[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.

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.

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.

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.

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.

Cholesterol-Modified Anti-Il6 siRNA Reduces the Severity of Acute Lung Injury in Mice.

Small interfering RNA (siRNA) holds significant therapeutic potential by silencing target genes through RNA interference. Current clinical applications of siRNA have been primarily limited to liver diseases, while achievements in delivery methods are expanding their applications to various organs, including the lungs. Cholesterol-conjugated siRNA emerges as a promising delivery approach due to its low toxicity and high efficiency. This study focuses on developing a cholesterol-conjugated anti-Il6 siRNA and the evaluation of its potency for the potential treatment of inflammatory diseases using the example of acute lung injury (ALI). The biological activities of different Il6-targeted siRNAs containing chemical modifications were evaluated in J774 cells in vitro. The lead cholesterol-conjugated anti-Il6 siRNA after intranasal instillation demonstrated dose-dependent therapeutic effects in a mouse model of ALI induced by lipopolysaccharide (LPS). The treatment significantly reduced Il6 mRNA levels, inflammatory cell infiltration, and the severity of lung inflammation. IL6 silencing by cholesterol-conjugated siRNA proves to be a promising strategy for treating inflammatory diseases, with potential applications beyond the lungs.

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.

piR-1919609 Is an Ideal Potential Target for Reversing Platinum Resistance in Ovarian Cancer.

PURPOSE: PIWI-interacting RNAs (piRNAs) are a type of noncoding small RNA that can interact with PIWI-like RNA-mediated gene silencing (PIWIL) proteins to affect biological processes such as transposon silencing through epigenetic effects. Recent studies have found that piRNAs are widely dysregulated in tumors and associated with tumor progression and a poor prognosis. Therefore, this study aimed to investigate the effect of piR-1919609 on the proliferation, apoptosis, and drug resistance of ovarian cancer cells. METHODS: In this study, we used small RNA sequencing to screen and identify differentially expressed piRNAs in primary ovarian cancer, recurrent ovarian cancer, and normal ovaries. A large-scale verification study was performed to verify the expression of piR-1919609 in different types of ovarian tissue, including ovarian cancer tissue and normal ovaries, by RT-PCR and to analyze its association with the clinical prognosis of ovarian cancer. The expression of PIWILs in ovarian cancer was verified by RT-PCR, Western blotting and immunofluorescence. The effects of piR-1919609 on ovarian cancer cell proliferation, apoptosis and drug resistance were studied through in vitro and in vivo models. RESULTS: (1) piR-1919609 was highly expressed in platinum-resistant ovarian cancer tissues (p < 0.05), and this upregulation was significantly associated with a poor prognosis and a shorter recurrence time in ovarian cancer patients (p < 0.05). (2) PIWIL2 was strongly expressed in ovarian cancer tissues (p < 0.05). It was expressed both in the cytoplasm and nucleus of ovarian cancer cells. (3) Overexpression of piR-1919609 promoted ovarian cancer cell proliferation, inhibited apoptosis, and promoted tumor growth in nude mice. (4) Inhibition of piR-1919609 effectively reversed ovarian cancer drug resistance. CONCLUSION: In summary, we showed that piR-1919609 is involved in the regulation of drug resistance in ovarian cancer cells and might be an ideal potential target for reversing platinum resistance in ovarian cancer.

Combinatory Nanovesicle with siRNA-Loaded Extracellular Vesicle and IGF-1 for Osteoarthritis Treatments.

Extracellular vesicles (EVs) have been found to have the characteristics of their parent cells. Based on the characteristics of these EVs, various studies on disease treatment using mesenchymal stem cell (MSC)-derived EVs with regenerative activity have been actively conducted. The therapeutic nature of MSC-derived EVs has been shown in several studies, but in recent years, there have been many efforts to functionalize EVs to give them more potent therapeutic effects. Strategies for functionalizing EVs include endogenous and exogenous methods. In this study, human umbilical cord MSC (UCMSC)-derived EVs were selected for optimum OA treatments with expectation via bioinformatics analysis based on antibody array. And we created a novel nanovesicle system called the IGF-si-EV, which has the properties of both cartilage regeneration and long-term retention in the lesion site, attaching positively charged insulin-like growth factor-1 (IGF-1) to the surface of the UCMSC-derived Evs carrying siRNA, which inhibits MMP13. The downregulation of inflammation-related cytokine (MMP13, NF-kB, and IL-6) and the upregulation of cartilage-regeneration-related factors (Col2, Acan) were achieved with IGF-si-EV. Moreover, the ability of IGF-si-EV to remain in the lesion site for a long time has been proven through an ex vivo system. Collectively, the final constructed IGF-si-EV can be proposed as an effective OA treatment through its successful MMP13 inhibition, chondroprotective effect, and cartilage adhesion ability. We also believe that this EV-based nanoparticle-manufacturing technology can be applied as a platform technology for various diseases.

Supramolecular Nanoparticles of Histone and Hyaluronic Acid for Co-Delivery of siRNA and Photosensitizer In Vitro.

Small interfering RNA (siRNA) has significant potential as a treatment for cancer by targeting specific genes or molecular pathways involved in cancer development and progression. The addition of siRNA to other therapeutic strategies, like photodynamic therapy (PDT), can enhance the anticancer effects, providing synergistic benefits. Nevertheless, the effective delivery of siRNA into target cells remains an obstacle in cancer therapy. Herein, supramolecular nanoparticles were fabricated via the co-assembly of natural histone and hyaluronic acid for the co-delivery of HMGB1-siRNA and the photosensitizer chlorin e6 (Ce6) into the MCF-7 cell. The produced siRNA-Ce6 nanoparticles (siRNA-Ce6 NPs) have a spherical morphology and exhibit uniform distribution. In vitro experiments demonstrate that the siRNA-Ce6 NPs display good biocompatibility, enhanced cellular uptake, and improved cytotoxicity. These outcomes indicate that the nanoparticles constructed by the co-assembly of histone and hyaluronic acid hold enormous promise as a means of siRNA and photosensitizer co-delivery towards synergetic therapy.

siRNA-based therapy for gastric adenocarcinoma: what's next step?

Gastric cancer continues to have a high death rate despite advancements in their diagnosis and treatment. Novel treatment techniques are thus desperately needed. This is where double-stranded RNA molecules known as small interfering RNA (siRNA), which may selectively target the mRNA of disease-causing genes, may find use in medicine. For siRNAs to function properly in the human body, they must be shielded from deterioration. Furthermore, in order to maintain organ function, they must only target the tumor and spare normal tissue. siRNAs have been designed using clever delivery mechanisms including polymers and lipids to achieve these objectives. Although siRNA protection is not hard to acquire, it is still challenging to target cancer cells with them. Here, we first discuss the basic characteristics of gastric cancer before describing the properties of siRNA and typical delivery methods created specifically for gastric tumors. Lastly, we provide a succinct overview of research using siRNAs to treat gastric tumors.

syn-tasiRnas targeting the coat protein of potato virus Y confer antiviral resistance in Nicotiana benthamiana.

Trans-acting small interfering RNAs (tasiRNAs) are 21-nt phased (phased siRNAs) resulting from successive DCL-catalyzed processing from the end of a double-stranded RNA substrate originating from the RDR of an AGO-catalyzed cleaved RNA at a micro RNA target site. Plant tasiRNAs have been synthesized to produce synthetic tasiRNAs (syn-tasiRNAs) targeting viral RNAs that confer viral resistance. In this study, we engineered syn-tasiRNAs to target potato virus Y (PVY) infection by replacing five native siRNAs of TAS1c with 210-bp fragments from the coat protein (CP) region of the PVY genome. The results showed that the transient expression of syn-tasiR-CPpvy2 in Nicotiana benthamiana (N. benthamiana) plants conferred antiviral resistance, supported by the absence of PVY infection symptoms and viral accumulation. This indicated that syn-tasiR-CPpvy2 successfully targeted and silenced the PVY CP gene, effectively inhibiting viral infection. syn-tasiR-CPpvy1 displayed attenuated symptoms and decreased viral accumulation in these plants However, severe symptoms of PVY infection and a similar amount of viral accumulation as the control were observed in plants expressing syn-tasiR-CPpvy3. syn-tasiR-CPpvy/pvx, which targets both PVY and potato virus X (PVX), was engineered using a single precursor. After the transient expression of syn-tasiR-CPpvy/pvx3 and syn-tasiR-CPpvy/pvx5 in N. benthamiana, the plants were resistant to both PVY and PVX. These results suggested that engineered syn-tasiRNAs could not only specifically induce antiviral resistance against one target virus but could also be designed for multi-targeted silencing of different viruses, thereby preventing complex virus infection in plants.

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.

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.

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.

Cell division cyclin 25C knockdown inhibits hepatocellular carcinoma development by inducing endoplasmic reticulum stress.

BACKGROUND: Cell division cyclin 25C (CDC25C) is a protein that plays a critical role in the cell cycle, specifically in the transition from the G2 phase to the M phase. Recent research has shown that CDC25C could be a potential therapeutic target for cancers, particularly for hepatocellular carcinoma (HCC). However, the specific regulatory mechanisms underlying the role of CDC25C in HCC tumorigenesis and development remain incompletely understood. AIM: To explore the impact of CDC25C on cell proliferation and apoptosis, as well as its regulatory mechanisms in HCC development. METHODS: Hepa1-6 and B16 cells were transduced with a lentiviral vector containing shRNA interference sequences (LV-CDC25C shRNA) to knock down CDC25C. Subsequently, a xenograft mouse model was established by subcutaneously injecting transduced Hepa1-6 cells into C57BL/6 mice to assess the effects of CDC25C knockdown on HCC development in vivo. Cell proliferation and migration were evaluated using a Cell Counting Kit-8 cell proliferation assays and wound healing assays, respectively. The expression of endoplasmic reticulum (ER) stress-related molecules (glucose-regulated protein 78, X-box binding protein-1, and C/EBP homologous protein) was measured in both cells and subcutaneous xenografts using quantitative real-time PCR (qRT-PCR) and western blotting. Additionally, apoptosis was investigated using flow cytometry, qRT-PCR, and western blotting. RESULTS: CDC25C was stably suppressed in Hepa1-6 and B16 cells through LV-CDC25C shRNA transduction. A xenograft model with CDC25C knockdown was successfully established and that downregulation of CDC25C expression significantly inhibited HCC growth in mice. CDC25C knockdown not only inhibited cell proliferation and migration but also significantly increased the ER stress response, ultimately promoting ER stress-induced apoptosis in HCC cells. CONCLUSION: The regulatory mechanism of CDC25C in HCC development may involve the activation of ER stress and the ER stress-induced apoptosis signaling pathway.

DCLK1 Expression and its Functional Significance in Intrahepatic Cholangiocarcinoma and Bil-IN Stage.

BACKGROUND/AIM: Although several studies in some neoplasms have reported correlation between the expression levels of Doublecortin-like kinase1(DCLK1) and carcinogenesis, its role in cholangiocarcinoma remains unknown. MATERIALS AND METHODS: DCLK1 expression in normal epithelium (NE), biliary intraepithelial neoplasia (BilIN)1∼3, and intrahepatic cholangiocarcinoma (ICC) were investigated immuno-histochemically. The molecular effects of DCLK1 were investigated by gene silencing using RNAi [DCLK1-tagrgeting (siDCLK1)]. The human ICC cell lines HuCCT1 and HuH28 were transfected with these siRNAs, and used for assays in the presence or absence of DCLK1 inhibitors. RESULTS: The positive ratio of DCLK1 expression in ICC was higher than that in NE, and equally distributed among BilIN1∼3 (NE: BilIN1: BilIN2: BilIN3: ICC=62%: 91%: 97%: 100%: 95%, p<0.05). In the wound healing assay, the migration of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the cell invasion assay, the invasion of the siDCLK1-treated cells was significantly inhibited compared to the NT-treated cells (p<0.05). In the presence of the DCLK1 inhibitor, cell proliferative capacity at 24 hours was decreased in a concentration-dependent manner. CONCLUSION: DCLK1 was highly expressed in the early stage of ICC carcinogenesis. Human ICC cell growth was suppressed in vitro by siRNA silencing of DCLK1 or after treatment with the DCLK1 inhibitor, indicating DCLK1 may be molecular target for ICC therapy.

RRM2 Is a CTNNB1 Transport Regulator Promoting Colon Cancer Progression.

BACKGROUND/AIM: The cytoplasmic retention and stabilization of CTNNB1 (ß-catenin) in response to Wnt is well documented in playing a role in tumor growth. Here, through the utilization of a multiplex siRNA library screening strategy, we investigated the modulation of CTNNB1 function in tumor cell progression by ribonucleoside-diphosphate reductase subunit M2 (RRM2). MATERIALS AND METHODS: We conducted a multiplex siRNA screening assay to identify targets involved in CTNNB1 nuclear translocation. In order to examine the effect of inhibition of RRM2, selected from the siRNA screening results, we performed RRM2 knockdown and assayed for colon cancer cell viability, sphere formation assay, and invasion assay. The interaction of RRM2 with CTNNB1 and its impact on oncogenesis was examined using immunoprecipitation, immunoblotting, immunocytochemistry, and RT-qPCR. RESULTS: After a series of screening and filtration steps, we identified 26 genes that were potentially involved in CTNNB1 nuclear translocation. All candidate genes were validated in various cell lines. The results revealed that siRNA-mediated knockdown of RRM2 reduces the nuclear translocation of CTNNB1. This reduction was accompanied by a decrease in cell count, resulting in a suppressive effect on tumor cell growth. CONCLUSION: High throughput siRNA screening is an attractive strategy for identifying gene functions in cancers and the interaction between RRM2 and CTNNB1 is an attractive drug target for regulating RRM2-CTNNB1-related pathways in cancers.

Electrospun composite-coated endotracheal tubes with controlled siRNA and drug delivery to lubricate and minimize upper airway injury.

Endotracheal Tubes (ETTs) maintain and secure a patent airway; however, prolonged intubation often results in unintended injury to the mucosal epithelium and inflammatory sequelae which complicate recovery. ETT design and materials used have yet to adapt to address intubation associated complications. In this study, a composite coating of electrospun polycaprolactone (PCL) fibers embedded in a four-arm polyethylene glycol acrylate matrix (4APEGA) is developed to transform the ETT from a mechanical device to a dual-purpose device capable of delivering multiple therapeutics while preserving coating integrity. Further, the composite coating system (PCL-4APEGA) is capable of sustained delivery of dexamethasone from the PCL phase and small interfering RNA (siRNA) containing polyplexes from the 4APEGA phase. The siRNA is released rapidly and targets smad3 for immediate reduction in pro-fibrotic transforming growth factor-beta 1 (TGFϐ1) signaling in the upper airway mucosa as well as suppressing long-term sequelae in inflammation from prolonged intubation. A bioreactor was used to study mucosal adhesion to the composite PCL-4APEGA coated ETTs and investigate continued mucus secretory function in ex vivo epithelial samples. The addition of the 4APEGA coating and siRNA delivery to the dexamethasone delivery was then evaluated in a swine model of intubation injury and observed to restore mechanical function of the vocal folds and maintain epithelial thickness when observed over 14 days of intubation. This study demonstrated that increase in surface lubrication paired with surface stiffness reduction significantly decreased fibrotic behavior while reducing epithelial adhesion and abrasion.

Construction of cryomicroneedles loaded with milk-derived exosomes encapsulated TNF-α siRNA and efficacy of percutaneous acupoint administration in rheumatoid arthritis.

Inhibiting the expression of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine widely distributed in the serum and synovial fluid, is important for managing rheumatoid arthritis (RA). Despite the good therapeutic effects of TNF-α small interfering RNA (TNF-α siRNA) in RA animal models, safe and efficient siRNA delivery systems that retain stability are lacking. We introduced a novel therapy using milk-derived exosomes(mEXOs)-encapsulated TNF-α siRNA-coated cryomicroneedle (cryoMN) patch and evaluated its efficacy via local transdermal administration through acupoints in RA treatment. The loading of TNF-α siRNAs into mEXOs was achieved by sonication, the loading rate, stability, and in vitro release of mEXOs-TNF-α siRNA were determined. The cryoMNs were prepared by micromolding, morphology, drug loading, and mechanical strength of the cryoMN array were analyzed. The loading efficiency of TNF-α siRNA was up to 21% and each cryoMN contained 39.6 ± 1.29 µg of TNF-α siRNA. Frozen sections penetrated 523 ± 63 µm deep. In vitro experiments have shown that mEXOs-TNF-α siRNA cryoMNs have good biocompatibility and inhibit the proliferation of HFLS-RA cells. In vivo pharmacodynamics studies found that general conditions, changes in microcirculation indexes, synovial histopathological changes, and expression of related proteins in the synovial tissue in RA rabbits were effectively alleviated by mEXOs-TNF-α siRNA cryoMNs. Improvement of each index at acupoints was greater than that at non-acupoints. Our findings facilitate the development of cryoMNs combined with exosomes and acupoints drug delivery for the treatment of RA. The combination of exosomes and cryoMNs will enable the development of new-generation microneedle-based treatments.