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Results for “MESSENGER-RNA”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 379 records · Page 21
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Chitosan oligosaccharides enhance lipid droplets via down-regulation of PCSK9 gene expression in HepG2 cells

Highlights: • COS enhance lipid droplets in HepG2 cells. • COS down-regulate the expression of PCSK9 to maintain hepatic LDLR abundance and activity. • COS inhibit HNF1α and SREBP-2 activity leading to down-regulation of PCSK9. Chitosan oligosaccharides (COS), linear polymers of N-acetyl-D-glucosamine and deacetylated glucosamine, exhibit diverse pharmacological effects such as antimicrobial, antitumor, antioxidant and anti-inflammatory activities. Here, we explored their hypocholesterolemic effects in vivo and the molecular mechanisms of COS in hepatic cells. Our in vivo study of dyslipidemic ApoE{sup -/-} male mice showed that COS treatment of 500 mg kg{sup −1} d{sup −1} for 4 weeks clearly reduced the lipid deposits in the aorta and significantly decreased the hepatic proprotein convertase subtilisin/kexin type 9 (PCSK9) protein levels versus HFD groups (p < 0.05). To elucidate the mechanisms behind these effects, the HepG2 cell line was treated with COS. We found that COS (200 μg/ml) increased the amount of cell-surface low-density lipoprotein receptor (LDLR) and enhanced the lipid droplets in HepG2 cells (p < 0.05). The mRNA levels of LDLR and HMG-CoA protein levels were not altered, and the mRNA levels of PCSK9 were down-regulated by COS treatment for 24 h. We also observed that the expression levels of SREBP-2 (125 kD) and HNF-1α were increased in total cell lysates, but nuclear SREBP-2 (nSREBP-2, 68 kD, the active subunit of SREBP-2) levels were decreased and FOXO3a levels increased in nuclear lysates after COS treatment for 24 h. We demonstrated that one of the reasons for regulation of lipid transfer with COS is that FOXO3a levels are up-regulated by COS, leading to a reduction in the PCSK9 promoter binding capacity of HNF-1α and thus suppressing PCSK9 gene expression, up-regulating LDLR levels, and enhancing the lipid droplets in HepG2 cells. In addition, decreased expression of the PCSK9 gene was also contributed to by down-regulation of SREBP-2 by COS. We further confirmed the effect of suppression of PCSK9 expression by COS by utilizing RNA interference to silence HNF-1α and SREBP-2. Finally, to the best of our knowledge, we are the first to demonstrate that PCSK9 expression and LDLR activity are synergistically changed by a combination of HNF-1α and SREBP-2 after COS treatment. Our findings indicate that COS may regulate PCSK9 to modulate hepatic LDLR abundance and activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Knockdown of MALAT1 enhances chemosensitivity of ovarian cancer cells to cisplatin through inhibiting the Notch1 signaling pathway

Highlights: • MALAT1 knockdown reversed CDDP resistance in CDDP-resistant ovarian cancer cells. • MALAT1 knockdown inhibits the Notch1 pathway and ABCC1 expression. • MALAT1 interacts with Notch1 in ovarian cancer cells. • Notch1 knockdown reverses CDDP resistance in CDDP-resistant ovarian cancer cells. • MALAT1 knockdown enhances cytotoxicity of CDDP in ovarian cancer cells in vivo. Long non-coding RNAs (lncRNAs) are critical regulators in chemoresistance of various tumors including ovarian cancer. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been reported to be upregulated and contributed to ovarian cancer tumorigenesis. The aim of this study was to explore the roles of MALAT1 and the underlying molecular regulatory mechanism in the chemoresistance of ovarian cancer cells. Our data demonstrated that MALAT1 and Notch1 mRNA were upregulated in ovarian cancer tissues, as well as cisplatin (CDDP)-resistant ovarian cancer cells. A positive correlation between MALAT1 and Notch1 mRNA expression was observed. MALAT1 knockdown significantly attenuated CDDP resistance, and enhanced CDDP-induced apoptosis in CDDP-resistant ovarian cancer cells. MALAT1 knockdown enhanced CDDP-induced apoptosis in vivo, as indicated by upregulation of Bax protein expression and downregulation of Bcl-2 protein expression. Additionally, MALAT1 knockdown inhibited the Notch1 pathway and ABCC1 expression in CDDP-resistant ovarian cancer cells. MALAT1 was demonstrated to interact with Notch1. Notch1 knockdown attenuated CDDP resistance, and downregulated the protein expression of ABCC1 in ovarian cancer cells. Taken together, our findings suggested that knockdown of MALAT-1 enhanced chemosensitivity of ovarian cancer cells to CDDP through inhibiting Notch1 signaling pathway.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Leptin up-regulates microRNA-27a/b-3p level in hepatic stellate cells

Highlights: • Leptin up-regulates miR-27a/b-3p levels in hepatic stellate cells (HSCs). • Multiple signaling pathways mediate the effect of leptin on miR-27a/b-3p. • miR-27a/b-3p reduce SREBP1c and LXRα levels, and inhibit HSC activation. • miR-27a/b-3p involved in leptin-associated liver fibrosis. Obese patients, often accompanied by hyperleptinemia, are prone to liver fibrogenesis. Leptin is an adipocyte-derived hormone and plays a promotion role in liver fibrosis. Sterol regulatory element binding protein-1c (SREBP1c) exerts a crucial role in inhibiting hepatic stellate cell (HSC) activation, a key step in liver fibrogenesis. Our previous studies indicated that leptin inhibited SREBP1c expression, contributing to leptin-induced HSC activation and liver fibrosis. microRNAs (miR) have emerged as important layers of regulatory control and regulate gene expression, and are implicated in numerous diseases. The present study revealed leptin up-regulation of miR-27a/b-3p levels in HSCs in vitro and in vivo. Three signaling pathways were required for leptin regulation of miR-27a/b-3p levels. miR-27a/b-3p could reduce SREBP1c and liver x receptor α (LXRα) levels, increased α-smooth muscle actin (α-SMA, a marker for HSC activation) and α1(I)collagen levels in cultured HSCs. miR-27a/b-3p regulation of SREBP1c and LXRα were independent of 3′-untranslated region of SREBP1c and LXRα mRNA. In vivo experiments further demonstrated the miR-27a/b-3p involved in leptin-associated decrease in SREBP1 level in HSCs, HSC activation, and liver fibrosis. These data might have potential implications for our understanding of molecular mechanisms underlying leptin roles in liver fibrogenesis of obese patients with hyperleptinaemia.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

SIRT1 and microRNAs: The role in breast, lung and prostate cancers

Highlights: • Breast, lung and prostate cancers are the most common and threatening cancers worldwide. • SIRT1 is elevated in human breast, lung and prostate cancers. • SIRT1 and microRNAs serve as a tumor promotor or tumor suppressor. • MicroRNAs and SIRT1 deregulation have been linked to cancer. Breast cancer and prostate cancer are the most common malignant tumors in female and men, respectively. Furthermore, lung cancer is the leading cause of cancer deaths worldwide. It is an emergency to develop a powerful strategy to treat these threatening cancers more effectively, because of low efficacy and high rates of chemotherapy effects. MicroRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression via induction of translational repression or mRNA degradation. MiRNA deregulation has been linked to cancer initiation and progression. Silent Inflammation Regulator 2 (SIR2) proteins-sirtuins- are a family of histone deacetylases (HDACs) that catalyze deacetylation of both histone and non- histone lysine residues. SIRT1 can act as an oncogene. It plays a role in tumorigenesis by anti-apoptotic activity and is implicated in diverse cellular process including autophagy, senescence, apoptosis, proliferation, and aging. MicroRNAs and SIRT1 serve as tumor suppressors or tumor promotors depending on the oncogenic pathway specific to particular tumors. MicroRNAs modulate cancer development by targeting SIRT1. In this review, we underlie the specific mechanisms involved in these threatening cancers by microRNAs/SIRT1 pathways.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The RNA binding protein tristetraprolin down-regulates autophagy in lung adenocarcinoma cells

Highlights: • TTP down-regulates autophagy to induce cell death in lung adenocarcinoma cells. • Induction of TTP did not result in cell apoptosis, but result in down-regulation of autophagy in lung adenocarcinoma cells. • A novel mechanism of tumor suppressor by TTP in lung adenocarcinoma is suggested. Tristetraprolin (TTP) is the most well-known member of RNA-binding zinc-finger protein that play a significant role in accelerating mRNA decay. Increasingly studies have reported that TTP was functioned as a tumor suppressor gene in several types of carcinomas, while its underlying mechanism is not clear yet. In the current study, we found that TTP overexpression decreased cell proliferation and increased cell death in lung adenocarcinoma cells, with the cell cycle arrest at the S phase. Remarkably, instead of inducing cell apoptosis directly, TTP overexpression alters cell autophagy. Our studies demonstrate that TTP overexpression has no effect on apoptosis related genes, but decreases the expression of autophagy-related genes, including Beclin 1 and LC3II. The level of autophagy flux assessed by infection with the mGFP–RFP–LC3 adenovirus construction has been blocked by TTP overexpression. Moreover, the autophagic vacuoles number detected by transmission electron microscopy decreased with TTP expression up-regulation. Our results indicate, for the first time, that TTP suppresses cell proliferation and increases cell death through cell autophagy pathway in lung cancer cells. Our study provides a new angle of view for TTP function as a tumor suppressor which could be targeted in tumor treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

NUSAP1 gene silencing inhibits cell proliferation, migration and invasion through inhibiting DNMT1 gene expression in human colorectal cancer

Highlights: • NUSAP1 was upregulated in human CRC tissues and cell lines. • NUSAP1 regulates CRC cell proliferation and cell apoptosis. • NUSAP1 mediates CRC cell invasion, migration and EMT. • NUSAP1 silencing suppress the DNMT1 gene expression. Colorectal cancer (CRC) is one of the most common cause of cancer-related death in both female and male patients, with a high capacity for tumor migration and invasion. Recently, aberrant nucleolar and spindle-associated protein 1 (NUSAP1) expression has been reported in several cancers. However, the biological function and molecular mechanism of NUSAP1 in CRC have not been reported. Here, we demonstrated that NUSAP1 gene expression was notably upregulated in CRC tissues and cell lines (Caco2, LS174T, SW480, and LoVo). Subsequently, SW480 and LoVo cells were transfected with NUSAP1 siRNA, respectively, and the biological function of NUSAP1 was investigated. Results indicated that NUSAP1 silencing by siRNA inhibited CRC cell proliferation, and induces cell apoptosis. Moreover, NUSAP1 knockdown suppressed cell migration, cell invasion, and epithelial-to-mesenchymal transition (EMT). Furthermore, NUSAP1 silencing notably inhibited the mRNA and protein expression level of DNA methyltransferase 1 (DNMT1). DNMT1 overexpression partly rescued the effect of NUSAP1 silencing on colorectal cancer biological function. Taken together, NUSAP1 gene silencing induced cell apoptosis, and inhibited cell proliferation, cell migration, cell invasion, and EMT in colorectal cancer through inhibiting DNMT1 gene expression. These findings indicat that NUSAP1 is a promising molecular target for CRC treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Diacylglycerol lipase alpha promotes tumorigenesis in oral cancer by cell-cycle progression

Highlights: • DAGLA was up-regulated in human oral squamous cell carcinoma (OSCC) cells compared with human normal oral keratinocytes (HNOKs). • Overexpression of DAGLA in OSCCs controlled cell proliferation through cell-cycle progression. • The tumoral volume of the orlistat-treated group was clearly smaller than that of the control group in vivo. • DAGLA expression level might be a useful diagnostic indicator and a new therapeutic target for OSCCs. Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Tripartite motif 31 promotes resistance to anoikis of hepatocarcinoma cells through regulation of p53-AMPK axis

Highlights: • TRIM31 was upregulated after anchorage deprival of HCC cells. • Overactivation of AMPK pathway was responsible for TRIM31-mediated resistance to anoikis of HCC cells. • TRIM31 directly targeted p53 for K-48 linked ubiquitous degradation. • TRIM31-p53-AMPK axis played a pivotal role in anoikis-resistance of metastatic liver cancer. Anoikis-resistance is an essential feature of cancer cells to obtain successful metastasis, whereas the molecular mechanism involved in this process of hepatocellular carcinoma (HCC) cells is not fully understood. Here we demonstrated that tripartite motif-containing (TRIM) 31, a new member of the TRIM family, was significantly upregulated in the anchorage-deprived HCC cells compared with their attached counterpart. When we blocked TRIM31 expression by its specific interference RNAs, the anoikis-resistance of HCC cells was significantly reversed. We further verified that overactivation of AMPK pathway was responsible for TRIM31-mediated resistance to anoikis of HCC cells; and TRIM31 could directly target p53, the upstream suppressor of AMPK pathway, and mediate K48-linked ubiquitous degradation of p53 in a RING-domain-dependent way. Therefore we demonstrated that TRIM31 promoted anoikis-resistance by targeting p53 for degradation and subsequently overactivating AMPK pathway. Thus our study defined for the first time the role of TRIM31 in the anoikis-resistant process of HCC cells and it may pave a new avenue for manipulation of metastatic cancer by targeting TRIM31.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Withaferin A and sulforaphane regulate breast cancer cell cycle progression through epigenetic mechanisms

Highlights: • Tumor suppressor p21protein is reactivated via withaferin A (WA) and sulforaphane (SFN). • Combinatorial nutritive compounds have the ability to impede cell cycle progression. • Mutant p53 is down-regulated via the studied compounds where wild type p53 is not. • Epigenetic mechanisms are regulated by WA and SFN. • The H3K4Me3 marker is increased at the p21 promoter after WA and SFN treatment. Little is known about the effects of combinatorial dietary compounds on the regulation of epigenetic mechanisms involved in breast cancer prevention. The human diet consists of a multitude of components, and there is a need to elucidate how certain compounds interact in collaboration. Withaferin A (WA), found in the Indian winter cherry and documented as a DNA methyltransferase (DNMT) inhibitor, and sulforaphane (SFN), a well-known histone deacetylase (HDAC) inhibitor found in cruciferous vegetables, are two epigenetic modifying compounds that have only recently been studied in conjunction. The use of DNMT and HDAC inhibitors to reverse the malignant expression of certain genes in breast cancer has shown considerable promise. Previously, we found that SFN + WA synergistically promote breast cancer cell death. Herein, we determined that these compounds inhibit cell cycle progression from S to G2 phase in MDA-MB-231 and MCF-7 breast cancer. Furthermore, we demonstrate that this unique combination of epigenetic modifying compounds down-regulates the levels of Cyclin D1 and CDK4, and pRB; conversely, the levels of E2F mRNA and tumor suppressor p21 are increased independently of p53. We find these events coincide with an increase in unrestricted histone methylation. We propose SFN + WA-induced breast cancer cell death is attributed, in part, to epigenetic modifications that result in the modulated expression of key genes responsible for the regulation of cancer cell senescence.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

ER-localized protein-Herpud1 is a new mediator of IL-4-induced macrophage polarization and migration

Highlights: • Herpud1 is induced by IL-4 in M2 macrophages. • Herpud1 is involved in IL-4 promoted polarization and migration of M2 macrophage. • Herpud1-associated ER stress play an important role in polarization and migration of M2 macrophage. ER-localized proteins have been reported function in endoplasmic reticulum, unfolded protein degradation and destruction of misfolded proteins by the ER-associated protein degradation (ERAD) system, but their function in the chemotaxis of macrophage cells remained un-addressed. Here, we showed that ER protein with ubiquitin like domain 1(Herpud1) was upregulated in IL-4-treated M2 macrophage cells and its expression pattern was similar with macrophage polarization markers, such as Arg1, Mrc1 and Fizz1. Inhibition of Herpud1 by using specific target shRNA decreased these marker's expression at mRNA and protein level in IL-4-treated or -untreated M2 macrophage cells. IL-4 treatment promoted M2 macrophage cell migration and polarization, but this promotion was weakened by Herpud1 depletion and we got similar results by inhibition of ER stress response with chemical molecule 4-phenylbutyric acid (4-PBA) in IL-4-treated or untreated-M2 macrophage cells with Herpud1 overexpression. These results indicated that depending on ER-associated protein degradation (ERAD) to help unfolded protein degradation or destruction is not the only function of Herpud1 and acting as a mediator of IL-4 induced macrophage activation and polarization maybe another unrevealed function, elucidating the role of Herpud1-associated M2 macrophage cell polarization and activation are helpful for exploration the function of macrophage cells in immune response.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Long non-coding RNA UCA1 upregulation promotes the migration of hypoxia-resistant gastric cancer cells through the miR-7-5p/EGFR axis

Highlights: • Two novel hypoxia-resistant gastric cancer (HRGC) cell lines were established. • UCA1 was upregulated in HRGC cells, which promoted their migration. • UCA1 directly interacted with miR-7-5p. • UCA1 suppressed the degradation of EGFR mRNA by sponging miR-7-5p. A variety of solid tumors are surrounded by a hypoxic microenvironment, which is known to be associated with high metastatic capability and resistance to various clinical therapies, contributing to a poor survival rate for cancer patients. Although the majority of previous studies on tumor-associated hypoxia have focused on acute hypoxia, chronic hypoxia more closely mimics the actual hypoxic microenvironment of a tumor. In this study, two novel hypoxia-resistant gastric cancer (HRGC) cell lines which could grow normally in 2% oxygen were established. The long non-coding RNA UCA1 was upregulated in HRGC cells, which promoted their migration. Bioinformatics analysis and a luciferase reporter assay showed that miR-7-5p could bind to specific sites of UCA1 to regulate the target EGFR through competitive endogenous RNA function. UCA1 directly interacted with miR-7-5p and decreased the binding of miR-7-5p to the EGFR 3′-untranslated region, which suppressed the degradation of EGFR mRNA by miR-7-5p. Therefore, long-term hypoxia induced UCA1 to promote cell migration by enhancing the expression of EGFR. This study thus reveals a new mechanism by which a hypoxic microenvironment promotes tumor metastasis, and highlights UCA1 as a potential biomarker for predicting the metastasis of gastric cancer to guide clinical treatment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

The change of nuclear LC3 distribution in acute myeloid leukemia cells

Highlights: • AML cells with Ara-c or Sorafenib resulted in autophagy enhancement. • Nuclear LC3 moved into the cytoplasm when autophagy was enhanced. • Sirt1 and DOR as regulatory molecules for the observed nuclear LC3 shift. • Sirt1 or DOR affected the expression of Beclin1, HMGB1, and AMPK. • Cytotoxicity of drugs was enhanced when nuclear LC3 shifting was blocked. Making sure the change of nuclear LC3 distribution in the autophagy of acute myeloid leukemia (AML) cell and finding out the regulation mechanism may lead to a breakthrough for killing AML cells. Western blots were performed to assess the expression of autophagy proteins. Changes in the LC3 distribution were monitored by immunofluorescence assays together with western blots, and the expression levels of Sirt1, DOR, Beclin1, HMGB1, and AMPK mRNA were detected via fluorescent quantitative PCR. The effects of Sirt1 and DOR on cell proliferation and survival were analyzed by MTT, flow cytometry, and western blotting assays. We found that treating AML cells with Ara-c or Sorafenib resulted in autophagy enhancement, and when autophagy was enhanced, nuclear LC3 moved into the cytoplasm. Notably, when autophagy was inhibited by blocking the nuclear LC3 shift, the cytotoxicity of drugs was enhanced. Our results also identified Sirt1 and DOR as regulatory molecules for the observed nuclear LC3 shift, and these molecules further affected the expression of Beclin1, HMGB1, and AMPK. Our results suggest the distribution of nuclear LC3 can be a novel way for further studying death of AML cells,and the regulatory molecules may be new targets for treating AML.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

PRP4 kinase induces actin rearrangement and epithelial-mesenchymal transition through modulation of the actin-binding protein cofilin

Highlights: • PRP4 is involved in pre-mRNA splicing and cell signalling. • PRP4 modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. • PRP4 induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway in HCT116 cells. • Dephosphorylation of cofilin results in F-actin stabilization, re-distribution of cytoplasmic actin, formation of actin stress fibers, and inhibition of cell motility. • PRP4 over-expression induces the expressions of PP1A, which directly or indirectly dephosphorylates cofilin, resulting in actin cytoskeleton rearrangement, downregulation of E-cadherin, and EMT induction. Cofilin activation may be associated with EMT properties, and promotes the progression of human colon cancer. Cell actin cytoskeleton is primarily modulated by Rho family proteins. RhoA regulates several downstream targets, including Rho-associated protein kinase (ROCK), LIM-Kinase (LIMK), and cofilin. Pre-mRNA processing factor 4B (PRP4) modulates the actin cytoskeleton of cancer cells via RhoA activity inhibition. In this study, we discovered that PRP4 over-expression in HCT116 colon cancer cells induces cofilin dephosphorylation by inhibiting the Rho-ROCK-LIMK-cofilin pathway. Two-dimensional gel electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) analysis indicated increased expression of protein phosphatase 1A (PP1A) in PRP4-transfected HCT116 cells. The presence of PRP4 increased the expression of PP1A both at the mRNA and protein levels, which possibly activated cofilin through dephosphorylation and subsequently modulated the cell actin cytoskeleton. Furthermore, we found that PRP4 over-expression did not induce cofilin dephosphorylation in the presence of okadaic acid, a potent phosphatase inhibitor. Moreover, we discovered that PRP4 over-expression in HCT116 cells induced dephosphorylation of migration and invasion inhibitory protein (MIIP), and down-regulation of E-cadherin protein levels, which were further restored by the presence of okadaic acid. These findings indicate a possible molecular mechanism of PRP4-induced actin cytoskeleton remodeling and epithelial-mesenchymal transition, and make PRP4 an important target in colon cancer.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

HuR translocation to the cytoplasm of cancer cells in actin-independent manner

Highlights: • Molecular transport system of HuR could be different in individual cancer. • Cancer cells show alternative HuR transport when cytoskeleton structure is impaired. • Oral and cervical cancer implicate a microfilament independent HuR transport. Human antigen R (HuR) is a RNA-binding protein, which binds to the AU-rich element (ARE) in the 3′-untranslated region (3′-UTR) of certain mRNA and is involved in the export and stabilization of ARE-mRNA. HuR constitutively relocates to the cytoplasm in many cancer cells, however the mechanism of intracellular HuR trafficking is poorly understood. To address this question, we examined the functional role of the cytoskeleton in HuR relocalization. We tested the effect of actin depolymerizing macrolide latrunculin A or myosin II ATPase activity inhibitor blebbistatin for HuR relocalization induced by the vasoactive hormone Angiotensin II in cancer and control normal cells. Western blot and confocal imaging data revealed that both inhibitors attenuated the cytoplasmic HuR in normal cells but no such alteration was observed in cancer cells. Concomitant with changes in intracellular HuR localization, both inhibitors markedly decreased the accumulation and half-lives of HuR target ARE-mRNAs in normal cells, whereas no change was observed in cancer cells. Furthermore, co-immunoprecipitation experiments with HuR proteins revealed clear physical interaction with ß-actin only in normal cells. The current study is the first to verify that cancer cells can implicate a microfilament independent HuR transport. We hypothesized that when cytoskeleton structure is impaired, cancer cells can acquire an alternative HuR trafficking strategy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Differentiation in stem/progenitor cells along fetal or adult hepatic stages requires transcriptional regulators independently of oscillations in microRNA expression

Highlights: • The role of microRNAs in differentiation of stem cells is not well understood. • This study found microRNA levels oscillated during differentiation of hepatocytes. • Many functions but not differentiation were regulated by microRNAs in hepatocytes. • Regulators of gene networks independently maintained hepatic differentiation. • These insights will advance how stem cells may contribute in disease mechanisms and therapies. Understanding mechanisms in lineage differentiation is critical for organ development, pathophysiology and oncogenesis. To determine whether microRNAs (miRNA) may serve as drivers or adjuncts in hepatic differentiation, we studied human embryonic stem cell-derived hepatocytes and primary hepatocytes representing fetal or adult stages. Model systems were used for hepatic lineage advancement or regression under culture conditions with molecular assays. Profiles of miRNA in primary fetal and adult hepatocytes shared similarities and distinctions from pluripotent stem cells or stem cell-derived early fetal-like hepatocytes. During phenotypic regression in fetal or adult hepatocytes, miRNA profiles oscillated to regain stemness-associated features that had not been extinguished in stem cell-derived fetal-like hepatocytes. These oscillations in stemness-associated features were not altered in fetal-like hepatocytes by inhibitory mimics for dominantly-expressed miRNA, such as hsa-miR-99b, -100, -214 and -221/222. The stem cell-derived fetal-like hepatocytes were permissive for miRNA characterizing mature hepatocytes, including mimics for hsa-miR-122, -126, -192, -194 and -26b, although transfections of the latter did not advance hepatic differentiation. Examination of genome-wide mRNA expression profiles in stem cell-derived or primary fetal hepatocytes indicated targets of highly abundant miRNA regulated general processes, e.g., cell survival, growth and proliferation, functional maintenance, etc., without directing cell differentiation. Among upstream regulators of gene networks in stem cell-derived hepatocytes included HNF4A, SNAI1, and others, which affect transcriptional circuits directing lineage development or maintenance. Therefore, miRNA expression oscillated in response to microenvironmental conditions, whereas lineage-specific transcriptional regulators, such as HNF4A, were necessary for directing hepatic differentiation. This knowledge will be helpful for understanding the contribution of stem cells in pathophysiological states and oncogenesis, as well as for applications of stem cell-derived hepatocytes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Aldolase promotes the development of cardiac hypertrophy by targeting AMPK signaling

Highlights: • Aldolase A (AldoA) is overexpressed in hypertrophic hearts. • AldoA promotes cardiomyocyte hypertrophy in vitro. • AldoA knockdown represses cardiac hypertrophy in vivo. • AMPK is critically involved in AldoA function in cardiac hypertrophy. Metabolic dysfunction is a hallmark of cardiac hypertrophy and heart failure. During cardiac failure, the metabolism of cardiomyocyte switches from fatty acid oxidation to glycolysis. However, the roles of key metabolic enzymes in cardiac hypertrophy are not understood fully. Here in the present work, we identified Aldolase A (AldoA) as a core regulator of cardiac hypertrophy. The mRNA and protein levels of AldoA were significantly up-regulated in transverse aortic constriction (TAC)- and isoproterenol (ISO)-induced hypertrophic mouse hearts. Overexpression of AldoA in cardiomyocytes promoted ISO-induced cardiomyocyte hypertrophy, whereas AldoA knockdown repressed cardiomyocyte hypertrophy. In addition, adeno-associated virus 9 (AAV9)-mediated in vivo knockdown of AldoA in the hearts rescued ISO-induced decrease in cardiac ejection fraction and fractional shortening and repressed cardiac hypertrophy. Mechanism study revealed that AldoA repressed the activation of AMP-dependent protein kinase (AMPK) signaling in a liver kinase B1 (LKB1)-dependent and AMP-independent manner. Inactivation of AMPK is a core mechanism underlying AldoA-mediated promotion of ISO-induced cardiomyocyte hypertrophy. By contrast, activation of AMPK with metformin and AICAR blocked AldoA function during cardiomyocyte hypertrophy. In summary, our data support the notion that AldoA-AMPK axis is a core regulatory signaling sensing energetic status and participates in cardiac hypertrophy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Commonly used thiol-containing antioxidants reduce cardiac differentiation and alter gene expression ratios of sarcomeric isoforms

Highlights: • β-mercaptoethanol (BME) and monothiolglycerol (MTG) reduce cardiac differentiation. • BME and MTG treatments decrease TNNI3/TNNI1 and MLC2V/MLC2A expression ratios. • BME and MTG treatments reduce beating rates and contraction/relaxation velocities. • Trolox, a strong ROS scavenger, does not inhibit cardiac differentiation. Reactive oxygen species (ROS) scavengers such as beta-mercaptoethanol (BME) and monothiol glycerol (MTG) are extensively used in stem cell research to prevent cellular oxidative stress. However, how these antioxidant supplements impact stem cell cardiac differentiation, a process regulated by redox-signaling remains unknown. In this study, we found that removal of BME from the conventional high-glucose, serum-based differentiation medium improved cardiac differentiation efficiency by 2-3 fold. BME and MTG treatments during differentiation significantly reduced mRNA expression of cardiac progenitor markers (NKX2.5 and ISL1) as well as sarcomeric markers (MLC2A, MLC2V, TNNI3, MYH6 and MYH7), suggesting reduced cardiomyogenesis by BME or MTG. Moreover, BME and MTG altered the expression ratios between the sarcomeric isoforms. In particular, TNNI3 to TNNI1 ratio and MLC2V to MLC2A ratio were significantly lower in BME or MTG treated cells than untreated cells, implying altered cardiomyocyte phenotype and maturity. Lastly, BME and MTG treatments resulted in less frequent beating, slower contraction and relaxation velocities than untreated cells. Interestingly, none of the above-mentioned effects was observed with Trolox, a non-thiol based antioxidant, despite its strong antioxidant activity. This work demonstrates that commonly used antioxidant supplements may cause considerable changes to cellular redox state and the outcome of differentiation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2018

Staphylococcal enterotoxin C2 stimulated the maturation of bone marrow derived dendritic cells via TLR-NFκB signaling pathway

Highlights: • SEC2 stimulate the maturation of BMDCs. • SEC2 induce the production of pro-inflammation cytokines in BMDCs. • SEC2 activate NF-κB signaling pathway in BMDCs. As a kind of superantigen, staphylococcal enterotoxin C2 (SEC2) is well known as a powerful immunomodulator. However, most previous studies about SEC2 focus on its T cell activating characters. But the direct effect of SEC2 on antigen-presenting cells (APCs) which are important for the T cell activation is not clearly. In this study, we investigated the effect of SEC2 on murine bone marrow-derived dendritic cells (BMDCs) which are known as the specialized professional APCs. Contrary to its effects on T cells, SEC2 could not induce proliferation or cytotoxicity to BMDCs even in high concentrations. While SEC2 could promote the mature of BMDCs with increased expression of co-stimulatory molecules on cell membrane such as CD80, CD86, and MHC II. The production of pro-inflammatory cytokines such as TNF-α, IFN-γ and IL-6 were also increased in BMDCs treated with SEC2. We also found that SEC2 enhanced the genes expression of pattern recognition receptors including toll-like receptors 2 (TLR2) and TLR4 in BMDCs, and up-regulated the key signal molecule MyD88 in both mRNA and protein levels. In addition, SEC2 also caused IκBα degradating and NFκB p65 translocating from the cytoplasm to the nucleus in BMDCs. The siRNAs for both TLR2 and TLR4, as well as NFκB specific inhibitor BAY 11–7085 could inhibit the co-stimulatory molecules expression and pro-inflammatory cytokines releasing induced by SEC2. Moreover, TLR2/4 specific siRNAs inhibited p65 and MyD88 upregulation induced by SEC2. In summary, all our results indicated that SEC2 could stimulate BMDCs maturation through TLR-NFκB signaling pathways.

60 APPLIED LIFE SCIENCES↗