shRNA Research Articles

Fighting drug-resistant lung cancer by induction of NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated ferroptosis.

Drug resistance is a major challenge in cancer treatment. The substrates of NAD(P)H:quinone oxidoreductase 1 (NQO1) show a promising anticancer effect in clinical trials. We previously identified a natural NQO1 substrate 2-methoxy-6-acetyl-7-methyljuglone (MAM) with a potent anticancer effect. The present study was designed to explore the efficacy of MAM in fighting against drug-resistant non-small cell lung cancer (NSCLC). The anticancer effect of MAM was evaluated in cisplatin-resistant A549 and AZD9291-resistant H1975 cells. The interaction of MAM with NQO1 was measured by cellular thermal shift assay and drug affinity responsive target stability assay. The NQO1 activity and expression were measured using NQO1 recombinant protein, Western blotting, and immunofluorescence staining assay. The roles of NQO1 were examined by NQO1 inhibitor, small interfering RNA (siRNA), and short hairpin RNA (shRNA). The roles of reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation were determined. MAM induced significant cell death in drug-resistant cells with similar potency to that of parental cells, which were completely abolished by NQO1 inhibitor, NQO1 siRNA, and iron chelators. MAM activates and binds to NQO1, which triggers ROS generation, LIP increase, and lipid peroxidation. MAM significantly suppressed tumor growth in the tumor xenograft zebrafish model. These results showed that MAM induced ferroptosis by targeting NQO1 in drug-resistant NSCLC cells. Our findings provided a novel therapeutic strategy for fighting against drug resistance by induction of NQO1-mediated ferroptosis.

WTAP Mediated the N6-methyladenosine Modification of PDK4 to Regulate the Malignant Behaviors of Colorectal Cancer Cells In Vitro and In Vivo.

The role of WT1-associated protein (WTAP) in mediating the N6-methyladenosine (m6A) modification of pyruvate dehydrogenase kinase 4 (PDK4) in colorectal cancer (CRC) has been previously reported. This research manages to unveil the function and mechanism of WTAP mediating the m6A modification in CRC. Expressions of PDK4 and WTAP in CRC were assessed by bioinformatics analysis and verified by Western blot. After the transfection with short hairpin RNAs (shRNAs) for WTAP (shWTAP) and PDK4 (shPDK4) to manipulate the expressions of PDK4 and WTAP, the viability, proliferation, migration, invasion, and levels of m6A, PDK4 and WTAP in CRC cells were determined by cell counting kit-8 (CCK-8), colony formation, transwell, Western blot, or M6A-RNA immunoprecipitation (MeRIP)-qPCR assays. M6A binding sites in PDK4 were additionally predicted through bioinformatics analysis, and the interaction of PDK4 and WTAP was confirmed using an RNA pull-down assay. Tumor volume and weight in the constructed xenograft-tumor mouse model were recorded. PDK4 expression was low, yet WTAP and m6A expressions were high in CRC cells. WTAP bound with the m6A binding sites in PDK4. PDK4 silencing facilitated the viability, proliferation, migration and invasion, inhibited the expression of PDK4 in CRC cells, and accelerated the growth of xenografts in vivo. However, the depletion of WTAP4 exerted the opposite effects and further offset the impact of PDK4 silencing. WTAP mediates the m6A modification of PDK4 to regulate the malignant behaviors of CRC cells in vitro and in vivo.

ATF3 regulates SPHK1 in cardiomyocyte injury via endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress is common in different human pathologies, including cardiac diseases. Sphingosine kinase-1 (SPHK1) represents an important player in cardiac growth and function. Nevertheless, its function in cardiomyocyte ER stress remains vague. This study sought to evaluate the mechanism through which SPHK1 might influence ER stress during myocardial infarction (MI). MI-related GEO data sets were queried to screen differentially expressed genes. Murine HL-1 cells exposed to oxygen-glucose deprivation (OGD) and mice with MI were induced, followed by gene expression manipulation using short hairpin RNAs and overexpression vectors. The activating transcription factor 3 (ATF3) and SPHK1 expression was examined in cells and tissues. Cell counting kit-8, TUNEL, DHE, HE, and Masson's staining were conducted in vitro and in vivo. The inflammatory factor concentrations in mouse serum were measured using ELISA. Finally, the transcriptional regulation of SPHK1 by ATF3 was validated. ATF3 and SPHK1 were upregulated in vivo and in vitro. ATF3 downregulation reduced the SPHK1 transcription. ATF3 and SPHK1 downregulation increased the viability of OGD-treated HL-1 cells and decreased apoptosis, oxidative stress, and ER stress. ATF3 and SPHK1 downregulation narrowed the infarction area and attenuated myocardial fibrosis in mice, along with reduced inflammation in the serum and ER stress in the myocardium. In contrast, SPHK1 reduced the protective effect of ATF3 downregulation in vitro and in vivo. ATF3 downregulation reduced SPHK1 expression to attenuate cardiomyocyte injury in MI.

Knockdown of PHLDA1 alleviates sepsis-induced acute lung injury by downregulating NLRP3 inflammasome activation.

To investigate the regulatory mechanism of pleckstrin homology-like domain, family A, member 1 (PHLDA1) in sepsis-induced acute lung injury (ALI). Mice model of sepsis were established by cecal ligation and puncture (CLP). The expression of PHLDA1 was reduced by injecting short hairpin RNA (shRNA)-PHLDA1 into the tail vein. The levels of PHLDA1, pro-inflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), IL-1β, IL-18, super-oxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH), molecular mechanism related to pyroptosis, such as caspase 1, adaptor apoptosis-associated speck-like protein containing a CARD (ASC), and gasdermin D (GSDMD)-N, and nucleotide oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) were tested by Western blot analysis, quantitative real-time polymerase chain reaction, and enzyme-linked-immunosorbent serologic assay. Pathological changes in lung tissues were examined by hematoxylin and eosin staining. Wet-dry weight ratio of lung tissues was observed. The expression of PHLDA1 was up-regulated in lung tissues from CLP-induced septic mice. Knockdown of PHLDA1 could reduce lung injury and wet-dry weight ratio in mice with sepsis-induced ALI. Moreover, silencing of PHLDA1 decreased the expressions of IL-1β, TNF-α, IL-18, IL-6, and MDA but increased SOD and GSH expressions in CLP-induced septic mice. The expressions of NLRP3, GSDMD-N, ASC, and caspase 1 were decreased by PHLDA1 silencing. Knockdown of PHLDA1 inhibited lung inflammation and pyroptosis in mice with sepsis-induced ALI by down-regulating NLRP3.

Knockdown of Long Noncoding RNA CCAT2 Suppresses Malignant Phenotype in Human Laryngeal Squamous Cell Carcinoma.

This study aimed to explore the biological role and mechanism underlying the effects of colon cancer-associated transcript 2 (CCAT2), a long noncoding RNA (lncRNA) in human laryngeal squamous cell carcinoma (LSCC). CCAT2 expression levels in clinical LSCC samples and TU-212 cell line were evaluated by quantitative real-time PCR. The correlation of CCAT2 expression level with clinical-pathological characteristics of patients and their prognosis was analyzed. The functional role of CCAT2 in human LSCC was assessed by Cell Counting Kit-8, Transwell assay, flow cytometric analysis, and LSCC xenograft experiment in vivo. The expression of potential targeted proteins was detected by Western blotting and immunohistochemistry. We found that expression of CCAT2 was significantly elevated in LSCC tissues and TU-212 cells (p<0.05). Survival analysis showed that LSCC patients with high expression of CCAT2 had a shorter 5-year overall survival rate than those with low expression (p<0.05). In addition, CCAT2 silencing with short hairpin RNA significantly decreased the proliferative and invasive potential of TU-212 cells (p<0.05) and promoted their apoptosis. In Nude mice, CCAT2 knockdown suppressed the growth of tumor and decreased its volume and weight in comparison with the controls (p<0.05). In TU-212 cells, CCAT2 silencing with short hairpin RNA significantly down-regulated the expression of β-catenin and CDK8 (p<0.05). Thus, knockdown of CCAT2 suppresses proliferation and invasion of the cells and inhibits Wnt/β-catenin signaling pathway in LSCC, which indicates novel therapeutic targets and prognostic indicators in patients with LSCC.

Angiopoietin-like 4 (ANGPTL4) Suppression Ameliorates Lupus Nephritis in MRL/lpr Mice by Inactivating NLRP3 Inflammasome and Inhibiting Inflammatory Response.

Lupus nephritis (LN) refers to the injury caused by systemic lupus erythematosus (SLE) involving the kidneys. A previous study identified angiopoietin-like protein 4 (ANGPTL4) as a novel urinary biomarker for tracking disease activity in LN. To investigate the detailed role and regulatory mechanism of ANGPTL4 in experimental models of LN. MRL/lpr mice 11-week-old were injected with adeno-associated virus (AAV)-mediated ANGPTL4 short hairpin RNA (shRNA). At 16 and 20 weeks of age, 24-h urine samples were harvested to measure proteinuria levels. After the mice were sacrificed, blood and kidney tissues were harvested to examine serum creatinine (cr) and blood urea nitrogen (BUN) levels, kidney histological changes, and pro-inflammatory cytokine production. Additionally, the levels of NLRP3 inflammasome-associated molecules in mouse renal tissues were detected to clarify the underlying mechanism. The AAV-sh-ANGPTL4 injection significantly reduced the proteinuria, cr, and BUN levels in MRL/lpr mice. ANGPTL4 silencing ameliorated glomerular, tubular, and interstitial damage in mice, mitigating the pathological alternations of LN. In addition, ANGPTL4 knockdown repressed pro-inflammatory cytokine production in the kidneys. Mechanically, ANGPTL4 suppression inhibited NLRP3 inflammasome expression in renal tissues of mice. ANGPTL4 silencing inhibits the NLRP3 inflammasome-mediated inflammatory response, thereby ameliorating LN in MRL/lpr mice.

Functional involvement of endothelial lipase in hepatitis B virus infection.

HBV infection causes chronic liver disease and leads to the development of HCC. To identify host factors that support the HBV life cycle, we previously established the HC1 cell line that maintains HBV infection and identified host genes required for HBV persistence. The present study focused on endothelial lipase (LIPG), which binds to heparan sulfate proteoglycans (HSPGs) in the cell membrane. We found HBV infection was impaired in humanized liver chimeric mouse-derived hepatocytes that were transduced with lentivirus expressing short hairpin RNA against LIPG. Long-term suppression of LIPG combined with entecavir further suppressed HBV replication. LIPG was shown to be involved in HBV attachment to the cell surface by using 2 sodium taurocholate cotransporting peptide (NTCP)-expressing cell lines, and the direct interaction of LIPG and HBV large surface protein was revealed. Heparin and heparinase almost completely suppressed the LIPG-induced increase of HBV attachment, indicating that LIPG accelerated HBV attachment to HSPGs followed by HBV entry through NTCP. Surprisingly, the attachment of a fluorescently labeled NTCP-binding preS1 probe to NTCP-expressing cells was not impaired by heparin, suggesting the HSPG-independent attachment of the preS1 probe to NTCP. Interestingly, attachment of the preS1 probe was severely impaired in LIPG knockdown or knockout cells. Inhibitors of the lipase activity of LIPG similarly impaired the attachment of the preS1 probe to NTCP-expressing cells. LIPG participates in HBV infection by upregulating HBV attachment to the cell membrane by means of 2 possible mechanisms: increasing HBV attachment to HSPGs or facilitating HSPG-dependent or HSPG-independent HBV attachment to NTCP by its lipase activity.

Hydrogen Peroxide Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation in Human Hepatoma Cells.

The hepatitis B virus (HBV) is constantly exposed to significant oxidative stress characterized by elevated levels of reactive oxygen species (ROS), such as H2O2, during infection in hepatocytes of patients. In this study, we demonstrated that H2O2 inhibits HBV replication in a p53-dependent fashion in human hepatoma cell lines expressing sodium taurocholate cotransporting polypeptide. Interestingly, H2O2 failed to inhibit the replication of an HBV X protein (HBx)-null HBV mutant, but this defect was successfully complemented by ectopic expression of HBx. Additionally, H2O2 upregulated p53 levels, leading to increased expression of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induced the ubiquitination-dependent proteasomal degradation of HBx. The inhibitory effect of H2O2 was nearly abolished not only by treatment with a representative antioxidant, N-acetyl-L-cysteine but also by knockdown of either p53 or Siah-1 using specific short hairpin RNA, confirming the role of p53 and Siah-1 in the inhibition of HBV replication by H2O2. The present study provides insights into the mechanism that regulates HBV replication under conditions of oxidative stress in patients.

Hsa_circ_0000585 promotes chemoresistance to cis-platin in epithelial cells of ovarian cancer by modulating autophagy

BackgroundChemoresistance, i.e., resistance to cisplatin (DDP), has been a major obstacle to ovarian cancer treatment. It has been found that circular RNAs (circRNAs) play vital roles in the tumorigenesis various cancers by regulating autophagy, while few studies focusing on cisplatin-resistance ovarian cancer (CROC). MethodsThe expressions of the circRNAs were detected by qRT-PCR. Short hairpin RNA targeting circRNA was used to explore the biological functions of the circRNA. Cell viability, autophagic flux, immunofluorescence, and xenograft tumors experiments were performed to further illustrate the underlying mechanisms. ResultsHsa_circ_0000585 was increased in cisplatin-resistant SKOV3/DDP cells. Stably knocking down hsa_circRNA_0000585 expression in SKOV3/DDP cells was established by RNA interference. We found that downregulation of hsa_circ_0000585 significantly enhanced the sensitivity of DDP/SkOV3 cells to DDP. In vivo study, hsa_circRNA_0000585 knockdown significantly decreased tumor volume in nude mice. Under the measurements of western blot and cellular immunofluorescence, hsa_circ_0000585 knockdown significantly inhibited the expression of Beclin1 and P62, indicating the autophagic flux was inhibited. Administrations with autophagic inhibitor “Chloroquine (CQ)” and autophagy activator “QX77” further confirmed that hsa_circ_0000585 knockdown resulted in autophagy inhibition. ConclusionsOverall, this study provided a new insight into the role of circRNAs in the mechanism of DDP-resistance in ovarian cancer. Hsa_circRNA_0000585 may be promising therapeutic targets for the enhancement of the sensitivity of ovarian cancer cells to cisplatin-mediated chemotherapy.

TPST2-mediated receptor tyrosine sulfation enhances leukocidin cytotoxicity and S. aureus infection.

An essential fact underlying the severity of Staphylococcus aureus (S. aureus) infection is the bicomponent leukocidins released by the pathogen to target and lyse host phagocytes through specific binding cell membrane receptors. However, little is known about the impact of post-transcriptional modification of receptors on the leukocidin binding. In this study, we used small interfering RNA library (Horizon/Dharmacon) to screen potential genes that affect leukocidin binding on receptors. The cell permeability was investigated through flow cytometry measuring the internalization of 4',6-diamidino-2-phenylindole. Expression of C5a anaphylatoxin chemotactic receptor 1 (C5aR1), sulfated C5aR1 in, and binding of 6x-His-tagged Hemolysin C (HlgC) and Panton-Valentine leukocidin (PVL) slow-component to THP-1 cell lines was detected and analyzed via flow cytometry. Bacterial burden and Survival analysis experiment was conducted in WT and myeloid TPST-cko C57BL/6N mice. After short hairpin RNA (shRNA) knockdown of TPST2 gene in THP-1, HL-60, and RAW264.7, the cytotoxicity of HlgAB, HlgCB, and Panton-Valentine leukocidin on THP-1 or HL-60 cells was decreased significantly, and the cytotoxicity of HlgAB on RAW264.7 cells was also decreased significantly. Knockdown of TPST2 did not affect the C5aR1 expression but downregulated cell surface C5aR1 tyrosine sulfation on THP-1. In addition, we found that the binding of HlgC and LukS-PV on cell surface receptor C5aR1 was impaired in C5aR1+TPST2- and C5aR1-TPST2- cells. Phagocyte knockout of TPST2 protects mice from S. aureus infection and improves the survival of mice infected with S. aureus. These results indicate that phagocyte TPST2 mediates the bicomponent leukocidin cytotoxicity by promoting cell membrane receptor sulfation modification that facilitates its binding to leukocidin S component.

Mechanism of Robo1 in the pentylenetetrazol-kindled epilepsy mouse model.

The neural network hypothesis is one of the important pathogenesis of drug-resistant epilepsy. Axons guide molecules through synaptic remodeling and brain tissue remodeling, which may result in the formation of abnormal neural networks. Therefore, axon guidance plays a crucial role in disease progression. However, although Robo1 is one of the important components of axon guidance, the role of Robo1 in epilepsy remains unclear. In this study, we aimed to explore the mechanism of Robo1 in epilepsy. Male adult C57BL/6 mice were intraperitoneally injected with pentylenetetrazol to establish an epilepsy model. Lentivirus (LV) was given via intracranial injection 2 weeks before pentylenetetrazol injection. Different expressions of Robo1 between the control group, LV-mediated Robo1 short hairpin RNA group, empty vector control LV group, and normal saline group were analyzed using Western blot, immunofluorescence staining, Golgi staining, and video monitoring. Robo1 was increased in the hippocampus in the pentylenetetrazol-induced epilepsy mouse model; lentiviral Robo1 knockdown prolonged the latency of seizure and reduced the seizure grade in mice and resulted in a decrease in dendritic spine density, while the number of mature dendritic spines was maintained. We speculate that Robo1 has been implicated in the development and progression of epilepsy through its effects on dendritic spine morphology and density. Epileptic mice with Robo1 knockdown virus intervention had lower seizure grade and longer latency. Follow-up findings suggest that Robo1 may modulate seizures by affecting dendritic spine density and morphology. Downregulation of Robo1 may negatively regulate epileptogenesis by decreasing the density of dendritic spines and maintaining a greater number of mature dendritic spines.

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells.

Xenotransplantation reemerged as a promising alternative to conventional transplantation enlarging the available organ pool. However, success of xenotransplantation depends on the design and selection of specific genetic modifications and on the development of robust assays allowing for a precise assessment of tissue-specific immune responses. Nevertheless, cell-based assays are often compromised by low proliferative capacity of primary cells. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we generated immortalized PTECs (imPTECs) by overexpression of simian virus 40 T large antigen. ImPTECs not only showed typical morphology and phenotype, but, in contrast to primary PTECs, they maintained steady cell cycling rates and functionality. Furthermore, swine leukocyte antigen (SLA) class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for short hairpin RNAs targeting β2-microglobulin and the class II transactivator. This contributed to reducing xenogeneic T-cell cytotoxicity (p < 0.01) and decreasing secretion of pro-inflammatory cytokines such as IL-6 and IFN-γ. This study showed the feasibility of generating highly proliferative PTECs and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTECs was demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation.

Photo-dependent cytosolic delivery of shRNA into a single blastomere in a mouse embryo

Single-cell-specific delivery of small RNAs, such as short hairpin RNA (shRNA) and small noncoding RNAs, allows us to elucidate the roles of specific upregulation of RNA expression and RNAi-mediated gene suppression in early embryo development. The photoinduced cytosolic dispersion of RNA (PCDR) method that we previously reported can introduce small RNAs into the cytosol of photoirradiated cells and enable RNA delivery into a single-cell in a spatiotemporally specific manner. However, the PCDR method has only been applied to planer cultured cells and not to embryos. This study demonstrated that the PCDR method can be utilized for photo-dependent cytosolic shRNA delivery into a single blastomere and for single blastomere-specific RNA interference in mouse embryos. Our results indicate that PCDR is a promising approach for studying the developmental process of early embryogenesis.

SINE-derived short noncoding RNAs: their evolutionary origins, molecular mechanisms, and physiological significance

Short Interspersed Elements (SINEs) comprise a significant portion of the genomes of higher eukaryotes, including humans and mice. This review focuses on SINE-derived noncoding RNAs (ncRNAs), particularly BC1, BC200, and 4.5SH RNA, which are expressed abundantly and in a species-specific manner. These ncRNAs seem to have independently evolved their functions during evolutionary processes: BC1 and BC200 have become cytoplasmic translation inhibitors, while 4.5SH RNA has developed into a nuclear ncRNA that regulates splicing. This review delves into the unique roles of these ncRNAs, with a special emphasis on the recently discovered splicing regulation function of 4.5SH RNA. Furthermore, we discuss their evolutionary trajectories and potential implications for understanding the complexities of gene regulation.

Macrophage-organoid co-culture model for identifying treatment strategies against macrophage-related gemcitabine resistance

BackgroundGemcitabine resistance (GR) is a significant clinical challenge in pancreatic adenocarcinoma (PAAD) treatment. Macrophages in the tumor immune-microenvironment are closely related to GR. Uncovering the macrophage-induced GR mechanism could help devise a novel strategy to improve gemcitabine treatment outcomes in PAAD. Therefore, preclinical models accurately replicating patient tumor properties are essential for cancer research and drug development. Patient-derived organoids (PDOs) represent a promising in vitro model for investigating tumor targets, accelerating drug development, and enabling personalized treatment strategies to improve patient outcomes.MethodsTo investigate the effects of macrophage stimulation on GR, co-cultures were set up using PDOs from three PAAD patients with macrophages. To identify signaling factors between macrophages and pancreatic cancer cells (PCCs), a 97-target cytokine array and the TCGA-GTEx database were utilized. The analysis revealed CCL5 and AREG as potential candidates. The role of CCL5 in inducing GR was further investigated using clinical data and tumor sections obtained from 48 PAAD patients over three years, inhibitors, and short hairpin RNA (shRNA). Furthermore, single-cell sequencing data from the GEO database were analyzed to explore the crosstalk between PCCs and macrophages. To overcome GR, inhibitors targeting the macrophage-CCL5-Sp1-AREG feedback loop were evaluated in cell lines, PDOs, and orthotopic mouse models of pancreatic carcinoma.ResultsThe macrophage-CCL5-Sp1-AREG feedback loop between macrophages and PCCs is responsible for GR. Macrophage-derived CCL5 activates the CCR5/AKT/Sp1/CD44 axis to confer stemness and chemoresistance to PCCs. PCC-derived AREG promotes CCL5 secretion in macrophages through the Hippo-YAP pathway. By targeting the feedback loop, mithramycin improves the outcome of gemcitabine treatment in PAAD. The results from the PDO model were corroborated with cell lines, mouse models, and clinical data.ConclusionsOur study highlights that the PDO model is a superior choice for preclinical research and precision medicine. The macrophage-CCL5-Sp1-AREG feedback loop confers stemness to PCCs to facilitate gemcitabine resistance by activating the CCR5/AKT/SP1/CD44 pathway. The combination of gemcitabine and mithramycin shows potential as a therapeutic strategy for treating PAAD in cell lines, PDOs, and mouse models.

AMPK Mediates Early Activation of the Unfolded Protein Response through a Positive Feedback Loop in Palmitate-Treated Muscle Cells.

Activation of the unfolded protein response (UPR) is closely related to the pathogenesis of many metabolic disorders. Accumulating evidence also shows that UPR and metabolic signaling pathways are interdependent. The AMP-activated protein kinase (AMPK) signal pathway controls the energy balance of eukaryotes. The aim of this study was therefore to investigate the possible interaction between AMPK signaling and UPR in muscle cells exposed to saturated fatty acids, as well as the potential mechanism. The saturated fatty acid palmitate was used to induce UPR in C2C12 myotubes. Compound C or knockdown of AMPKα with short hairpin RNA (shRNA) were used to inhibit the AMPK signaling pathway in palmitate-treated muscle cells. AMPK signaling in myotubes was activated using 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICAR) or ex229. C2C12 myotubes were pre-treated with taurourdodeoxycholic acid (TUDCA) to inhibit UPR before adding palmitate. Real-time PCR and Western blotting were performed to evaluate the expression of UPR markers and activation of AMPK. Palmitate treatment induced UPR in C2C12 myotubes while activating AMPK signaling. Inhibition of the AMPK pathway with compound C or AMPK shRNA reduced palmitate-induced activation of UPR, while inhibition of UPR with TUDCA reduced palmitate-induced AMPK activation. This indicates a positive feedback loop between UPR and AMPK. Furthermore, activation of the AMPK pathway with AICAR or ex229 caused a dose-dependent upregulation of UPR markers, including activating transcription factor 4 (ATF4), binding immunoglobulin protein (BIP), and growth arrest and DNA damage-inducible 34 (GADD34) protein. These results provide the first evidence that AMPK signaling is involved in the early activation of UPR caused by saturated fatty acids in skeletal muscle. Furthermore, they indicate that physiological or pharmacological activation of the AMPK pathway (e.g., by exercise or phenformin, respectively) can promote muscle health and function, thereby improving the quality of life in individuals with metabolic disorders due to a high-fat diet or obesity.

Upregulation of girdin delays endothelial cell apoptosis via promoting engulfment of platelets.

Endothelial cells are crucial in maintaining the homeostasis of the blood-brain barrier. Girders of actin filament (Girdin) and phosphor (p)-Girdin are essential for the engulfment of human brain microvascular endothelial cells (HBMECs) into platelets (PLTs), but the potential mechanism remains unclear and requires further study. Following PLT and cytochalasin D treatment, Hoechst 33,342 detected apoptosis. The transfection efficiency of the short hairpin RNA targeting Girdin (sh-Girdin) or overexpressing Girdin (OE-Girdin) was determined using western blotting. Sh-Girdin, OE-Girdin, mutated Girdin (m-Girdin), and microfilament binding region deleted Girdin (Del-Girdin) were transfected into HBMECs under PLT conditions. Subsequently, the engulfment of HBMECs by PLTs was detected by flow cytometry and transmission electron microscopy. Girdin and phosphorylated (p)-Girdin levels were quantified by western blot. The positive expression of Girdin was measured by immunohistochemistry (IHC). The localization of PLT, Girdin, and p-Girdin and the engulfment of HBMECs in PLTs were analyzed by confocal microscopy. Cytochalasin D overturned the inhibitory effect of PLT on cell apoptosis. OE-Girdin enhanced the fluorescent intensity of PLT-labelling and the engulfment of HBMECs by PLTs, while sh-Girdin, m-Girdin, and Del-Girdin ran reversely. OE-Girdin elevated the Girdin and p-Girdin levels, while sh-Girdin and Del-Girdin were the opposite, but m-Girdin did not affect the p-Girdin and Girdin levels. Girdin and p-Girdin were co-located with PLTs in HBMECs. The over-expression of Girdin was identified as being associated with the increasing engulfment of PTLs. Girdin may be an effective target to alleviate endothelial cell apoptosis.

High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells.

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.

SIX1 Downregulation Suppresses Self-renewal Capacity and THY1 Expression in Hepatocellular Carcinoma and SIX1 Dominate the Survival in Liver Cancer.

Sine oculis homeoprotein 1 exerts an essential role in embryonic development, and it was also identified to be reactivated in various types of mammalian cancer. The sine oculis homeoprotein 1 transcription factor was demonstrated to induce epithelial-mesenchymal transition, regulate crucial genes associated with cancer progression, and increase the oncogenic potential of cells. Therefore, the present study aimed to identify the role of sine oculis homeoprotein 1 in cancer. Sine oculis homeoprotein 1 gene expression was tested with real-time quantitative polymerase chain reaction (PCR) in different cancer types. Sine oculis homeoprotein 1 expression was suppressed by short hairpin RNA transduction in the SNU398 hepatocellular carcinoma cell line. The effects of sine oculis homeoprotein 1 on cell proliferation, drug resistance, and sphere formation were assessed in shSIX1 cells. Immunohistochemical and in silico analyses were performed to determine the prognostic role of sine oculis homeoprotein 1 expression. The upregulated expression levels of sine oculis homeoprotein 1 were revealed to be correlated with the stage of the disease in breast, colon, and liver cancer, with liver cancer exhibiting the highest expression profile. Sine oculis homeoprotein 1 downregulation significantly affected cell proliferation and suppressed sorafenib resistance and sphere-forming ability. Furthermore, sine oculis homeoprotein 1 knockdown cells were identified to have decreased CD90 levels, essential for cancer stem cell properties. Finally, sine oculis homeoprotein 1 expression was a CD90-independent biomarker for the clinical prognosis of liver cancer. The results of this study showed that the knockdown of sine oculis homeoprotein 1 expression might help to prevent hepatocarcinogenesis by increasing drug sensitivity and controlling tumor sphere formation. Overall, these results indicated that sine oculis homeoprotein 1 expression might be useful as a diagnostic marker for patients with hepatocellular carcinoma.

Pharmacological targeting of netrin-1 inhibits EMT in cancer.

Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFβ1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.