siRNA Constructs Research Articles

PB2171: THE NOVEL CSN5/JAB1 INHIBITOR CSN5I-3 SUPPRESSES CELL GROWTH AND UPREGULATES GENE EXPRESSION OF TYPE-I INTERFERONS (IFN) IN ANAPLASTIC LARGE CELL LYMPHOMA (ALCL)

Background: The CSN5/Jab1 (cJun activation domain-binding protein 1), initially discovered as a cJun coactivator, represents the fifth component of an evolutionary highly conserved 8 subunit protein complex, named COP9 signalosome (CSN5). Accumulating evidence suggests that the CSN5/Jab1 gene operates as an oncogene in cancer through multiple mechanisms including cell cycle control via CDK inhibitors. We have previously shown that CSN5/Jab1 protein is overexpressed in T-cell lymphomas with the highest frequency of positive tumors being observed in both ALK+ and ALK- anaplastic large cell lymphomas (ALCL). Targeting CSN5/Jab1 activity in cancer is now possible since a novel inhibitor, namely CSN5i-3 (Novartis), has been developed for clinical use. The potential role of CSN5/Jab1 in modulation of anti-tumor immune responses in cancer, and particularly in ALCL, is unknown to date. Aims: To investigate the effects of the selective CSN5i-3 inhibitor in cell growth and type-I interferon - associated anti-tumor immune responses in ALCL. Methods: The ALCL in vitro system included 5 cell lines (3 ALK+ and 2 ALK- ALCL cell lines). Cell growth and viability were assessed by trypan blue and MTT assays. The effects of the CSN5i-3 on G1-S cell cycle progression were assessed by BrdU incorporation assay and on apoptosis by Annexin V/AAD staining and flow cytometry. Expression of various cell cycle regulating proteins at baseline and experimental conditions were analysed by Westem blot analysis. Gene expression of type I interferons, including interferonbeta (IFN-β), CXCL10 and interferon gamma (IFN-y), as well as, STING and a control gene (GAPDH) at the RNA level, were analysed with qRT-PCR. The ALCL cell lines were treated with the specific CSN5i-3 inhibitor at different concentrations. Silencing of the CSN5/Jab1 gene was performed using transient transfection with specific siRNA construct using the Amaxa Nucleofector System (Lonza). Results: Treatment of ALCL cell lines with increasing concentrations of CSN5i-3 resulted in decreased protein levels of CSN5/Jab1, which was associated with significantly decreased cell growth but slight increase in apoptosis (10-15%). The effects on cell growth linked to downregulation of cell cycle progression were associated with upregulation of the CDK inhibitors p21, p27 and p57. Similarly, CSN5/Jab1 gene silencing resulted in decreased cell growth associated with increased expression of similar cell cycle inhibiting proteins. In addition, inhibition of CSN5/Jab1 activity by CSN5i-3 resulted in increased IFN-β and IFN-y mRNA levels in ALK+ ALCL cells, and CXCL10, IFN-y and STING mRNA levels in ALK- ALCL cells. Similarly, knocking down CSN5/Jab1 gene by transient transfection with siRNA led to upregulation of type-I interferons, particularly IFN-β and CXCL10 in ALCL, suggesting that the effects of CSN5i-3 can be attributed to specific inhibition of CSN5/Jab1 activity. Summary/Conclusion: The novel inhibitor CSN5i-3 sufficiently downregulates expression of CSN5/Jab1 oncogene, inhibits cell cycle progression and contributes to type-I interferon - associated anti-tumor immune response in ALK+ and ALK- ALCL. These preclinical findings suggest a potential role of CSN5i-3 in molecular therapeutics and immunotherapy in T-cell lymphomas.

Virus-like siRNA construct dynamically responsive to sequential microenvironments for potent RNA interference

Cytoplasmic transportation of therapeutic nucleic acids is deemed as an onerous task with aim of precise knockdown towards the targeted genes. Pertaining to the programed functionalities of natural virus in circumventing the biological barriers, we tailored multifaceted chemistries into manufacture of synthetic siRNA delivery vehicles in resembling the functionalities of viral vectors to dynamically tackle with a sequential of biological obstacles encountered in the journey of systemic anti-tumor RNAi therapy. Once harnessing ligands with RGD motif for specific internalization into subcellular endosomal compartments of the tumor cells, the architecture of the proposed delivery vehicles was subjected to facile transformation responsive to pH stimuli in acidic endosomal compartments. The external biocompatible PEGylation palisade was consequently detached, unveiling the cytomembrane-lytic cationic components to commit disruptive potencies to the anionic endosomal membranes for translocation of siRNA conjugates into cytosol. Eventually, liberation of active siRNA could be accomplished due to its responsiveness to the strikingly high level of glutathione in cytosol, thereby contributing to potent RNAi. Hence, our elaborated virus-mimicking platform has demonstrated significant anti-tumor efficacy through systemic administration of anti-angiogenic RNAi payloads, which inspired prosperous potentials in a variety of therapeutic applications.

M5C-Related lncRNA Predicts Lung Adenocarcinoma and Tumor Microenvironment Remodeling: Computational Biology and Basic Science.

Purpose: Epigenetic RNA modification regulates gene expression post-transcriptionally. The aim of this study was to construct a prognostic risk model for lung adenocarcinoma (LUAD) using long non-coding RNAs (lncRNAs) related to m5C RNA methylation. Method: The lncRNAs regulated by m5C methyltransferase were identified in TCGA-LUAD dataset using Pearson correlation analysis (coefficient > 0.4), and clustered using non-negative matrix decomposition. The co-expressing gene modules were identified by WGCNA and functionally annotated. The prognostically relevant lncRNAs were screened by LASSO regression and a risk model was constructed. LINC00628 was silenced in the NCI-H460 and NCI-H1299 cell lines using siRNA constructs, and migration and invasion were assessed by the Transwell and wound healing assays respectively. Results: We identified 185 m5C methyltransferase-related lncRNAs in LUAD, of which 16 were significantly associated with overall survival. The lncRNAs were grouped into two clusters on the basis of m5C pattern, and were associated with significant differences in overall and disease-free survival. GSVA revealed a close relationship among m5C score, ribosomes, endolysosomes and lymphocyte migration. Using LASSO regression, we constructed a prognostic signature consisting of LINC00628, LINC02147, and MIR34AHG. The m5C-lncRNA signature score was closely related to overall survival, and the accuracy of the predictive model was verified by the receiver operating characteristic curve and decision curve analysis. Knocking down LINC00628 in NCI-H460 and NCI-H1299 cells significantly reduced their migration and invasion compared to that of control cells. Conclusion: We constructed a prognostic risk model of LUAD using three lncRNAs regulated by m5C methyltransferase, which has potential clinical value.

Circular RNA Profiling Identifies Novel circPPARA that Promotes Intramuscular Fat Deposition in Pigs.

Intramuscular fat (IMF) content plays an important role in pork quality. Circular RNAs (circRNAs) implicate various biological processes; however, the regulatory mechanisms and functions of circRNAs in porcine IMF remains elusive. Hence, the study assessed the circRNA expression profiling in the longissimus dorsi muscle of pigs with high (H) and low (L) IMF content to unravel their regulatory functions in improving meat quality. The RNA sequencing analysis identified 29,732 circRNAs from six sampled pigs, most of which were exon-derived. In the muscle, 336 were differentially expressed (DE) between the H and L IMF groups; 196 circRNAs were upregulated, and 140 were downregulated. Subsequent qRT-PCR validation of 10 DE circRNAs revealed expression patterns consistent with the RNA-seq data. Gene ontology and KEGG enrichment analysis revealed that most significantly enriched DE circRNAs' host genes were linked to lipid metabolism and adipogenesis processes. The circRNA-miRNA regulatory network analysis found several circRNAs targeting miRNAs associated with adipogenesis. Finally, a novel circRNA, circPPARA, was identified with the expression positively correlated with the IMF content. Detailed analysis revealed that circPPARA was formed via head-to-tail splicing and was more stable than the linear PPARA, predominantly located in the cytoplasm. Functional studies using overexpression and siRNA constructs demonstrated that circPPARA promotes differentiation and hinders the proliferation of porcine intramuscular preadipocytes. Moreover, the dual-luciferase assay revealed that circPPARA adsorbed miR-429 and miR-200b, thereby promoting intramuscular adipogenesis in pigs. Our results identified a candidate circRNA, circPPARA, that affects porcine IMF content. The study provides knowledge of the regulatory functions of circRNAs in intramuscular adipogenesis and abundant resource for future research on circRNAs in pigs.

Knockdown of capsid protein encoding novel ATPase domain inhibits genome packaging in potato leafroll virus.

Potato leafroll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the CP with ATPase domain exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. In addition, molecular docking study reveals that the PLRV CP protein has ATP-binding pocket at the interface of each monomer. This further confirms that knockdown of the CP harboring ATP-binding domain could hamper the process of viral genome packaging and assembly. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses.

RNAi Mediated Silencing of ftf1 Gene Enhances Resistance in Banana to Fusarium Wilt

Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is a devastating disease of banana. Race 1 of Fusarium wilt is a serious threat to Musa spp. cv. Rasthali (AAB, Silk subgroup), which is a choice variety traditionally grown in most of the South East Asian countries. The investigation was an attempt to study whether RNAi-mediated expression of small interfering RNAs (siRNAs) targeted against ftf1 (fusarium transcription factor1) gene in transgenic banana could impart resistance against Foc race 1. Partial sequences comprising sense and antisense sequences of ftf1 gene were assembled as siRNA construct in a binary vector background and transformed into embryogenic cell suspensions of banana cv. Rasthali by Agrobacterium mediated genetic transformation. Presence of RNAi construct in transgenic banana plants were confirmed by polymerase chain reaction assay. Forty- six transformed lines derived from RNAi-FTF1 were found to be free of external symptoms of Foc after 3 months of challenge inoculation. Seventeen transgenic lines continued to resist the Foc race 1 even after six months of post inoculation. The present study confirmed that the host induced RNAi-mediated gene silencing of a fungal gene could confer resistance against Foc in banana.

Targeting the splicing factor NONO inhibits GBM progression through GPX1 intron retention.

Background: Splicing factors are essential for nascent pre-mRNA processing and critical in cancer progression, suggesting that proteins with splicing functions represent potential molecular targets for cancer therapy. Here, we investigate the role of splicing factors in glioblastoma multiforme (GBM) progression and the possibility of targeting them for the treatment of the disease.Methods: The TCGA and CGGA public databases were used to screen for differentially expressed mRNA splicing factors. Immunohistochemistry and qRT-PCR were used to analyze the expression of non-POU domain-containing octamer-binding protein (NONO), a Drosophila behavior human splicing (DBHS) protein. Knockdown/overexpression of NONO with siRNA and lentiviral expression constructs was used to examine cell growth, apoptosis, and invasion in GBM cells. RNA sequencing was used to identify potential downstream molecular targets of NONO. RIP-PCR and RNA pulldown were used to determine the interaction between NONO and pre-mRNA. JC-1 staining and the seahorse assay were performed to assess redox homeostasis.Results: Expression of NONO was increased in GBM samples and associated with poor survival in patients (P = 0.04). Knockdown of NONO suppressed GBM growth, and overexpression of NONO promoted GBM tumorigenesis in vitro and in vivo. RNA sequencing-based transcriptomic profiling confirmed that knockdown of NONO in U251 and P3 cells resulted in global intron retention of pre-mRNA and led to abnormal splicing of specific pre-mRNAs for GPX1 and CCN1. NONO bound to a consensus motif in the intron of GPX1 pre-mRNA in association with another DBHS protein family member, PSPC1. Knockdown of NONO impaired tumor growth, invasion, and redox homeostasis through aberrant splicing of GPX1. Finally, Auranofin, a small molecule inhibitor of NONO, suppressed GBM tumor growth in an orthotopic xenograft model in mice.Conclusions: We demonstrated that intron retention was a critical alternative RNA splicing event to occur in GBM progression, and that NONO was a key regulator of mRNA splicing in GBM. Targeting NONO represents a novel, potential therapeutic strategy for GBM treatment.

The lncRNA VPS9D1-AS1 Promotes Hepatocellular Carcinoma Cell Cycle Progression by Regulating the HuR/CDK4 Axis.

Long noncoding RNAs (lncRNAs) represent promising therapeutic targets associated with hepatocellular carcinoma (HCC). lncRNA VPS9D1 antisense RNA 1 (VPS9D1-AS1) regulates colon and prostate cancer, but its relevance in HCC remains to be clarified. Using microarray data from the NCBI Gene Expression Omnibus (GEO) database (GSE65485) and The Cancer Genome Atlas (TCGA) database, VPS9D1-AS1 expression in HCC and normal liver tissue sample HCC were compared. Relative lncRNA expression was also measured through real-time quantitative PCR (qPCR) in 80 pairs of HCC tumor and paracancerous tissues and in human HCC cell lines. VPS9D1-AS1 knockdown was achieved by transfecting these HCC cells with a specific siRNA construct in vitro, and the proliferation of these cells was quantified through cell proliferation assays and colony formation assays, while flow cytometry was employed to assess their cell cycle progression. The role of the VPS9D1-AS1 lncRNA as a regulator of HCC tumorigenesis was also assessed in vivo by subcutaneously implanting BALB/c nude mice with HepG2 cells stably expressing either sh-VPS9D1-AS1 or a control shRNA construct. Mechanistic analyses were additionally conducted by examining in vitro CDK4 and HuR expression through western blotting and qPCR. VPS9D1-AS1 expression was significantly increased in HCC tissues in the analyzed databases and our independent tissue samples. Elevated VPS9D1-AS1 expression was related to larger tumor size and more advanced tumor, node, metastasis (TNM) stage, and HCC patients expressing higher levels of this lncRNA exhibited poorer survival outcomes. Knocking down VPS9D1-AS1 impaired the proliferative and colony formation activity of HepG2 cells while promoting their apoptotic death. Consistently, VPS9D1-AS1 silencing suppressed HCC tumor growth in vivo. Mechanistically, VPS9D1-AS1 was able to bind to the HuR protein and thereby influence the stability and expression of the CDK4 mRNA, thus impacting HCC cell proliferation. The VPS9D1-AS1/HuR/CDK4 signaling axis regulates HCC tumor cell oncogenic activity, highlighting this pathway as a promising therapeutic target.

Abstract P498: Polycystin-1 Regulates Excitation-contraction Coupling In Atrial Cardiomyocytes

Patients with Autosomal Dominant Polycystic Kidney disease (ADPKD) have multiple cardiovascular manifestations, including increased susceptibility to arrhythmias. Mutations in polycystin-1 (PC1) encoding gene accounts for 85% cases of ADPKD, whereas mutations in polycystin-2 (PC2) only accounts for 15%. In kidney cells, PC1 interacts with PC2 to form a protein complex at the primary cilia to regulate calcium influx via PC2. However, cardiomyocytes are non-ciliated cells and the role of both PC1 and PC2 in atrial cardiomyocytes remains unknown. We have previously demonstrated that PC1 regulates action potentials and calcium handling to fine-tune ventricular cardiomyocyte contraction. Here, we hypothesize that PC1 regulates action potentials and calcium handling in atrial cardiomyocytes independent of PC2 actions. To test this hypothesis, we differentiated human induced pluripotent stem cells (iPSC) into atrial cardiomyocytes (iPSC-aCM) using previously published protocols. To determine the contribution of PC1/PC2 in atrial excitation-contraction coupling, protein expression was knocked down utilizing specific siRNA constructs, for each protein, or a universal control siRNA transfected using lipofectamine RNAiMAX. We measured action potentials using the potentiometric dye FluoVolt and intracellular calcium with Fura-2 AM or Fluo-4. Changes in fluorescence were monitored using a multiwavelength IonOptix system. iPSC-aCM were paced at 2 Hz to synchronize the beating pattern using field electrical stimulation. Our data shows that PC1 ablation significantly decreased action potential duration at 50% and 80% of repolarization, by 24% and 23%, respectively. Moreover, we observed that PC1 knockdown significantly reduced calcium transient amplitude elicited by field electrical stimulation without changes in calcium transient decay. Interestingly, PC2 knockdown did not modify calcium transients in atrial cardiomyocytes (iPSC-aCM). Our data suggest that PC1 regulates atrial excitation-contraction coupling independent of PC2 actions. This study warrants further investigation into atrial dysfunction in ADPKD patients with PC1 mutations.

Expression Silencing of Glutathione Peroxidase 4 in Mouse Erythroleukemia Cells Delays In Vitro Erythropoiesis.

Among the eight human glutathione peroxidase isoforms, glutathione peroxidase 4 (GPX4) is the only enzyme capable of reducing complex lipid peroxides to the corresponding alcohols. In mice, corruption of the Gpx4 gene leads to embryonic lethality and more detailed expression silencing studies have implicated the enzyme in several physiological processes (e.g., embryonal cerebrogenesis, neuronal function, male fertility). Experiments with conditional knockout mice, in which expression of the Gpx4 gene was silenced in erythroid precursors, indicated a role of Gpx4 in erythropoiesis. To test this hypothesis in a cellular in vitro model we transfected mouse erythroleukemia cells with a Gpx4 siRNA construct and followed the expression kinetics of erythropoietic gene products. Our data indicate that Gpx4 is expressed at high levels in mouse erythroleukemia cells and that expression silencing of the Gpx4 gene delays in vitro erythropoiesis. However, heterozygous expression of a catalytically inactive Gpx4 mutant (Gpx4+/Sec46Ala) did not induce a defective erythropoietic phenotype in different in vivo and ex vivo models. These data suggest that Gpx4 plays a role in erythroid differentiation of mouse erythroleukemia cells but that heterozygous expression of a catalytically inactive Gpx4 is not sufficient to compromise in vivo and ex vivo erythropoiesis.

Tumor-Targeted Anti-VEGF RNAi Capable of Sequentially Responding to Intracellular Microenvironments for Potent Systemic Tumor Suppression.

Selective intracellular transportation of RNA interference (RNAi) into the cytosol of tumor cells is deemed as an intriguing strategy for treatment of intractable tumors. Pertaining to sequential biological barriers, polymeric RNAi therapeutics were engineered by covalent conjugation of multiple small interference RNA (siRNA) molecules onto a polylysine (PLys) segment of cyclic Arg-Gly-Asp (RGD)-poly(ethylene glycol)-block-PLys [RGD-PEG-PLys(siRNA)] through a redox-responsive disulfide linkage. Furthermore, the constructed polyanionic siRNA conjugates were designed to precipitate with inorganic CaPO3 (CaP) for manufacturing siRNA delivery nanoassemblies. The subsequent investigations validated their appreciable colloidal stability in physiological conditions. Moreover, the RGD ligand facilitated cellular endocytosis in cancerous cells, and internalized nanoassemblies could readily dissociate in the acidic endosomal microenvironment due to CaP dissolution. Simultaneously, the elevated osmotic pressure owing to CaP dissolution provoked disruption of endosomes, thereby accounting for release of RGD-PEG-PLys(siRNA) into the cytosol. Eventually, the disulfide linkage in RGD-PEG-PLys(siRNA) could cleave in the reducing cytoplasmic microenvironment, eliciting siRNA liberation for RNAi. Ultimately, the proposed siRNA constructs, attempting to encapsulate antiangiogenic RNAi payloads, exhibited potent in vivo RNAi to the targeted glioma cells and antitumor efficacy via systemic administration.

Inhibition of potato leafroll virus multiplication and systemic translocation by siRNA constructs against putative ATPase fold of movement protein

Viruses cause many severe plant diseases, resulting in immense losses of crop yield worldwide. Therefore, developing novel approaches to control plant viruses is crucial to meet the demands of a growing world population. Recently, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virion particles is completed inside the host plant, mature virions or sometimes naked viral genomes spread cell-to-cell through plasmodesmata by interacting with the virus-encoded movement protein (MP). We used the RNAi approach to suppress MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with MP siRNA constructs exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of MP gene expression is an efficient method for generating PLRV-resistant potato plants. Further, we identified novel ATPase motifs in MP that may be involved in DNA binding and translocation through plasmodesmata. We also showed that the ATPase activity of MP was stimulated in the presence of DNA/RNA. Overall, our findings provide a robust technology to generate PLRV-resistant potato plants, which can be extended to other species. Moreover, this approach also contributes to the study of genome translocation mechanisms of plant viruses.

Knockout of miR-21-5p alleviates cartilage matrix degradation by targeting Gdf5 in temporomandibular joint osteoarthritis.

AimsThe study aimed to determine whether the microRNA miR21-5p (MiR21) mediates temporomandibular joint osteoarthritis (TMJ-OA) by targeting growth differentiation factor 5 (Gdf5).MethodsTMJ-OA was induced in MiR21 knockout (KO) mice and wild-type (WT) mice by a unilateral anterior crossbite (UAC) procedure. Mouse tissues exhibited histopathological changes, as assessed by: Safranin O, toluidine blue, and immunohistochemistry staining; western blotting (WB); and quantitative real-time polymerase chain reaction (RT-qPCR). Mouse condylar chondrocytes were transfected with a series of MiR21 mimic, MiR21 inhibitor, Gdf5 siRNA (si-GDF5), and flag-GDF5 constructs. The effects of MiR-21 and Gdf5 on the expression of OA related molecules were evaluated by immunofluorescence, alcian blue staining, WB, and RT-qPCR.ResultsUAC altered the histological structure and extracellular matrix content of cartilage in the temporomandibular joint (TMJ), and KO of MiR21 alleviated this effect (p < 0.05). Upregulation of MiR21 influenced the expression of TMJ-OA related molecules in mandibular condylar chondrocytes via targeting Gdf5 (p < 0.05). Gdf5 overexpression significantly decreased matrix metalloproteinase 13 (MMP13) expression (p < 0.05) and reversed the effects of MiR21 (p < 0.05).ConclusionMiR21, which acts as a critical regulator of Gdf5 in chondrocytes, regulates TMJ-OA related molecules and is involved in cartilage matrix degradation, contributing to the progression of TMJ-OA.Cite this article: Bone Joint Res 2020;9(10):689–700.

MiR-548a-3p Weakens the Tumorigenesis of Colon Cancer Through Targeting TPX2.

Background: MicroRNA (miRNA) has been verified a significant factor to participate in the progression of colon cancer (CC). In this study, the authors investigated the mechanism and function of miR-548a-3p in CC. Materials and Methods: Bioinformatics analysis was used to analyze the mRNA expression profile and miRNA expression profile from GEO data series. The expression of miRNA and mRNA was analyzed by real-time quantification polymerase chain reaction in 43 pairs of CC clinical tissue samples and CC cells. The Western blot assay was used to detect the TPX2 protein. Then, SW480 and HCT116 cells were stably transfected with miR-548a-3p mimic, miR-548a-3p inhibitor, TPX2 overexpression, and TPX2 siRNA constructs to study the effects of miR-548a-3p and TPX2. Cellular functional experiments included cell counting kit-8 assay, BrdU incorporation assay, and wound healing assay. In addition, luciferase reporter assay was applied to detect the regulatory association between miR-548a-3p and TPX2. Results: TPX2 and miR-548a-3p were identified as the interested mRNA and miRNA by microarray analysis. In CC tissues and cell lines, miR-548a-3p with low expression and TPX2 with high expression were observed. What's more, exogenous overexpressed miR-548a-3p impaired the cell viability, cell proliferation, and cell migration, while TPX2 overexpression enhanced the malignancy phenotypes. However, the promotion effect of TPX2 on CC cells was impaired by miR-548a-3p. Conclusion: This study revealed that miR-548a-3p attenuated the development of CC by targeting TPX2.

LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling.

Controlled cell growth and proliferation are essential for tissue homeostasis and development. Wnt and Hippo signaling are well known as positive and negative regulators of cell proliferation, respectively. The regulation of Hippo signaling by the Wnt pathway has been shown, but how and which components of Wnt signaling are involved in the activation of Hippo signaling during nutrient starvation are unknown. Here, we report that a reduction in the level of low-density lipoprotein receptor-related protein 6 (LRP6) during nutrient starvation induces phosphorylation and cytoplasmic localization of YAP, inhibiting YAP-dependent transcription. Phosphorylation of YAP via loss of LRP6 is mediated by large tumor suppressor kinases 1/2 (LATS1/2) and Merlin. We found that O-GlcNAcylation of LRP6 was reduced, and the overall amount of LRP6 was decreased via endocytosis-mediated lysosomal degradation during nutrient starvation. Merlin binds to LRP6; when LRP6 is less O-GlcNAcylated, Merlin dissociates from it and becomes capable of interacting with LATS1 to induce phosphorylation of YAP. Our data suggest that LRP6 has unexpected roles as a nutrient sensor and Hippo signaling regulator.

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.

Cullin-7 (CUL7) is overexpressed in glioma cells and promotes tumorigenesis via NF-\u03baB activation

BackgroundCullin-7 (CUL7) is a member of the DOC domain-containing cullin family and is involved in the regulation of cell transformation. However, the clinical significance, potential mechanism and upstream regulators of CUL7 in malignant gliomas remain to be determined.MethodsExpression level data and clinical information were obtained via the Cancer Genome Atlas (TCGA) database, the Chinese Glioma Genome Atlas (CGGA) database, immunohistochemistry (IHC) and western blot analysis. Gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of CUL7. RNA silencing was performed using siRNA or lentiviral constructs in U87MG and U251 glioma cell lines and GSC267 glioma stem cells. CUL7 overexpression was performed using the GV141-CUL7 plasmid construct. In addition, overexpression of miR-3940-5p was performed and validated by quantitative real-time PCR (qRT-PCR). Cells were characterized in vitro or in vivo to evaluate their molecular status, cell proliferation, invasion, and migration by Cell Counting Kit (CCK)-8, EdU, flow cytometry, colony formation, Transwell and 3D tumour spheroid invasion assays. Coimmunoprecipitation (co-IP) and western blotting were performed to test the mechanisms of activation of the NF-κB signalling pathway.ResultsHigh CUL7 expression was associated with a high tumour grade, a mesenchymal molecular glioma subtype and a poor prognosis in patients. Gene silencing of CUL7 in U87MG and U251 cells significantly inhibited tumour growth, invasion and migration in vitro and in vivo. Western blot analysis revealed that cyclin-dependent kinase inhibitors and epithelial-mesenchymal transition (EMT) molecular markers changed under CUL7 silencing conditions. In contrast, CUL7 overexpression promoted tumour growth, invasion and migration. Gene set enrichment analysis (GSEA) and western blot analysis revealed that CUL7 was positively associated with the NF-κB pathway. Moreover, with coimmunoprecipitation assays, we discovered that CUL7 physically associated with MST1, which further led to ubiquitin-mediated MST1 protein degradation, which promoted activation of the NF-κB signalling pathway. Finally, CUL7 was found to be downregulated by miR-3940-5p, which suppressed the development of gliomas.ConclusionsOur findings indicate that CUL7 plays a significant role in promoting tumorigenesis via NF-κB activation and that it can be negatively regulated by miR-3940-5p in human gliomas. Furthermore, CUL7 might be a candidate molecular target for the treatment of glioma.

MiR-187* Enhances SiHa Cervical Cancer Cell Oncogenicity Via Suppression of WWOX.

Cervical cancer is one of the most common cancers worldwide and a major cause of cancer-related mortality among women. Previous studies have reported that microRNA-miR-187*, which is one of the non-coding parts of the genome producing small conserved ribonucleic acids, is associated with various cancers. In this study, we explored the function of miR-187* in cervical cancer cells. miR-187* mimic, WWOX reporter constructs, siRNA and overexpression constructs were transfected into SiHa cells to investigate the function and regulatory mechanisms of miR-187*. Exogenous miR-187* was found to increase the oncogenic phenotypes of SiHa cells. The tumor suppressor gene WWOX is a novel target of miR-187* in SiHa cells. WWOX siRNA suppressed endogenous WWOX expression and increased the oncogenic phenotypes of SiHa cells. Exogenous WWOX expression was able to suppress the oncogenic phenotypes of SiHa cells and rescue cells from miR-187*-induced migration. miR-187* seems to enhance SiHa cervical cancer cell oncogenicity via suppression of the WWOX pathway.

Inhibition of PHLDA2 transcription by DNA methylation and YY1 in goat placenta.

Pleckstrin homology-like domain family A member 2 (PHLDA2) is essential for placental development in mammals. This study was conducted to investigate transcriptional regulation of goat PHLDA2 in the placenta. Real-time PCR and Western blot analyses showed different expression of the PHLDA2 in goat placentas during gestation with highest expression at 30 and 45days post coitus (P<0.05). Luciferase reporter assays demonstrated the highest promoter activity in the region of -1023/+20 (P<0.05). A CpG island was defined within -631/+379 region, where lower level of CpG-methylation was detected with bisulfite sequencing PCR in the placenta than that in the spleen and liver (P<0.05). Meanwhile, in vitro experiments showed that 5-AzaC enhanced the gene expression in a dose-dependent manner. Site-directed mutation in vitro demonstrated that transcription factor Ying-yang 1 (YY1) had an inhibitory effect on the PHLDA2 expression, and the inhibition was further confirmed with overexpression and siRNA constructs of YY1. ChIP and RE-ChIP analyses further identified the binding of YY1 to the PHLDA2 promoter by interaction with histone deacetylase 1 (HDAC1) and HDAC3. This study uncovers the negative regulation of the CpG-methylation and YY1 on goat PHLDA2 expression. YY1 prefers binding to CpG-methylation sequences, and inhibits goat PHLDA2 expression via recruiting HDAC1 and 3.

Cell-to-Cell Variation in Gene Expression for Cultured Human Cells Is Controlled in Trans by Diverse Genes: Implications for the Pathobiology of Aging.

Cell-to-cell variation in gene expression increases among homologous cells within multiple tissues during aging. We call this phenomenon variegated gene expression (VGE). Long, healthy life requires robust and coordinated gene expression. We posit that nature may have evolved VGE as a bet-hedging mechanism to protect reproductively active populations. The price we may pay is accelerated aging. That hypothesis will require the demonstration that genetic loci are capable of modulating degrees of VGE. While loci controlling VGE in yeast and genes controlling interindividual variation in gene expression in Caenorhabditis elegans have been identified, there has been no compelling evidence for the role of specific genetic loci in modulations of VGE of specific targets in humans. With the assistance of a core facility, we used a customized library of siRNA constructs to screen 1,195 human genes to identify loci contributing to the control of VGE of a gene with relevance to the biology of aging. We identified approximately 50 loci controlling VGE of the prolongevity gene, SIRT1. Because of its partial homology to FOXO3A, a variant of which is enriched in centenarians, our laboratory independently confirmed that the knockdown of FOXF2 greatly diminished VGE of SIRT1 but had little impact upon the VGE of WRN. While the role of these VGE-altering genes on aging in vivo remains to be determined, we hypothesize that some of these genes can be targeted to increase functionality during aging.