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DOE OSTI2018

Who and how in the regulation of mitochondrial cristae shape and function

Highlights: • Mitochondrial ultrastructure defines (patho)physiology. • Description of morphometric parameters to define cristae shape. • Dissection of OPA1, MICOS and ATPase dimers contribution in the modulation of mitochondrial ultrastructure. Mitochondrial adaptation to different physiological conditions highly relies on the regulation of mitochondrial ultrastructure, particularly at the level of cristae compartment. Cristae represent the membrane hub where most of the respiratory complexes embed to account for OXPHOS and energy production in the form of adenosine triphosphate (ATP). Changes in cristae number and shape define the respiratory capacity as well as cell viability. The identification of key regulators of cristae morphology and the understanding of their contribution to the mitochondrial ultrastructure and function have become an strategic goal to understand mitochondrial disorders and to exploit as therapeutic targets. This review summarizes the known regulators of cristae ultrastructure and discusses their contribution and implications for mitochondrial dysfunction.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Rac1 regulates sepsis-induced formation of platelet-derived microparticles and thrombin generation

Dysfunctional coagulation aggravates clinical outcome in patients with sepsis. The aim of this study was to define the role of Rac-1 in the formation of platelet-derived microparticles (PMPs) and thrombin generation (TG) in abdominal sepsis. Male C57BL/6 mice underwent cecal ligation and puncture (CLP). Scanning electron microscopy and flow cytometry were used to quantify PMPs. TG was determined by use of a fluorimetric assay. It was found that CLP increased Rac1 activity in platelets, which was abolished by administration of the Rac1 inhibitor NSC23766. Sepsis-induced TG in vivo was reflected by reduced capacity of plasma from septic animals to generate thrombin ex vivo. Administration of NSC23766 increased peak and total TG in plasma from CLP mice indicating that Rac-1 regulates sepsis-induced formation of thrombin. The number of circulating PMPs was markedly elevated in animals with abdominal sepsis. Treatment with NSC23766 significantly decreased formation of PMPs in septic mice. Platelet activation in vitro caused release of numerous MPs. Notably, NSC23766 abolished PMP formation in activated platelets in vitro. These findings suggest that Rac-1 regulates PMP formation and TG in sepsis and that inhibition of Rac1 activity could be a useful target to inhibit dysfunctional coagulation in abdominal sepsis. - Highlights: • Rac-1 regulates platelet-derived microparticles formation in abdominal sepsis. • Rac-1 mediates formation of thrombin generation in abdominal sepsis. • Targeting Rac-1 activity might protect against coagulation dysfunction in sepsis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Macrophage infiltration regulates the adipose ECM reconstruction and the fibrosis process after fat grafting

Background: Fat grafting experiences a regeneration process from free lipoaspirate to intact adipose tissue. The adipose extracellular matrixes (ECM) provide the structure and biochemical support for surrounding cells; inflammatory cells, like macrophages, regulate the process. Our hypothesis states that transferred fat undergoes ECM remodeling after fat grafting and this process is regulated by macrophage infiltration. Methods: Lipoaspirate was injected subcutaneously into the back of nude mice. The micro-structure of the fat grafts was observed and evaluated using scanning electron microscope (SEM) and collagen I immunohistostaining. The gene transcription level of collagen proteins and the matrix metalloproteinases (MMPs) were assessed by qRT-PCR. Local injection of clodronate-encapsulated liposome was used to evaluate the role of macrophages of fat grafts at different stages in ECM remodeling, depletion of macrophages, at different time points (Week 1 and Week 4). Results: Results from the SEM analysis showed that liposuction caused severe damage to the ECM structure in freshly aspirated adipose tissue. On Day 1 post-transplantation, the surface of adipocytes was covered with platelets and this secreted fibrin network on the fat grafts. An integral adipose structure was already established with an intact ECM at the end of Week 1. The early depletion of macrophages remarkably hindered ECM reconstruction process by down-regulating the expression of collagen proteins and MMPs. Expression of Collagen I was significantly decreased after depletion of macrophages in both gene and protein levels. Results also showed that the depletion of macrophages at the later stage of fat grafting resulted in less fibrosis and capsule formation. Conclusion: Free fat aspirates undergo a prompt ECM reconstruction process and completed in the first week; this process can be initiated with platelets and mainly modulated by inflammatory cells such as macrophages. It was also observed that prolonged macrophage infiltration contributes to fibrosis and capsule formation in fat grafts. - Highlights: • Free fat aspirates undergo a prompt ECM reconstruction process in the first week. • The ECM reconstruction process is initiated with platelets and modulated by macrophages. • Depletion of macrophage in transferred fat resulted in delayed ECM reconstruction.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Gadolinium chloride ameliorates acute lung injury associated with severe acute pancreatitis in rats by regulating CYLD/NF-κB signaling

The present study was embarked on an investigation of the mechanisms behind the effects of Gadolinium chloride (GdCl{sub 3}) on lung injury associated with severe acute pancreatitis (SAP) in rats. Rats were randomly distributed into three groups: sham operation group (SO), SAP group and SAP treated with GdCl{sub 3} group (SAP + GdCl{sub 3}). Retrograde injection of 5% sodium taurocholate into the biliopancreatic duct was adopted to induce SAP. Lung tissue specimens were harvested for histological study, wet-to-dry weight ratio calculation and myeloperoxidase examination. Meanwhile, bronchoalveolar lavage fluid was analyzed for TNF-α and IL-1β activity and proteins content. Then the apoptosis ratio of alveolar macrophages (AMs) was detected. NF-κB activation and cylindromatosis (CYLD) expression in AMs were measured respectively. Results showed that GdCl{sub 3} treatment notably ameliorated lung injury induced by SAP, and simultaneously, the apoptosis ratio of AMs was significantly promoted. The NF-κB activation was obviously inhibited when CYLD expression was markedly up-regulated in AMs of SAP + GdCl{sub 3}. Negative correlation was analyzed between CYLD and NF-κB in both SAP and SAP + GdCl{sub 3}. These data demonstrate that GdCl{sub 3} ameliorates lung injury secondary to SAP in rats mainly by up-regulating CYLD expression and inhibiting NF-κB activation in AMs, which may play a vital role in lung injury. - Highlights: • GdCl{sub 3} ameliorates lung injury associated with SAP by inducing the apoptosis of AMs. • NF-kB activation can inhibit the apoptosis of AMs. • CYLD negatively regulates NF-kB activation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

CREPT regulated by miR-138 promotes breast cancer progression

CREPT (also known as RPRD1B) function as an oncogene and is highly expressed in several kinds of cancers. However, the distribution and clinical significance of CREPT in breast cancer (BC) still not clarified. In this study, we found that the CREPT expression is greatly upregulated in BC tissues and cell lines. Moreover, the CREPT expression was significantly associated with tumor differentiation and metastasis. Next, the functional assay of CREPT showed that CREPT could promote BC proliferation and invasion both in vitro and in vivo. Dual-luciferase reporter assay indicated that miR-138 regulated the expression of CREPT by binding to its 3′-UTR. miR-138 is downregulated and inversely correlated with CREPT expression in BCs. Overexpression of miR-138 suppressed tumor growth and invasion, these effects could be reversed by re-expressing CREPT. Mechanistically, CREPT regulated β-catenin/TCF4/cyclin D1 pathway in BC. In conclusion, the data suggested that miR-138/CREPT involved BC progression, providing potential therapeutic targets for BC. - Highlights: • CREPT expression is greatly upregulated in BC tissues and cell lines. • CREPT could promote BC proliferation and invasion both in vitro and tumor growth in vivo. • CREPT is a direct target of miR-138 in breast cancer. • MiR-138 suppressed BC progression by repressing CREPT. • CREPT regulates β-catenin/TCF4/cyclin D1 pathway in BC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

mir-67 regulates P. aeruginosa avoidance behavior in C. elegans

Highlights: • The MicroRNA mir-67 expression is up-regulated after a pathogenic bacterium P. aeruginosa infection. • mir-67 regulates P. aeruginosa avoidance behavior by inhibiting SAX-7 expression. • The mir-67-SAX-7 signaling is independent on the known signaling pathways that regulate pathogen avoidance. Pathogen avoidance behaviors are found throughout the animal kingdom and are important for animal's survival in nature. As a free-living nematode, C. elegans is exposed to a variety of microorganisms, including toxic or pathogenic bacteria, in soil. C. elegans can develop efficient avoidance responses to pathogenic bacteria to minimize the infection risk. However, the role of microRNAs (miRNAs) in pathogen avoidance in C. elegans remains unclear. In this report, we showed that the miRNA mir-67 was involved in a behavioral avoidance response to P. aeruginosa PA14. Exposure to P. aeruginosa PA14 induced the expression of mir-67 in worms. mir-67(n4899) mutants exhibited a reduced ability to avoid P. aeruginosa PA14. By combining quantitative proteomic analysis with miRNA target prediction algorithms, we identified SAX-7/L1CAM, which is transmembrane cell adhesion receptor molecule, as the target of mir-67. Silencing of sax-7 by RNAi on mir-67 mutants rescued avoidance behavioral. Our data demonstrate that the mir-67-SAX-7 pathway modulate the behavioral avoidance response to pathogens, thus providing a new perspective in the role of miRNAs in host–microbe interactions.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2017

Redox regulation of plant S-nitrosoglutathione reductase activity through post-translational modifications of cysteine residues

Highlights: • Plant GSNORs are inhibited by cysteine oxidative modification by H{sub 2}O{sub 2}. • GSNORs are inhibited by NO and nitroxyl though reversible cysteine modifications. • Oxidative and nitrosative inhibition of GSNOR can be reversed by reducing agents. • S-nitrosation of conserved cysteine 271 was confirmed in inhibited GSNOR enzyme. Nitric oxide (NO) is considered as a signalling molecule involved in a variety of important physiological and pathological processes in plant and animal systems. The major pathway of NO reactions in vivo represents S-nitrosation of thiols to form S-nitrosothiols. S-nitrosoglutathione reductase (GSNOR) is the key enzyme in the degradation pathway of S-nitrosoglutathione (GSNO), a low-molecular weight adduct of NO and glutathione. GSNOR indirectly regulates the level of protein S-nitrosothiol in the cells. This study was focused on the dynamic regulation of the activity of plant GSNORs through reversible S-nitrosation and/or oxidative modifications of target cysteine residues. Pre-incubation with NO/NO{sup −} donors or hydrogen peroxide resulted in a decreased reductase and dehydrogenase activity of all studied plant GSNORs. Incubation with thiol reducing agent completely reversed inhibitory effects of nitrosative modifications and partially also oxidative inhibition. In biotin-labelled samples, S-nitrosation of plant GSNORs was confirmed after immunodetection and using mass spectrometry S-nitrosation of conserved Cys271 was identified in tomato GSNOR. Negative regulation of constitutive GSNOR activity in vivo by nitrosative or oxidative modifications might present an important mechanism to control GSNO levels, a critical mediator of the downstream signalling effects of NO, as well as for formaldehyde detoxification in dehydrogenase reaction mode.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Chloroplast protein PLGG1 is involved in abscisic acid-regulated lateral root development and stomatal movement in Arabidopsis

Highlights: • PLGG1, a chloroplast protein, regulates lateral root growth under ABA treatment. • PLGG1 is also involved in ABA-inhibited seed germination, ABA-mediated stomatal movement, and drought tolerance. • The deficiency of PLGG1 alters the expression of a set of ABA-responsive genes. The plant hormone abscisic acid (ABA) plays a crucial role in root architecture; however, the molecular mechanism of ABA-regulated lateral root (LR) growth is not well known. We screened an Arabidopsis thaliana mutant with LR growth that was sensitive to ABA from a T-DNA insertion mutant library, which was an allelic mutant of plgg1-1, termed plgg1-2. PLGG1 encodes a chloroplast protein that transports plastidic glycolate and glycerate. The length and number of LRs at the root-hypocotyl junction of plgg1-1 and plgg1-2 were significantly impaired under exogenous ABA treatment, and the transgenic plant complementary lines of plgg1-2 restored LR growth in response to ABA. In addition, we found that PLGG1 is involved in other major ABA responses, including ABA-inhibited seed germination, ABA-mediated stomatal movement, and drought tolerance. These findings open new perspectives on elucidating the mechanism of ABA response, and provide clues for analysing the functions of chloroplast proteins in regulating root growth.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

SENP1 attenuates the liver fibrosis through down-regulating the expression of SMAD2

To investigate whether SENP1 could play a regulating role in the liver fibrosis process, the Sprague-Dawley (SD) rats were used to establish the liver fibrosis rat models by intraperitoneally injecting with 1 ml/kg of 10% CCl{sub 4}, while the control normal rats were injected with olive oil. Then confirmation experiments to verify the successful establishment of these models were conducted by detecting the cellular and lobular architecture, and liver function indexes using hematoxylin-eosin staining, Masson's trichrome staining and microplate method, respectively. In addition, the expression levels of fibrosis markers including collagen I, collagen III, α-SMA and TGF-β1 were inspected using quantitative real-time PCR (qRT-PCR), as well as SMAD2. Subsequently, the relative mRNA and protein level of SENP1 was also determined via qRT-PCR and western blot analysis. Next, the HSC-T6 cells of SENP1 knock-down were constructed and used to test the relative protein expression levels of α-SMA and SMAD2 in these cells. The results of hematoxylin-eosin staining, Masson's trichrome staining and microplate method turned out that the rat liver fibrosis models were constructed successfully, which was further confirmed by the increased expression of collagen I, collagen III, α-SMA and TGF-β1 in mRNA and protein level, as well as SMAD2. Then the expression of SENP1 was overexpressed in the rat liver fibrosis models induced by CCl{sub 4} and the TGF-β1 treatment could increase the protein expression level of collagen I, collagen III and α-SMA. Lastly, the SENP1 knockdown HSC-T6 cells were successfully constructed, while the silence of SENP1 down-regulated the protein expression of α-SMA and SMAD2. In conclusion, this study provided a new regulation mechanism about the liver fibrosis process.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Intracellular cholesterol level regulates sensitivity of glioblastoma cells against temozolomide-induced cell death by modulation of caspase-8 activation via death receptor 5-accumulation and activation in the plasma membrane lipid raft

Highlights: • Intracellular cholesterol positively regulates temozolomide-induced glioma apoptosis. • DR5-caspase-8 axis is essential for enhancement of glioma apoptosis by cholesterol. • HMG-CoA reductase inhibitors suppress temozolomide-induced glioma apoptosis. • Intracellular cholesterol loading overcomes temozolomide resistance of glioma cells. Development of resistance against temozolomide (TMZ) in glioblastoma (GBM) after continuous treatment with TMZ is one of the critical problems in clinical GBM therapy. Intracellular cholesterol regulates cancer cell biology, but whether intracellular cholesterol is involved in TMZ resistance of GBM cells remains unclear. The involvement of intracellular cholesterol in acquired resistance against TMZ in GBM cells was investigated. Intracellular cholesterol levels were measured in human U251 MG cells with acquired TMZ resistance (U251-R cells) and TMZ-sensitive control U251 MG cells (U251-Con cells), and found that the intracellular cholesterol level was significantly lower in U251-R cells than in U251-Con cells. In addition, treatment by intracellular cholesterol remover, methyl-beta cyclodextrin (MβCD), or intracellular cholesterol inducer, soluble cholesterol (Chol), regulated TMZ-induced U251-Con cell death in line with changes in intracellular cholesterol level. Involvement of death receptor 5 (DR5), a death receptor localized in the plasma membrane, was evaluated. TMZ without or with MβCD and/or Chol caused accumulation of DR5 into the plasma membrane lipid raft and formed a complex with caspase-8, an extrinsic caspase cascade inducer, reflected in the induction of cell death. In addition, treatment with caspase-8 inhibitor or knockdown of DR5 dramatically suppressed U251-Con cell death induced by combination treatment with TMZ, MβCD, and Chol. Combined treatment of Chol with TMZ reversed the TMZ resistance of U251-R cells and another GBM cell model with acquired TMZ resistance, whereas clinical antihypercholesterolemia agents at physiological concentrations suppressed TMZ-induced cell death of U251-Con cells. These findings suggest that intracellular cholesterol level affects TMZ treatment of GBM mediated via a DR5-caspase-8 mechanism.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Negative regulation of lncRNA GAS5 by miR-196a inhibits esophageal squamous cell carcinoma growth

Highlights: • lncRNA GAS5 is a novel target of miR-196a. • miR-196a decreased the expression of GAS5 possibly through RISC. • GAS5 expression is significantly decreased in patients with ESCC. • The reduced expression of GAS5 in ESCC may be not related to DNA methylation but the high level of miR-196a. • GAS5 functions as a tumor suppressor gene by inhibiting cell growth. MiR-196a could play important roles in carcinogenesis by targeting many protein coding genes. However, little is known about whether miR-196a can target any long non-coding RNAs (lncRNAs). In the present study, we screen lncRNAs which are regulated by miRNA-196a in human esophageal squamous cell carcinoma (ESCC). We found that miR-196a could suppress the expression of lncRNA growth arrest-specific 5(GAS5). GAS5 is frequently down-regulated in 86 paired human ESCC tissues. Importantly, there was lower GAS5 expression in the late stage of ESCC patients. The reduced expression of GAS5 in ESCC may not be related to DNA methylation but related to the high expression of miR-196a. In vitro and in vivo studies indicated that GAS5 could inhibit the growth of ESCC cells. Using Chromatin Isolation by RNA Purification–qPCR, we found that miR-196a could bind to GAS5. The Luciferase Reporter Assay indicated that miR-196a could bind to the seventh exon of GAS5. Additionally, both GAS5 and miR-196a could bind to Ago2 which is a key component of the RNA-induced silencing complex (RISC). Together, these results suggest that GAS5 functions as a tumor suppressor gene in ESCC and is regulated by miR-196a involved in RISC.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Down-regulation of lncRNA MEG3 promotes hypoxia-induced human pulmonary artery smooth muscle cell proliferation and migration via repressing PTEN by sponging miR-21

Highlights: • Hypoxia induced cell proliferation and migration in PASMCs. • MEG3 expression was down-regulated in PASMCs hypoxia cell models. • MEG3 exerts its role via miR-21/PTEN axis in PASMCs hypoxia cell models. Hypoxia-induced pulmonary hypertension is a life-threatening disease arising from a progressive increase in pulmonary vascular resistance, irreversible pulmonary vascular remodeling and resulting in right ventricular failure. Recent studies suggested that pulmonary artery smooth muscle cell proliferation and migration played an important role in the pathogenesis of hypoxia-induced pulmonary hypertension. However, the mechanisms of hypoxia-induced pulmonary hypertension are complicated and largely unclear. In this study, we discovered that lncRNA MEG3 was down-regulated in human pulmonary artery smooth muscle cell in hypoxia, and inhibition of MEG3 promoted the cell proliferation and cell migration in both normal and hypoxia condition. Further study demonstrated that MEG3 exerted its function via regulation of miR-21 expression in both normal and hypoxia condition. In addition, we displayed the modulation of PTEN by miR-21 and their role in hypoxia. Ultimately, our study illustrated that MEG3 exerts its role via miR-21/PTEN axis in human pulmonary artery smooth muscle cell under both normal and hypoxia conditions.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

IL-13 regulates IL-17C expression by suppressing NF-κB-mediated transcriptional activation in airway epithelial cells

Highlights: • IL-1β markedly elevated IL-17C expression viathe NF-κB-signaling pathway in normal human bronchial epithelial cells. • IL-13 attenuated IL-1β-mediated IL-17C expression via a STAT6-signaling pathway. • IL-1β enhanced binding of the NF-κB p65 sub unit to two IL-17C promoter regions (−130/−120 and −157/−147). • IL-13 attenuated IL-1β-mediated p65 binding to IL-17C promoter regions, resulting in lower IL-17C expression. The cytokine interleukin (IL)-17C is highly expressed in epithelial tissues and involved in innate immune responses; however, the regulation of IL-17C expression in the airways remains poorly understood. Here, we show that IL-1β strongly induces both IL-17C mRNA and protein expression in primary normal human bronchial epithelial cells. Conversely, IL-13 significantly reduced the IL-1β-induced IL-17C expression. Attenuation of the nuclear factor (NF)-κB-signaling pathway using an NF-κB-subunit p65-specific small-interfering RNA (siRNA), reduced IL-1β-induced IL-17C expression, demonstrating the importance of NF-κB signaling in IL-17C regulation. The inhibitory effects of IL-13 on IL-17C expression were abolished when the Janus kinase (JAK)/signal transducer and activator of transcription 6 (STAT6)-signaling pathway was impaired, using either the JAK inhibitor ruxolitinib or a STAT6-specific siRNA. Western blot analysis demonstrated that IL-1β promoted both IκB-α phosphorylation and degradation, and p65 nuclear translocation. Although IL-13 induced STAT6 phosphorylation and nuclear translocation, it did not affect the activation of the IL-1β-mediated NF-κB-pathway. Using chromatin immunoprecipitation, we confirmed that IL-1β enhanced p65 binding to regions within the IL-17C promoter that flank putative NF-κB-binding sites (−130/−120 and −157/−147). Interestingly, IL-13 treatment reduced the IL-1β-mediated p65 binding to these regions. These findings demonstrate that NF-κB-mediated transcriptional mechanisms are critically involved in the IL-1β-mediated IL-17C induction, and that IL-13 negatively regulates this induction by suppressing NF-κB-based transcriptional activation.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

The splicing factor transformer2 (tra2) functions in the Drosophila fat body to regulate lipid storage

Highlights: • Transformer2 (tra2) is a splicing protein whose metabolic functions are unknown. • Tra2 functions in the Drosophila fat body to modulate triglyceride levels. • Decreasing tra2 in the fat body promotes starvation resistance. • Tra2 alters CPT1 splicing in the fly fat body. Excess nutrients are stored as triglycerides mainly in the adipose tissue of an animal and these triglycerides are located in structures called lipid droplets. Previous genome-wide RNAi screens in Drosophila cells identified splicing factors as playing a role in lipid droplet formation. Our lab has recently identified the SR protein, 9G8, as an important factor in fat storage as decreasing its levels results in augmented triglyceride storage in the fat body. Previous in vitro studies have implicated 9G8 in the regulation of splicing of the sex determination gene doublesex (dsx) by binding to transformer (tra) and transformer2 (tra2); however, any function of these sex determination proteins in regulating metabolism is unknown. In this study, we have uncovered a role of tra2 to regulate fat storage in vivo. Inducing tra2{sup dsRNA} in the adult fat body resulted in an increase in triglyceride levels but had no effect on glycogen storage. Consistent with the triglyceride phenotype, tra2 knockdown flies lived longer under starvation conditions. In addition, this increase in triglycerides is due to more fat storage per cell and not an increase in the number of fat cells. Interestingly, the splicing of CPT1, an enzyme involved in the breakdown of lipids, was altered in flies with decreased tra2. The less-catalytically active isoform of CPT1 accumulated in tra2{sup dsRNA} flies suggesting a decrease in lipid breakdown, which is consistent with the increased triglyceride levels observed in these flies. Together, these results suggest a link between mRNA splicing, sex determination and lipid metabolism and may provide insight into the mechanisms underlying tissue-specific splicing and nutrient storage.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

High glucose up-regulates microRNA-34a-5p to aggravate fibrosis by targeting SIRT1 in HK-2 cells

Highlights: • The expression of SIRT1 is declined and the level of miR-34a-5p is up-regulated in the kidneys of DN mice. • SIRT1 prevents TGF-β1-induced pro-fibrotic gene expression. • SIRT1 expression was declined time-dependently under high glucose in HK-2 cells. • MiR-34a-5p aggravate fibrosis through directly suppressing SIRT1. • MiR-34a-5p inhibitor reverse the expressions of downstream genes through SIRT1 in HK-2 cells. Tubulointerstitial fibrosis (TIF) is crucial in the development of renal fibrosis in diabetic nephropathy(DN). Previous data shows that SIRT1 plays an important role on fibrosis, but the effect on TIF in DN and underlying mechanisms remains uncertain. In this study, we evaluated the vital role of SIRT1 and identified SIRT1 as a downstream target gene of microRNA-34a-5p (miR-34a-5p) in TIF of DN. The result revealed that expression of miR-34a-5p, fibronectin(FN),collagen type I (COL1) and transforming growth factor β1 (TGF-β1) were up-regulated accompanied by the corresponding down-regulation of SIRT1 in renal tissues of high fat diet and streptozotocin(HFD/STZ)induced diabetic mice with DN, and that the SIRT1 mRNA level was negatively correlated with miR-34a-5p expression in high glucose stimulated human proximal tubule cell line(HK-2) cells. We then demonstrated that overexpression of SIRT1 reduced, whereas small interfering RNA targeting SIRT1 enhanced the expressions of TGF-β1 and fibrosis-related genes including FN and COL1 in HK-2 cells. Furthermore, we identified that miR-34a-5p directly suppressed SIRT1 to increase the profibrogenic effects of TGFβ1 through targeting the 3'untranslated region of SIRT1. The functional correlation of miR-34a-5p induced SIRT1 decrease was supported by overexpression and inhibition of miR-34a-5p in HK-2 cells. All the results reveal that SIRT1 which is vital in the evolution of renal TIF in DN can be directly suppressed by miR-34a-5p, and suggest that miR-34a-5p is a new target for DN treatment.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Angiotensin II type 1 receptor-associated protein regulates carotid intimal hyperplasia through controlling apoptosis of vascular smooth muscle cells

Highlights: • ATRAP as a negative regulator of AT1 receptor plays an inhibitory role in VSMCs proliferation both in vitro and in vivo. • ATRAP triggers the marked apoptosis of VSMCs in balloon-injuried carotid arteries and depresses neointimal formation. • ATRAP induces VSMCs apoptosis through the influence of the PI3K/Akt signaling pathway. Intimal hyperplasia is the main cause of restenosis after carotid artery injury, and the underlying mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). Angiotensin II Type 1 Receptor-Associated Protein (ATRAP) has been reported to withstand intimal hyperplasia by inhibiting VSMCs proliferation and migration; however, whether the beneficial effect of ATRAP associates with VSMCs apoptosis remains unclarified. We demonstrated that the adenoviral-mediated overexpression of ATRAP induced VSMC apoptosis, alleviating the balloon injury-induced neointima formation in rats. Under the condition of Angiotensin-II stimulation, ATRAP overexpression induced the apoptosis of rat VSMCs by depressing the PI3K-Akt signaling; whereas up-regulation of Akt by PTEN inhibitor abolished the apoptotic death. Thus, ATRAP regulates carotid intimal hyperplasia through controlling the PI3K-Akt signal-mediated VSMCs apoptosis.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Netrin-1 promotes metastasis of gastric cancer by regulating YAP activity

Yes-associated protein (YAP) is a major downstream molecular of the Hippo pathway, which plays important role in cancer development. Netrin-1 conveys oncogenic activity in many types of malignant tumors. However, the downstream signaling of netrin-1 mediating its oncogenic effects in gastric cancer (GC) is not well defined. Here, we aim to investigate the role of netrin-1 in metastasis potential of GC by regulating YAP. In this study, we showed that netrin-1 inhibition significantly decreased migration and invasion abilities of GC cells, while netrin-1 overexpression effectively reversed this effect. We also demonstrated that netrin-1 upregulated YAP expression via its transmembrane receptor neogenin. Furthermore, our in vitro and in vivo results showed that the effect of netrin-1 on GC cells migration and invasion abilities was regulated by YAP. Collectively, our results defined netrin-1 as a positive regulator of malignant tumor metastasis in GC by activating the YAP signaling, with potential implications for new approaches to GC therapy.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Glucagon-like peptide-1 contributes to increases ABCA1 expression by downregulating miR-758 to regulate cholesterol homeostasis

Abnormal regulation of lipid metabolism is associated with type 2 diabetes mellitus (T2DM). GLP-1 as a new treatment for T2DM, has unique effects in modulating cholesterol homeostasis. However, the mechanism of this effect is largely missing. The aim of this study was to determine the effects of GLP-1 on cholesterol-induced lipotoxicity in hepatocytes and examine the underlying mechanisms. The cell viability was determined, and caspase-3 was used to detect the effects of GLP-1 on cholesterol-induced apoptosis. The alterations of miR-758 and ATP-binding cassette transporter A1 (ABCA1) resulting from cholesterol incubation or GLP-1 were detected by qRT-PCR and Western blot assays. Overexpression of miR-758 abrogated the GLP-1-mediated ABCA1 expression, and conversely, down-regulation of miR-758 aggravated GLP-1's action and revealed significant promotion effects. BODIPY-Cholesterol efflux assay revealed that treatment with miR-758 inhibitor significantly enhanced ABCA1-dependent cholesterol efflux, resulting in reduced total cholesterol. Furthermore, Oil red O staining and cholesterol measurement were used to detect lipid accumulation. As a result, cholesterol significantly attenuated cell viability, promoted cell apoptosis, and facilitated lipid accumulation, and these effects were reversed by GLP-1. This study provides evidence that, in HepG2 cells, GLP-1 may affect cholesterol homeostasis by regulating the expression of miR-758 and ABCA1. These data can inform the development of biomarkers for miR-758, and potentially other drugs, on the key pathways of lipid metabolism.

60 APPLIED LIFE SCIENCES↗