Targeted silencing of GNAS in a human model of osteoprogenitor cells results in the deregulation of the osteogenic differentiation program.

Introduction: The dysregulation of cell fate toward osteoprecursor cells associated with most GNAS-based disorders may lead to episodic de novo extraskeletal or ectopic bone formation in subcutaneous tissues. The bony lesion distribution suggests the involvement of abnormal differentiation of mesenchymal stem cells (MSCs) and/or more committed precursor cells. Data from transgenic mice support the concept that GNAS is a crucial factor in regulating lineage switching between osteoblasts (OBs) and adipocyte fates. The mosaic nature of heterotopic bone lesions suggests that GNAS genetic defects provide a sensitized background for ectopic osteodifferentiation, but the underlying molecular mechanism remains largely unknown. Methods: The effect of GNAS silencing in the presence and/or absence of osteoblastic stimuli was evaluated in the human L88/5 MSC line during osteodifferentiation. A comparison of the data obtained with data coming from a bony lesion from a GNAS-mutated patient was also provided. Results: Our study adds some dowels to the current fragmented notions about the role of GNAS during osteoblastic differentiation, such as the premature transition of immature OBs into osteocytes and the characterization of the differences in the deposed bone matrix. Conclusion: We demonstrated that our cell model partially replicates the in vivo behavior results, resulting in an applicable human model to elucidate the pathophysiology of ectopic bone formation in GNAS-based disorders.

Growth inhibition of subcutaneous tumor with glioma cells in nude mice by silencing TLR4.

This study investigated the regulatory impact of Toll-like receptor 4 (TLR4) gene on glioma cell proliferation and apoptosis, elucidating the molecular mechanisms underlying TLR4-induced growth inhibition in vivo. U-87MG-Sh and U-87MG-NC cells, with silenced TLR4 and negative control plasmid respectively, were established. Eighteen nude mice, divided into transfection, negative control, and blank control groups, were inoculated with corresponding cells. Over four weeks, the transfection group exhibited significantly reduced tumor growth rates, smaller mass and volume, and lower growth activity compared to controls. Histological analysis revealed sparse tumor cells, increased fibrous connective tissue, and slower angiogenesis in the transfection group. Flow cytometry demonstrated a lower proliferation index and increased G0/1 cell count in the transfection group. mRNA levels of TLR4, NF-κB, and CyclinD1 were significantly lower in the transfection group. TLR4 silencing correlated with U-87MG cell proliferation regulation, growth inhibition, NF-κB and CyclinD1 modulation, and induction of cell cycle arrest and apoptosis. These findings suggest TLR4 as a potential gene therapy target for glioma.

Molecular cloning, identification, transcriptional analysis, and silencing of enolase on the life cycle of Haemaphysalis longicornis (Acari, Ixodidae) tick.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

shRNA-mediated gene silencing of HDAC11 empowers CAR-T cells against prostate cancer.

Epigenetic mechanisms are involved in several cellular functions, and their role in the immune system is of prime importance. Histone deacetylases (HDACs) are an important set of enzymes that regulate and catalyze the deacetylation process. HDACs have been proven beneficial targets for improving the efficacy of immunotherapies. HDAC11 is an enzyme involved in the negative regulation of T cell functions. Here, we investigated the potential of HDAC11 downregulation using RNA interference in CAR-T cells to improve immunotherapeutic outcomes against prostate cancer. We designed and tested four distinct short hairpin RNA (shRNA) sequences targeting HDAC11 to identify the most effective one for subsequent analyses. HDAC11-deficient CAR-T cells (shD-NKG2D-CAR-T) displayed better cytotoxicity than wild-type CAR-T cells against prostate cancer cell lines. This effect was attributed to enhanced activation, degranulation, and cytokine release ability of shD-NKG2D-CAR-T when co-cultured with prostate cancer cell lines. Our findings reveal that HDAC11 interference significantly enhances CAR-T cell proliferation, diminishes exhaustion markers PD-1 and TIM3, and promotes the formation of T central memory TCM populations. Further exploration into the underlying molecular mechanisms reveals increased expression of transcription factor Eomes, providing insight into the regulation of CAR-T cell differentiation. Finally, the shD-NKG2D-CAR-T cells provided efficient tumor control leading to improved survival of tumor-bearing mice in vivo as compared to their wild-type counterparts. The current study highlights the potential of HDAC11 downregulation in improving CAR-T cell therapy. The study will pave the way for further investigations focused on understanding and exploiting epigenetic mechanisms for immunotherapeutic outcomes.

Silencing PCMT1 enhances the sensitivity of breast cancer cells to paclitaxel through the PI3K/Akt/STMN1 pathway.

This study aimed to investigate whether silencing Protein L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) expression can enhance the sensitivity of breast cancer cells to paclitaxel and its possible mechanism. Tumor tissues and adjacent histologically normal tissues were collected from patients with breast cancer admitted to our hospital. Human normal breast epithelial cells MCF10A, human breast cancer cells MCF-7, and paclitaxel-resistant breast cancer cells MCF-7/PR were purchased. MCF-7/PR cells were further grouped into negative control (NC) group, si-PCMT1 group (transfected with si-PCMT1), 740Y-P group (treated with 740Y-P, an activator of phosphatidylinositol 3-kinase (PI3K)/ v-Akt Murine Thymoma Viral Oncogene (AKT) signaling pathway), and si-PCMT1 + 740Y-P group (transfected with si-PCMT1 and then treated with 740Y-P). The expression level of PCMT1 in tissues and cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot analysis was used to detect the protein expression level of PCMT1 in tissues and cells as well as the protein level of p-PI3K, PI3K, p-Akt, Akt, and Stathmin1 (STMN1) in cells. 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and colony formation assays were used to determine cell viability, scratch assay was used to assess the migration ability of cells, and Transwell assay was used to assess the invasion ability of cells. The expression of PCMT1 was remarkably up-regulated in breast cancer tissues and MCF-7/PR cells. Silencing PCMT1 expression significantly inhibited the proliferation, migration, and invasion of MCF-7/PR cells, and alleviated the resistance of cancer cells to paclitaxel. Additionally, silencing PCMT1 expression also inhibited the activation of PI3K/Akt/STMN1 pathway in MCF-7/PR cells, while activating PI3K/Akt/STMN1 pathway significantly reversed the effect of silencing PCMT1 expression on MCF-7/PR cells. PCMT1 is highly expressed in breast cancer tissues and MCF-7/PR cells, and silencing PCMT1 expression can not only inhibit the development of breast cancer but also enhance paclitaxel sensitivity. Its mechanism of action may be achieved by inhibiting PI3K/Akt/STMN1 signaling.

Pepper immunity against Ralstonia solanacearum is positively regulated by CaWRKY3 through modulation of different WRKY transcription factors.

BACKGROUND: Several WRKY transcription factors (TFs), including CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 are known to govern the resistance of pepper (Capsicum annuum L.) plants to Ralstonia solanacearum infestation (RSI) and other abiotic stresses. However, the molecular mechanisms underlying these processes remain elusive. METHODS: This study functionally described CaWRKY3 for its role in pepper immunity against RSI. The roles of phytohormones in mediating the expression levels of CaWRKY3 were investigated by subjecting pepper plants to 1 mM salicylic acid (SA), 100 µM methyl jasmonate (MeJA), and 100 µM ethylene (ETH) at 4-leaf stage. A virus-induced gene silencing (VIGS) approach based on the Tobacco Rattle Virus (TRV) was used to silence CaWRKY3 in pepper, and transiently over-expressed to infer its role against RSI. RESULTS: Phytohormones and RSI increased CaWRKY3 transcription. The transcriptions of defense-associated marker genes, including CaNPR1, CaPR1, CaDEF1, and CaHIR1 were decreased in VIGS experiment, which made pepper less resistant to RSI. Significant hypersensitive (HR)-like cell death, H2O2 buildup, and transcriptional up-regulation of immunological marker genes were noticed in pepper when CaWRKY3 was transiently overexpressed. Transcriptional activity of CaWRKY3 was increased with overexpression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40, and vice versa. In contrast, Pseudomonas syringae pv tomato DC3000 (Pst DC3000) was easily repelled by the innate immune system of transgenic Arabidopsis thaliana that overexpressed CaWRKY3. The transcriptions of defense-related marker genes like AtPR1, AtPR2, and AtNPR1 were increased in CaWRKY3-overexpressing transgenic A. thaliana plants. CONCLUSION: It is concluded that CaWRKY3 favorably regulates phytohormone-mediated synergistic signaling, which controls cell death in plant and immunity of pepper plant against bacterial infections.

Effect of Silencing subolesin and enolase impairs gene expression, engorgement and reproduction in Haemaphysalis longicornis (Acari: Ixodidae) ticks.

IMPORTANCE: Haemaphysalis longicornis is an obligate blood-sucking ectoparasite that has gained attention due its role of transmitting medically and veterinary significant pathogens and it is the most common tick species in Republic of Korea. The preferred strategy for controlling ticks is a multi-antigenic vaccination. Testing the efficiency of a combination antigen is a promising method for creating a tick vaccine. OBJECTIVE: The aim of the current research was to analyze the role of subolesin and enolase in feeding and reproduction of H. longicornis by gene silencing. METHODS: In this study, we used RNA interference to silence salivary enolase and subolesin in H. longicornis. Unfed female ticks injected with double-stranded RNA targeting subolesin and enolase were attached and fed normally on the rabbit's ear. Real-time polymerase chain reaction was used to confirm the extent of knockdown. RESULTS: Ticks in the subolesin or enolase dsRNA groups showed knockdown rates of 80% and 60% respectively. Ticks in the combination dsRNA (subolesin and enolase) group showed an 80% knockdown. Knockdown of subolesin and enolase resulted in significant depletion in feeding, blood engorgement weight, attachment rate, and egg laying. Silencing of both resulted in a significant (p < 0.05) reduction in tick engorgement, egg laying, egg hatching (15%), and reproduction. CONCLUSIONS AND RELEVANCE: Our results suggest that subolesin and enolase are an exciting target for future tick control strategies.

POLR1D silencing suppresses lung cancer cells proliferation and migration via inhibition of PI3K-Akt pathway.

AIM: The most common type of cancer that leads to death worldwide is lung cancer. Despite significant surgery and chemotherapy improvements, lung cancer patient's survival rate is still poor. The RNA polymerase I subunit D (POLR1D) gene can induce various cancers. A current study reported that POLR1D plays a vital role in cancer prognosis. However, its biological function in the development of lung cancer remains unclear. METHODS: Reverse transcription PCR (RT-PCR) measured the relative POLR1D protein expression level in lung cancer cell lines. Lung cancer cell proliferation, migration, and invasion were analyzed by performing cell counting kit-8 (CCK-8), and transwell. The phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/AKT) signaling pathway-related protein expressions were examined by Western blotting assay. RESULTS: POLR1D protein expression was elevated in lung cancer. Lung cancer cell loss-of-function tests showed that POLR1D silencing could attenuate cell viability both in SK-MES-1 and in H2170 cells. Furthermore, silencing POLR1D inhibited SK-MES-1 and H2170 cells proliferation, migration, and invasion. Moreover, SK-MES-1 and H2170 cells' migration and invasion capacity were potentially suppressed by the knockdown of POLR1D. The progression of multiple cancers has been implicated in the PI3K/AKT pathway. Here, we observed that POLR1D silencing suppressed lung cancer progression by inhibition of the PI3K-Akt pathway. CONCLUSIONS: The study speculated that POLR1D might provide a new potential therapeutic possibility for treating lung cancer patients via targeting PI3K/AKT.

Epigenetic silencing of LDHB promotes hepatocellular carcinoma by remodeling the tumor microenvironment.

Lactate dehydrogenase B (LDHB) reversibly catalyzes the conversion of pyruvate to lactate or lactate to pyruvate and expressed in various malignancies. However, the role of LDHB in modulating immune responses against hepatocellular carcinoma (HCC) remains largely unknown. Here, we found that down-regulation of lactate dehydrogenase B (LDHB) was coupled with the promoter hypermethylation and knocking down the DNA methyltransferase 3A (DNMT 3A) restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Moreover, immune checkpoint inhibitors (ICIs) have shown considerable promise for HCC treatment and patients with higher LDHB expression responded better to ICIs. Finally, we found that overexpression of LDHB suppressed HCC growth in immunocompetent but not in immunodeficient mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and therapeutic target for HCC immunotherapy.

Targeted degradation of oncogenic BCR-ABL by silencing the gene of NEDD8 E3 ligase RAPSYN.

Tyrosine kinase inhibitors have been the standard treatment for patients with Philadelphia chromosome-positive (Ph+) leukemia. However, a series of issues, including drug resistance, relapse and intolerance, are still an unmet medical need. Here, we report the targeted siRNA-based lipid nanoparticles in Ph+ leukemic cell lines for gene therapy of Ph+ leukemia, which specifically targets a recently identified NEDD8 E3 ligase RAPSYN in Ph+ leukemic cells to disrupt the neddylation of oncogenic BCR-ABL. To achieve the specificity for Ph+ leukemia therapy, a single-chain fragment variable region (scFv) of anti-CD79B monoclonal antibody was covalently conjugated on the surface of OA2-siRAPSYN lipid nanoparticles to generate the targeted lipid nanoparticles (scFv-OA2-siRAPSYN). Through effectively silencing RAPSYN gene in leukemic cell lines by the nanoparticles, BCR-ABL was remarkably degraded accompanied by the inhibition of proliferation and the promotion of apoptosis. The specific targeting, therapeutic effects and systemic safety were further evaluated and demonstrated in cell line-derived mouse models. The present study has not only addressed the clinical need of Ph+ leukemia, but also enabled gene therapy against a less druggable target.

Tripartite Motif-containing Protein 11 Silencing Inhibits Proliferation and Glycolysis and Promotes Apoptosis of Esophageal Squamous Cell Carcinoma Cells by Inactivating Signal Transduction and Activation of Transcription Factor 3/c-Myc Signaling.

Esophageal squamous cell carcinoma (ESCC) is a common type of human digestive tract cancer with poor survival. Tripartite motif-containing protein 11 (TRIM11) is an oncogene in certain cancers that can regulate glycolysis and signal transduction and activation of transcription factor 3 (STAT3) signaling. This study was designed to investigate the role and the mechanism of TRIM11 in ESCC. First, TRIM11 expression in ESCC tissues and the correlation between TRIM11 expression and prognosis were analyzed using bioinformatics tools. After TRIM11 expression was detected by Western blot in ESCC cells, TRIM11 was silenced to evaluate its effect on the malignant phenotypes of ESCC cells. Cell proliferation and apoptosis were assessed by cell counting kit-8 assay, ethynyl-2'- deoxyuridine staining, and flow cytometry, respectively. The glucose uptake and lactate secretion were detected to examine glycolysis. In addition, Western blot was employed to detect the expression of proteins related to apoptosis, glycolysis, and STAT3/c-Myc signaling. Then, ESCC cells were treated with STAT3 activator further to clarify the regulatory effect of TRIM11 on STAT3/c-Myc signaling. TRIM11 was upregulated in ESCC tissues and cells, and high expression of TRIM11 was associated with a poor prognosis. TRIM11 knockdown inhibited the proliferation and glycolysis while facilitating apoptosis of ESCC cells. Besides, the expression of p-STAT3 and c-Myc was significantly downregulated by TRIM11 silencing. Of note, the STAT3 activator partially reversed the effects of TRIM11 depletion on the proliferation, apoptosis, and glycolysis in ESCC cells. Collectively, TRIM11 loss-of-function affects the proliferation, apoptosis, and glycolysis in ESCC cells by inactivating STAT3/c-Myc signaling.

HER2 siRNA Facilitated Gene Silencing Coupled with Doxorubicin Delivery: A Dual Responsive Nanoplatform Abrogates Breast Cancer.

The present study investigated the concurrent delivery of antineoplastic drug, doxorubicin, and HER2 siRNA through a targeted theranostic metallic gold nanoparticle designed using polysaccharide, PSP001. The as-synthesized HsiRNA@PGD NPs were characterized in terms of structural, functional, physicochemical, and biological properties. HsiRNA@PGD NPs exposed adequate hydrodynamic size, considerable ζ potential, and excellent drug/siRNA loading and encapsulation efficiency. Meticulous exploration of the biocompatible dual-targeted nanoconjugate exhibited an appealing biocompatibility and pH-sensitive cargo release kinetics, indicating its safety for use in clinics. HsiRNA@PGD NPs deciphered competent cancer cell internalization, enhanced cytotoxicity mediated via the induction of apoptosis, and excellent downregulation of the overexpressing target HER2 gene. Further in vivo explorations in the SKBR3 xenograft breast tumor model revealed the appealing tumor reduction properties, selective accumulation in the tumor site followed by significant suppression of the HER2 gene which contributed to the exclusive abrogation of breast tumor mass by the HsiRNA@PGD NPs. Compared to free drugs or the monotherapy constructs, the dual delivery approach produced a synergistic suppression of breast tumors both in vitro and in vivo. Hence the drawings from these findings implicate that the as-synthesized HsiRNA@PGD NPs could offer a promising platform for chemo-RNAi combinational breast cancer therapy.

Co-localization and analysis of miR477b with fiber length quantitative trait loci in cotton.

Cotton is an important cash crop for the textile industry. However, the understanding of natural genetic variation of fiber elongation in relation to miRNA is lacking. A miRNA gene (miR477b) was found to co-localize with a previously mapped fiber length (FL) quantitative trait locus (QTL). The miR477b was differentially expressed during fiber elongation between two backcross inbred lines (BILs) differing in FL and its precursor sequences. Bioinformatics and qRT-PCR analysis were further used to analyse the miRNA genes, which could produce mature miR477b. Cotton plants with virus-induced gene silencing (VIGS) constructs to over-express the allele of miR477b from the BIL with longer fibers had significantly longer fibers as compared with negative control plants, while the VIGS plants with suppressed miRNA expression had significantly shorter fibers. The expression level of the target gene (DELLA) and related genes (RDL1 and EXPA1 for DELLA through HOX3 protein) in the two BILs and/or the VIGS plants were generally congruent, as expected. This report represents one of the first comprehensive studies to integrate QTL linkage mapping and physical mapping of small RNAs with both small and mRNA transcriptome analysis, followed by VIGS, to identify candidate small RNA genes affecting the natural variation of fiber elongation in cotton.

CsSHMT3 gene enhances the growth and development in cucumber seedlings under salt stress.

Salt stress is one of the major factors limiting plant growth and productivity. Many studies have shown that serine hydroxymethyltransferase (SHMT) gene play an important role in growth, development and stress response in plants. However, to date, there have been few studies on whether SHMT3 can enhance salt tolerance in plants. Therefore, the effects of overexpression or silencing of CsSHMT3 gene on cucumber seedling growth under salt stress were investigated in this study. The results showed that overexpression of CsSHMT3 gene in cucumber seedlings resulted in a significant increase in chlorophyll content, photosynthetic rate and proline (Pro) content, and antioxidant enzyme activity under salt stress condition; whereas the content of malondialdehyde (MDA), superoxide anion (H2O2), hydrogen peroxide (O2·-) and relative conductivity were significantly decreased when CsSHMT3 gene was overexpressed. However, the content of chlorophyll and Pro, photosynthetic rate, and antioxidant enzyme activity of the silenced CsSHMT3 gene lines under salt stress were significantly reduced, while MDA, H2O2, O2·- content and relative conductivity showed higher level in the silenced CsSHMT3 gene lines. It was further found that the expression of stress-related genes SOD, CAT, SOS1, SOS2, NHX, and HKT was significantly up-regulated by overexpressing CsSHMT3 gene in cucumber seedlings; while stress-related gene expression showed significant decrease in silenced CsSHMT3 gene seedlings under salt stress. This suggests that overexpression of CsSHMT3 gene increased the salt tolerance of cucumber seedlings, while silencing of CsSHMT3 gene decreased the salt tolerance. In conclusion, CsSHMT3 gene might positively regulate salt stress tolerance in cucumber and be involved in regulating antioxidant activity, osmotic adjustment, and photosynthesis under salt stress. KEY MESSAGE: CsSHMT3 gene may positively regulate the expression of osmotic system, photosynthesis, antioxidant system and stress-related genes in cucumber.

OTUB2 silencing promotes ovarian cancer via mitochondrial metabolic reprogramming and can be synthetically targeted by CA9 inhibition.

Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.

Novel role of LLGL2 silencing in autophagy: reversing epithelial-mesenchymal transition in prostate cancer.

PURPOSE: Prostate cancer (PCa) is a major urological disease that is associated with significant morbidity and mortality in men. LLGL2 is the mammalian homolog of Lgl. It acts as a tumor suppressor in breast and hepatic cancer. However, the role of LLGL2 and the underlying mechanisms in PCa have not yet been elucidated. Here, we investigate the role of LLGL2 in the regulation of epithelial-mesenchymal transition (EMT) in PCa through autophagy in vitro and in vivo. METHODS: PC3 cells were transfected with siLLGL2 or plasmid LLGL2 and autophagy was examined. Invasion, migration, and wound healing were assessed in PC3 cells under autophagy regulation. Tumor growth was evaluated using a shLLGL2 xenograft mouse model. RESULTS: In patients with PCa, LLGL2 levels were higher with defective autophagy and increased EMT. Our results showed that the knockdown of LLGL2 induced autophagy flux by upregulating Vps34 and ATG14L. LLGL2 knockdown inhibits EMT by upregulating E-cadherin and downregulating fibronectin and α-SMA. The pharmacological activation of autophagy by rapamycin suppressed EMT, and these effects were reversed by 3-methyladenine treatment. Interestingly, in a shLLGL2 xenograft mouse model, tumor size and EMT were decreased, which were improved by autophagy induction and worsened by autophagy inhibition. CONCLUSION: Defective expression of LLGL2 leads to attenuation of EMT due to the upregulation of autophagy flux in PCa. Our results suggest that LLGL2 is a novel target for alleviating PCa via the regulation of autophagy.

Silencing Ditylenchus destructor cathepsin L-like cysteine protease has negative pleiotropic effect on nematode ontogenesis.

Ditylenchus destructor is a migratory plant-parasitic nematode that severely harms many agriculturally important crops. The control of this pest is difficult, thus efficient strategies for its management in agricultural production are urgently required. Cathepsin L-like cysteine protease (CPL) is one important protease that has been shown to participate in various physiological and pathological processes. Here we decided to characterize the CPL gene (Dd-cpl-1) from D. destructor. Analysis of Dd-cpl-1 gene showed that Dd-cpl-1 gene contains a signal peptide, an I29 inhibitor domain with ERFNIN and GNFD motifs, and a peptidase C1 domain with four conserved active residues, showing evolutionary conservation with other nematode CPLs. RT-qPCR revealed that Dd-cpl-1 gene displayed high expression in third-stage juveniles (J3s) and female adults. In situ hybridization analysis demonstrated that Dd-cpl-1 was expressed in the digestive system and reproductive organs. Silencing Dd-cpl-1 in 1-cell stage eggs of D. destructor by RNAi resulted in a severely delay in development or even in abortive morphogenesis during embryogenesis. The RNAi-mediated silencing of Dd-cpl-1 in J2s and J3s resulted in a developmental arrest phenotype in J3 stage. In addition, silencing Dd-cpl-1 gene expression in female adults led to a 57.43% decrease in egg production. Finally, Dd-cpl-1 RNAi-treated nematodes showed a significant reduction in host colonization and infection. Overall, our results indicate that Dd-CPL-1 plays multiple roles in D. destructor ontogenesis and could serve as a new potential target for controlling D. destructor.

Silencing of the T-type voltage-gated calcium channel α1 subunit by fungus-mediated RNAi altered the structure of F-actin and caused defective behaviors in Ditylenchus destructor.

BACKGROUND: T-type calcium channels, characterized as low-voltage activated (LVA) calcium channels, play crucial physiological roles across a wide range of tissues, including both the neuronal and nonneuronal systems. Using in situ hybridization and RNA interference (RNAi) techniques in vitro, we previously identified the tissue distribution and physiological function of the T-type calcium channel α1 subunit (DdCα1G) in the plant-parasitic nematode Ditylenchus destructor. METHODS AND RESULTS: To further characterize the functional role of DdCα1G, we employed a combination of immunohistochemistry and fungus-mediated RNAi and found that DdCα1G was clearly distributed in stylet-related tissue, oesophageal gland-related tissue, secretory-excretory duct-related tissue and male spicule-related tissue. Silencing DdCα1G led to impairments in the locomotion, feeding, reproductive ability and protein secretion of nematodes. To confirm the defects in behavior, we used phalloidin staining to examine muscle changes in DdCα1G-RNAi nematodes. Our observations demonstrated that defective behaviors are associated with related muscular atrophy. CONCLUSION: Our findings provide a deeper understanding of the physiological functions of T-type calcium channels in plant-parasitic nematodes. The T-type calcium channel can be considered a promising target for sustainable nematode management practices.

Gene Silencing via RNA Interference in Cryptococcus.

RNA interference (RNAi) is a molecular biology technique for silencing specific eukaryotic genes without altering the DNA sequence in the genome. The silencing effect occurs because of decreased levels of mRNA that then result in decreased protein levels for the gene. The specificity of the silencing is dependent upon the presence of sequence-specific double-stranded RNA (dsRNA) that activates the cellular RNAi machinery. This chapter describes the process of silencing a specific target gene in Cryptococcus using a dual promoter vector. The plasmid, pIBB103, was designed with two convergent GAL7 promoters flanking a ura5 fragment that acts as a reporter for efficient RNAi. The target gene fragment is inserted between the promoters to be transcribed from both directions leading to the production of dsRNA in cells that activate the RNAi pathway.

Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes.

BACKGROUND: Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines. RESULTS: We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved. CONCLUSIONS: The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.