Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood–brain barrier injury through the Wnt/β-catenin signalling pathway

Background Disruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear. Methods Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test. Results Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R. Conclusion This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

Chorionic somatomammotropin RNA interference alters fetal liver glucose utilization

Chorionic somatomammotropin (CSH) is a placenta-specific hormone associated with fetal growth, and fetal and maternal metabolism in both humans and sheep. We hypothesized that CSH deficiency could impact sheep fetal liver glucose utilization. To generate CSH-deficient pregnancies, day 9 hatched blastocysts were infected with lentiviral particles expressing CSH-specific shRNA (RNAi) or scramble control shRNA (SC) and transferred to synchronized recipients. CSH RNAi generated two distinct phenotypes at 135 days of gestational age (dGA); pregnancies with IUGR (RNAi-IUGR) or with normal fetal weight (RNAi-NW). Fetal body, fetal liver and placental weights were reduced (P < 0.05) only in RNAi-IUGR pregnancies compared to SC. Umbilical artery plasma insulin and insulin-like growth factor 1 (IGF1) concentrations were decreased, whereas insulin receptor beta (INSR) concentration in fetal liver was increased (P < 0.05) in both RNAi phenotypes. The mRNA concentrations of IGF1, IGF2, IGF binding protein 2 (IGFBP2) and IGFBP3 were decreased (P < 0.05) in fetal livers from both RNAi phenotypes. Fetal liver glycogen concentration and glycogen synthase 1 (GYS1) concentration were increased (P < 0.05), whereas fetal liver phosphorylated-GYS (inactive GYS) concentration was reduced (P < 0.05) in both RNAi phenotypes. Lactate dehydrogenase B (LDHB) concentration was increased (P < 0.05) and IGF2 concentration was decreased (P < 0.05) in RNAi-IUGR fetal livers only. Our findings suggest that fetal liver glucose utilization is impacted by CSH RNAi, independent of IUGR, and is likely tied to enhanced fetal liver insulin sensitivity in both RNAi phenotypes. Determining the physiological ramifications of both phenotypes, may help to differentiate direct effect of CSH deficiency or its indirect effect through IUGR.

Atpase Family AAA Domain-Containing Protein 2 (ATAD2) As a Novel Target in Multiple Myeloma

Multiple myeloma (MM) is a genomically heterogenous malignancy characterized by a number of copy number alterations (CNA). Moreover, there is a clear evolution of genomic changes that may affect prognosis. To identify the drivers of this inherent genomic instability, we applied an integrated genomics approach utilizing genomic data for copy number changes and transcriptomic profile. We first identified genes whose expression correlated with total copy number events in a patient dataset (gse26863, n=246). We then applied those genes to identify the genes whose elevated expression correlated with both overall and event free survival in two different datasets (IFM70, n=170; gse2408, n=559). Elevated expression of this 30 gene signature also correlated with poor overall as well as event free survival in a third myeloma dataset (MMRF; P&lt;0.0005 for both EFS and OS). We have begun to validate these genes for impact on genomic instability and on MM cell growth and survival. Here, we present the functional validation of AAA domain containing protein 2 (ATAD2), which is one of the top-most genes in our 30 gene signature in MM and has also been part of a chromosomal instability signature representing six different cancer types (Nature Genetics, 38: 9, 2006) and a mitotic chromosomal instability signature identified in breast cancer [Sci Transl Med, 2013]. Using The Cancer Genome Atlas data we have also correlated elevated ATAD2 expression with poor OS in pulmonary and pancreatic cancers (P&lt;0.02). ATAD2 is a member of the AAA ATPase family of proteins containing two conserved ATPase domains and a bromodomain. Bromodomain containing proteins are involved in the regulation of gene expression and are frequently dysregulated in MM as well as other cancers. ATAD2 bromodomain selectively recognizes acetylated histone 4 (acetylated at K5 and K12) and has been shown to regulate many cellular processes including cell proliferation. ATAD2 has been shown to interact with and stimulate transcriptional activity of MYC and has also been shown to contribute to invasion and migration in several cancers. We have confirmed elevated ATAD2 expression relative to normal PBMC by Western blotting in all twelve myeloma cell lines tested. To further delineate its role in MM, we evaluated the impact of its knockdown on various parameters of growth and genome maintenance using shRNAs. Relative to control shRNA, knockdown with multiple different shRNAs resulted in near complete cell death in 3 MM cell lines (JJN3, H929 and RPMI) in five days. Reduced cell viability was accompanied by increased apoptosis as seen by annexin V/PI staining. Relative to control, ATAD2 knockdown in RPMI cells led to increase in the apoptotic cell fraction by 56% and in H929 cells by 42%. To investigate genomic impact of elevated ATAD2 expression, the live cell fraction from control and ATAD2-knockdown cells was evaluated, right after selection, for impact on the expression of γ-H2AX (a DNA break marker), pRPA32 (a marker of DNA end resection, a distinct step in the initiation of homologous recombination; HR) and recombinase RAD51 (a key player in HR). ATAD2-knockdown in H929 cells inhibited spontaneous DNA breaks, DNA end resection as well as RAD51 expression, suggesting that elevated ATAD2 contributes to increased spontaneous DNA damage and HR activity observed in MM cells. To further confirm its impact on genome stability, the live cell fraction from control and knockdown cells was evaluated for micronuclei (a marker of genomic instability). Relative to control shRNA-transduced cells, knockdown of ATAD2 cells reduced micronuclei by 58% in H929 and 53% in JJN3 cells. In summary, we demonstrate that elevated ATAD2 contributes to dysregulation of DNA repair and genome stability and is required for MM cell survival, indicating that it is a promising target to inhibit growth and reduce genomic evolution in myeloma. Disclosures Munshi: BMS: Consultancy; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; C4: Current equity holder in private company; Janssen: Consultancy; Adaptive: Consultancy; Legend: Consultancy; Amgen: Consultancy; AbbVie: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.

PSII-39 Trophectoderm-specific RNA interference of chorionic somatomammotropin alters glucose metabolism in sheep fetal liver

Chorionic somatomammotropin (CSH) is a placenta-specific hormone and secreted into both fetal and maternal circulation. Reduced maternal CSH is observed with intrauterine growth restriction (IUGR) in both humans and sheep, and it has long been held that CSH modulates maternal and fetal metabolism. We hypothesized that CSH deficiency, created by RNA interference (RNAi), could impact fetal liver glucose metabolism. To generate CSH-deficient pregnancies, day 9 hatched blastocysts were infected with lentiviral particles expressing CSH-specific shRNA (RNAi) or scramble control shRNA (SC) and transferred to synchronized recipients. CSH RNAi generated two distinct phenotypes at 135 dGA; CSH RNAi pregnancies with IUGR (RNAi-IUGR; n = 8) or without IUGR (RNAi; n = 8). Data from both RNAi phenotypes were compared separately with SC using Welch’s t-test. Liver and placental weights were reduced (P &lt; 0.05) in RNAi-IUGR pregnancies, but not in RNAi pregnancies, as compared to SC (n = 8). Umbilical artery plasma insulin and insulin-like growth factor 1 (IGF1) concentrations were decreased (P &lt; 0.05), whereas insulin receptor beta (IRβ) concentration, as determined by Western immunoblot analysis, in fetal liver was increased (P &lt; 0.05) in both RNAi phenotypes. Fetal liver glycogen quantity was also increased (P &lt; 0.05) in both RNAi phenotypes. Glycogen synthase-1 (GYS-1) concentration in fetal liver was increased (P &lt; 0.05) in both RNAi phenotypes, whereas there was no change in GYS-2 concentration. Phosphorylated-GYS (inactive GYS) was reduced (P &lt; 0.05) in fetal livers for both RNAi phenotypes. Lactate dehydrogenase beta (LDHβ) concentration was increased (P &lt; 0.05) and IGF2 concentration was decreased (P &lt; 0.05) in RNAi-IUGR fetal livers only. From these results we conclude that fetal liver glucose metabolism is impacted by CSH RNAi, independent of IUGR, and is likely tied to enhanced insulin sensitivity in both CSH RNAi phenotypes. Differences between the two phenotypes may help differentiate direct and indirect effects of CSH. Supported by NIH R01 HD093701.

Long Noncoding RNAs Coregulated by Annexin A7 and JNK in Hepatocellular Carcinoma Cells Identified by Whole-Genome Expression Profiling

Knockdown of Annexin A7 (ANXA7) or C-Jun N-terminal kinase (JNK) inhibits the proliferation, migration, invasion, and lymphatic adhesion of hepatocellular carcinoma (HCC) cells, suggesting that ANXA7 and JNK signaling pathways contribute to HCC growth and lymph node metastasis (LNM). While the intervening molecular pathways are largely unknown, emerging evidence suggests that long noncoding RNAs (lncRNAs) participate in ANXA7 and JNK signaling. To identify potential therapeutic targets for HCC, we screened for lncRNAs differentially expressed among Hca-P cells stably expressing shRNA-ANXA7, shRNA-JNK, or control-shRNA. RNA sequencing identified 216 lncRNAs differentially expressed between shRNA-ANXA7 and control-shRNA cells, of which 101 were downregulated and 115 upregulated, as well as 436 lncRNAs differentially expressed between shRNA-JNK and control-shRNA cells, of which 236 were downregulated and 200 upregulated. Fifty-six lncRNAs were differentially expressed under both ANXA7 and JNK knockdown. We selected 4 of these for verification based on putative involvement in cancer regulation according to GO and KEEG analyses of target genes. Knockdown of ANXA7 or JNK suppressed expression of NONMMUT012084.2, NONMMUT024756.2, and ENSMUST00000130486, and enhanced expression of ENSMUST00000197932. These lncRNAs are intriguing candidate targets for mechanistic analysis of HCC progression and therapeutic intervention.

Targeted Inhibition of STAT3 in Neural Stem Cells Promotes Neuronal Differentiation and Functional Recovery in Rats with Spinal Cord Injury

BackgroundSignal transducer and activator of transcription protein 3 (STAT3) is expressed in neural stem cells (NSCs), and some studies have shown that STAT3 is involved in regulating NSC differentiation. However, the possible molecular mechanism and the role of STAT3 in spinal cord injury (SCI) are unknown. Thus, in the present study, we identified possible molecular mechanisms by which STAT3 regulates NSC differentiation in vitro and investigated the potential therapeutic effect of transplanting STAT3-silenced NSCs in rat SCI models in vivo.MethodsIn vitro, NSCs were divided into the following three groups: control, control shRNA, and STAT3-shRNA lentivirus groups. NSCs in each treatment group were examined for neuronal differentiation via immunofluorescence, and Western blot analysis was used to investigate the possible molecular mechanisms. In vivo, the rats were divided into four groups that underwent laminectomy and complete spinal cord transection accompanied by transplantation of control-shRNA-treated or STAT3-shRNA-treated NSCs at the injured site. Spinal cord-evoked potentials and the Basso-Beattie-Bresnahan score were used to examine functional recovery after SCI. Axonal regeneration and tissue repair were assessed via retrograde tracing using Fluorogold, hematoxylin-eosin staining and immunofluorescence.ResultsKnockdown of STAT3 promoted neuronal differentiation in NSCs and mechanistic target of mammal rapamycin (mTOR) activation in vitro, and transplantation of STAT3-RNAi-treated NSCs enhanced rat functional recovery and tissue repair, as well as neuronal differentiation of the transplanted NSCs in vivo.ConclusionsWe have provided in vitro and in vivo evidence that STAT3 is a negative regulator of NSC neuronal differentiation. Transplantation of STAT3-inhibited NSCs appears to be a promising potential strategy for enhancing the benefit of NSC-mediated regenerative cell therapy for SCI.

Examination of the Role of miR-23a in the Development of Thermotolerance

Background: Thermotolerance is an acquired state of increased heat resistance that occurs following exposure to non-lethal proteotoxic stress. A large body of evidences implicates that molecular chaperon members belonging to the heat shock protein family could be acting as potential mediators of the thermotolerant state. Objective: Recent evidence has demonstrated heat shock proteins HSP90, HSP70 and HSP27 have inhibited heat-induced cell death by intervening at various steps in stressinduced apoptotic pathways. Previous studies have shown that HSP70 prevented heatinduced apoptosis by preventing the NOXA dependent decrease in MCL-1 levels leading to both BAX activation and cytochrome c release from mitochondria. We have also demonstrated that HSP70 expressing cells have enhanced levels of miR-23a prevent heat-induced increase in NOXA levels and suppress apoptosis. Methods: Stably transfected cell lines expressing either a control shRNA or a miR-23a targeting shRNA are quantified using both RT-PCR and semi-quantitative RT-PCR to determine the effect of different hyperthermic exposure treatment on miR-23a and Noxa mRNA expression levels. Results: This study shows that thermotolerant-induced pre-heat shock treatment is capable of increasing miR-23a levels. Furthermore, stable cell clones expressing a miR- 23a targeting shRNA having reduced miR-23a levels are incapable of developing a thermotolerance state, leading to apoptosis. Conclusion: These results demonstrate the novel finding that miR-23a is an important factor in the development of the thermotolerant state.

Lenti-shRNA-mediated FAM54A knockdown suppresses the proliferation and induces apoptosis of human Burkitt lymphoma cells

Background Burkitt lymphoma is a kind of non-Hodgkin B-cell-derived malignancy, derived from germinal center B cells. FAM54A has been proved to be involved in various physiological and pathological processes of cancers, but the biological function of FAM54A in Burkitt lymphoma remains unclear. Thus, the aim of our research was to elucidate the roles of FAM54A in proliferation, apoptosis and cell cycle of Burkitt lymphoma.Methods Burkitt lymphoma cell line (Namalwa) was chosen to perform the following experiments. FAM54A-shRNA and negative control-shRNA lentivirus that were synthesized, respectively by Qiagen were used to transfect targeted cells in order to knockdown FAM54A or as negative control. Then, cell proliferation, cell cycle and cell apoptosis were detected by using MTS assay, propidium iodide staining and Annexin V-APC staining, respectively.Results Our results showed that high expression of FAM54A protein was found in Namalwa cell line. Furthermore, MTS analysis exhibited that knockdown of FAM54A obviously inhibited cell proliferation in Namalwa cells. What’s more, cell cycle analysis showed that knockdown of FAM54A induced Namalwa cell apoptosis and arrested cell cycle in G2/M phase.Conclusions These findings suggest that FAM54A is essential for Namalwa cell proliferation and may be a potential therapeutic target for the treatment of Burkitt lymphoma.

CARMA1 Is Required for Notch1-Induced NF-κb Activation in T-Cell Acute Lymphoblastic Leukemia

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm originated from early T cell progenitors. Constitutive activation of the Notch1-signaling pathway plays an important role in T-ALL. After three shears, Notch1 generates intracellular fragment of Notch1 (Notch1-IC), which translocates to the necleus and exerts its biological functions. NF-κB pathway is an important regulator of cell survival and a major downstream target of Nothc1 in T-ALL. However, the molecular mechanism of Notch1 activating NF-κB pathway is still unknown. CARMA1 forms a complex with MALT1 and BCL10 (CBM complex), which plays a role in activation of NF-κB pathway in leukomonocyte. Therefore, we asked whether CARMA1 mediates Notch1-induced NF-κB pathway. Methods Microarray gene expression data were obtained from GEO database (accession number GSE26713) to assess expression of CARMA1, BCL10, MALT1 in T-ALL patients. In addition, we apply microarray dataset (GEO accession number GSE42328) to examine the relationship between gene expression and patients outcome. Western blot was used to analyze CRAMA1 expression in different cell lines. CCK-8 assay, EdU assay, cell cycle and colony formation experiments were performed in CARMA1-shRNA and control-shRNA T-ALL cell lines to investigate the effect of CARMA1 in T-ALL. We intravenously injected the Control-shRNA and CARMA1-shRNA MOLT-4 cells into NOD-SCID/IL2Rgnull (NSG) mice to evaluate the influence of CARMA1 knockdown on overall survival. Dual luciferase reporter assay was applied to detect transcriptional activity of NF-κB. Results We found that CARMA1 was highly expressed in T-ALL cell lines, but not observed in B-ALL and AML cell lines. The T-ALL patients with high CARMA1 expression had better outcome compared with the low expression group. However, we failed to observe any significant differences in the expression of BCL10 and MALT1 between primary normal and T-ALL cells or relationship of survival with MALT1 and BCL10 in T-ALL. A previous finding showed that Notch1 mutations are correlated with an improved long-term prognosis (J Clin Oncol 2013;31:4333). The above results suggested both high expression of CARMA1 and activating Notch1 mutaions relate to favorable prognosis, indicating the possible relevance between CARMA1 expression and Notch1 activation. To elucidate the effect of CARMA1 on cell proliferation, we performed CARMA1 knockdown experiment with shRNAs in T-ALL cells. CARMA1 knockdown in MOLT-4 cells significantly decrease the quantity and size of cell colonies. MOLT-4 and Jurkat cell lines showed significant reductions in cell viability through CARMA1 silencing compared with control-shRNA cells. The EdU proliferation assay demonstrated that knockdown of CARMA1 in MOLT-4 and Jurkat cell lines markedly reduced the number of EdU-positive cells. Interestingly, CARMA1 had no significant effect on cell proliferation in CCRF-CEM cells. This finding is consistent with a recent report, which demonstrated that CARMA1 knockdown showed no difference in proliferation of CCRF-CEM cells, but only one cell line was used (Leukemia. 2017;31:255). CCRF-CEM is a SIL-TAL1 fusion gene positive cell line and its survival depends on the activation of TAL1 transcriptional complex. Therefore, the proliferation of SIL-TAL1 fusion gene positive cell lines may be independent of CARMA1 expression. We next explored the significance of CARMA1 in cell cycle and in vivo tumorigenic ability. Compared to the control-shRNA cells, CARMA1 knockdown arrested T-ALL cell lines in G1 phase. CARMA1 knockdown remarkably led to the downregulation of the percentage of CD45 positive cells and prolonged overall survival of NSG mice. To further determine the role of CARMA1 in Notch1-induced NF-Κb activation, we transfected Notch1-IC into control-shRNA and CARMA1-shRNA cells, respectively. We found that Notch1-IC significantly upregulated the activity of NF-κB in control-shRNA cells. However, the Notch1-induced NF-κB was abolished in CARMA1 knockdown T-ALL cells. The results demonstrate that CARMA1 is involved in Notch1-mediated NF-κB activation. Conclusion Taken together, CARMA1 markedly contributes to cell survival in SIL-TAL1 negative T-ALL cells and may play a crucial role in Notch1-induced activation of NF-κB pathway. Furthermore, CRAMA1 might be a prognostic marker and a prospective therapeutic target for T-ALL. Disclosures No relevant conflicts of interest to declare.

Brain-Derived Neurotrophic Factor and Supraoptic Vasopressin Neurons in Hyponatremia

Hyponatremia due to elevated arginine vasopressin (AVP) secretion increases mortality in liver failure patients. The mechanisms causing dysregulation of AVP secretion are unknown. Our hypothesis is that inappropriate AVP release associated with liver failure is due to increased brain-derived neurotrophic factor (BDNF) in the supraoptic nucleus (SON). BDNF diminishes GABAA inhibition in SON AVP neurons by increasing intracellular chloride through tyrosine receptor kinase B (TrkB) activation and downregulation of K+/Cl– cotransporter 2 (KCC2). This loss of inhibition could increase AVP secretion. This hypothesis was tested using shRNA against BDNF (shBDNF) in the SON in bile duct ligated (BDL) male rats. All BDL rats had significantly increased liver weight (p < 0.05; 6–9) compared to shams. BDL rats with control ­shRNA injections (BDL scrambled [SCR]) developed hyponatremia with increased plasma AVP and copeptin (CPP; all p < 0.05; 6–9) compared to sham groups. This is the first study to show that phosphorylation of TrkB is significantly increased along with significant decrease in phosphorylation of KCC2 in BDL SCR rats compared to the sham rats (p < 0.05;6–8). Knockdown of BDNF in the SON of BDL rats (BDL shBDNF) significantly increased plasma osmolality and hematocrit compared to BDL SCR rats (p < 0.05; 6–9). The BDL shBDNF rats had significant (p < 0.05; 6–9) decreases in plasma AVP and CPP concentration compared to BDL SCR rats. The BDNF knockdown also significantly blocked the increase in TrkB phosphorylation and decrease in KCC2 phosphorylation (p < 0.05; 6–8). The results indicate that BDNF produced in the SON contributes to increased AVP secretion and hyponatremia during liver failure.