junction formation in merozoites, is also important for sporozoite invasion of both target cells. With the aim of comprehensively describing the mechanisms of sporozoite invasion, the expression and localization profiles of rhoptry proteins were investigated in Plasmodium berghei sporozoites. Of 12 genes representing merozoite rhoptry molecules, nine are transcribed in oocyst-derived sporozoites at a similar or higher level compared to those in blood-stage schizonts. Immuno-electron microscopy demonstrates that eight proteins, namely RON2, RON4, RON5, ASP/RON1, RALP1, RON3, RAP1, and RAMA, localize to rhoptries in sporozoites. It is noteworthy that most rhoptry neck proteins in merozoites are localized throughout rhoptries in sporozoites. This study demonstrates that most rhoptry proteins, except components of the high-molecular mass rhoptry protein complex, are commonly expressed in merozoites and sporozoites in Plasmodium spp., which suggests that components of the invasion mechanisms are basically conserved between infective forms independently of their target cells. Combined with sporozoite-stage specific gene silencing strategies, the contribution of rhoptry proteins in invasion mechanisms can be described.

Nanoparticle-siRNA: a potential strategy for ovarian cancer therapy?

Ovarian cancer is one of the most common causes of mortality throughout the world. Unfortunately, chemotherapy has failed to cure advanced cancers developing multidrug resistance (MDR). Moreover, it has critical side effects because of nonspecific toxicity. Thanks to specific silencing of oncogenes and MDR-associated genes, nano-siRNA drugs can be a great help address the limitations of chemotherapy. Here, we review the current advances in nanoparticle-mediated siRNA delivery strategies such as polymeric- and lipid-based systems, rigid nanoparticles and nanoparticles coupled to specific ligand systems. Nanoparticle-based codelivery of anticancer drugs and siRNA targeting various mechanisms of MDR is a cutting-edge strategy for ovarian cancer therapy, which is completely discussed in this review.

Jack of Many Trades: The Multifaceted Role of miR528 in Monocots

Jack of Many Trades: The Multifaceted Role of miR528 in Monocots

Lipidoid-polymer hybrid nanoparticles loaded with TNF siRNA suppress inflammation after intra-articular administration in a murine experimental arthritis model

Rheumatoid arthritis (RA) is a common autoimmune disease, which is characterized by painful chronic inflammation in the joints, and novel safe and efficacious treatments are urgently needed. RNA interference (RNAi) therapy based on small interfering RNA (siRNA) is a promising approach for silencing specific genes involved in inflammation. However, delivery of siRNA to the target site, i.e. the cytosol of immune cells, is a challenge. Here, we designed lipid-polymer hybrid nanoparticles (LPNs) composed of lipidoid and poly(DL-lactic-co-glycolic acid) loaded with a therapeutic cargo siRNA directed against the proinflammatory cytokine tumor necrosis factor (TNF), which plays a key role in the progression of RA. We compared their efficacy and safety with reference lipidoid-based stable nucleic acid lipid particles (SNALPs) in vitro and in vivo. Cryogenic transmission electron microscopy, atomic force microscopy and small-angle X-ray scattering revealed that the mode of loading of siRNA in lamellar structures differs between the two formulations. Thus, siRNA was tightly packed in LPNs, while LPNs displayed lower adhesion than SNALPs. The LPNs mediated a higher TNF silencing effect in vitro than SNALPs in the RAW 264.7 macrophage cell line activated with lipopolysaccharide. For both types of delivery systems, macropinocytosis was involved in cellular uptake. In addition, clathrin-mediated endocytosis contributed to uptake of SNALPs. LPNs loaded with TNF siRNA mediated sequence-specific suppression of inflammation in a murine experimental arthritis model upon intra-articular administration. Hence, the present study demonstrates that LPN-mediated TNF knockdown constitutes a promising approach for arthritis therapy of TNF-mediated chronic inflammatory conditions.

Self-assembling Nanotechnology for Cancer Personalized Medicine

domenico.marson@dia.units.itTheranostics is a new field of medicine, which combines specific targeted therapies and diagnostic tests. With a key focus on patient centered care, theranostics provides a transition from conventional medicine to a contemporary personalized and precision medicine approach. The theranostic paradigm in cancer involves nanoscience to unite diagnostic and therapeutic applications to form nanosized agents for diagnosis, drug/gene delivery and treatment response monitoring. These nanocarriers can indeed be engineered to precisely control drug/gene/sensor-release rate and/or target specific organs/tissues within the body with a specific amount of therapeutic/diagnostic agent. In order to fulfill these expectations, any nanovector system must be designed to transport the optimum amount of therapeutic/diagnostic cargo to the desired target site where the active principle is to be released at an optimal rate during a specific time window.Keeping the promises of theranostics is a current formidable challenge in (bio)nanotechnology, and major efforts are devoted to the design of integrated multifunctional and multivalent nanovectors able to provide selective recognition combined with sustained release and/or diagnostic reporting. In this contribution, the pathway leading to two types of nanosystems obtained by exploiting the quintessence of nanotechnology, i.e., the self-assembling process of small, amphiphilic molecules, is reported. Depending on the specific chemistry adopted, these nanomicelles are able to perform specific and effective gene silencing via targeted small interfering RNA (siRNA) delivery, and provide PET images with significantly superior imaging quality relating to sensitivity, specificity and accuracy when compared to the clinical standard [18F]FDG.

Xanthine oxidase-mediated oxidative stress promotes cancer cell-specific apoptosis

The natural compound Alternol was shown to induce profound oxidative stress and apoptotic cell death preferentially in cancer cells. In this study, a comprehensive investigation was conducted to understand the mechanism for Alternol-induced ROS accumulation responsible for apoptotic cell death. Our data revealed that Alternol treatment moderately increased mitochondrial superoxide formation rate, but it was significantly lower than the total ROS positive cell population. Pre-treatment with mitochondria-specific anti-oxidant MitoQ, NOX or NOS specific inhibitors had no protective effect on Alternol-induced ROS accumulation and cell death. However, XDH/XO inhibition by specific small chemical inhibitors or gene silencing reduced total ROS levels and protected cells from apoptosis induced by Alternol. Further analysis revealed that Alternol treatment significantly enhanced XDH oxidative activity and induced a strong protein oxidation-related damage in malignant but not benign cells. Interestingly, benign cells exerted a strong spike in anti-oxidant SOD and catalase activities compared to malignant cells after Alternol treatment. Cell-based protein-ligand engagement and in-silicon docking analysis showed that Alternol interacts with XDH protein on the catalytic domain with two amino acid residues away from its substrate binding sites. Taken together, our data demonstrate that Alternol treatment enhances XDH oxidative activity, leading to ROS-dependent apoptotic cell death.

Evaluation of the therapeutic potential effect of Fas receptor gene knockdown in experimental model of non-alcoholic steatohepatitis

Aim: Stimulation of Fas death receptor is introduced as a major cause of non-alcoholic steatohepatitis (NASH) progression through suppression of cell viability. Therefore, the blocking of death pathways is hypothesised to be express new approaches to NASH therapy. For this purpose, current experiment applied synthetic small interference RNA (SiRNA) to trigger Fas death receptor and to show its potential therapeutic role in designed NASH model.Methods: Male mice were placed on a western diet (WD) for 8 weeks and exposed to cigarette smoke during the last 4 weeks of feeding to induce NASH model. In the next step, Fas SiRNA was injected to mice aiming to examine specific Fas gene silencing, after 8 weeks. As a control, mice received scrambled SiRNA. Reversible possibility of disease was examined by 3 weeks of recovery.Results: Analysis of data is accompanied with the significant histopathological changes (steatosis, ballooning and inflammation), increased lipid profile and hepatic enzyme activities (AST, ALT, ALP) plus TBARS as well as decreased antioxidants levels in NASH model. Upon Fas-SiRNA injection, almost all measured parameters of NASH such as overexpression of Fas receptor, caspase3, NF-kB genes and marked increase of hepatic TNF-α were significantly restored and were remained nearly unchanged following recovery liking as scrambled groups.Conclusions: The suppression of Fas receptor signalling subsequent RNAi therapy may represent an applicable strategy to decline hepatocyte damages and so NASH progression in mice.

Cathepsin B Affects the Activation of Nucleotide-binding Domain and Leucine-rich-repeat-containing Family and Pyrin Domain-containing 3 Inflammasome via Transient Receptor Potential Mucolipin-1

Objective To explore the effects of cathepsin B(CTSB)on the activation of nucleotide-binding domain and leucine-rich-repeat-containing family and pyrin domain-containing 3(NLRP3)inflammasome via transient receptor potential mucolipin-1(TRPML1)in cell oxidative stress model and specific gene silencing cell model. Methods BV2 cells cultured in vivo were treated separately or simultaneously with hydrogen peroxide(H2O2),calcium-sensitive receptor agonist gadolinium trichloride(GdCl3),and CTSB inhibitor CA-074Me,and interleukin-1(IL-1)beta and caspase-1 protein were detected by enzyme-linked immunosorbent assay.The growth activity of BV2 cells in each group was measured by MTT.BV2 cells were treated with different concentrations of H2O2.Cystatin C mRNA and TRPML1 mRNA in BV2 cells were detected by real-time quantitative polymerase chain reaction and the proteins of TRPML1,CTSB,cathepsin D(CTSD),cathepsin L(CTSL)and cathepsin V(CTSV)were detected by Western blot.Specific small interfering RNA was designed for TRPML1 gene target sequence.TRPML1 gene silencing cell lines(named Tr-si-Bv2 cells)were established in BV2 cells and treated with or without H2O2.TRPML1,CTSB and transcription factor EB(TFEB)proteins in Tr-si-Bv2 cells or control cells were detected by Western blot. Results After treatment with H2O2,the expression of caspase-1 protein and NLRP3 mRNA in BV2 cells was increased,and IL-1beta protein in BV2 cells was significantly increased after treatment with GdCl3(P=0.0036).After treatment with CA-074Me,the doses of NLRP3 mRNA(P=0.037),caspase-1(P=0.021),and IL-1β(P= 0.036)were significantly reduced.Cells in the H2O2 group and H2O2+GdCl3 group grew more slowly.The expressions of CTSB mRNA and TRPML1 mRNA,or CTSB and TRPML1 proteins in BV2 cells in the treatment group with 200 μmol/L of H2O2 concentration were similar.H2O2-induced CTSB protein expression was inhibited after silencing TRPML1 gene.The changes of other cathepsins were not affected for the different concentration of H2O2.In the BV2 cells treated with TRPML1 gene silencing,the expression of CTSB protein was significantly reduced and the difference was statistically significant(P=0.021)between the H2O2 +siRNA treatment group and the H2O2 treatment group.<b>Conclusion</b> CTSB regulates the activation of NLRP3 inflammasome in the oxidative stress model of microglia cells,probably mediated by calcium channel protein TRPML1.