siRNA-mediated Gene Knockdown Research Articles

#2050 APRT deficiency: insights from in vitro modelling of DHA-induced kidney injury and identification of therapeutic target

Abstract Background and Aims Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in renal excretion of 2,8-dihydroxyadenine (DHA) in excessive amounts, leading to kidney stones and crystal nephropathy with associated inflammation and fibrosis. The effects of other kidney stone constituents, such as calcium oxalate and uric acid, have been thoroughly studied in cell culture models, both in monolayer and three-dimensional (3D) assays. However, the effect of DHA in cell culture models has not yet been investigated. This study aimed to establish a comprehensive cell culture model to investigate DHA crystal-induced kidney injury and to identify therapeutic targets for clinical intervention. Method Three lines of kidney cells (MDCK, HK-2, and HEK293) were utilized in monolayer and 3D assays, employing both liquid-liquid interface (LLI) and “on-top” of Matrigel culture models. DHA exposure, matching the quantities found in the urine of untreated patients with APRT deficiency, was used in conjunction with siRNA-mediated APRT gene knockdown to assess the changes in cellular phenotypes. Assessments included cell viability and migration assays, RT-PCR, western blotting, phase-contrast microscopy, and immunostaining. Paraffin-embedded kidney tissue samples from patients with APRT deficiency and healthy controls were obtained from the Landspitali National University Hospital Biobank (Icelandic IRB permit: VSN 21-117-V1) and phenotypically analyzed using immunohistochemistry (IHC) staining for collagen I (Col I), collagen III (Col III), and APRT. Results After 72 h of incubation with DHA at concentrations of 60, 120, 240, and 480 μg/mL, the viability of all cell lines decreased. Proliferation was evaluated using a scratch assay after treatment with DHA at two different concentrations (120 and 480 μg/mL) for 24 h, and all cell lines showed decreased migration at the highest concentration. Enhanced CD44 expression was observed in both HEK293 and MDCK cells with increasing DHA concentration. In a 3D environment “on-top” of Matrigel, MDCK cells maintained polarized structures despite accumulation of DHA and did not show an increased EMT phenotype compared with TGFβ-treated cells. HEK293 and HK-2 cells formed solid colonies with DHA accumulation within the colony. MDCK cells formed a polarized cell layer grown on Transwell polyester membranes and demonstrated trans-epithelial electrical resistance (TEER) in LLI, which decreased when the cells were treated with DHA at 120 and 480 μg/mL. APRT expression was significantly reduced in all cell lines after successful knockdown. Analysis of kidney tissue specimens using IHC showed increased expression of Col III in patients with APRT deficiency compared to healthy controls. Enhanced Col I expression was also observed in these patients, which, together with Col III, was consistent with increased fibrosis. Conclusion This study revealed that incubation with varying concentrations of DHA led to decreased viability and impaired migration in all cell lines tested. Increased CD44 expression suggests potential crystal binding to renal tubular epithelial cells when exposed to increasing DHA concentrations. In a 3D environment, MDCK cells maintained polarized structures with DHA accumulation without enhancement of the EMT phenotype. Treatment with DHA resulted in a decrease in TEER, indicating a defective barrier function. Increased expression of Col III and Col I was observed in kidney tissue samples from APRT-deficient patients, suggesting increased fibrosis. Overall, these findings highlight the impact of DHA on cell viability, proliferation, EMT, and barrier function, highlighting the role of DHA in kidney fibrosis in patients with APRT-deficiency.

Efficient polymeric nanoparticles for RNAi in macrophage reveal complex effects on polarization markers upon knockdown of STAT3/STAT6

Tumor associated macrophages (TAMs) are the most abundant immune cell type in the tissue microenvironment, affecting tumor progression, metastasis and therapeutic response. Different macrophage activation (“polarization”) states can be distinguished: resting (M0; non-activated), pro-inflammatory/anti-tumorigenic (M1) and anti-inflammatory/pro-tumorigenic (M2). When exploring macrophages as targets in novel cancer immunotherapy approaches, TAM repolarization from the M2 into the M1 phenotype is an intriguing strategy to block their pro-tumoral and enhance their anti-tumoral properties.In the context of RNAi-based gene knockdown of M2 promoting genes, major bottlenecks include cellular siRNA delivery and correct intracellular processing. This is particularly true in case of macrophages as a cell type well-known to be notoriously hard-to-transfect. Among polymeric nanocarriers, the cationic polymer polyethylenimine (PEI) is widely explored for delivering nucleic acids. Further advanced nanocarriers are tyrosine-modified polymers based on PEI or polypropylenimine dendrimers (PPI) for highly efficient siRNA delivery in vitro and in vivo.In this paper, we explored a panel of PEI- or PPI-based nanoparticle systems for siRNA-mediated gene knockdown efficacy in macrophages and subsequent TAM repolarization. The tyrosine-modified linear 10 kDa PEI (LP10Y) or branched 5 kDa PEI (P5Y) as well as a tyrosine-modified PPI (PPI-Y) were found most efficient for gene knockdown in macrophage cell lines or primary macrophages, independent of their polarization. Knockdown of STAT6 or STAT3 led to repolarization of M2 macrophages, as indicated by alterations in various M2 and M1 marker levels. This highly specific approach also demonstrated non-redundant functions of STAT3 and STAT6. Importantly, macrophage re-polarization from M2 to M1 upon PPI-Y/siRNA-mediated STAT6 knockdown increased tumor cell phagocytosis in a co-culture model.In conclusion, we identify certain tyrosine-modified PEI- or PPI-based nanoparticles as particularly efficient for macrophage transfection, and the specific, siRNA-mediated STAT6 knockdown as a promising approach for macrophage repolarization and enhancement of their tumor cell suppressive role.

Development of a novel senescence-related gene signature to predict clinical outcomes, immune landscape, and chemotherapeutic sensitivity in oral squamous cell carcinoma.

Cellular senescence significantly associates with tumor initiation, progression, and therapeutic response across multiple cancers. Here, we sought to develop a novel senescence-related genes (SRGs)-derived signature for oral squamous cell carcinoma (OSCC) prognostication and therapeutic response prediction. OSCC-specific SRG prognostic signature was established with univariate Cox regression, Kaplan-Meier survival, and LASSO-penalized multivariate Cox regression analyses. A SRG nomogram integrating this signature and selected clinicopathological parameters were constructed by multivariate Cox regression. SiRNA-mediated gene knockdown was exploited to validate its function in vitro. The utilities of SRG signature in predicting immune status and chemotherapeutic sensitivities were analyzed. The prognostic performance of SRG signature/nomogram was satisfactory in multiple independent cohorts. CDK1 knockdown induced senescence phenotype in vitro. Moreover, SRG signature scores negatively correlated with tumor-infiltrating immune cells and associated with multiple chemotherapeutic drug sensitivities. Our results established SRG-derived signature/nomogram as powerful predictors for prognosis and chemotherapeutic response for OSCC.

ANGI-09. UNRAVELING THE INVOLVEMENT OF BAIAP2 AND CDC42 IN MEDULLOBLASTOMA PATHOGENESIS

Abstract The most common malignant pediatric brain tumor is medulloblastoma. Despite advancements in treatment, medulloblastoma survivors have significant long-term medical side effects and increased all-cause mortality. That is why novel, more effective, and safe treatments are direly necessary. One treatment challenge is that medulloblastoma tumors can disseminate via the bloodstream and cerebrospinal fluid leading to a poorer prognosis. The underlying mechanisms that promote metastasis and invasion in medulloblastoma are not clearly understood. One method we used to identify potential key regulators in the pathogenesis of medulloblastoma is by analyzing microarray and single-cell RNA sequencing data. We mined publicly-available datasets along with our data from genetically engineered models of medulloblastoma to identify differentially expressed genes compared to non-tumor brain cells. We identified BAIAP2 and CDC42 to be differentially expressed and have higher expression in medulloblastoma compared to other brain cancers. BAIAP2 and CDC42 are known to be implicated in migration and invasion. Extensive literature demonstrates an interaction between BAIAP2 and CDC42 in multiple cell types. However, this interaction has yet to be thoroughly studied in medulloblastoma. We hypothesize that CDC42 interacts with BAIAP2 in medulloblastoma cells to facilitate tumor pathogenesis. To further investigate this, we conducted siRNA-mediated gene knockdown of BAIAP2 and CDC42 in ONS-76 cells. We determined that BAIAP2 and CDC42 are highly expressed in medulloblastoma, and knockdown of BAIAP2 modulates CDC42 expression. Further, we determined that cell proliferation and migration were reduced upon BAIAP2 and CDC42 knockdown. Our findings suggest that BAIAP2 and CDC42 have a role in the migration and proliferation of medulloblastoma ONS-76 cells. Altogether, these findings indicate BAIAP2 and CDC42 as possible targets for future studies.

Differentially expressed chaperone genes reveal a stress response required for unidirectional regeneration in the basal chordate Ciona

BackgroundUnidirectional regeneration in the basal chordate Ciona intestinalis involves the proliferation of adult stem cells residing in the branchial sac vasculature and the migration of progenitor cells to the site of distal injury. However, after the Ciona body is bisected, regeneration occurs in the proximal but not in the distal fragments, even if the latter include a part of the branchial sac with stem cells. A transcriptome was sequenced and assembled from the isolated branchial sacs of regenerating animals, and the information was used to provide insights into the absence of regeneration in distal body fragments.ResultsWe identified 1149 differentially expressed genes, which were separated into two major modules by weighted gene correlation network analysis, one consisting of mostly upregulated genes correlated with regeneration and the other consisting of only downregulated genes associated with metabolism and homeostatic processes. The hsp70, dnaJb4, and bag3 genes were among the highest upregulated genes and were predicted to interact in an HSP70 chaperone system. The upregulation of HSP70 chaperone genes was verified and their expression confirmed in BS vasculature cells previously identified as stem and progenitor cells. siRNA-mediated gene knockdown showed that hsp70 and dnaJb4, but not bag3, are required for progenitor cell targeting and distal regeneration. However, neither hsp70 nor dnaJb4 were strongly expressed in the branchial sac vasculature of distal fragments, implying the absence of a stress response. Heat shock treatment of distal body fragments activated hsp70 and dnaJb4 expression indicative of a stress response, induced cell proliferation in branchial sac vasculature cells, and promoted distal regeneration.ConclusionsThe chaperone system genes hsp70, dnaJb4, and bag3 are significantly upregulated in the branchial sac vasculature following distal injury, defining a stress response that is essential for regeneration. The stress response is absent from distal fragments, but can be induced by a heat shock, which activates cell division in the branchial sac vasculature and promotes distal regeneration. This study demonstrates the importance of a stress response for stem cell activation and regeneration in a basal chordate, which may have implications for understanding the limited regenerative activities in other animals, including vertebrates.

Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival.

5-Hydroxytryptamine receptor 1E (5-HTR1E) is reported to activate cyclic AMP (cAMP) and extracellular-signal related kinases (ERK) pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin-induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin-induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gβγ and Gq were not associated with 5-HTR1E activation, while blocking protein kinase A (PKA) inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, β-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockdown studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

A robust, flow-based, microfluidic device for siRNA-mediated gene knockdown in glioblastoma spheroids

Glioblastoma (GBM) is a deadly disease with a poor prognosis, there is therefore a crucial need for novel therapeutic targets. Current preclinical models of GBM fail to predict clinical outcomes, thus, new translationally relevant models are urgently needed for reliable therapeutic target validation. 3D spheroid culture of cancer cells has been shown to better reflect tumour biology than 2D monolayer culture, as has culturing cells in flow-based microfluidic devices, which mimic key aspects of the tumour microenvironment. Gene knockdown by siRNA is a key preclinical target validation tool, however, siRNA-mediated knockdown of cancer spheroids in microfluidic culture has not yet been demonstrated. Here we describe a simple and robust microfluidic device that can maintain GBM spheroids (U87 cells) for at least 7 days. Via RNA sequencing analysis, we demonstrate that spheroids grown in microfluidic culture are more proliferative than spheroids grown in static plate culture and downregulate genes associated with cell adhesion, potentially offering insights into the metastatic process. Comparison of target gene (PRMT2 and RAB21) knockdown using siRNA between 2D monolayer cultured cells, static spheroid culture and spheroids maintained in the microfluidic device showed that gene expression (as measured by quantitative-PCR) was significantly reduced in all culture systems. Knockdown was most efficient in cells grown in 2D monolayer culture followed by static spheroid culture, but we also demonstrate [Formula: see text] knockdown efficiency using the microfluidic device. In summary, this study describes an easy-to-use microfluidic culture platform and provides evidence that pre-clinical siRNA-mediated target validation studies will be possible in flow systems that mimic tumour physiology.

Adiponectin Stimulates Apolipoprotein A-1 Gene Expression in HepG2 Cells via AMPK, PPARα, and LXRs Signaling Mechanisms.

Adiponectin is an adipose tissue hormone, participating in energy metabolism and involved in atherogenesis. Previously, it was found that adiponectin increases expression of the APOA1 (apolipoprotein A-1) gene in hepatocytes, but the mechanisms of this effect remained unexplored. Our aim was to investigate the role of adiponectin receptors AdipoR1/R2, AMP-activated protein kinase (AMPK), nuclear peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRs) in mediating the action of adiponectin on hepatic APOA1 expression in human hepatoma HepG2 cells. The level of APOA1 expression was determined by RT-qPCR and ELISA. We showed that the siRNA-mediated knockdown of genes coding for AdipoR1, AdipoR2, AMPK, PPARα, and LXRα and β prevented adiponectin-induced APOA1 expression in HepG2 cells and demonstrated that interaction of PPARα and LXRs with the APOA1 gene hepatic enhancer is important for the adiponectin-dependent APOA1 transcription. The results of this study point out to the involvement of both types of adiponectin receptors, AMPK, PPARα, and LXRs in the adiponectin-dependent upregulation of the APOA1 expression.

Small-molecule Wnt inhibitors are a potential novel therapy for intestinal fibrosis in Crohns disease.

Intestinal fibrosis and stricture formation is an aggressive complication of Crohns disease (CD), linked to increased morbidity and costs. The present study investigates the contribution of Wingless-Int-1 (Wnt) signalling to intestinal fibrogenesis, considers potential cross-talk between Wnt and transforming growth factor β1 (TGFβ) signalling pathways, and assesses the therapeutic potential of small-molecule Wnt inhibitors.β-catenin expression was explored by immunohistochemistry (IHC) in formalin-fixed paraffin embedded (FFPE) tissue from patient-matched nonstrictured (NSCD) and strictured (SCD) intestine (n=6 pairs). Functional interactions between Wnt activation, TGFβ signalling, and type I collagen (Collagen-I) expression were explored in CCD-18Co cells and primary CD myofibroblast cultures established from surgical resection specimens (n=16) using small-molecule Wnt inhibitors and molecular techniques, including siRNA-mediated gene knockdown, immunofluorescence (IF), Wnt gene expression arrays, and western blotting. Fibrotic SCD tissue was marked by an increase in β-catenin-positive cells. In vitro, activation of Wnt-β-catenin signalling increased Collagen-I expression in CCD-18Co cells. Conversely, ICG-001, an inhibitor of β-catenin signalling, reduced Collagen-I expression in cell lines and primary CD myofibroblasts. TGFβ increased β-catenin protein levels but did not activate canonical Wnt signalling. Rather, TGFβ up-regulated WNT5B, a noncanonical Wnt ligand, and the Wnt receptor FZD8, which contributed directly to the up-regulation of Collagen-I through a β-catenin-independent mechanism. Treatment of CCD-18Co fibroblasts and patient-derived myofibroblasts with the FZD8 inhibitor 3235-0367 reduced extracellular matrix (ECM) expression. Our data highlight small-molecule Wnt inhibitors of both canonical and noncanonical Wnt signalling, as potential antifibrotic drugs to treat SCD intestinal fibrosis. They also highlight the importance of the cross-talk between Wnt and TGFβ signalling pathways in CD intestinal fibrosis.

Thermal preconditioning in a reef-building coral alleviates oxidative damage through a BI-1-mediated antioxidant response

Global coral reef decline is largely driven by the breakdown of the coral-algal symbiosis during temperature stress. Corals can acclimatize to higher temperatures, but the cellular processes underlying this ability are poorly understood. We show that preconditioning-based improvements in thermal tolerance in Pocillopora acuta are accompanied by increases in host glutathione reductase (GR) activity and gene expression, which prevents DNA damage. A strong correlation between GR and BI-1 expressions in heat-stressed preconditioned corals and the presence of an antioxidant response element (ARE) in the GR promoter suggest BI-1 could regulate GR expression through Nrf2/ARE pathway. To fortify this link, we developed and GFP-validated an siRNA-mediated gene knockdown protocol and targeted the coral BI-1 gene. BI-1 knock-down specifically decreased GR expression and activity and increased oxidative DNA damage in heat-stressed preconditioned corals, showing that a BI-1-mediated, enhanced antioxidant response during acute heat stress is a key mechanism that prevents oxidative DNA damage after preconditioning.

SiRNA-mediated gene knockdown via electroporation in hydrozoan jellyfish embryos

As the sister group to bilaterians, cnidarians stand in a unique phylogenetic position that provides insight into evolutionary aspects of animal development, physiology, and behavior. While cnidarians are classified into two types, sessile polyps and free-swimming medusae, most studies at the cellular and molecular levels have been conducted on representative polyp-type cnidarians and have focused on establishing techniques of genetic manipulation. Recently, gene knockdown by delivery of short hairpin RNAs into eggs via electroporation has been introduced in two polyp-type cnidarians, Nematostella vectensis and Hydractinia symbiolongicarpus, enabling systematic loss-of-function experiments. By contrast, current methods of genetic manipulation for most medusa-type cnidarians, or jellyfish, are quite limited, except for Clytia hemisphaerica, and reliable techniques are required to interrogate function of specific genes in different jellyfish species. Here, we present a method to knock down target genes by delivering small interfering RNA (siRNA) into fertilized eggs via electroporation, using the hydrozoan jellyfish, Clytia hemisphaerica and Cladonema paciificum. We show that siRNAs targeting endogenous GFP1 and Wnt3 in Clytia efficiently knock down gene expression and result in known planula phenotypes: loss of green fluorescence and defects in axial patterning, respectively. We also successfully knock down endogenous Wnt3 in Cladonema by siRNA electroporation, which circumvents the technical difficulty of microinjecting small eggs. Wnt3 knockdown in Cladonema causes gene expression changes in axial markers, suggesting a conserved Wnt/β-catenin-mediated pathway that controls axial polarity during embryogenesis. Our gene-targeting siRNA electroporation method is applicable to other animals, including and beyond jellyfish species, and will facilitate the investigation and understanding of myriad aspects of animal development.

MiR-1224-5p modulates osteogenesis by coordinating osteoblast/osteoclast differentiation via the Rap1 signaling target ADCY2

MicroRNAs (miRNAs) broadly regulate normal biological functions of bone and the progression of fracture healing and osteoporosis. Recently, it has been reported that miR-1224-5p in fracture plasma is a potential therapy for osteogenesis. To investigate the roles of miR-1224-5p and the Rap1 signaling pathway in fracture healing and osteoporosis development and progression, we used BMMs, BMSCs, and skull osteoblast precursor cells for in vitro osteogenesis and osteoclastogenesis studies. Osteoblastogenesis and osteoclastogenesis were detected by ALP, ARS, and TRAP staining and bone slice resorption pit assays. The miR-1224-5p target gene was assessed by siRNA-mediated target gene knockdown and luciferase reporter assays. To explore the Rap1 pathway, we performed high-throughput sequencing, western blotting, RT-PCR, chromatin immunoprecipitation assays and immunohistochemical staining. In vivo, bone healing was judged by the cortical femoral defect, cranial bone defect and femoral fracture models. Progression of osteoporosis was evaluated by an ovariectomy model and an aged osteoporosis model. We discovered that the expression of miR-1224-5p was positively correlated with fracture healing progression. Moreover, in vitro, overexpression of miR-1224-5p slowed Rankl-induced osteoclast differentiation and promoted osteoblast differentiation via the Rap1-signaling pathway by targeting ADCY2. In addition, in vivo overexpression of miR-1224-5p significantly promoted fracture healing and ameliorated the progression of osteoporosis caused by estrogen deficiency or aging. Furthermore, knockdown of miRNA-1224-5p inhibited bone regeneration in mice and accelerated the progression of osteoporosis in elderly mice. Taken together, these results identify miR-1224-5p as a key bone osteogenic regulator, which may be a potential therapeutic target for osteoporosis and fracture nonunion.

Identification of Adamts4 as a novel adult cardiac injury biomarker with therapeutic implications in patients with cardiac injuries

Pathological cardiac remodeling as an aftermath of a severe cardiac injury can lead to ventricular dysfunction and subsequent heart failure. Adamts4, a metalloproteinase, and disintegrin with thrombospondin-like motif, involved in the turnover of certain extracellular matrix molecules and pathogenesis of osteoarthritis, also plays a role in cardiac remodeling although little is presently known about its expression and function in the heart. Here, we have investigated the dynamic expression pattern of Adamts4 during cardiogenesis and also in the adult heart. To our surprise, adult cardiac injury reactivated Adamts4 expression concomitant with fibrosis induction. To better understand the mechanism, cultured H9c2 cardiomyocyte cells were subjected to ROS injury and Hypoxia. Moreover, through combinatorial treatment with SB431542 (an inhibitor of Tgf-β1), and Adamts4 siRNA mediated gene knockdown, we were able to decipher a regulatory hierarchy to the signal cascade being at the heart of Tgf-β regulation. Besides the hallmark expression of Adamts4 and Tgf-β1, expression of other fibrosis-related markers like Collagen-III, alpha-SMA and Periostin were also assessed. Finally, increased levels of Adamts4 and alpha-SMA proteins in cardiac patients also resonated well with our animal and cell culture studies. Overall, in this study, we highlight, Adamts4 as a novel biomarker of adult cardiac injury.

Kinesin-7 CENP-E is essential for chromosome alignment and spindle assembly of mouse spermatocytes

Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome alignment, which contribute to genome stability in mitosis. However, the functions and mechanisms of CENP-E in the meiotic division of male spermatocytes remain largely unknown. In this study, by combining the use of chemical inhibitors, siRNA-mediated gene knockdown, immunohistochemistry, and high-resolution microscopy, we have found that CENP-E inhibition results in chromosome misalignment and metaphase arrest in dividing spermatocyte during meiosis. Strikingly, we have revealed that CENP-E regulates spindle organization in metaphase I spermatocytes and cultured GC-2 spd cells. CENP-E depletion leads to spindle elongation, chromosome misalignment, and chromosome instability in spermatocytes. Together, these findings indicate that CENP-E mediates the kinetochore recruitment of BubR1, spindle assembly checkpoint and chromosome alignment in dividing spermatocytes, which finally contribute to faithful chromosome segregation and chromosome stability in the male meiotic division.

Girdin influences pancreatic cancer progression by regulating c-MYC expression.

Pancreatic ductal adenocarcinoma is a complex gastrointestinal tumor with high metastatic potential and poor prognosis. Actin-binding protein Girdin is highly expressed in a variety of tumors and promotes tumorigenesis and progression. However, the mechanisms underlying the involvement of Girdin in pancreatic cancer have not been clarified. In this study, we observed that the expression of Girdin was upregulated in pancreatic cancer cells. The siRNA-mediated gene knockdown experiments showed that reduced expression of Girdin in pancreatic cancer cells inhibited cell proliferation, migration, and invasion while promoting cell apoptosis. Functional assays revealed that c-MYC overexpression in pancreatic cancer cells could significantly increase the cell proliferation ability and rates of cell migration and invasion while decreasing the apoptosis rate. It has been shown that phosphorylation plays a role in the functional regulation of the c-MYC gene. Subsequently, we examined the expression level of c-MYC in cells with manipulated expression of Girdin and identified a positive correlation between Girdin expression and c-MYC expression. Moreover, we found that Girdin knockdown in c-MYC-overexpressing pancreatic cancer cells slowed cell growth, blocked the cell cycle progression, significantly promoted apoptosis, and markedly decreased the cell migration and invasion. This finding indicated that silencing Girdin could mitigate the effect of c-MYC on promoting proliferation and metastasis of pancreatic cancer. Overall, this study provided evidence that Girdin promoted pancreatic cancer development presumably by regulating the c-MYC overexpression.

SLC7A9 as a Potential Biomarker for Lymph Node Metastasis of Esophageal Squamous Cell Carcinoma.

The expression of solute carrier (SLC) 7 family genes is reportedly associated with several malignancies. Here, we focused on SLC7A9 and investigated its expression, function, and clinical significance in esophageal squamous cell carcinoma (ESCC). SLC7A9 transcription levels were evaluated in 13 ESCC cell lines, and polymerase chain reaction (PCR) array analysis was conducted to detect coordinately expressed genes with SLC7A9. SLC7A9 contributions to proliferation, invasion, and migration were evaluated in ESCC cells subjected to siRNA-mediated gene knockdown and pCMV6-entry plasmid-mediated overexpression. SLC7A9 expression was detected in 189 ESCC tissues by quantitative reverse-transcription (qRT)-PCR and correlated with clinicopathological parameters. The expression levels of SLC7A9 varied widely in ESCC cell lines and correlated with FGFBP1 expression. Knockdown of SLC7A9 significantly suppressed the proliferation, invasion, and migration of the ESCC cell lines. Moreover, overexpression of SLC7A9 enhanced cell proliferation and migration. In analyses of clinical specimens, SLC7A9 mRNA was overexpressed in the ESCC tissues compared with the adjacent normal esophageal tissues. High mRNA expression was significantly associated with high levels of squamous cell carcinoma-related antigen and carcinoembryonic antigen, advanced disease stage, and lymph node metastasis. High SLC7A9 expression was also significantly associated with poor disease-specific and disease-free survival, and lymph node recurrence after radical surgery, but not with the other recurrence patterns. On multivariate analysis, high SLC7A9 expression was an independent predictor of lymph node recurrence. SLC7A9 influences the malignant behavior of ESCC cells. Tumor SLC7A9 expression may serve as a novel biomarker for predicting lymph node metastasis and recurrence in ESCC patients.

A design approach for layer-by-layer surface-mediated siRNA delivery

The ability to coat scaffolds and wound dressings with therapeutic short interfering RNA (siRNA) holds much potential for applications in wound healing, cancer treatment, and regenerative medicine. Layer-by-layer (LbL) technology is an effective method to formulate polyelectrolyte thin films for local delivery of siRNA; however, the formation and efficacy of LbL coatings as drug delivery systems are highly contingent on the assembly conditions. Here, we investigate the effects of LbL assembly parameters on film composition and consequent siRNA-mediated gene knockdown efficiency in vitro. Films comprising poly(β-amino ester) (PBAE) and siRNA were built on polyglactin 910 (Vicryl) sutures consisting of poly(10% L-lactide, 90% glycolide). A fractional factorial design was employed, varying the following LbL assembly conditions: pH, ionic strength, PBAE concentration, and siRNA concentration. Effects of these parameters on PBAE loading, siRNA loading, their respective weight ratios, and in vitro siRNA-mediated knockdown were elucidated. The parameter effects were leveraged to create a rationally designed set of solution conditions that was predicted to give effective siRNA-mediated knockdown, but not included in any of the original experimental conditions. This level of knockdown with our rationally designed loading conditions (47%) is comparable to previous formulations from our lab while being simpler in construction and requiring fewer film layers, which could save time and cost in manufacturing. This study highlights the importance of LbL solution conditions in the preparation of surface-mediated siRNA delivery systems and presents an adaptable methodology for extending these electrostatically-assembled coatings to the delivery of other therapeutic nucleic acids. Statement of significanceShort interfering RNA (siRNA) therapeutics are powerful tools to silence aberrant gene expression in the diseased state; however, the clinical utility of these therapies relies on effective controlled delivery approaches. Electrostatic self-assembly through the layer-by-layer (LbL) process enables direct siRNA release from surfaces, but this method is highly dependent upon the specific solution conditions used. Here, we use a fractional factorial design to illustrate how these assembly conditions impact composition of siRNA-eluting LbL thin films. We then elucidate how these properties mediate in vitro transfection efficacy. Ultimately, this work presents a significant step towards understanding how optimization of assembly conditions for surface-mediated LbL delivery can promote transfection efficacy while reducing the processing and material required.

Apoptosis is a generator of Wnt-dependent regeneration and homeostatic cell renewal in the ascidian Ciona.

ABSTRACTIn the ascidian Ciona intestinalis, basal body parts regenerate distal structures but distal body parts do not replace basal structures. Regeneration involves the activity of adult stem cells in the branchial sac, which proliferate and produce migratory progenitor cells for tissue and organ replacement. Branchial sac-derived stem cells also replenish recycling cells lining the pharyngeal fissures during homeostatic growth. Apoptosis at injury sites occurs early during regeneration and continuously in the pharyngeal fissures during homeostatic growth. Caspase 1 inhibitor, caspase 3 inhibitor, or pan-caspase inhibitor Z-VAD-FMK treatment blocked apoptosis, prevented regeneration, and suppressed branchial sac growth and function. A pharmacological screen and siRNA-mediated gene knockdown indicated that regeneration requires canonical Wnt signaling. Wnt3a protein rescued both caspase-blocked regeneration and branchial sac growth. Inhibition of apoptosis did not affect branchial sac stem cell proliferation but prevented the survival of progenitor cells. After bisection across the mid-body, apoptosis occurred only in the regenerating basal fragments, although both fragments contained a part of the branchial sac, suggesting that apoptosis is unilateral at the wound site and the presence of branchial sac stem cells is insufficient for regeneration. The results suggest that apoptosis-dependent Wnt signaling mediates regeneration and homeostatic growth in Ciona.

TRIP6 is required for tension at adherens junctions.

Hippo signaling mediates influences of cytoskeletal tension on organ growth. TRIP6 and LIMD1 have each been identified as being required for tension-dependent inhibition of the Hippo pathway LATS kinases and their recruitment to adherens junctions, but the relationship between TRIP6 and LIMD1 was unknown. Using siRNA-mediated gene knockdown, we show that TRIP6 is required for LIMD1 localization to adherens junctions, whereas LIMD1 is not required for TRIP6 localization. TRIP6, but not LIMD1, is also required for the recruitment of vinculin and VASP to adherens junctions. Knockdown of TRIP6 or vinculin, but not of LIMD1, also influences the localization of myosin and F-actin. In TRIP6 knockdown cells, actin stress fibers are lost apically but increased basally, and there is a corresponding increase in the recruitment of vinculin and VASP to basal focal adhesions. Our observations identify a role for TRIP6 in organizing F-actin and maintaining tension at adherens junctions that could account for its influence on LIMD1 and LATS. They also suggest that focal adhesions and adherens junctions compete for key proteins needed to maintain attachments to contractile F-actin.

BBS Proteins Affect Ciliogenesis and Are Essential for Hedgehog Signaling, but Not for Formation of iPSC-Derived RPE-65 Expressing RPE-Like Cells.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1−/−-defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.