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.

MLKL regulates radiation-induced death in breast cancer cells: an interplay between apoptotic and necroptotic signals.

Resistance to caspase-dependent apoptosis is often responsible for treatments failure in cancer. Necroptosis is a type of programmed necrosis that occurs under caspase-deficient conditions that could overcome apoptosis resistance. Our purpose was to investigate the interrelationship between apoptotic and necroptotic death pathways and their influence on the response of breast cancer cells to radiotherapy in vitro. Human BC cell lines MCF-7 and MDA-MB-231 were treated with ionizing radiation, and then several markers of apoptosis, necroptosis, and survival were assessed in the presence and absence of necroptosis inhibition. MLKL knockdown was achieved by siRNA transfection. Our main findings emphasize the role of necroptosis in cellular response to radiation represented in the dose- and time-dependent elevated expression of necroptotic markers RIPK1, RIPK3, and MLKL. Knockdown of necroptotic marker MLKL by siRNA led to a significant elevation in MDA-MB-231 and MCF-7 survival with a dose modifying factor (DMF) of 1.23 and 1.61, respectively. Apoptotic markers Caspase 8 and TRADD showed transitory or delayed upregulation, indicating that apoptosis was not the main mechanism by which cells respond to radiation exposure. Apoptotic markers also showed a significant elevation following MLKL knockdown, suggesting its role either as a secondary or death alternative pathway. The result of our study emphasizes the critical role of the necroptotic pathway in regulating breast cancer cells responses to radiotherapy and suggests a promising utilization of its key modulator, MLKL, as a treatment strategy to improve the response to radiotherapy.

Synergistic Immunoregulation: harnessing CircRNAs and PiRNAs to Amplify PD-1/PD-L1 Inhibition Therapy.

The utilization of PD-1/PD-L1 inhibitors marks a significant advancement in cancer therapy. However, the efficacy of monotherapy is still disappointing in a substantial subset of patients, necessitating the exploration of combinational strategies. Emerging from the promising results of the KEYNOTE-942 trial, RNA-based therapies, particularly circRNAs and piRNAs, have distinguished themselves as innovative sensitizers to immune checkpoint inhibitors (ICIs). These non-coding RNAs, notable for their stability and specificity, were once underrecognized but are now known for their crucial roles in regulating PD-L1 expression and bolstering anti-cancer immunity. Our manuscript offers a comprehensive analysis of selected circRNAs and piRNAs, elucidating their immunomodulatory effects and mechanisms, thus underscoring their potential as ICIs enhancers. In conjunction with the recent Nobel Prize-awarded advancements in mRNA vaccine technology, our review highlights the transformative implications of these findings for cancer treatment. We also discuss the prospects of circRNAs and piRNAs in future therapeutic applications and research. This study pioneers the synergistic application of circRNAs and piRNAs as novel sensitizers to augment PD-1/PD-L1 inhibition therapy, demonstrating their unique roles in regulating PD-L1 expression and modulating immune responses. Our findings offer a groundbreaking approach for enhancing the efficacy of cancer immunotherapy, opening new avenues for treatment strategies. This abstract aims to encapsulate the essence of our research and the burgeoning role of these non-coding RNAs in enhancing PD-1/PD-L1 inhibition therapy, encouraging further investigation into this promising field.

Complete genome sequence and genetic characterization of a novel segmented RNA virus infecting Nilaparvata lugens.

The brown planthopper (BPH), Nilaparvata lugens, is a significant agricultural pest capable of long-distance migration and transmission of viruses that cause severe disease in rice. In this study, we identified a novel segmented RNA virus in a BPH, and this virus exhibited a close relationship to members of a recently discovered virus lineage known as "quenyaviruses" within the viral kingdom Orthornavirae. This newly identified virus was named "Nilaparvata lugens quenyavirus 1" (NLQV1). NLQV1 consists of five positive-sense, single-stranded RNAs, with each segment containing a single open reading frame (ORF). The genomic characteristics and phylogenetic analysis support the classification of NLQV1 as a novel quenyavirus. Notably, all of the genome segments of NLRV contained the 5'-terminal sequence AUCUG. The characteristic virus-derived small interfering RNA (vsiRNA) profile of NLQV1 suggests that the antiviral RNAi pathway of the host BPH was activated in response to virus infection. These findings represent the first documented report of quenyaviruses in planthoppers, contributing to our understanding of quenyaviruses and expanding our knowledge of insect-specific viruses in planthoppers.

[Expression of DARS2 in colorectal cancer and its clinical significance].

Objective: To investigate the expression of DARS2 and its clinical significance in colorectal cancer. Methods: In this study, bioinformatics tools, especially gene expression profile interactive analysis 2 (GEPIA2), were used to conduct an in-depth analysis of DARS2 expression in colorectal cancer tissues. Immunohistochemical staining was carried out in 108 colorectal cancer specimens and 30 normal colorectal tissues obtained from the First Affiliated Hospital of Nanchang University, Nanchang, China. Colorectal cancer cell lines (HCT116 and SW480) were transfected with small interfering RNA (siRNA) and DARS2 overexpression plasmid to examine the effects of DARS2 knockdown and overexpression on cell function. To assess the effects on cell function, CCK8 and transwell migration assays were used to assess proliferation and cell motility, respectively. Additionally, protein immunoblotting was employed to scrutinize the expression of proteins associated with the epithelial-mesenchymal transition of colorectal cancer cells. Results: DARS2 exhibited a pronounced upregulation in expression within colorectal cancer tissues compared to their normal epithelial counterparts. Furthermore, DARS2 expression was higher in colorectal cancer of stage Ⅲ-Ⅳ than those of stage Ⅰ-Ⅱ, exhibiting a significant correlation with N staging, M staging, and pathological staging (P<0.05). Kaplan-Meier analyses showed a decreased overall survival rate in colorectal cancer with DARS2 expression compared to those without DARS2 expression (P<0.05). In the siRNA transfection group, there was a significant reduction in cell proliferation and migration (P<0.01 and P<0.05, respectively). Conversely, the transfection of DARS2 overexpression plasmids substantially increased both cell proliferation and migration (P<0.05). Additionally, immunoblotting revealed that DARS2 knockdown led to an upregulation of E-cadherin expression and a downregulation of N-cadherin and vimentin expression. In contrast, DARS2 overexpression resulted in increased N-cadherin and vimentin expression, coupled with reduction in E-cadherin expression. Conclusions: There is a strong association between DARS2 expression and colorectal cancer progression. Silencing DARS2 inhibits cell proliferation and migration, exerting a discernible influence on the epithelial-mesenchymal transition process.

Advancements in small interfering RNAs therapy for acute lymphoblastic leukemia: promising results and future perspectives.

Acute lymphoblastic leukemia (ALL) is the most common type of cancer among children, presenting significant healthcare challenges for some patients, including drug resistance and the need for targeted therapies. SiRNA-based therapy is one potential solution, but problems can arise in administration and the need for a delivery system to protect siRNA during intravenous injection. Additionally, siRNA encounters instability and degradation in the reticuloendothelial system, off-target effects, and potential immune system stimulation. Despite these limitations, some promising results about siRNA therapy in ALL patients have been published in recent years, showing the potential for more effective and precise treatment, reduced side effects, and personalized approaches. While siRNA-based therapies demonstrate safety and efficacy, addressing the mentioned limitations is crucial for further optimization. Advancements in siRNA-delivery technologies and combination therapies hold promise to improve treatment effectiveness and overcome drug resistance. Ultimately, despite its challenges, siRNA therapy has the potential to revolutionize ALL treatments and improve patient outcomes.

Discovery of a new class of cell-penetrating peptides by novel phage display platform.

The primary hurdles for small interference RNA (siRNA) in clinical use are targeted and cytosolic delivery. To overcome both challenges, we have established a novel platform based on phage display, called NNJA. In this approach, a lysosomal cathepsin substrate is engineered within the flexible loops of PIII, that is displaying a unique random sequence at its N-terminus. NNJA library selection targeting cell-expressed targets should yield specific peptides localized in the cytoplasm. That is because phage internalization and subsequent localization to lysosome, upon peptide binding to the cell expressed target, will result in cleavage of PIII, rendering phage non-infective. Such phage will be eliminated from the selected pool and only peptide-phage that escapes lysosomes will advance to the next round. Proof of concept studies with the NNJA library demonstrated cytosolic localization of selected peptide-phage and peptide-siRNA, confirmed through confocal microscopy. More importantly, conjugation of siHPRT to monomeric or multimeric NNJA peptides resulted in significant reduction in HPRT mRNA in various cell types without significant cytotoxicity. Sequence similarity and clustering analysis from NGS dataset provide insights into sequence composition facilitating cell penetration. NNJA platform offers a highly efficient peptide discovery engine for targeted delivery of oligonucleotides to cytosol.

[Role and mechanism of cysteine and glycine-rich protein 2 in the malignant progression of neuroblastoma].

OBJECTIVE: To investigate the function and underlying mechanism of cysteine and glycine-rich protein 2 (CSRP2) in neuroblastoma (NB). METHODS: The correlation between the expression level of CSRP2 mRNA and the prognosis of NB children in NB clinical samples was analyzed in R2 Genomics Analysis and Visualization Platform. The small interfering RNA (siRNA) targeting CSRP2 or CSRP2 plasmid were transfected to NB cell lines SK-N-BE(2) and SH-SY5Y. Cell proliferation was observed by crystal violet staining and real-time cellular analysis. The ability of colony formation of NB cells was observed by colony-forming unit assay. Immunofluorescence assay was used to detect the expression of the proliferation marker Ki-67. Flow cytometry analysis for cell cycle proportion was used with cells stained by propidium iodide (PI). Annexin V/7AAD was used to stain cells and analyze the percentage of cell apoptosis. The ability of cell migration was determined by cell wound-healing assay. The level of protein and mRNA expression of CSRP2 in NB primary tumor and NB cell lines were detected by Western blot and quantitative real-time PCR (RT-qPCR). RESULTS: By analyzing the NB clinical sample databases, it was found that the expression levels of CSRP2 in high-risk NB with 3/4 stages in international neuroblastoma staging system (INSS) were significantly higher than that in low-risk NB with 1/2 INSS stages. The NB patients with high expression levels of CSRP2 were shown lower overall survival rate than those with low expression levels of CSRP2. We detected the protein levels of CSRP2 in the NB samples by Western blot, and found that the protein level of CSRP2 in 3/4 INSS stages was significantly higher than that in 1/2 INSS stages. Knockdown of CSRP2 inhibited cell viability and proliferation of NB cells. Overexpression of CSRP2 increased the proliferation of NB cells. Flow cytometry showed that the proportion of sub-G1, G0/G1 and S phase cells and Annexin V positive cells were increased after CSRP2 deficiency. In the cell wound-healing assay, the healing rate of NB cells was significantly attenuated after knockdown of CSRP2. Further mechanism studies showed that the proportion of the proliferation marker Ki-67 and the phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) were significantly decreased after CSRP2 knockdown. CONCLUSION: CSRP2 is highly expressed in high-risk NB with 3/4 INSS stages, and the expression levels of CSRP2 are negatively correlated with the overall survival of NB patients. CSRP2 significantly increased the proliferation and cell migration of NB cells and inhibited cell apoptosis via the activation of ERK1/2. All these results indicate that CSRP2 promotes the progression of NB by activating ERK1/2, and this study will provide a potential target for high-risk NB therapy.

The role of cyclin-dependent kinase inhibitor 3 in the proliferation and migration of renal cell carcinoma.

The cyclin-dependent kinase inhibitor 3 (CDKN3) gene, is over expressed in renal cell carcinoma (RCC). However, the cell biology functions of RCC are not well understood. The present study aimed to verify the ability of CDKN3 to promote the proliferation and migration of RCC through in vitro experiments. Subsequently, the clinical prognostic effects were analyzed using The Cancer Genome Atlas (TCGA; https://www.cancer.gov/) and Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/). The chelators, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), an analogue of the anti-tumor agent, were screened through bioinformatics analysis. The expression of CDKN3 is positively correlated with the IC50 of Dp44mT. In two RCC cell lines, 786-0 and Caki-1, we conducted small interfering RNA (siRNA) knockdown of CDKN3 and overexpression of CDKN3 by transfection plasmid. Subsequently, we administered Dp44mT to examine the resulting alterations in cell proliferation, migration, and apoptosis, thereby elucidating the role of CDKN3 and Dp44mT in these processes. The results of the experiment revealed a positive association between CDKN3 expression and the proliferation of RCC cell lines. Down-regulating CDKN3 significantly increased the apoptosis rate and inhibited cell migration in 786-0 and Caki-1 cells. Furthermore, bioinformatics analysis revealed a high expression of CDKN3 in RCC and a negative association between CDKN3 expression and survival. Gene set enrichment analysis (GSEA) revealed a significant association between high CDKN3 expression and the cell cycle pathway. Furthermore, we identified Dp44mT as a drug highly correlated with CDKN3 through the database. Subsequent addition of Dp44mT resulted in similar findings to those observed upon CDKN3 knockdown. Our findings have important implications for the diagnosis and treatment of CDKN3 in RCC. Additionally, Dp44mT is likely to be a promising candidate for future clinical applications.

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.

[Effect of SIRT7 on inhibiting the epithelial-mesenchymal transformation in pancreatic cancer cells and related mechanism].

Objective: To investigate the effect and mechanism of SIRT7 in epithelial mesenchymal transformation (EMT) of pancreatic cancer cells. Methods: The pancreatic cancer cells were divided into siControl, siSIRT7, over-expression SIRT7, siSIRT7+siCOL4A1, and siSIRT7+siSLUG groups using siRNA or plasmid transfection. The proliferation, migration and invasion of pancreatic cancer cells were detected by EdU, wound healing assay and Transwell experiments, respectively. The expression of EMT and cancer stem cell (CSC) markers were detected by quantitative real-time reverse transcription polymerase chain reaction assay (qRT-PCR) and western blot. RNA sequencing (RNA-seq) in SIRT7 knockdown PANC-1 cells was performed to explore the signaling pathways and target genes regulated by SIRT7. Then the target genes directly regulated by SIRT7 were identified with quantitative chromatin immunoprecipitation experiment (q-ChIP) and chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR). The expressions of SIRT7 and target genes were detected by immunohistochemical (IHC) in pancreatic cancer tissues, and the correlation between SIRT7 and target gene expression was analyzed using TCGA dataset. The correlation between expression of SIRT7 or target genes and survival was analyzed on KM-plotter website. Finally, GeneMANIA, STRING and ENCORI were used to predict SIRT7-related proteins and miRNAs. Results: EdU assay showed that the cell proliferation rates in SIRT7-overexpressed PANC-1 [(19.33±0.35)%] and BxPC-3 cells [(17.00±1.89)%] were lower than those in the control group [(31.60±1.37)% and (24.33±0.78)%, respectively, P＜0.05]. The proliferation rates of SIRT7-knockdown PANC-1 [(23.94±1.00)% and (27.08±0.97)%] and BxPC-3 cells [(22.00±1.86)% and (25.96±1.61)%] were higher than those of the siControl group [(11.80±1.86)% and (13.42±1.39)%, respectively, P＜0.05]. In PANC-1 cells, the wound healing assay showed that the relative migration rate of SIRT7-overexpression cells [(76.67±2.74)%] was lower than that of control cells [(100.00±2.13)%, P＜0.05]; the relative migration rate of cells with SIRT7 knockdown [(134.22±4.08)% and (199.82±9.20)%, respectively] was higher than that of siControl group [(102.24±3.13)%, P＜0.05]. Compared with the control group, SIRT7 overexpression decreased the number of migrated BxPC-3 cells (45.66±1.69 vs 28.33±2.62, P＜0.05); while SIRT7 knockdown increased these numbers (65.66±2.86 and 82.00±2.94 versus 33.00±0.81, P＜0.01). Transwell experiment revealed that the number of invaded cells in SIRT7 overexpression groups (16.33±2.05 and 34.66±1.69) was lower than that control groups (54.33±4.64 and 58.66±5.90, P＜0.05); with SIRT7 knockdown, the numbers of invaded PANC-1 (63.66±2.49 and 69.33±3.29) and BxPC-3 cells (134.33±3.09 and 181.66±4.02) were higher than those in control groups (35.33±2.49 and 42.00±0.81, P˂0.05). Also, SIRT7 knockdown decreased the expressions of epithelial markers and increased the expressions of mesenchymal and CSC markers. RNA-seq analysis showed that SIRT7 was involved in regulating a variety of cancer-related signaling pathways, including the pancreatic cancer pathway and the EMT pathway. Furthermore, SIRT7 could directly bind to the promoter regions of target genes, such as COL4A1 and SLUG. SIRT7 was negatively correlated with the expression and function of COL4A1 and SLUG in pancreatic cancer cells. The expressions of SIRT7, COL4A1, SLUG and SOX2 were verified in pancreatic cancer tissues by IHC. Finally, SIRT7 was predicted to be associated with many proteins and miRNAs based on GeneMANIA, STRING, and ENCORI online tools. Conclusions: SIRT7 can inhibit the EMT of pancreatic cancer cells through transcriptionally inhibiting the expression of target genes, such as COL4A1 and SLUG. Thus, SIRT7 may serve as a potential tumor suppressor gene in pancreatic cancer.

Identification of Arabidopsis thaliana small RNAs responsive to the fungal pathogen Botrytis cinerea at an early stage of interaction.

In plants, small RNAs (sRNAs), mainly microRNAs (miRNAs) and small interfering RNAs (siRNAs), have been described as key regulators of plant development, growth, and abiotic and biotic responses. Despite reports indicating the involvement of certain sRNAs in regulating the interaction between Botrytis cinerea (a major necrotrophic fungal phytopathogen) and host plants, there remains a lack of analysis regarding the potential regulatory roles of plant sRNAs during early stages of the interaction despite early immune responses observed then during infection. We present the first transcriptome-wide analysis of small RNA expression on the early interaction between the necrotrophic fungus Botrytis cinerea and the model plant Arabidopsis thaliana. We found that evolutionary conserved A. thaliana miRNAs were the sRNAs that accumulated the most in the presence of B. cinerea. The upregulation of miR167, miR159 and miR319 was of particular interest because these, together with their target transcripts, are involved in the fine regulation of the plant hormone signaling pathways. We also describe that miR173, which triggers the production of secondary siRNAs from TAS1 and TAS2 loci, as well as secondary siRNAs derived from these loci, is upregulated in response to B. cinerea. Thus, at an early stage of the interaction there are transcriptional changes of sRNA-guided silencing pathway genes and of a subset of sRNAs that targeted genes from the PPR gene superfamily, and these may be important mechanisms regulating the interaction between A. thaliana and B. cinerea. This work provides the basis for a better understanding of the regulation mediated by sRNAs during early B. cinerea-plant interaction and may help in the development of more effective strategies for its control.

Targeting dependency on a paralog pair of CBP/p300 against de-repression of KREMEN2 in SMARCB1-deficient cancers.

SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, is the causative gene of rhabdoid tumors and epithelioid sarcomas. Here, we identify a paralog pair of CBP and p300 as a synthetic lethal target in SMARCB1-deficient cancers by using a dual siRNA screening method based on the "simultaneous inhibition of a paralog pair" concept. Treatment with CBP/p300 dual inhibitors suppresses growth of cell lines and tumor xenografts derived from SMARCB1-deficient cells but not from SMARCB1-proficient cells. SMARCB1-containing SWI/SNF complexes localize with H3K27me3 and its methyltransferase EZH2 at the promotor region of the KREMEN2 locus, resulting in transcriptional downregulation of KREMEN2. By contrast, SMARCB1 deficiency leads to localization of H3K27ac, and recruitment of its acetyltransferases CBP and p300, at the KREMEN2 locus, resulting in transcriptional upregulation of KREMEN2, which cooperates with the SMARCA1 chromatin remodeling complex. Simultaneous inhibition of CBP/p300 leads to transcriptional downregulation of KREMEN2, followed by apoptosis induction via monomerization of KREMEN1 due to a failure to interact with KREMEN2, which suppresses anti-apoptotic signaling pathways. Taken together, our findings indicate that simultaneous inhibitors of CBP/p300 could be promising therapeutic agents for SMARCB1-deficient cancers.

ATP-dependent chromatin remodeller brahma related gene 1 promotes keratinocyte migration and modulates cell Signalling during wound healing in human skin.

Skin wound healing is driven by proliferation, migration and differentiation of several cell types that are controlled by the alterations in the gene expression programmes. Brahma Gene 1 (BRG1) (also known as SMARCA4) is a core ATPase in the BRG1 Associated Factors (BAF) ATP-dependent chromatin remodelling complexes that alter DNA-histone interaction in chromatin at the specific gene regulatory elements resulting in increase or decrease of the target gene transcription. Using siRNA mediated suppression of BRG1 during wound healing in a human ex vivo and in vitro (scratch assay) models, we demonstrated that BRG1 is essential for efficient skin wound healing by promoting epidermal keratinocytes migration, but not their proliferation or survival. BRG1 controls changes in the expression of genes associated with gene transcription, response to wounding, cell migration and cell signalling. Altogether, our data revealed that BRG1 play positive role in skin repair by promoting keratinocyte migration and impacting the genes expression programmes associated with cell migration and cellular signalling.

Transcriptional landscape of small non-coding RNAs reveals diversity of categories and functions in molluscs.

Small non-coding RNAs (sncRNAs) are non-coding RNA molecules that play various roles in metazoans. Among the sncRNAs, microRNAs (miRNAs) guide post-translational gene regulation during cellular development, proliferation, apoptosis, and differentiation, while PIWI-interacting RNAs (piRNAs) suppress transposon activity to safeguard the genome from detrimental insertion mutagenesis. While an increasing number of piRNAs are being identified in the soma and germlines of various organisms, they are scarcely reported in molluscs. To unravel the small RNA (sRNA) expression patterns and genomic function in molluscs, we generated a comprehensive sRNA dataset by sRNA sequencing (sRNA-seq) of eight mollusc species. Abundant miRNAs were identified and characterized in all investigated molluscs, and ubiquitous piRNAs were discovered in both somatic and gonadal tissues in six of the investigated molluscs, which are more closely associated with transposon silencing. Tens of piRNA clusters were also identified based on the genomic mapping results, which varied among different tissues and species. Our dataset serves as important reference data for future genomic and genetic studies on sRNAs in these molluscs and related species, especially in elucidating the ancestral state of piRNAs in bilaterians.

Effect of Clemizole on Alpha-Synuclein-Preformed Fibrils-Induced Parkinson's Disease Pathology: A Pharmacological Investigation.

Parkinson's disease (PD) is a neurodegenerative disorder associated with mitochondrial dysfunctions and oxidative stress. However, to date, therapeutics targeting these pathological events have not managed to translate from bench to bedside for clinical use. One of the major reasons for the lack of translational success has been the use of classical model systems that do not replicate the disease pathology and progression with the same degree of robustness. Therefore, we employed a more physiologically relevant model involving alpha-synuclein-preformed fibrils (PFF) exposure to SH-SY5Y cells and Sprague Dawley rats. We further explored the possible involvement of transient receptor potential canonical 5 (TRPC5) channels in PD-like pathology induced by these alpha-synuclein-preformed fibrils with emphasis on amelioration of oxidative stress and mitochondrial health. We observed that alpha-synuclein PFF exposure produced neurobehavioural deficits that were positively ameliorated after treatment with the TRPC5 inhibitor clemizole. Furthermore, Clemizole also reduced p-alpha-synuclein and diminished oxidative stress levels which resulted in overall improvements in mitochondrial biogenesis and functions. Finally, the results of the pharmacological modulation were further validated using siRNA-mediated knockdown of TRPC5 channels, which also decreased p-alpha-synuclein expression. Together, the results of this study could be superimposed in the future for exploring the beneficial effects of TRPC5 channel modulation for other neurodegenerative disorders and synucleopathies.

Identification of hub genes and therapeutic siRNAs to develop novel adjunctive therapy for Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a devastating X-linked neuromuscular disorder caused by various defects in the dystrophin gene and still no universal therapy. This study aims to identify the hub genes unrelated to excessive immune response but responsible for DMD progression and explore therapeutic siRNAs, thereby providing a novel treatment. METHODS: Top ten hub genes for DMD were identified from GSE38417 dataset by using GEO2R and PPI networks based on Cytoscape analysis. The hub genes unrelated to excessive immune response were identified by GeneCards, and their expression was further verified in mdx and C57 mice at 2 and 4 months (M) by (RT-q) PCR and western blotting. Therapeutic siRNAs were deemed as those that could normalize the expression of the validated hub genes in transfected C2C12 cells. RESULTS: 855 up-regulated and 324 down-regulated DEGs were screened from GSE38417 dataset. Five of the top 10 hub genes were considered as the candidate genes unrelated to excessive immune response, and three of these candidates were consistently and significantly up-regulated in mdx mice at 2 M and 4 M when compared with age-matched C57 mice, including Col1a2, Fbn1 and Fn1. Furthermore, the three validated up-regulated candidate genes can be significantly down-regulated by three rational designed siRNA (p < 0.0001), respectively. CONCLUSION: COL1A2, FBN1 and FN1 may be novel biomarkers for DMD, and the siRNAs designed in our study were help to develop adjunctive therapy for Duchenne muscular dystrophy.

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.

Small extracellular vesicles (sEVs)-based gene delivery platform for cell-specific CRISPR/Cas9 genome editing.

Small extracellular vesicles (sEVs) are naturally occurring vesicles that have the potential to be manipulated to become promising drug delivery vehicles for on-demand in vitro and in vivo gene editing. Here, we developed the modular safeEXO platform, a prototype sEV delivery vehicle that is mostly devoid of endogenous RNA and can efficaciously deliver RNA and ribonucleoprotein (RNP) complexes to their intended intracellular targets manifested by downstream biologic activity. We also successfully engineered producer cells to produce safeEXO vehicles that contain endogenous Cas9 (safeEXO-CAS) to effectively deliver efficient ribonucleoprotein (RNP)-mediated CRISPR genome editing machinery to organs or diseased cells in vitro and in vivo. We confirmed that safeEXO-CAS sEVs could co-deliver ssDNA, sgRNA and siRNA, and efficaciously mediate gene insertion in a dose-dependent manner. We demonstrated the potential to target safeEXO-CAS sEVs by engineering sEVs to express a tissue-specific moiety, integrin alpha-6 (safeEXO-CAS-ITGA6), which increased their uptake to lung epithelial cells in vitro and in vivo. We tested the ability of safeEXO-CAS-ITGA6 loaded with EMX1 sgRNAs to induce lung-targeted editing in mice, which demonstrated significant gene editing in the lungs with no signs of morbidity or detectable changes in immune cell populations. Our results demonstrate that our modular safeEXO platform represents a targetable, safe, and efficacious vehicle to deliver nucleic acid-based therapeutics that successfully reach their intracellular targets. Furthermore, safeEXO producer cells can be genetically manipulated to produce safeEXO vehicles containing CRISPR machinery for more efficient RNP-mediated genome editing. This platform has the potential to improve current therapies and increase the landscape of treatment for various human diseases using RNAi and CRISPR approaches.

Administration of Liposomal-Based Pde3b Gene Therapy Protects Mice Against Collagen-Induced Rheumatoid Arthritis via Modulating Macrophage Polarization.

Background: Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation and joint destruction. Despite progress in RA therapy, it remains difficult to achieve long-term remission in RA patients. Phosphodiesterase 3B (Pde3b) is a member of the phosphohydrolyase family that are involved in many signal transduction pathways. However, its role in RA is yet to be fully addressed. Methods: Studies were conducted in arthritic DBA/1 mice, a suitable mouse strain for collagen-induced rheumatoid arthritis (CIA), to dissect the role of Pde3b in RA pathogenesis. Next, RNAi-based therapy with Pde3b siRNA-loaded liposomes was assessed in a CIA model. To study the mechanism involved, we investigated the effect of Pde3b knockdown on macrophage polarization and related signaling pathway. Results: We demonstrated that mice with CIA exhibited upregulated Pde3b expression in macrophages. Notably, intravenous administration of liposomes loaded with Pde3b siRNA promoted the macrophage anti-inflammatory program and alleviated CIA in mice, as indicated by the reduced inflammatory response, synoviocyte infiltration, and bone and cartilage erosion. Mechanistic study revealed that depletion of Pde3b increased cAMP levels, by which it enhanced PKA-CREB-C/EBPß pathway to transcribe the expression of anti-inflammatory program-related genes. Conclusion: Our results support that Pde3b is involved in the pathogenesis of RA, and Pde3b siRNA-loaded liposomes might serve as a promising therapeutic approach against RA.