siRNA-mediated Gene Silencing Research Articles

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

BackgroundElectrotransfection 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.ResultsWe 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.ConclusionsThe 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.

Cardiac commitment driven by MyoD expression in pericardial stem cells.

Cellular therapy holds immense promise to remuscularize the damaged myocardium but is practically hindered by limited allogeneic sources of cardiac-committed cells that engraft stably in the recipient heart after transplantation. Here, we demonstrate that the pericardial tissue harbors myogenic stem cells (pSCs) that are activated in response to inflammatory signaling after myocardial infarction (MI). The pSCs derived from the MI rats (MI-pSCs) show in vivo and in vitro cardiac commitment characterized by cardiac-specific Tnnt2 expression and formation of rhythmic contraction in culture. Bulk RNA-seq analysis reveals significant upregulation of a panel of genes related to cardiac/myogenic differentiation, paracrine factors, and extracellular matrix in the activated pSCs compared to the control pSCs (Sham-pSCs). Notably, we define MyoD as a key factor that governs the process of cardiac commitment, as siRNA-mediated MyoD gene silencing results in a significant reduction of myogenic potential. Injection of the cardiac-committed cells into the infarcted rat heart leads to long-term survival and stable engraftment in the recipient myocardium. Therefore, these findings point to pericardial myogenic progenitors as an attractive candidate for cardiac cell-based therapy to remuscularize the damaged myocardium.

Abstract 74: Exploring the expression of cancer-intrinsic PD1 in osteosarcoma cells: A comprehensive proteomic analysis of its functional landscape

Abstract Cancer treatment was revolutionized by immune checkpoint inhibitors (ICIs) targeting the PD1/PDL1 axis - a brake to the immune system. Despite remarkable success, some patients do not respond to therapy or rapidly deteriorate due to an unclear mechanism. As reported for several types of tumors, PD1 receptor is not solely expressed on immune cells but also on cancer cells. Depending on the tumor type, it may act either as a suppressor or a tumor promoter. The limited response to ICIs was also reported in osteosarcoma (OSA), the most common type of bone cancer, mostly affecting children and young adults. With no significant improvement in therapy over the past 30 years, there is an urgent need for therapeutic advancement. This study aimed to determine if PD1 protein is expressed in OSA and whether it has a functional role, potentially affecting the response to ICIs. Strikingly, our results revealed both surface and intracellular PD1 expression in U2OS osteosarcoma cells, confirmed by Western Blot, flow cytometry and immunofluorescence analyses. siRNA-mediated PDCD1 gene silencing significantly increased cell migration and viability in scratch and viability assays. LC-MS-based global proteomics showed that PDCD1 knockdown markedly affects U2OS cells’ proteomic landscape compared to the control. To dissect PD1 receptor downstream signaling effects, we subjected the identified proteins to GO and GSEA large-scale data interpretation tools. The results strongly suggested that PDCD1 silencing leads to the enrichment of proteins involved in cell growth, migration and motility, corresponding to the cellular effects we initially observed. Importantly, the analysis clearly indicated positive regulation of MAPK cascade in cells with perturbed PD1 expression, aligning with PD1 mediated MAPK regulation in T cells. To reveal the mechanism underlying PD1 signaling, we focused on proteins with significantly changed expression in response to the knockdown. Using the STRING database, we created their interacting network, revealing that numerous proteins are closely related, and the majority of them are phosphoproteins, indicating significance in cellular signaling. Remarkably, many of the STRING-analyzed proteins belong to intersecting pathways, such as AKT1S1 and LAMTOR proteins, the critical components for mTORC1 signaling, or selected members of the Src family kinases along with their negative regulator CSK. In summary, we report previously undiscovered functional expression of PD1 in OSA cells, providing insights into the landscape of PD1 downstream signaling. Our studies indicate the role of PD1 in regulating cell growth, proliferation, and motility, suggesting osteosarcoma-expressed PD1 as a tumor suppressor. Further studies are urgently needed to investigate its therapeutic significance and explore if we could make ICIs more effective to treat osteosarcoma. Citation Format: Katarzyna Joanna Dziubek, Jakub Faktor, Ted Hupp, Maciej Parys, Sachin Kote. Exploring the expression of cancer-intrinsic PD1 in osteosarcoma cells: A comprehensive proteomic analysis of its functional landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 74.

Abstract 1080: Using genome-wide CRISPR screening to develop novel combination therapies for high-risk Ewing’s sarcoma

Abstract Ewing’s sarcoma (EWS) is an aggressive bone and soft-tissue cancer, characterized by a hallmark fusion oncoprotein comprising the amino terminus of a FET gene and the carboxy terminus of an ETS gene, most commonly (85%), the EWSR1-FLI1 t (11,22) fusion.TK-216 is a first-in-class EWSR1-FLI1 inhibitor currently in phase II clinical trials for EWS. Although TK-216 was designed to target the fusion protein interaction directly, it has demonstrated limited efficacy as a monotherapy. Thus, more effective TK-216 combination therapies are needed. We have used genome wide Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Knockout screening in EWS cells treated with TK-216 to identify novel synthetic lethality. We have identified 97 genes whose deletion potentially enhances the action of TK-216 in EWS cells. We have also identified several druggable targets, such as USP9X and the oxidative phosphorylation (OXPHOS) pathway as synthetic lethal interactions for TK-216. USP9X is known to deubiquitinate and stabilize the EWS-FLI1 fusion protein. USP9X and EWS-FLI1 protein expression levels were found to be positively correlated in EWS clinical specimens. USP9X siRNA-mediated gene silencing reduced EWS cell viability. Most importantly, USP9X siRNA-mediated gene silencing sensitized EWS cells to TK-216 treatment. In addition, inhibition of USP9X with a small-molecule inhibitor WP1130, led to significant cell death in EWS alone and in combination with TK-216 with wide therapeutic indices. The molecular mechanism of synergy and in vivo efficacy of the novel TK-216 combination will be determined in our study for future clinical translation. Citation Format: Daenikka Ravindrarajah, Sukriti Krishan, Kayleen Simpson, Twishi Gulati, Belamy B. Cheung, Glenn M. Marshall. Using genome-wide CRISPR screening to develop novel combination therapies for high-risk Ewing’s sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1080.

Abstract 4430: Metabolic reprogramming by ASPSCR1::TFE3 and VCP

Abstract Alveolar soft part sarcoma (ASPS) is a lethal soft tissue malignancy typically incident among adolescents and young adults, distinguished by the t(X;17) ASPSCR1::TFE3 fusion gene (AT3). Conventional chemotherapy has shown limited efficacy against ASPS, necessitating exploration of alternative strategies. Previous investigations have shown the pivotal role of the fusion gene in sarcomagenesis in mice, particularly in lactate-rich environments. This study aims to elucidate the metabolic alterations actuated by the AT3 fusion gene, both in vitro and in vivo. To model in vitro metabolic profiles, ASPS (ASPS-1) and renal cell carcinoma (FU-UR1) cell lines, each expressing AT3, were employed. Utilizing siRNA-mediated gene silencing, AT3 and VCP, a cofactor, were targeted in vitro. Metabolomic profiling through GC-MS analyzed the impact of depletion of AT3 or its transcriptional co-factor, VCP. C13-labeled metabolites were introduced and traced in vitro and in vivo to enhance comprehension of carbon utilization in aerobic respiration. While metabolic activity and O2 consumption differed between cell lines, inhibition of AT3 and VCP led to similar metabolite alterations. Significant changes in amino acid metabolism, notably glutamate, aspartate, and alanine were observed in both cell lines. There was notable diversion of glucose carbon utilization away from typical entry into the tricarboxylic acid (TCA) cycle, with minimal carbon incorporation beyond alpha-ketoglutarate. Despite prior data indicating heightened mitophagy activity in AT3 driven cells, inhibition of lysosomal activity did not yield significant changes in cellular respiration or O2 consumption. Citation Format: Rahi Patel, Amir Pozner, Kyle Dunlap, Lara Carroll, Jeff Brown, Greg Ducker, Kevin Jones. Metabolic reprogramming by ASPSCR1::TFE3 and VCP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4430.

Abstract 427: A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype

Abstract Background: Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive. Objective: We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes. Methods: In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-β-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype. Results: Higher expression in G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-β-induced EMT biomarker SNAIL and cell chemotaxis. Conclusion: Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets. Citation Format: Sima Torabidastgerdooei, Borhane Annabi. A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 427.

The impact of wound-healing assay, phorbol myristate acetate (PMA) stimulation and siRNA-mediated FURIN gene silencing on endogenous retroviral ERVW-1 expression level in U87-MG astrocytoma cells

PurposeHuman endogenous retroviruses (HERVs) are ubiquitous genomic sequences. Normally dormant HERVs, undergo reactivation by environmental factors. This deregulation of HERVs’ transcriptional equilibrium correlates with medical conditions such as multiple sclerosis (MS). Here we sought to explore whether exposing the U-87 MG astrocytoma cells to traumatic injury deregulates the expression of HERV-W family member ERVW-1 encoding syncytin-1. We also examined the expression of FURIN gene that is crucial in syncytin-1 synthesis. Material and methodsScratch assay was used as a model of cells injury in U-87 MG cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and migration assay using Boyden chamber were used. Phorbol 12-myristate 13-acetate (PMA) and small interfering RNA (siRNA) were used for cell stimulation and gene expression inhibition, respectively. ResultsResults revealed reduced ERVW-1 expression in cells exposed to injury (p ​< ​0.05) while GFAP gene - a marker of active astrocytes, was upregulated (p ​< ​0.01). These findings were confirmed by both WB and RT-qPCR. Expression of FURIN gene was not altered after injury, but cell stimulation by PMA strongly increased FURIN expression, simultaneously downregulating ERVW-1 (p ​< ​0.01). SiRNA-mediated expression inhibition of ERVW-1 and FURIN influenced the mRNA level for SLC1A5 (ASCT2) - primary syncytin-1 receptor, that was significantly lower. FURIN inhibition by siRNA caused strong upregulation of ERVW-1 expression (p ​< ​0.01). ConclusionResults showed that mechanical impact affects the expression of endogenous retroviruses in U-87 MG astrocytoma cells by scratch assay. Regulation of FURIN, a crucial enzyme in ERVW-1 turnover may support the therapy of some neurological conditions.

The impact of calcium-binding protein S100P on temozolomide sensitivity in glioblastoma cells

AimGlioblastoma (GBM), a highly aggressive primary brain tumor, often displays resistance to the standard chemotherapy agent Temozolomide (TMZ), presenting a formidable treatment challenge. This study aims to explore the impact of S100P, a calcium-binding protein linked to TMZ resistance in GBM cells, by investigating the consequences of S100P knockdown on TMZ resistance. The research, employing bioinformatics analysis and siRNA-mediated gene silencing, reveals a significant relationship between S100P and TRIM29 in the context of TMZ resistance. Material and methodsHuman GBM cells underwent transfection with S100P siRNA to achieve targeted suppression of S100P expression. The assessment of TMZ resistance was conducted through MTT cell viability assays, and the molecular mechanisms underlying the observed effects of S100P knockdown on TMZ resistance were explored using real-time PCR. ResultsOur findings reveal a significant reduction in TMZ resistance upon S100P knockdown in GBM cells. This effect is demonstrated by an increase in cell death and a concurrent decrease in the expression of proteins associated with TMZ resistance. ConclusionThe study establishes S100P as a promising therapeutic target to overcome TMZ resistance in GBM. Silencing S100P expression emerges as a strategy to enhance the sensitivity of GBM cells to TMZ chemotherapy. These results advocate further investigations to unveil the clinical viability of targeting S100P in the treatment of GBM.

Depletion of the m1A writer TRMT6/TRMT61A reduces proliferation and resistance against cellular stress in bladder cancer.

Bladder cancer (BLCA) is a common and deadly disease that results in a reduced quality of life for the patients and a significant economic burden on society. A better understanding of tumorigenesis is needed to improve clinical outcomes. Recent evidence places the RNA modification m1A and its regulatory proteins TRMT6/TRMT61A and ALKBH3 in BLCA pathogenesis. TRMT6/TRMT61A, ALKBH1, and ALKBH3 expression was examined in human BLCA cell lines and a normal urinary tract epithelium cell line through qRT-PCR and western blot analysis. Prestoblue Cell Viability Reagent, wound-healing assay, and live-cell imaging-based cell displacement analysis, were conducted to assess proliferation, migration, and displacement of this BLCA cell line panel. Cell survival was assessed after inducing cellular stress and activating the unfolded protein response (UPR) with tunicamycin. Moreover, siRNA-mediated gene silencing in two BLCA cell lines (5637 and HT1197) was conducted to investigate the biological roles of TRMT6/TRMT61A. Heterogeneous morphology, proliferation, displacement, tunicamycin sensitivity, and expression levels of m1A regulators were observed among the panel of cell lines examined. In general, TRMT61A expression was increased in BLCA cell lines when compared to SV-HUC-1. Depletion of TRMT6/TRMT61A reduced proliferation capacity in both 5637 and HT1197 cell lines. The average cell displacement of 5637 was also reduced upon TRMT6/TRMT61A depletion. Interestingly, TRMT6/TRMT61A depletion decreased mRNA expression of targets associated with the ATF6-branch of the UPR in 5637 but not in HT1197. Moreover, cell survival after induction of cellular stress was compromised after TRMT6/TRMT61A knockdown in 5637 but not in HT1197 cells. The findings suggest that TRMT6/TRMT61A plays an oncogenic role in BLCA and is involved in desensitizing BLCA cells against cellular stress. Further investigation into the regulation of TRMT6/TRMT61A expression and its impact on cellular stress tolerance may provide insights for future BLCA treatment.

Role of INPP4B in the proliferation, migration, invasion, and survival of human endometrial cancer cells.

Inositol polyphosphate 4-phosphatase type II (INPP4B) has been identified as a tumor repressor in several human cancers while its role in endometrial cancer has not been investigated yet. Therefore, the current study was designed to determine whether INPP4B participates in the progression of endometrial cancer by utilizing clinical data and experimental determination. We first include six chemotherapy-treated patients with recurrent and metastatic endometrioid carcinoma to determine the relationship between INPP4B mutation and relative tumor burden. By using siRNA-mediated gene silencing and vector-mediated gene overexpression, we further determined the effect of manipulating INPP4B expression on the proliferation, invasion, and survival of endometrial cancer cells. Furthermore, the repressing effect of INPP4B together with its role in chemotherapy was further validated by xenograft tumor-bearing mice models. Western blot analysis was used to explore further downstream signaling modulated by INPP4B expression manipulation. Two of the patients were found to have INPP4B mutations and the mutation frequency of INPP4B increased during the progression of chemotherapy resistance. Endometrial cancer cells with silenced INPP4B expression were found to have promoted tumor cell proliferation, invasion, and survival. Endometrial cancer cells overexpressing INPP4B were found to have decreased tumor cell proliferation, invasion, and survival. An in vivo study using six xenograft tumor-bearing mice in each group revealed that INPP4B overexpression could suppress tumor progression and enhance chemosensitivity. Furthermore, INPP4B overexpression was found to modulate the activation of Wnt3a signaling. The current study suggested that INPP4B could be a suppressor in endometrial cancer progression and might be a target for endometrial cancer treatment. Also, INPP4B might serve as a predictor of chemosensitivity determination.

A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype.

Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive. We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes. In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-β-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype. Higher expression in G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-β-induced EMT biomarker SNAIL and cell chemotaxis. Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets.

Targeting FGFR3 is a Useful Therapeutic Strategy for Rheumatoid Arthritis Treatment.

Rheumatoid arthritis (RA) is a systemic inflammatory disease in which TNF-α plays an important role. Fibroblast growth factor receptor 3 (FGFR3) is reportedly involved in RA by regulating the expression of inflammatory cytokines. This study examined the expression profile of FGFR3 in human synovial biopsy tissues and evaluated its gene-silencing effects on behaviors of synovial cells. Immunohistochemical staining was used to measure FGFR3 expression in human RA joint tissues. Cell proliferation, migration, and apoptosis assays were used to monitor behavioral changes in cultured synovial SW-982 cells with siRNA-mediated FGFR3 gene silencing. Immunofluorescent staining and western blotting were used to detect molecular changes in the FGFR3 gene-silenced cells. FGFR3 up-regulation was noted in both cytoplasms and nuclei of synovial cells in human RA joints. FGFR3 siRNA delivery experiments corroborated that FGFR3 knockdown decreased proliferation and migration, and triggered apoptosis of synovial cells. The FGFR3 gene knockdown enhanced constitutive expression of epithelial marker E-cadherin and conversely suppressed expression of epithelial-mesenchymal transition (EMT) markers, including Snail, fibronectin, and vimentin. In addition, FGFR3 silencing significantly reduced the constitutive expressions of TNF-α, transcription factor NF-κΒ, and downstream COX-2 protein and collagenolytic enzyme MMP-9. MAPK inhibition markedly suppressed constitutive levels of NF-κΒ, COX-2, and MMP-9. Genetic interference of FGFR3 could modulate the expression of inflammatory mediators and EMT markers in the synovial cells. Targeting the FGFR3/MAPK signal axis may be considered a useful therapeutic strategy to ameliorate the development of RA.

Curcumin attenuates replicative senescence in human dental follicle cells and restores their osteogenic differentiation

ObjectiveThis study aimed to examine the therapeutic effects of curcumin against replicative senescence in dental follicle cells (DFCs). MethodsHuman DFCs were cultured in Dulbecco's Modified Eagle Medium with growth supplements. Replicative senescence in DFCs at different passages was assessed using β-galactosidase activity assay. Cell proliferation and size of DFCs at different passages were determined by CCK-8 kit and microscopy method, respectively. In addition, curcumin's effect on replicative senescence, cell proliferation, and size of DFCs at different passages was analyzed. Using western-blot analysis and siRNA-mediated gene silencing, we determined the molecular mechanisms involved in curcumin's effect against replicative senescence and osteogenic differentiation in DFCs at different passages. ResultsWe observed decreased proliferation and increased cell size and replicative senescence in cultured human DFCs at higher passages. Intriguingly, despite not showing any effect on cell size, curcumin (50 μM) significantly restored proliferation ability in DFCs and inhibited their replicative senescence. Concerning mechanisms, we found that curcumin inhibits replicative senescence in DFCs via down-regulation of senescence markers (P16 & P21) and restoration of proliferation markers (E2F1 & P53). Additionally, curcumin also rescued the osteogenic differentiation potential in higher-passage DFCs via restoration of osteogenic markers RUNX2 and OPN. ConclusionOur findings reveal for the first time that curcumin could act as a potential anti-senescence therapeutic for DFCs via regulation of proliferation, senescence, and osteogenic differentiation markers.

Repositioning the antihistamine ebastine as an intracellular siRNA delivery enhancer

Small interfering RNAs (siRNAs) are promising therapeutics for the treatment of human diseases via the induction of sequence-specific gene silencing. To be functional, siRNAs require cytosolic delivery into target cells. However, state-of-the-art delivery systems mediate cellular entry through endocytosis and suffer from ineffective endosomal escape, routing a substantial fraction of the siRNA towards the lysosomal compartment. Cationic amphiphilic drugs (CADs) have been described to improve cytosolic siRNA delivery by the transient induction of lysosomal membrane permeabilization. In this work, we evaluated ebastine, an antihistamine CAD, for its ability to enhance cytosolic release of siRNA in a non-small cell lung cancer model. In particular, we demonstrated that ebastine can improve the siRNA-mediated gene silencing efficiency of a polymeric nanogel by 40-fold, outperforming other CAD compounds. Additionally, ebastine substantially enhanced gene knockdown of a cholesterol-conjugated siRNA, in two-dimensional (2D) cell culture as well as in three-dimensional (3D) tumor spheroids. Finally, ebastine could strongly promote siRNA delivery of lipid nanoparticles (LNPs) composed of a pH-dependent switchable ionizable lipid and with stable PEGylation, in contrast to state-of-the-art LNP formulations. Altogether, we identified ebastine as a potent and versatile siRNA delivery enhancer in cancer cells, which offers opportunities for drug combination therapy in oncology.

Phosphorylation of USP20 on Ser334 by IRAK1 promotes IL-1β-evoked signaling in vascular smooth muscle cells and vascular inflammation

Reversible lysine-63 (K63) polyubiquitination regulates proinflammatory signaling in vascular smooth muscle cells (SMCs) and plays an integral role in atherosclerosis. Ubiquitin-specific peptidase 20 (USP20) reduces NFκB activation triggered by proinflammatory stimuli, and USP20 activity attenuates atherosclerosis in mice. The association of USP20 with its substrates triggers deubiquitinase activity; this association is regulated by phosphorylation of USP20 on Ser334 (mouse) or Ser333 (human). USP20 Ser333 phosphorylation was greater in SMCs of atherosclerotic segments of human arteries as compared with nonatherosclerotic segments. To determine whether USP20 Ser334 phosphorylation regulates proinflammatory signaling, we created USP20-S334A mice using CRISPR/Cas9-mediated gene editing. USP20-S334A mice developed ∼50% less neointimal hyperplasia than congenic WT mice after carotid endothelial denudation. WT carotid SMCs showed substantial phosphorylation of USP20 Ser334, and WT carotids demonstrated greater NFκB activation, VCAM-1 expression, and SMC proliferation than USP20-S334A carotids. Concordantly, USP20-S334A primary SMCs invitro proliferated and migrated less than WT SMCs in response to IL-1β. An active site ubiquitin probe bound to USP20-S334A and USP20-WT equivalently, but USP20-S334A associated more avidly with TRAF6 than USP20-WT. IL-1β induced less K63-linked polyubiquitination of TRAF6 and less downstream NFκB activity in USP20-S334A than in WT SMCs. Using invitro phosphorylation with purified IRAK1 and siRNA-mediated gene silencing of IRAK1 in SMCs, we identified IRAK1 as a novel kinase for IL-1β-induced USP20 Ser334 phosphorylation. Our findings reveal novel mechanisms regulating IL-1β-induced proinflammatory signaling: by phosphorylating USP20 Ser334, IRAK1 diminishes the association of USP20 with TRAF6 and thus augments NFκB activation, SMC inflammation, and neointimal hyperplasia.

Galectin-3 Mediates Tumor Progression in Astrocytoma by Regulating Glycogen Synthase Kinase-3β Activity

Numerous studies have considered galectin-3 or Glycogen synthase kinase 3 beta (GSK3B) as a potential prognosis marker for various cancers. However, the correlation between the protein expression of galectin-3/GSK3B and the clinical parameters of astrocytoma has not been reported. This study aims to validate the correlation between the clinical outcomes and protein expression of galectin-3/GSK3B in astrocytoma. Immunohistochemistry staining was performed to detect galectin-3/GSK3B protein expression in patients with astrocytoma. The Chi-square test, Kaplan-Meier evaluation, and Cox regression analysis were used to determine the correlation between clinical parameters and galectin-3/GSK3B expression. Cell proliferation, invasion, and migration were compared between a non-siRNA group and a galectin-3/GSK3B siRNA group. Protein expression in galectin-3 or GSK3B siRNA-treated cells was evaluated using western blotting. Galectin-3 and GSK3B protein expression were significantly positively correlated with the World Health Organization (WHO) astrocytoma grade and overall survival time. Multivariate analysis revealed that WHO grade, galectin-3 expression, and GSK3B expression were independent prognostic factors for astrocytoma. Galectin-3 or GSK3B downregulation induced apoptosis and decreased cell numbers, migration, and invasion. siRNA-mediated gene silencing of galectin-3 resulted in the downregulation of Ki-67, cyclin D1, VEGF, GSK3B, p-GSK3B Ser9 (p-GSK3B S9), and β-catenin. In contrast, GSK3B knockdown only decreased Ki-67, VEGF, p-GSK3B S9, and β-catenin protein expression but did not affect cyclin D1 and galectin-3 protein expression. The siRNA results indicated that GSK3B is downstream of the galectin-3 gene. These data support that galectin-3 mediated tumor progression by upregulating GSK3B and β-catenin protein expression in glioblastoma. Therefore, galectin-3 and GSK3B are potential prognostic markers, and their genes may be considered to be anticancer targets for astrocytoma therapy.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural β-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and β-anomeric linkages, N-glycosides with β-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Full activation of thermogenesis in brown adipocytes requires Basigin action.

Exploring mechanisms responsible for brown adipose tissue's (BAT) high metabolic activity is crucial to exploit its energy-dissipating ability for therapeutic purposes. Basigin (Bsg), a multifunctional highly glycosylated transmembrane protein, was recently proposed as one of the 98 critical markers allowing to distinguish 'white' and 'brown' adipocytes, yet its function in thermogenic brown adipocytes is unknown. Here, we report that Bsg is negatively associated with obesity in mice. By contrast, Bsg expression increased in the mature adipocyte fraction of BAT upon cold acclimation. Additionally, Bsg levels were highly induced during brown adipocyte maturation in vitro and were further increased upon β-adrenergic stimulation in a HIF-1α-dependent manner. siRNA-mediated Bsg gene silencing in cultured brown adipocytes did not impact adipogenesis nor mitochondrial function. However, a significant decrease in mitochondrial respiration, lipolysis and Ucp1 transcription was observed in adipocytes lacking Bsg, when activated by norepinephrine. Furthermore, using gas chromatography/mass spectrometry-time-of-flight analysis to assess the composition of cellular metabolites, we demonstrate that brown adipocytes lacking Bsg have lower levels of intracellular lactate and acetoacetate. Bsg was additionally required to regulate intracellular AcAc and tricarboxylic acid cycle intermediate levels in NE-stimulated adipocytes. Our study highlights the critical role of Bsg in active brown adipocytes, possibly by controlling cellular metabolism.

SiRNA Mediated Gene Silencing in the Pancreatic Cancer Capan-1 Cell Line

Today, pancreatic cancer ranks second after the cardiovascular system among the causes of death. The pancreatic cancer which is an aggressive type progressing without giving too many symptoms. In addition, factors such as difficulty in early diagnosis, rapid metastasis and non-response to traditional treatments cause a low survival rate in this type of cancer. Therefore, many studies are carried out to develop alternative diagnosis and treatment methods. Focus is put on target-oriented studies to increase the survival rate and eliminate other negative effects. The gene therapy method is at the forefront of such studies. RNA interference (RNAi) molecules are crucial in this method. From among these molecules, small non-coding RNA (siRNA) are used in studies as a therapeutic agent by its delivery to the target gene by various mechanisms. Studies are carried out with different biological and chemical agents to deliver the molecules to the target cell efficiently.&#x0D; This study aimed to silence the c-Myc gene using gold nanoparticle (AuNP)-siRNA in the Capan-1 cell line. We performed Real-Time PCR, Dual Staining and gel electrophoresis using gold nanoparticles (2X, 4X, 8X) and siRNA (25 nM) at concentrations determined in the Capan-1 cell line. The data obtained from the analysis suggested that the nanoparticle siRNA complex could be used effectively in silencing the target gene. Nevertheless, effective results could be obtained with more knowledge on this subject.

Effect of LDHA Inhibition on TNF-α-Induced Cell Migration in Esophageal Cancers.

Cell migration is an essential part of the complex and multistep process that is the development of cancer, a disease that is the second most common cause of death in humans. An important factor promoting the migration of cancer cells is TNF-α, a pro-inflammatory cytokine that, among its many biological functions, also plays a major role in mediating the expression of MMP9, one of the key regulators of cancer cell migration. It is also known that TNF-α is able to induce the Warburg effect in some cells by increasing glucose uptake and enhancing the expression and activity of lactate dehydrogenase subunit A (LDHA). Therefore, the aim of the present study was to investigate the interrelationship between the TNF-α-induced promigratory activity of cancer cells and their glucose metabolism status, using esophageal cancer cells as an example. By inhibiting LDHA activity with sodium oxamate (SO, also known as aminooxoacetic acid sodium salt or oxamic acid sodium salt) or siRNA-mediated gene silencing, we found using wound healing assay and gelatin zymography that LDHA downregulation impairs TNF-α-dependent tumor cell migration and significantly reduces TNF-α-induced MMP9 expression. These effects were associated with disturbances in the activation of the ERK1/2 signaling pathway, as we observed by Western blotting. We also reveal that in esophageal cancer cells, SO effectively reduces the production of lactic acid, which, as we have shown, synergizes the stimulating effect of TNF-α on MMP9 expression. In conclusion, our findings identified LDHA as a regulator of TNF-α-induced cell migration in esophageal cancer cells by the ERK1/2 signaling pathway, suggesting that LDHA inhibitors that limit the migration of cancer cells caused by the inflammatory process may be considered as an adjunct to standard therapy in esophageal cancer patients.