Scramble siRNA Research Articles

NLRC5 silencing ameliorates cardiac fibrosis by inhibiting the TGF‑β1/Smad3 signaling pathway.

The proliferation of cardiac fibroblasts (CFs) and excessive deposition of extracellular matrix are the predominant pathological characteristics of cardiac fibrosis. As the largest member of the nucleotide‑binding domain and leucine‑rich repeat (NLR) family, NLRC5 has been shown to be pivotal in the development of hepatic fibrosis. However, whether NLRC5 is involved in the pathogenesis of cardiac fibrosis remains to be elucidated. The present study aimed to investigate the role of NLRC5 and its mechanisms in regulating cardiac fibrosis. CFs were stimulated with transforming growth factor (TGF)‑β1 for various times and the mRNA and protein expression of NLRC5 was assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. In addition, CFs were transfected with small interfering (si)RNA targeting NLRC5 or scramble siRNA for 24h and then stimulated with TGF‑β1 for 24h. Subsequently, cell proliferation was measured using an MTT assay, whereas cell migration was evaluated using a Transwell migration assay. The protein expression levels of α‑smooth muscle actin, collagen I, connective tissue growth factor, phosphorylated‑Smad3 and Smad3 were measured using western blot analysis. The results demonstrated that NLRC5 was upregulated in TGF‑β1‑induced CFs. The knockdown of NLRC5 significantly inhibited cell proliferation and migration, and suppressed myofibroblast differentiation and the expression of pro‑fibrotic molecules in TGF‑β1‑treated CFs. Furthermore, the knockdown of NLRC5 attenuated TGF‑β1‑induced phosphorylation of small mothers against decapentaplegic (Smad)3 in the CFs. The results of the present study indicated that NLRC5 acted as a key regulator of pathological cardiac fibrosis, and NLRC5 silencing ameliorated cardiac fibrosis by inhibiting the TGF‑β1/Smad3 signaling pathway. These results suggested that NLRC5 may be a novel target for attenuating cardiac fibrosis.

Rs10719 Polymorphism Located within DROSHA 3'-Untranslated Region is Responsible for Development of Primary Hypertension by Disrupting Binding with microRNA-27b.

BackgroundMiR-27b is reportedly involved with many diseases (e.g., gastric cancer) by acting on different signaling pathways. In this study, we aimed at understanding the relationship between miR-27b and hypertension and its underlying molecular mechanism.Material/MethodsPeripheral blood was collected from patients with hypertension, and statistical analysis was performed to study the association between rs10719 and risk of hypertension. Tissue samples were collected from patients with lung cancer, and the expression of miR-27b and DROSHA was determined using Western blot analysis and real-time PCR.ResultsWe first searched the miRNA database online, and identified DROSHA as a virtual target of miR-27b with the “seed sequence” located within the 3′-UTR of the target gene, and then validated DROSHA to be the direct gene via luciferase reporter assay system. We also established the negative regulatory relationship between miR-27b and DROSHA via studying the relative luciferase activity. We also conducted real-time PCR to study the mRNA and protein expression level of miR-27b among different groups. Furthermore, we conducted real-time PCR and densitometry analysis to study the mRNA and protein expression level of DROSHA among different groups of cells treated with scramble control, miR-27b mimics, DROSHA siRNA, and miR-27b inhibitors to verify the negative regulatory relationship between MiR-27b and DROSHA.ConclusionsThe presence of rs10719 disrupted the interaction between miR-27b and DROSHA, which might be the underlying mechanism of the observation that rs10719 is significantly associated with risk of primary hypertension.

Dysregulation of miRNA-146a contributes to the development of lupus nephritis via targeting of TRAF6.

The objective of this study was to identify the association between genotypes of miR-146a rs2910164 and expression of TRAF6 as well as the risk of lupus nephritis (LN). A total of 567 systemic lupus erythematosus patients both with and without LN were included in the study. The luciferase activity of cells that carried miR-146a mimics was much lower than control and the miR-146a mRNA expression with the GG SNP was significantly overexpressed compared with that in GC and CC groups. Expressions of TRAF6 mRNA and protein with GG were markedly lower than those in GC and CC groups. Mesangial cells treated with miR-146a inhibitors displayed higher expression of TRAF6 mRNA and protein compared with scramble control, miR-146a mimics and TRAF6 siRNA groups. Rs2910164 is associated with the risk of LN and could function as a therapeutic target of the disease.

Mir-150 Up-Regulates Glut1 and Increases Glycolysis in Osteosarcoma Cells

Objective:Osteosarcoma (OS) is the most common malignant bone tumor in children and young adults. Many studies have shown that microRNAs play a critical role in proliferation and metastasis with this tumour type. However, whether aberrant expression might contribute to a metabolism switch in osteosarcoma cases is not clearly understood. In this study, we explored expression and function of miR-150 in osteosarcoma cells.Materials and methods:Expression of miR-150 was assessed by real-time PCR in cell lines and human patient tissues. Scramble siRNA, miR-150 inhibitor, and miR-150 mimics were transfected into osteosarcoma cells to determine their effects on proliferation rate, glucose uptake and lactate secretion. Finally, the relationship between Glut1 and the miR-150 level was explored by luciferase reporter assay and western blotting.Result:miR-150 was consistently decreased in cell lines and osteosarcoma tissues as compared to osteoblast cells and normal bone. Ectopic overexpression of miR-150 inhibited osteosarcoma cell proliferation and suppressed glucose uptake and lactate secretion. Loss of function of miR-150, on the other hand, enhanced osteosarcoma cell proliferation and increased glucose uptake and lactate secretion. Western blot and luciferase reporter assays showed that miR-150 may function by regulating Glut1 expression.Conclusion:These data suggest that miR-150 is involved in regulation of glycolysis in osteosarcoma cells by influencing Glut1 expression.

Role of GPx4 in human vascular endothelial cells, and the compensatory activity of brown rice on GPx4 ablation condition

Oxidative stress is implicated in the pathologies of vascular endothelial cells. However, the importance of specific antioxidant enzymes in vascular endothelial cells is not fully understood. The purpose of this study was to elucidate the importance of Glutathione peroxidase 4 (GPx4), and the involvement of ferroptosis on cell death induced by GPx4 loss in human vascular endothelial cells. In addition, we examined the compensatory activity of brown rice on GPx4 ablation condition.Human umbilical vein endothelial cells were transfected with GPx4 or scramble control siRNA. GPx4 knockdown caused the increase in the levels of lipid oxidation, and induced cytotoxicity. On the other hand, α-tocopherol (vitamin E) and extract of brown rice, ameliorated lipid peroxidation, cytotoxicity, and delay of proliferation induced by GPx4 knockdown. Furthermore, ferrostatin-1, inhibitor of ferroptosis, also prevented cytotoxicity and delay of proliferation.In conclusion, our data demonstrated that GPx4 is an essential antioxidant enzyme for protecting lipid peroxidation, and is a regulator of ferroptosis in vascular endothelial cells. Furthermore, vitamin E rich food, such as brown rice, can compensate for GPx4 loss by protecting cells against lipid peroxidation.

Aberrant Expression of microRNA-9 Contributes to Development of Intracranial Aneurysm by Suppressing Proliferation and Reducing Contractility of Smooth Muscle Cells.

BackgroundMiR-9 is reportedly involved with many diseases, such as acute myeloid leukemia and liver oncogenesis. In the present study we investigated the molecular mechanism, including the potential regulator and signaling pathways, of MYOCD, which is the gene that in humans encodes the protein myocardin.Material/MethodsWe searched the online miRNA database (www.mirdb.org) with the “seed sequence” located within the 3′-UTR of the target gene, and then validated MYOCD to be the direct gene via luciferase reporter assay system, and further confirmed it in cultured cells by using Western blot analysis and realtime PCR.ResultsWe established the negative regulatory relationship between miR-9 and MYOCD via studying the relative luciferase activity. We also conducted realtime PCR and Western blot analysis to study the mRNA and protein expression level of MYOCD between different groups (intracranial aneurysm vs. normal control) or cells treated with scramble control, miR-9 mimics, MYOCD siRNA, and miR-9 inhibitors, indicating the negative regulatory relationship between miR-9 and MYOCD. We also investigated the relative viability of smooth muscle cells when transfected with scramble control, miR-9 mimics, MYOCD siRNA, and miR-9 inhibitors to validate that miR-9 t negatively interferes with the viability of smooth muscle cells. We then investigated the relative contractility of smooth muscle cells when transfected with scramble control, miR-9 mimics, MYOCD siRNA, and miR-9 inhibitors, and the results showed that miR-9 weakened contractility.ConclusionsOur findings show that dysregulation of miR-9 is responsible for the development of IA via targeting MYOCD. miR-9 and its direct target, MYOCD, might novel therapeutic targets in the treatment of IA.

Down-Regulation of MicroRNA-133b Suppresses Apoptosis of Lens Epithelial Cell by Up-Regulating BCL2L2 in Age-Related Cataracts.

BackgroundMicroRNA-133b (miR-133b) has been reported to be involved in many diseases, including ovarian cancer and osteosarcoma. Accumulating evidence suggests that miR-133b plays important roles in human disease. In this study, we aimed to investigate the molecular mechanism, including the potential regulator and signaling pathways, of BCL2L2.Material/MethodsWe first searched the online miRNA database (www.mirdb.org) using the “seed sequence” located within the 3′-UTR of the target gene, and then performed luciferase assay to test the regulatory relationship between miR-133b and BCL2L2. Western blot and real-time PCR were used to determine the expression of BCL2L2 in human samples or cells treated with miRNA mimics or inhibitors. Flow cytometry was conducted to evaluate the apoptosis status of the cells.ResultsWe validated BCL2L2 to be the direct gene using a luciferase reporter assay. We also conducted real-time PCR and Western blot analyses to study the mRNA and protein expression level of BCL2L2 among different groups (control: n=29, cataract: n=33) or cells treated with scramble control, miR-133b mimics, BCL2L2 siRNA, and miR-133b inhibitors, and identified the negative regulatory relationship between miR-133b and BCL2L2. We also conducted experiments to investigate the influence of miR-133b and BCL2L2 on the viability and apoptosis of cells. The results showed that miR-133b positively interfered with the viability of cells, while BCL2L2 negatively interfered with the viability of cells, and that miR-133b inhibited apoptosis while BCL2L2 accelerated apoptosis.ConclusionsBCL2L2 was the virtual target of miR-133b, and we found a negative regulatory relationship between miR-133b and BCL2L2. MiR-133b and BCL2L2 interfered with the viability and apoptosis of cells.

Inhibitory Effect of TLR4 Gene Silencing on Intimal Hyperplasia of Vein Grafting.

The present study aimed to explore the regulating effect of Toll-like receptor 4 (TLR4) on intimal hyperplasia in rat vein grafts. Rat models of external jugular vein carotid artery bypass grafting were established. Afterward, TLR4 small interfering RNA (siRNA) recombinant plasmids were constructed, which were transfected into rat vein graft bypass to study the effect of TLR4 silencing on intimal hyperplasia and to explore the underlying mechanisms. Real-time polymerase chain reaction and Western blot were used to detect the expression levels of TLR4 and inflammatory factors in TLR4 siRNA-transfected vein graft bypass. The intimal thickness was evaluated using hematoxylin-eosin staining. Compared with the scramble siRNA group, the intimal thickness of vein grafting was decreased significantly, while the inflammatory factors including interleukin (IL) 1β, IL-6, and tumor necrosis factor α in grafted vein were dramatically downregulated in the TLR4 siRNA group. These results showed that local silencing of TLR4 in the vein grafts could inhibit intimal hyperplasia by downregulating the expression of inflammatory factors in the vein grafts, suggesting that TLR4 can be used as a new target for therapy of vascular intimal hyperplasia.

SH003 selectively induces p73‑dependent apoptosis in triple‑negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a breast cancer subtype that has an aggressive phenotype, is highly metastatic, has limited treatment options and is associated with a poor prognosis. In addition, metastatic TNBC has no preferred standard chemotherapy due to resistance to anthracyclines and taxanes. The present study demonstrated that a herbal extract, SH003, reduced cell viability and induced apoptosis in TNBC without cell cytotoxicity. Cell viability was examined using trypan blue exclusion and colony formation assays, which revealed a decrease in the cell viability. Additionally, apoptosis was determined using flow cytometry and a sub‑G1 assay, which revealed an increase in the proportion of cells in the sub‑G1 phase. The present study investigated the anticancer effect of SH003 in the Hs578T, MDA‑MB‑231 and ZR‑751 TNBC cell lines, and in the MCF7 and T47D non‑TNBC cell lines. Western blot analysis revealed that the expression levels of poly‑ADP‑ribose polymerase (PARP) cleavage protein in cells treated with SH003 were increased dose‑dependent manner, indicating that SH003 induced apoptosis via a caspase‑dependent pathway. Pre‑treatment with the caspase inhibitor Z‑VAD reduced SH003‑induced apoptosis was examined using trypan blue exclusion. Moreover, SH003 treatment enhanced the p73 levels in MDA‑MB‑231 cells but not in MCF7 cells. Transfection of p73 small interfering RNA (siRNA) in MDA‑MB0231 cells revealed that the apoptotic cell death induced by SH003 was significantly impaired in comparison with scramble siRNA transfected MDA‑MB‑231 cells. This was examined using trypan blue exclusion and flow cytometry analysis (sub‑G1). In addition, SH003 and paclitaxel exhibited synergistic anticancer effects on TNBC cells. The results indicate that SH003 exerts its anticancer effect via p73 protein induction and exhibits synergistic anticancer effects when combined with paclitaxel.

OS 15-07 ROLE OF (PRO) RENIN RECEPTOR IN THE ACTIVATION OF WNT/ β-CATENIN PATHWAY IN COLON CANCER

Objective: Constitutive activation of Wnt/β-catenin pathway plays an important role in the pathogenesis of colon cancer; however, recent studies have also indicated that elevated Wnt levels are essential. In the present study, we investigated the potential role of (pro) renin receptor in the pathophysiology of colon cancer. Design and Method: (P) RR expression in human colon tissues was measured by immunohistochemistry and Western blot analysis. Human colon cancer cell lines DLD-1 with adenomatous polyposis coli (APC) truncated mutation, and HCT116 withβ-catenin activating mutation, were transfected with scramble siRNA and siRNA against (pro) renin receptor. Results: (Pro) renin receptor expression was significantly increased in colon cancer tissue. (Pro) renin receptor knockdown significantly decreased protein levels of the main components of Wnt/β-catenin pathway (Wnt, phosphorylated-LRP6, active β-catenin and cyclin D1) in both DLD-1 and HCT116 cells. Wnt signaling activity, measured by Tcf/Wnt signaling reporter assay, was also significantly decreased by (pro) renin receptor knockdown. WST-1 assay showed that (pro) renin receptor knockdown markedly reduced cell proliferation activity. Conclusions: These data suggest that (pro) renin receptor promotes colon cancer through Wnt/β-catenin pathway despite of intrinsic its constitutive activation.

Hypoxia-induced microRNA-301b regulates apoptosis by targeting Bim in lung cancer.

Worldwide, lung cancer accounts for the majority of cancer-related deaths. Aberrant expression of miRNAs has increasingly been reported to be associated with tumour progression. This study aimed to explore the role of miR-301b in regulating apoptosis in lung cancer. Expression of miR-301b was assessed by real-time PCR in cell lines, human patient tissues and cells treated under hypoxia and DMOG. Scramble siRNA, miR-301b inhibitor and miR-301b mimics were transfected into lung cancer cells to determine their effects on apoptosis. Additionally, a mouse xenograft model was used to explore functions of miR-301b on apoptosis, in vivo. Finally, relationships between Bim and miR-301b levels were explored by luciferase reporter assay and Western blotting. We found that miR-301b was highly expressed in lung cancer tissues and cell lines. Expression of miR-301b was induced by hypoxia, and miR-301b suppressed expression of Bim by targeting its 3'UTR. Functionally, ectopic expression of miR-301b enhanced cell population growth, reduced apoptosis and reduced sensitivity of cells to chemotherapy. In the xenograft model, overexpression of miR-301b promoted tumour growth. Additionally, miR-301b and Bim expression were inversely correlated in clinical lung cancer samples. This study provides new insights into the function of miRNA-301b in lung cancer and suggests that miRNA-301b could be a potential molecular target for chemotherapy.

Role of PDGF-D and PDGFR-β in neuroinflammation in experimental ICH mice model

ObjectiveInflammation plays a key role in the pathophysiological processes after intracerebral hemorrhage (ICH). Post-ICH macrophages infiltrate the brain and release pro-inflammatory factors (tumor necrosis factor-α), amplifying microglial activation and neutrophil infiltration. Platelet-derived growth factor receptor-β (PDGFR-β) is expressed on macrophages and it's activation induces the recruitment of macrophages. Platelet-derived growth factor-D (PDGF-D) is an agonist with a significantly higher affinity to the PDGFR-β compared to another isoform of the receptor. In this study, we investigated the role of PDGF-D in the pro-inflammatory response after ICH in mice. MethodsA blood injection model of ICH was used in eight-week old male CD1 mice (weight 30g). Some mice received an injection of plasmin or PDGF-D. Gleevec, a PDGFR inhibitor, was administered at 1, 3 or 6h post-ICH. Plasmin was administered with or without PDGF-D siRNAs mixture or scramble siRNA. A plasmin-antagonist, ε-Aminocaproic acid (EACA), was co-administrated with the blood. The effects of ICH and treatment on the brain injury and post-ICH inflammation were investigated. ResultsICH resulted in the overexpression of PDGF-D, associated with the infiltration of macrophages. PDGFR-inhibition decreased ICH-induced brain injury, attenuating macrophage and neutrophil infiltration, reducing microglial activation and TNF-α production. Administration of recombinant PDGF-D induced TNF-α production, and PDGFR-inhibition attenuated it. A plasmin-antagonist suppressed PDGFR-β activation and microglial activation. Plasmin increased PDGF-D expression, and PDGF-D inhibition reduced neutrophil infiltration. ConclusionICH-induced PDGF-D accumulation contributed to post-ICH inflammation via PDGFR activation and enhanced macrophage infiltration. The inhibition of PDGFR had an anti-inflammatory effect. Plasmin is a possible upstream effector of PDGF-D. The targeting of PDGF-D may provide a novel way to decrease brain injury after ICH.

Role of hepcidin and its downstream proteins in early brain injury after experimental subarachnoid hemorrhage in rats.

Early brain injury (EBI) is a major cause of mortality from subarachnoid hemorrhage (SAH). We aimed to study the pathophysiology of EBI and explore the role of hepcidin, a protein involved in iron homeostatic regulation, and its downstream proteins. One hundred and thirty-two male Sprague-Dawley rats were assigned into groups (n=24/group): sham, SAH, SAH+hepcidin, SAH+hepcidin-targeting small interfering ribonucleic acid (siRNA), and SAH+scramble siRNA. Three hepcidin-targeting siRNAs and one scramble siRNA for hepcidin were injected 24h before hemorrhage induction, and hepcidin protein was injected 30min before induction. The rats were neurologically evaluated at 24h and euthanized at 72h. Hepcidin, ferroportin-1, and ceruloplasmin protein expression were measured by immunohistochemistry and Western blotting. Brain water content, blood-brain barrier (BBB) leakage, non-heme tissue iron and Garcia scale were evaluated. Hepcidin expression increased in the cerebral cortex and hippocampus after experimental SAH (P<0.05 compared to sham), while ferroportin-1 and ceruloplasmin decreased (P<0.05). Hepcidin injection lowered the expression of ferroportin-1 and ceruloplasmin further but siRNA reduced the levels of hepcidin (P<0.05 compared to SAH) resulting in recovery of ferroportin-1 and ceruloplasmin levels. Apoptosis was increased in SAH rats compared to sham (P<0.05) and increased slightly more by hepcidin, but decreased by siRNA (P<0.05 compared to SAH). SAH rats had lower neurological scores, high brain water content, BBB permeability, and non-heme tissue iron (P<0.05). In conclusion, downregulation of ferroportin-1 and ceruloplasmin caused by hepcidin enhanced iron-dependent oxidative damage and may be the potential mechanism of SAH.

Down-regulation of anti-apoptotic genes in tumor cell lines is facilitated by suppression of OCT4B1

PurposeThe OCT4B1 as a variant of OCT4 is expressed in both cancer cells and tissues. The anti-apoptotic property of this variant aid cancer cells to escape from apoptosis. Therefore, the aim of the present study was to determine the effects of OCT4B1 suppression on regulation of 25 genes involved in anti-apoptotic pathway in tumor cell lines. Material and methodsAGS (gastric adenocarcinoma), 5637 (bladder tumor) and U-87MG (brain tumor) cells were transfected with specific OCT4B1 siRNA and a scramble siRNA by siRNA silencing gene technology, using Lipofectamine 2000 commercial kit. The real-time PCR technique was employed to examine and calculate fold changes of evaluated genes using the 2−ΔΔCT formula. ResultsPresent results demonstrated that 22 (88%) of interested genes were similarly down-regulated in all three examined cell lines. Our results also indicated that three genes (CASP2, IGF1R,TNF) were up-regulated. The CFLAR gene was down-regulated in AGS, while it was inversely up-regulated in 5637 and U87MG cells. ConclusionsIt may possibly be concluded that suppression of OCT4B1 can lead to apoptosis in tumor cell lines and this is at least facilitated via down-regulation of examined anti-apoptotic genes. Accordingly, suppression of OCT4B1 may probably be considered as useful tool in cancer therapy and research.

TLR4 siRNA inhibits proliferation and invasion in colorectal cancer cells by downregulating ACAT1 expression

AimsToll-like receptor 4 (TLR4) is involved in tumor development. Numerous studies have confirmed that TLR4 mediates processes in tumorigenesis, for example, inflammation, proliferation and invasion. However, the effects of TLR4 on colorectal cancer development have not been fully elucidated. The present study aimed to evaluate the effects and mechanisms of TLR4 on colorectal cancer development. Main methodsThe expression of TLR4 and Acyl coenzyme A cholesterol acyltransferase 1 (ACAT1) in colorectal cancer tissues and cell lines was detected using RT-PCR and Western blot. The nude mouse xenograft model was established by subcutaneous injection of SW480 cells transfected with TLR4 scramble or TLR4 siRNA. CCK8 and transwell assays were used to evaluate the effects of TLR4 silencing on cell proliferation, migration and invasion in HT29 and SW480. RT-PCR and Western blot was used to determine the regulation between TLR4 and ACAT1. Key findingsBoth TLR4 and ACAT1 were highly expressed in colorectal cancer tissues and cell lines. Inhibition of TLR4 reduced tumor growth, suppressed cell proliferation, migration and invasion in HT29 and SW480. TLR4 siRNA decreased ACAT1 expression in HT29, and that overexpression of ACAT1 by pLV-Neo-ACAT1 abolished the effects of TLR4 on colorectal cancer cell lines. SignificanceTLR4 siRNA inhibits cell proliferation, migration and invasion by suppressing ACAT1 expression, suggesting that TLR4 may be a potential therapeutic target for the treatment of colorectal cancer.

Knockdown of versicanV1 induces a severe inflammatory response in LPS-induced acute lung injury via the TLR2-NF-κB signaling pathway in C57BL/6J mice.

The versican family is important in the modulation of inflammation, however, the role of versican V1 (V1) in lipo-polysaccharide (LPS)-induced acute lung injury (ALI) and the underlying mechanisms remain to be elucidated. To investigate this, the present study performed experiments in male C57BL/6J mice, which were randomly divided into a normal control group (control; n=6), an LPS-stimulated ALI group (LPS; n=6), a scramble small interfering (si)RNA group (scramble; n=6), a V1-siRNA group (V1-siRNA; n=6), a scramble siRNA and LPS-stimulated group (scramble+LPS; n=6) and a V1-siRNA and LPS-stimulated group (V1-siRNA+LPS; n=6). On day 1, the mice were anesthetized, and 5 nmol scramble siRNA or V1-siRNA were administered intratracheally. On day 3, LPS (1 mg/kg) or phosphate-buffered saline (50 µl per mouse) were injected intratracheally. All the mice were anesthetized and sacrificed on day 4, and samples were collected and analyzed. The mRNA and protein expression levels were examined using reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemical staining and western blot analysis. ALI was evaluated based on lung injury scores, cell counts and total protein concentrations in the bronchoalveolar lavage fluid (BALF). Inflammatory mediators were detected using an enzyme-linked immunosorbend assay. V1 was increased by LPS in the mouse ALI model, whereas specific V1 knockdown induced higher lung injury scores, and higher total cell counts and protein concentrations in the BALF. Tumor necrosis factor-α (TNF)-α was upregulated, and interleukin-6 exhibited an increasing trend. The expression of toll-like receptor 2 (TLR2), but not TLR4, increased, and the nuclear factor (NF)-κB pathway subunit, P65, was phosphorylated. Taken together, the expression of V1 was upregulated by LPS, and V1 inhibition resulted in the aggravation of LPS-induced ALI via the activation of TLR2-NF-κB and release of TNF-α.

Human islet cells are killed by BID-independent mechanisms in response to FAS ligand.

Cell death via FAS/CD95 can occur either by activation of caspases alone (extrinsic) or by activation of mitochondrial death signalling (intrinsic) depending on the cell type. The BH3-only protein BID is activated in the BCL-2-regulated or mitochondrial apoptosis pathway and acts as a switch between the extrinsic and intrinsic cell death pathways. We have previously demonstrated that islets from BID-deficient mice are protected from FAS ligand-mediated apoptosis in vitro. However, it is not yet known if BID plays a similar role in human beta cell death. We therefore aimed to test the role of BID in human islet cell apoptosis immediately after isolation from human cadaver donors, as well as after de-differentiation in vitro. Freshly isolated human islets or 10-12day cultured human islet cells exhibited BID transcript knockdown after BID siRNA transfection, however they were not protected from FAS ligand-mediated cell death in vitro as determined by DNA fragmentation analysis using flow cytometry. On the other hand, the same cells transfected with siRNA for FAS-associated via death domain (FADD), a molecule in the extrinsic cell death pathway upstream of BID, showed significant reduction in cell death. De-differentiated islets (human islet-derived progenitor cells) also demonstrated similar results with no difference in cell death after BID knockdown as compared to scramble siRNA transfections. Our results indicate that BID-independent pathways are responsible for FAS-dependent human islet cell death. These results are different from those observed in mouse islets and therefore demonstrate potentially alternate pathways of FAS ligand-induced cell death in human and mouse islet cells.

LncRNA uc.48+ is involved in diabetic neuropathic pain mediated by the P2X3 receptor in the dorsal root ganglia.

Some long non-coding RNAs (lncRNAs) participate in physiological processes that maintain cellular and tissue homeostasis, and thus, the dysregulated expression of lncRNAs is involved in the onset and progression of many pathological conditions. Research has indicated that the genetic knockout of some lncRNAs in mice resulted in peri- or postnatal lethality or developmental defects. Diabetes mellitus (DM) is a major cause of peripheral neuropathy. Our studies showed that the expression levels of lncRNA uc.48+ in the diabetic rat dorsal root ganglia (DRG) and the DM patients' serum samples were increased. It suggested that lncRNA uc.48+ was involved in the pathophysiological process of DM. The aim of this study was to investigate the effects of lncRNA uc.48+ small interfering RNA (siRNA) on diabetic neuropathic pain (DNP) mediated by the P2X3 receptor in the DRG. The values of the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured by the von Frey test and Hargreaves' test, respectively. The levels of P2X3 protein and messenger RNA (mRNA) in the DRG were detected by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and western blotting. The experiments showed that the MWT and TWL values in DM rats were lower than those in the control rats. The MWT and TWL values in DM rats treated with lncRNA uc.48+ siRNA were increased compared to those in DM rats, but there was no significant difference between the DM rat group and the DM + scramble siRNA group. The levels of P2X3 protein and mRNA in the DM DRG were higher than those in the control, while the levels of P2X3 protein and mRNA in the DG of DM rats treated with uc.48+ siRNA were significantly decreased compared to those in DM rats. The expression levels of TNF-α in the DRG of DM rats treated with uc.48+ siRNA were significantly decreased compared to those in the DM group. The phosphorylation and activation of ERK1/2 in the DM DRG were decreased by uc.48+ siRNA treatment. Therefore, uc.48+ siRNA treatment may alleviate the DNP by inhibiting the excitatory transmission mediated by the P2X3 receptor in DRG.

PTK7 Is Involved in the Chemotherapy Resistance of T-Cell Acute Lymphoblastic Leukemia through Interaction with FADD-Death Domain

Apoptosis-based therapies have been used as a powerful weapon against hematologic malignancies (Reed JC et al, Blood 2005 15; 408-418). One strategy for attacking cancer is to overcome its resistance to apoptosis via specific targeting molecules. In this study, we are focusing on protein tyrosine kinase 7 (PTK7) as a new target for cancer therapy. PTK7, also known as CCK4, is a member of transmembrane tyrosine kinase family proteins that has been implicated in cell migration, polarity and apoptosis. PTK7 is overexpressed in acute T-cell lymphoblastic leukemia cells, and a recent report indicated that PTK7 expression was associated with poor prognosis. However, the mechanism by which PTK7 elicits its anti-apoptotic effects and directly promotes tumor proliferation has not yet been clarified. In addition, no data has been reported regarding the relationship between doxorubicin resistance and PTK7 expression. In this study, we investigated the role of PTK7 in chemotherapy resistance. Two different shRNA lentiviruses targeting PTK7 were stably infected in Jurkat cells (Jurkat tet-shPTK7#1 and #2), and gene knockdown assays against PTK7 were performed. In the presence of doxycycline, we observed a decrease in the viability of PTK7 knockdown cells following stimulation with αFAS-Ab (Jurkat tet-shPTK7#1: 28.6% ± 5.2%, #2: 8.7% ± 0.3%) compared to control cells (considered to be 100%). Similar results of decreased cell viability were also obtained with TRAIL stimulation (Jurkat tet-shPTK7#1: 22.5% ± 5.9%, #2: 14.7% ± 9.8%) compared to control cells (considered to be 100%). Furthermore, we observed a decrease in the viability of PTK7 knockdown cells following treatment with Doxorubicin compared to control cells treated with scramble siRNA. The reduction of cell viability in PTK7 knockdown cells could not be attributed to PTK7 knockdown itself since annexin-V and PI staining showed limited apoptotic changes in Jurkat tet-shPTK7 cells. These changes were also not the result of Fas receptor (CD95) and Trail receptor (DR4, DR5) expression up-regulation as flow cytometric analysis revealed no differences in the expression rates of Fas receptor and Trail receptor between scramble control cells and PTK7 knockdown cells. Very interestingly, PTK7 knockdown had no effect on viability when non-cancer cell lines were stimulated with Fas, TRAIL and Doxorubicin, suggesting that the synergistic effect of PTK7 knockdown and TNF family cytokine treatment is cancer specific. Next, to identify interaction partners of the PTK7 cytoplasmic domain (PTK7-cyto), we performed a yeast two-hybrid assay, and showed that FADD-DD was the only molecule found to bind to PTK7-cyto in both LEU2 reporter and LacZ reporter assays. This protein interaction was further verified by co-immunoprecipitation assays. Finally, to demonstrate the activation of Caspase-8 and PARP, which is induced by TNF-family death receptors, immunoblotting was performed. The shRNA-mediated silencing of PTK7 in Jurkat cells indeed increased Fas- and TRAIL-induced processing of caspase-8 and PARP compared to control RNA. These data suggest that PTK7 appears to be important for a proximal step in Fas and TRAIL signaling, which is consistent with the selective sensitization of tumor cells to the extrinsic apoptosis pathway. Together, our findings reveal an unexpected role for PTK7 in terms of death receptor-mediated apoptosis, suggesting that PTK7 could provide a new target for cancer therapy. DisclosuresNo relevant conflicts of interest to declare.

Abstract A62: PARP-1 regulates NF-κB-mediated IL-8 expression in HER2 positive breast cancer

Abstract Background: We have previously reported that HER2+ breast tumors are sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) independent of a basal or induced homologous recombination (HR) repair deficiency. In addition to its DNA repair functions, PARP-1 acts as a coactivator for NF-κB, a tumor-promoting signaling pathway. In this study we investigated the effect of PARP inhibition on trastuzumab resistant HER2+ breast cancer cells as well as the role of PARP-1 in NF-κB-mediated transcription. Methods: Two trastuzumab resistant (TR) HER2+ breast cancer cell lines (BT-474 and UACC-812 TR) were used in these studies. Cell survival was determined by colony formation assays. PARP-1 knockdown was performed using siRNA and results were compared to cells treated with scrambled (SCR) siRNA. Cell proliferation after PARP-1 knockdown was measured using a coulter counter. Gene expression levels of NF-κB regulated genes were measured via the nCounter Gene Expression Assay and qRT-PCR. We also investigated gene expression levels in PARP-1 knockdown cells stimulated with human recombinant TNF-α, a ligand used to induce NF-κB activation. To validate these results, a chromatin immunoprecipitation (ChIP) assay was utilized to detect NF-κB (p65) at the IL-8 promoter in trastuzumab resistant HER2+ breast cancer cells. IL-8 protein expression levels were also determined via an ELISA assay. Results: HER2+ trastuzumab resistant breast cancer cells retained sensitivity to increasing concentrations of PARPi (ABT-888) as compared to their parental counterparts (survival fraction of more than 70% at 10 μM). Cell proliferation was also reduced by 40% after PARP-1 knockdown. NanoString analysis revealed that PARP-1 knockdown had variable effects on NF-κB-regulated genes, including IL-8, BRCA2, NFKBIZ, VEGFC, PIM1, and FASLG. Interestingly, PARP-1 knockdown had the greatest effect on IL-8 and strongly inhibited its gene expression levels by 90% compared to cells treated with SCR siRNA. These results were also validated by qRT-PCR analysis. PARP-1 knockdown also significantly inhibited TNF-α induced IL-8 gene expression (30%) as compared to cells treated with SCR siRNA. Further, there was an increased amount of p65 detected at the IL-8 promoter after TNF-α stimulation, which was then decreased by 30% after PARP-1 knockdown. IL-8 protein expression was also inhibited by 40% in TNF-α treated PARP-1 knockdown cells. Conclusions: Trastuzumab resistant HER2+ breast cancer cells remain sensitive to PARP inhibition. Further, PARP-1 regulates the expression of IL-8, an NF-κB regulated gene. These results suggest that inhibition of the interaction between PARP-1 and the NF-κB signaling pathway may be a potential mechanism behind the sensitivity to PARPi in HER2+ trastuzumab resistant breast cancer cells. Citation Format: Monicka E. Wielgos, Rajani Rajbhandari, Susan Nozell, C. Kent Osborne, Rachel Schiff, Eddy S. Yang. PARP-1 regulates NF-κB-mediated IL-8 expression in HER2 positive breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A62.