miR-143 Interferes with ERK5 Signaling, and Abrogates Prostate Cancer Progression in Mice

Abstract Background: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. Results: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. Conclusions: miR-143 is as a new target for prostate cancer treatment

Intranasal sufentanil versus intravenous morphine for acute severe trauma pain: A double-blind randomized non-inferiority study.

BACKGROUND: Intravenous morphine (IVM) is the most common strong analgesic used in trauma, but is associated with a clear time limitation related to the need to obtain an access route. The intranasal (IN) route provides easy administration with a fast peak action time due to high vascularization and the absence of first-pass metabolism. We aimed to determine whether IN sufentanil (INS) for patients presenting to an emergency department with acute severe traumatic pain results in a reduction in pain intensity non-inferior to IVM. METHODS AND FINDINGS: In a prospective, randomized, multicenter non-inferiority trial conducted in the emergency departments of 6 hospitals across France, patients were randomized 1:1 to INS titration (0.3 μg/kg and additional doses of 0.15 μg/kg at 10 minutes and 20 minutes if numerical pain rating scale [NRS] > 3) and intravenous placebo, or to IVM (0.1 mg/kg and additional doses of 0.05 mg/kg at 10 minutes and 20 minutes if NRS > 3) and IN placebo. Patients, clinical staff, and research staff were blinded to the treatment allocation. The primary endpoint was the total decrease on NRS at 30 minutes after first administration. The prespecified non-inferiority margin was -1.3 on the NRS. The primary outcome was analyzed per protocol. Adverse events were prospectively recorded during 4 hours. Among the 194 patients enrolled in the emergency department cohort between November 4, 2013, and April 10, 2016, 157 were randomized, and the protocol was correctly administered in 136 (69 IVM group, 67 INS group, per protocol population, 76% men, median age 40 [IQR 29 to 54] years). The mean difference between NRS at first administration and NRS at 30 minutes was -4.1 (97.5% CI -4.6 to -3.6) in the IVM group and -5.2 (97.5% CI -5.7 to -4.6) in the INS group. Non-inferiority was demonstrated (p < 0.001 with 1-sided mean-equivalence t test), as the lower 97.5% confidence interval of 0.29 (97.5% CI 0.29 to 1.93) was above the prespecified margin of -1.3. INS was superior to IVM (intention to treat analysis: p = 0.034), but without a clinically significant difference in mean NRS between groups. Six severe adverse events were observed in the INS group and 2 in the IVM group (number needed to harm: 17), including an apparent imbalance for hypoxemia (3 in the INS group versus 1 in the IVM group) and for bradypnea (2 in the INS group versus 0 in the IVM group). The main limitation of the study was that the choice of concomitant analgesics, when they were used, was left to the discretion of the physician in charge, and co-analgesia was more often used in the IVM group. Moreover, the size of the study did not allow us to conclude with certainty about the safety of INS in emergency settings. CONCLUSIONS: We confirm the non-inferiority of INS compared to IVM for pain reduction at 30 minutes after administration in patients with severe traumatic pain presenting to an emergency department. The IN route, with no need to obtain a venous route, may allow early and effective analgesia in emergency settings and in difficult situations. Confirmation of the safety profile of INS will require further larger studies. TRIAL REGISTRATION: ClinicalTrials.gov NCT02095366. EudraCT 2013-001665-16

Regulator of G protein signaling-4 controls fatty acid and glucose homeostasis.

International audienceCirculating free fatty acids are a reflection of the balance between lipogenesis and lipolysis that takes place mainly in adipose tissue. We found that mice deficient for regulator of G protein signaling (RGS)-4 have increased circulating catecholamines, and increased free fatty acids. Consequently, RGS4-/- mice have increased concentration of circulating free fatty acids; abnormally accumulate fatty acids in liver, resulting in liver steatosis; and show a higher degree of glucose intolerance and decreased insulin secretion in pancreas. We show in this study that RGS4 controls adipose tissue lipolysis through regulation of the secretion of catecholamines by adrenal glands. RGS4 controls the balance between adipose tissue lipolysis and lipogenesis, secondary to its role in the regulation of catecholamine secretion by adrenal glands. RGS4 therefore could be a good target for the treatment of metabolic diseases

miR-143 Interferes with ERK5 Signaling, and Abrogates Prostate Cancer Progression in Mice

Abstract Background: Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. Results: Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. Conclusions: miR-143 is as a new target for prostate cancer treatment

Expression of miR-143 in prostate cancer.

<p><i>A</i>, Quantitative real-time PCR (QPCR) of miR-143, normalized to the amount of RNU48 target in prostate cancer cell lines. The relative levels of miRNA expression were measured by determining the ΔCt values of the indicated cell line versus PNT2 cells. Data are means ± SEM for three independent experiments. Statistical significance, here and in subsequent figures, *<0,05; **<0,01; ***<0,001. <i>B</i>, QPCR of miR-143, normalized to the amount of RNU48 target in prostate tissue samples. The relative levels of miRNA expression were measured by determining the ΔCt values of the indicated gleason prostate cancer versus normal prostate. Data are means ± SEM of eleven prostate samples for each group. <i>C</i>, Representative <i>in situ</i> hybridization staining of miR-143 in human non-tumor and tumor prostate tissue. TMA presents 40 prostate cancer tissues vs. 10 normal prostate tissues. (TMA, magnification, 400×).</p

miR-143 overexpression in C4-2 cells and LNCaP cells.

<p><i>A</i>, QPCR of miR-143 in C4-2 cells (white bars) and LNCaP cells (black bars), normalized to RNU48 48 h after transfection with scrambled miR (control), miR-143 precursor or antimiR-143. Data are means ± SEM for five independent experiments. <i>B–C</i>, Cell growth in LNCaP (B) or C4-2 (C) cells during 48 h after transfection with scrambled miR (black rhombus), miR-143 precursor (black cross) or antimiR-143 (white triangle). Data are means ± SEM for three independent experiments. <i>D</i>, Quantification of BrdU incorporation 48 h after transfection in C4-2 and LNCaP cells in absence of miR-143 (top), in presence of miR-143 (middle) or antimiR-143 (bottom). Data are representative for three independent experiments. <i>E</i>, Blue trypan incorporation in C4-2 (white bars) and LNCaP (black bars) cells 48 h after transfection in absence or presence of miR-143 or in presence of antimiR-143. <i>F</i>, Percentage of cells in different phases of cell cycle in LNCaP and C4-2 cell lines 36 hours following transfection with control 5 (non relevant), miR-143 or antimiR-143. Similar results were obtained in two independent experiments. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007542#s2" target="_blank">Results</a> are expressed as mean ± sem (n = 2–4). <i>G</i>, FACS analysis of apoptosis in C4-2 (white bars) and LNCaP (black bars) cells 48 h after transfection in absence or presence of miR-143 or in presence of antimiR-143. <i>H</i>, Relative active Caspase 3 concentration in C4-2 (white bars) and LNCaP (black bars) cells 48 h after transfection in absence or presence of miR-143 or in presence of antimiR-143. The concentration of active caspases 3 is normalized with the global protein concentration.</p

Identification of ERK5 as a miR-143 target.

<p><i>A</i>, Schematic representation of the predicted target site of miR-143 in the 3′ UTR of ERK5 mRNA. Seed-matching sequence is indicated. H.s ERK5 3′UTR is the Homo Sapiens 3′UTRsequence. <i>B</i>, Western blot analysis of endogenous ERK5 expression in prostate normal and cancer cell lines. The relative expressions, normalized to GAPDH, were measured by Image J software as a fold of the indicated situation versus scrambled miR. Data are means ± SEM for three independent experiments. C, Representative immunohistochemical staining of ERK5, and in situ hybridization of miR-143 in consecutive sections of high-density tissue array. <i>D</i>, Western blot analysis of endogenous ERK5 expression in C4-2 and LNCaP cells 48 h after transient transfection of the indicated miR-143. Relative expression, quantified by Image J software is normalized to GAPDH, and measured by fold of the indicated situation versus scrambled miR. Data are means ± SEM for three independent experiments. <i>E</i>, Measure of the luciferase activity in COS cells transfected with a reporter luciferase gene pGL3 fused to the ERK-5 3′ UTR mutated or not with increasing concentrations or miR-143 (0; 25; 50; 100 nM). Values are normalized to beta-galactosidase activity and expressed in fold versus absence of miR-143. Black bars, ERK5 3′UTR, white bars, mutant 3′ UTR; data are means ±SEM for four experiments conducted in triplicate. <i>F</i>, Western blot analysis of endogenous c-Jun expression in LNCaP cells 48 h after transient transfection of the indicated miR-143. Data are representative for three independent experiments. <i>G</i>, QPCR of ERK5 in C4-2 cells (white bars) and LNCaP cells (black bars), normalized to 18 s gene 48 h after transfection with pSuper-shNeo (control) or pSuper-shERK5. Data are means ± SEM for three independent experiments. <i>H</i>, Quantification of BrdU incorporation in C4-2 cells (white bars) and LNCaP cells (black bars) 48 after transfection with pSuper-shNeo (control) or pSuper-shERK5. Data are representative for three independent experiments.</p

CXC Ligand 5 Is an Adipose-Tissue Derived Factor that Links Obesity to Insulin Resistance

International audienceWe show here high levels of expression and secretion of the chemokine CXC ligand 5 (CXCL5) in the macrophage fraction of white adipose tissue (WAT). Moreover, we find that CXCL5 is dramatically increased in serum of human obese compared to lean subjects. Conversely, CXCL5 concentration is decreased in obese subjects after a weight reduction program, or in obese non-insulin-resistant, compared to insulin-resistant, subjects. Most importantly we demonstrate that treatment with recombinant CXCL5 blocks insulin-stimulated glucose uptake in muscle in mice. CXCL5 blocks insulin signaling by activating the Jak2/STAT5/SOCS2 pathway. Finally, by treating obese, insulin-resistant mice with either anti-CXCL5 neutralizing antibodies or antagonists of CXCR2, which is the CXCL5 receptor, we demonstrate that CXCL5 mediates insulin resistance. Furthermore CXCR2-/- mice are protected against obesity-induced insulin resistance. Taken together, these results show that secretion of CXCL5 by WAT resident macrophages represents a link between obesity, inflammation, and insulin resistance