R1 mRNA Research Articles

Circ-PGC increases the expression of FOXR2 by targeting miR-532-3p to promote the development of non-small cell lung cancer

ABSTRACT This study was to explore the function of circular progastricsin (circ-PGC) in NSCLC. The histological morphology of tumor tissues was observed by hematoxylin and eosin (HE) staining. The expression of circ-PGC, miR-532-3p and forkhead box R2 (FOXR2) mRNA was measured by real-time quantitative polymerase chain reaction (RT-qPCR). The protein level of FOXR2 was checked by western blot. In functional analyses, cell viability, colony formation, cell apoptosis, cell migration and cell invasion were investigated using cell counting kit-8 (CCK-8) assay, colony formation assay, flow cytometry assay, wound healing assay and transwell assay. Besides, glycolysis metabolism was assessed by the levels of glucose consumption, lactate production and adenosine triphosphate (ATP) production. The predicted relationship between miR-532-3p and circ-PGC and FOXR2 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The results showed that circ-PGC and FOXR2 were upregulated in NSCLC tissues and cells. Circ-PGC knockdown or FOXR2 knockdown inhibited NSCLC cell viability, colony formation, cell migration, invasion and glycolysis metabolism, and FOXR2 overexpression rescued these inhibitory effects caused by circ-PGC knockdown. MiR-532-3p harbored the same binding site with circ-PGC and FOXR2, and circ-PGC positively regulated FOXR2 expression by targeting miR-532-3p. The expression of β-catenin and c-Myc was decreased in cells after circ-PGC knockdown but recovered with miR-532-3p inhibition or FOXR2 overexpression. Circ-PGC downregulation also inhibited tumor growth in vivo. In conclusion, circ-PGC positively regulated FOXR2 expression by competitively binding to miR-532-3p, thereby promoting the development of NSCLC, and the Wnt/β-catenin signaling pathway might be activated by the circ-PGC/miR-532-3p/FOXR2 network.

Novel lncRNA-HZ04 promotes BPDE-induced human trophoblast cell apoptosis and miscarriage by upregulating IP3 R1 /CaMKII/SGCB pathway by competitively binding with miR-hz04.

Normal pregnancy is essential for human reproduction. However, BaP (benzo(a)pyrene) and its metabolite BPDE (benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide) could cause dysfunctions of human trophoblast cells and might further induce miscarriage. Yet, the underlying mechanisms remain largely unknown. Herein, we identified a novel upregulated lnc-HZ04 and a novel downregulated miR-hz04 in villous tissues of unexplained recurrent miscarriage (RM) relative to those in healthy control tissues and also in BPDE-treated human trophoblast cells. Lnc-HZ04 directly and specifically bound with miR-hz04, diminished the reduction effects of miR-hz04 on IP3 R1 mRNA expression level and on IP3 R1 mRNA stability, and then activated the Ca2+ -mediated IP3 R1 /p-CaMKII/SGCB pathway, which further promoted trophoblast cell apoptosis. The miR-hz04 target site on lnc-HZ04 played crucial roles in these regulations. In normal trophoblast, relatively less lnc-HZ04 and more miR-hz04 suppressed this apoptosis pathway and gave normal pregnancy. After exposure to BPDE or in RM tissues, p53 was upregulated, which might promote p53-mediated lnc-HZ04 transcription. Relatively more lnc-HZ04 and less miR-hz04 activated this apoptosis pathway and might further induce miscarriage. BaP could also induce mice miscarriage by upregulating its corresponding murine apoptosis pathway. Therefore, BPDE-induced apoptosis of human trophoblast cells was associated with the occurrence of miscarriage. This work discovered the regulation roles of lnc-HZ04 and miR-hz04 and provided scientific and clinical understanding of the occurrence of unexplained miscarriage.

Binding of CCCTC-Binding Factor (CTCF) to the Minute Virus of Mice Genome Is Important for Proper Processing of Viral P4-Generated Pre-mRNAs.

Specific chromatin immunoprecipitation of salt-fractionated infected cell extracts has demonstrated that the CCCTC-binding factor (CTCF), a highly conserved, 11-zinc-finger DNA-binding protein with known roles in cellular and viral genome organization and gene expression, specifically binds the genome of Minute Virus of Mice (MVM). Mutations that diminish binding of CTCF to MVM affect processing of the P4-generated pre-mRNAs. These RNAs are spliced less efficiently to generate the R1 mRNA, and definition of the NS2-specific exon upstream of the small intron is reduced, leading to relatively less R2 and the generation of a novel exon-skipped product. These results suggest a model in which CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of the P4-generated pre-mRNA.

The effects of graded hyperthermic (32, 20 ℃) on spinal cord protection during ischemia-reperfusion in rat

Objective To study the neuroprotective effects of moderate hypothermia (32 ℃) and profound hypothermia (20 ℃) on spinal cord ischemia-reperfusion in rats. Methods A subcutaneous heat exchanger is used to produce a low temperature of the spinal cord. The balloon blocked the thoracic aorta to induce spinal cord ischemia for 10 minutes. 42 SD rats were randomly divided into 4 groups, group Ⅰ. Non-blocking group (37 ℃, 6); Ⅱ. Normal temperature ischemia group (37 ℃, 12); Ⅲ. moderate hypothermia group (32 ℃, 12); Ⅳ. Profound Hyperthermia group (20℃, 12). Six rats in groups Ⅱ, Ⅲ and Ⅳ were given ischemia for 10 minutes and the lumbar spinal cord was taken. The other 6 rats in each group were given reperfusion, and the neurological function was evaluated at 1, 2, 4, and 8 hours. All the samples from all three rats were examined by histological examination and analyzed. The release of neurotransmitter amino acids was observed by high pressure liquid chromatography, and N-Methyl-D-Aspartate R1 (NMDAR1) mRNA was detected by in situ hybridization. The test results were analyzed by t test. Results Hard palate and neuron necrosis occurred in the normal temperature ischemia-reperfusion group. In terms of histopathological findings, the Moderate Hyperthermia (32 ℃) group and the profound hypothermia (20 ℃) group were significantly better than normal temperature ischemia after reperfusion. After 10 minutes of spinal cord ischemia, The glutamate concentration and NMDAR1 mRNA expression in the profound hypothermia in the Moderate Hyperthermia (32 ℃) group and (20 ℃) group were significantly lower than those in the normal temperature ischemia group. After 8 hours of reperfusion, the concentrations of glutamate and NMDAR1 mRNA in the Moderate Hyperthermia (32 ℃) group and the profound hypothermia (20 ℃) group were significantly higher than those in the normal temperature control group. Spinal cord ischemia was 10 min. The glutamate concentration in the medium-low temperature (32 ℃) group was 10.88±1.17 (t=27.292, P 0.05), and that in the low temperature (20 ℃) group was (12.57±2.59) cells (t=4.154, P<0.01), which was significantly lower than that in the normal temperature ischemia group [(17.36±2.56) cells]. After reperfusion for 8 hours, the glutamate concentration in the medium-low temperature (32 ℃) group was 49.38±13.26 (t=-8.407, P<0.01), and the deep hypothermia (20 ℃) group was 44.95±2.12 (t=-23.933, P<0.01), which was significantly higher than the normal temperature ischemia group (14.08±3.70); the number of NMDAR1 mRNA positive cells in the low temperature (32 ℃) group was (22.47±5.40) cells (t=-5.437, P<0.01), and that in the deep hypothermia (20 ℃) group was (18.89±3.98) cells (t=-4.042, P<0.01), which was significantly higher than that in the normal temperature ischemia group [(12.06±3.45) cells]. Conclusion Mode moderate hyperthermia (32 ℃) and profound hypothermia (20 ℃) have protective effects on histopathology of spinal ischemia-reperfusion cord. The results of spinal ischemia-reperfusion after 8 hours indicate that spinal cord protection are still needed during reperfusion. Key words: Spinal cord ischemia-reperfusion; Moderate hyperthermia; Profound hyperthermia; Glutamate

The effect of leptin and resveratrol on JAK/STAT pathways and Sirt-1 gene expression in the renal tissue of ischemia/reperfusion induced rats.

Our study aimed to investigate the possible modifying effects of leptin and combined use of resveratrol on rat renal I/R injury and their relationship on signal pathways and apoptosis-related mechanisms. Renal ischemia-reperfusion (I/R) injury is an important cause of acute renal failure. Male Sprague Dawley rats were divided into 5 groups: Control, I/R, I/R+leptin, I/R+resveratrol and I/R+leptin+resveratrol. Leptin (10 μg/kg BW) was administered (i.p.) 30 min prior to I/R. Resveratrol was administered by gavage at 20 mg/kg BW per d for 12 d prior to I/R. The left renal artery was exposed to 1 h of ischemia and 1 h of reperfusion. Resveratrol treatment alone increased TNF-α, TNF-α R1, NF-κB, SIRT-1, STAT1 and STAT3 mRNA levels and decreased caspase 3 protein levels. Leptin treatment alone significantly decreased the caspase 3 protein levels. The combined use of resveratrol and leptin significantly increased STAT3, and caspase 3 mRNA levels, and decreased the caspase 3 protein levels. Apoptosis was significantly decreased especially in the leptin and leptin+resveratrol groups. The present study suggest that a combined use of resveratrol and leptin has preventive and regulatory effects on renal I/R injury; the mechanism involves decreasing apoptosis, likely by altering the JAK/STAT pathway and SIRT1 expression (Fig. 8, Ref. 24).

Ketamine alleviates bradykinin-induced disruption of the mouse cerebrovascular endothelial cell-constructed tight junction barrier via a calcium-mediated redistribution of occludin polymerization.

Following brain injury, a sequence of mechanisms leads to disruption of the blood-brain barrier (BBB) and subsequent cerebral edema, which is thought to begin with activation of bradykinin. Our previous studies showed that ketamine, a widely used intravenous anesthetic agent, can suppress bradykinin-induced cell dysfunction. This study further aimed to evaluate the protective effects of ketamine against bradykinin-induced disruption of the mouse cerebrovascular endothelial cell (MCEC)-constructed tight junction barrier and the possible mechanisms. Exposure of MCECs to bradykinin increased intracellular calcium (Ca2+) concentrations in a time-dependent manner. However, pretreatment of MCECs with ketamine time- and concentration-dependently lowered the bradykinin-induced calcium influx. As to the mechanisms, although exposure of MCECs to ketamine induced bradykinin R1 receptor protein and mRNA expression, this anesthetic did not change levels of the bradykinin R2 receptor, a major receptor that responds to bradykinin stimulation. Bradykinin increased amounts of soluble occludin in MCECs, but pretreatment with ketamine alleviated this disturbance in occludin polymerization. Consequently, exposure to bradykinin decreased the transendothelial electronic resistance in the MCEC-constructed tight junction barrier. However, pretreatment with ketamine attenuated the bradykinin-induced disruption of the tight junction barrier. Taken together, this study shows that ketamine at a therapeutic concentration can protect against bradykinin-induced breakage of the BBB via suppressing calcium-dependent redistribution of occludin tight junctions. Thus, ketamine has the potential for maintaining the BBB in critically ill patients with severe brain disorders.

Non-covalent complexes of folic acid and oleic acid conjugated polyethylenimine: An efficient vehicle for antisense oligonucleotide delivery

Polyethylenimine (PEI) was conjugated to oleic acid (PEI-OA) and evaluated as a delivery agent for LOR-2501, an antisense oligonucleotide against ribonucleotide reductase R1 subunit. PEI-OA/LOR-2501 complexes were further coated with folic acid (FA/PEI-OA/LOR-2501) and evaluated in tumor cells. The level of cellular uptake of FA/PEI-OA/LOR-2501 was more than double that of PEI/LOR-2501 complexes, and was not affected by the expression level of folate receptor (FR) on the cell surface. Efficient delivery was seen in several cell lines. Furthermore, pathway specific cellular internalization inhibitors and markers were used to reveal the principal mechanism of cellular uptake. FA/PEI-OA/LOR-2501 significantly induced the downregulation of R1 mRNA and R1 protein. This novel formulation of FA/PEI-OA provides a reliable and highly efficient method for delivery of oligonucleotide and warrants further investigation.

CD33-Targeted Lipid Nanoparticles (aCD33LNs) for Therapeutic Delivery of GTI-2040 to Acute Myelogenous Leukemia.

CD33-targeted lipid nanoparticles (aCD33LNs) were synthesized for delivery of GTI-2040, an antisense oligonucleotide (ASO) against the R2 subunit of ribonucleotide reductase, to acute myelogenous leukemia (AML). These LNs incorporated a deoxycholate-polyethylenimine (DOC-PEI) conjugate, which has shown significant activity to facilitate oligonucleotide delivery. Anti-CD33 scFv (aCD33) was added as a targeting ligand. The delivery efficiency of this system was investigated both in vitro and in vivo. When cells were treated with aCD33LN/GTI-2040, significant uptake was observed in CD33 positive Kasumi-1 cells. aCD33LNs loaded with GTI-2040 induced significant down-regulation of R2 mRNA and protein levels in AML cells. Moreover, aCD33LN/GTI-2040 showed a 15-fold reduction in the IC50 of antileukemic drug Ara-C in Kasumi-1 cells. In Kasumi-1 xenograft model, aCD33LN/GTI-2040 showed significant R2 downregulation compared to LN/GTI-2040. Furthermore, aCD33LN/GTI-2040 coadministered with Ara-C was shown to be highly effective in tumor growth inhibition and to greatly increase survival time of mice bearing Kasumi-1 xenograft tumors. The conjugate DOC-PEI has shown an ability to include calcein release from lipid nanoparticles, suggesting a potential mechanism contributing to efficient endosome release by DOC-PEI2K. These results indicate that aCD33LNs are a highly effective vehicle for the therapeutic delivery of antisense agents to AML.

Enhanced antisense oligonucleotide delivery using cationic liposomes incorporating fatty acid-modified polyethylenimine.

Antisense oligonucleotides (ASOs) have promising therapeutic potential in oncotherapy. However, low stability and efficacy limit their application in the clinic. Cationic liposomes have been investigated as delivery vehicles for ASOs. Here, we report the synthesis and evaluation of an ASO delivery vehicle comprising cationic liposomes incorporating fatty acid-modified polyethylenimine. An oleic acid derivative of branched polyethylenimine (PEI-OA) and a linoleic acid derivative of branched polyethylenimine (PEI-LA) were synthesized and incorporated into liposomes. The PEI-modified liposomes were synthesized by an ethanol injection method with composition of PEI-modified lipid/Chol/TPGS. The properties of these liposomes, including cytotoxicity, cellular uptake, ASO target silencing activity, based on mRNA and protein downregulation, were investigated. LOR-2501, an ASOs targeting ribonucleotide reductase R1 subunit (R1) was used as the therapeutic cargo. The PEI-modified liposomes showed relatively compact particle size and excellent colloidal stability for at least 25 days. PEI-modified liposomes effectively delivered LOR-2501 into KB cells and efficiently induced down-regulation of R1 mRNA and protein. Compared with regular cationic liposomes, PEI-modified liposomes was more effective, reducing R1 mRNA and protein by ~10%.

Microfluidic assembly of lipid nanoparticles for delivery of antisense oligonucleotides.

Microfluidically (MF) synthesized lipid nanoparticles (LNPs) for antisense oligonucleotides (ODN) delivery have been shown to be superior to those prepared by bulk mixing (BM). In this study, a 5-inlet MF chip was used to synthesize LNPs loaded with LOR-2501, an antisense ODN targeting the ribonucleotide reductase R1 subunit. The size distribution of ODN- LNPs was measured by dynamic light scattering. The cytotoxicity of ODN- LNPs was determined by MTS assay. Gene silencing activity of ODN- LNPs was investigated by qRT-PCR and by Western blot. Results showed that MF synthesis produced ODN-LNPs that have lower average size and polydispersity values. The highest antisense activity was shown by LNs synthesized by the MF T2 chip, with downregulation of R1 mRNA by 32.5%. In conclusion, given their simplicity, affordability and reproducibility, MF is an attractive method for synthesis of LNs for ODN delivery.

A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia.

The siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for the targeted delivery of LOR-1284 siRNA into acute myeloid leukemia (AML) cells. The in vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4-11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than the free LOR-1284 in reducing the R2 mRNA and protein levels. The Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to the free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with the decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific therapeutic delivery of LOR-1284 into AML cells.

Role of Pregnane X Receptor in Obesity and Glucose Homeostasis in Male Mice

Clinical obesity is a complex metabolic disorder affecting one in three adults. Recent reports suggest that pregnane X receptor (PXR), a xenobiotic nuclear receptor important for defense against toxic agents and for eliminating drugs and other xenobiotics, may be involved in obesity. Noting differences in ligand specificities between human and mouse PXRs, the role of PXR in high fat diet (HFD)-induced obesity was examined using male PXR-humanized (hPXR) transgenic and PXR-knock-out (PXR-KO) mice in comparison to wild-type (WT) mice. After 16 weeks on either a control diet or HFD, WT mice showed greater weight gain, whereas PXR-KO mice gained less weight due to their resistance to HFD-induced decreases in adipose tissue peroxisome proliferator-activated receptor α and induction of hepatic carnitine palmitoyltransferase 1, suggesting increased energy metabolism. Interestingly, control-fed PXR-KO mice exhibited hepatomegaly, hyperinsulinemia, and hyperleptinemia but hypoadiponectinemia and lower adiponectin receptor R2 mRNA levels relative to WT mice. Evaluation of these biologic indicators in hPXR mice fed a control diet or HFD revealed further differences between the mouse and human receptors. Importantly, although HFD-fed hPXR mice were resistant to HFD-induced obesity, both PXR-KO and hPXR mice exhibited impaired induction of glucokinase involved in glucose utilization and displayed elevated fasting glucose levels and severely impaired glucose tolerance. Moreover, the basal hepatic levels of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 were increased in hPXR mice compared with WT mice. Altogether, although the mouse PXR promotes HFD-induced obesity, the hPXR mouse carries a genetic predisposition for type 2 diabetes and thus provides a model for exploring the role of human PXR in the metabolic syndrome.

The effect of capsaicin on circulating biomarkers, soluble tumor necrosis factor and soluble tumor necrosis factor-receptor-1 and -2 levels in vivo using lipopolysaccharide-treated mice

The circulating soluble tumor necrosis factor (sTNF) and sTNF-receptor (R) 1 and -R2 have known as septic biomarker. The pungent component of capsicum, capsaicin (Cap), has several associated physiological activities, including anti-oxidant, anti-bacterial and anti-inflammatory effects. The aim of this study was to elucidate the effect of Cap on circulating sTNF and sTNF-R1 and -R2 in vivo using lipopolysaccharide (LPS)-treated mice. LPS (20 mg/kg, ip)-treated group was significantly increased circulating sTNF, sTNF-R1, and -R2 and TNF-α mRNA expression levels compared to the vehicle group. Treatment with LPS (20 mg/kg, ip) + Cap (4 mg/kg, sc)-treated group was significantly decreased both circulating sTNF levels (after 1 h only) and TNF-α mRNA expression (after 6 h) compared to the LPS-treated group. There is an early increase in circulating sTNF, sTNR-R1, and -R2 observed in the LPS-treated mice. Since Cap inhibits this initial increase as biomarkers, circulating sTNF, it is considered a potent treatment option for TNF-α-related diseases, such as septicemia. In conclusion, Cap interferes with TNF-α mRNA transcription and exerts an inhibiting effect on TNF-α release from macrophages in the early phase after LPS stimulation. Thus, Cap is considered a potent agent for the treatment of TNF-α-related diseases, such as septicemia.

Akt2- and ETS1-Dependent IP3 Receptor 2 Expression in Dendritic Cell Migration

Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca²⁺ signaling. We thus explored whether Akt2 regulates DC Ca²⁺ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2^-/-) and their wild type littermates (akt2^+/+). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca²⁺ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2^+/+ but not akt2^-/- DCs. CCL21-induced increase in cytosolic Ca²⁺ concentration, thapsigargin-induced release of Ca²⁺ from intracellular stores with subsequent store-operated Ca²⁺ entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release as well as Ca²⁺ release-activated Ca²⁺ (CRAC) channel activity were all significantly lower in mature akt2^-/- than in mature akt2^+/+ DCs. Transcript levels of IP₃ receptor IP₃R2 and of IP₃R2 regulating transcription factor ETS1 were significantly higher in akt2^+/+ than in akt2^-/- DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2^+/+ and to a lower extent in akt2^-/- DCs. Following maturation, protein abundance of IP₃R2 and ETS1 were similarly higher in akt2^+/+ than in akt2^-/- DCs. The IP₃R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2^+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP₃R2 mRNA abundance, thapsigargin- and ATP-induced Ca²⁺ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP₃R2 transcription.

Molecular analysis-directed, international, phase III trial in patients with advanced non-small-cell lung cancer.

8001 Background: ERCC1 (E1) and RRM1 (R1) are predictive markers for platinum agents and gemcitabine (G) respectively. In a phase II trial that utilized E1 and R1 mRNA expression levels for therapeutic decision-making, a response rate of 44%, PFS of 6.6 m, and OS of 13.3 m was achieved. Methods: Pts with advanced, chemo-naive NSCLC, PS 0-1, measurable disease, and a FFPE histological or cell block tumor specimen were eligible. E1 and R1 were analyzed by AQUA and categorized as high or low. Pts were randomized 2:1 to arm A, which received G and carboplatin (Cb) for R1/E1 low, docetaxel (D) and Cb for R1 high and E1 low, G and D for R1 low and E1 high, and D and vinorelbine (V) for R1/E1 high, or arm B, which received GC. Pts received up to 6 cycles. Efficacy was assessed every 6-8 weeks until 1 year from treatment initiation. The primary goal was to improve the 6-m PFS from 38% in arm B (median PFS 4.3 m) to 50% in arm A (median PFS 6.0 m). Secondary goals were improvements in OS (8.0 to 12.0 m) and RR (25% to 50%). Results: Of 275 eligible pts, 183 randomized to arm A. 56 were assigned to GCb, 26 to DCb, 37 to GD, and 64 to DV, which was not significantly different (p=.2) from the expected assignment (30%, 20%, 20%, 30%). In arm B, all 92 pts received GCb. Protein analysis was successful in 91% of pts. The median time from consent to completed gene analysis was 11 days. A tumor rebiopsy for the specific purpose of gene analysis was required in 17% of pts. The 6-m PFS rate and median PFS were 52.0% and 6.1 m in arm A and 56.5% and 6.9 m in arm B (p = 0.18). PFS was not significantly different among the treatment groups in arm A (p = 0.1). A significant survival advantage was found for pts with low E1 and low R1 in arm B compared to the same group in arm A (p = 0.02) although both received GCb therapy. OS and RR were not significantly different between both groups. A comparison between protein and mRNA levels for both genes revealed no significant correlation. Conclusions: This demonstrates that gene expression analysis for therapeutic decision making is feasible in newly diagnosed advanced-stage NSCLC pts. A tumor rebiopsy is safe, required in 17%, and acceptable to 89% pts. The survival results are false negative based on the internal control included in the trial. Clinical trial information: NCT00499109.

Association of Serum Adipocytokines With Insulin Resistance and Liver Injury in Patients With Chronic Hepatitis C Genotype 4

Hepatitis C virus (HCV) infection, especially genotypes 1 and 4, is associated with metabolic dysfunction. We investigated the potential role of adipocytokines in HCV-induced insulin resistance (IR) and modulating the progression of liver disease in patients with HCV-4. Serum adiponectin, high molecular weight adiponectin, leptin, tumor necrosis factor-α, interluekin-6, homeostasis model for the assessment of insulin resistance, and M30 protein were measured in 147 HCV patients and 89 controls. Liver biopsies were evaluated for steatosis/inflammation/fibrosis, adiponectin mRNA/protein, AdipoR1/-R2 mRNA, and phosphoenolpyruvate carboxykinase gene expression, and adiponectin and CD95 immunoreactivity. CD95 immunoreactivity and adiponectin immunoreactivity were detected in all biopsies examined. Hepatic adiponectin immunostaining correlated positively with the intensity of hepatic CD95/Fas immunostaining (r=0.424; P=0.001). Hepatocyte CD95/Fas upregulation correlated with fibrosis, inflammation, and steatosis (r=0.52, P=0.0001; r=0.16, P=0.04; r=0.24, P=0.0001; respectively). Significant correlations of serum adiponectin, its receptors mRNA expression, hepatic adiponectin immunostaining, and mRNA transcription for phosphoenolpyruvate carboxykinase were identified with steatosis. A positive association between adiponectin and hepatic inflammation and fibrosis was identified. This correlation remained significant even after adjusting for age, sex, and body mass index. Among body mass index, age, and sex-matched HCV-negative controls, patients with HCV-4 have higher serum leptin, adiponectin, and high molecular weight adiponectin, and these changes are independently correlated with IR. Our findings in patients with HCV-4 show that adiponectin correlates with IR and with the different stages of liver injury. Steatosis upregulates hepatocyte CD95/Fas and thus increases apoptosis, which facilitates inflammation and fibrosis. These findings may provide potential clues for novel therapeutic intervention.

Arterial Stiffness Is Associated With Increased Monocyte Expression of Adiponectin Receptor mRNA and Protein in Patients With Coronary Artery Disease

Arterial stiffness and carotid intima-media thickness (IMT) constitute validated cardiovascular prognostic markers. Adiponectin and its receptors 1 (AdipoR1) and 2 (AdipoR2) are involved in coronary artery disease (CAD). We investigated whether AdipoR1 and R2 mRNA and protein expression are associated with arterial stiffness, IMT and extent of coronary atherosclerosis. We studied 71 patients (61 men, 10 women) with angiographically proven CAD. We measured: (i) monocyte expression of AdipoR1 and AdipoR2 mRNA (quantitative real-time PCR) and protein expression (flow cytometry) (iii) adiponectin, metalloproteinase-9 (MMP-9) and C-reactive protein (CRP) blood levels, (iv) carotid-femoral artery pulse wave velocity (PWV) and carotid IMT. Patients with multi-vessel CAD had higher AdipoR1 and AdipoR2 mRNA than those with single-vessel (P < 0.05). PWV was associated with AdipoR1 mRNA (r = 0.474), AdipoR1 protein (r = 0.228), AdipoR2 mRNA (r = 0.716), AdipoR2-protein (r = 0.261), adiponectin (r = 0.236), and MMP-9 (r = 0.350) (P < 0.05, for all correlations). After adjustment for age, sex, waist-hip ratio, and mean blood pressure both AdipoR1 and AdipoR2 mRNA remained independent determinants of PWV (R(2) = 0.35 and R(2) = 0.57, P < 0.05). IMT was also associated with AdipoR2 mRNA, AdipoR2 protein, and MMP-9 (P < 0.05). Increased expression of ADR2 mRNA significantly related to MMP-9 (r = 0.210), and CRP (r = 0.531) (P < 0.05). Increased mRNA and protein expression of adiponectin receptors is related with increased aortic stiffness, coronary and peripheral atherosclerosis in patients with CAD. The interrelation of AdipoR2 with inflammatory markers, PWV and IMT suggests a compensatory increase of these receptors to counteract the excess inflammatory and atherogenic process in CAD. Thus, adiponectin receptors may provide a potential therapeutic target of agents activating their beneficial action.American Journal of Hypertension 2012; doi:10.1038/ajh.2012.42.

Elevated Plasma Adiponectin Level and Peripheral Blood Leukocyte Adiponectin Receptor Expression in Dogs Suffering from Insulin Deficiency

The aim of this study is to determine and compare the effect of obesity or insulin deficiency on plasma adiponectin level and peripheral blood leukocyte (PBL) adiponectin receptor 1 and 2 (ADIPOR1 and -R2) expression in dogs. Obese and insulin deficient (ID) dogs demonstrated a significant reduction (60% lower) and significant increase (2x higher) in circulating adiponectin level, respectively, as compared to normal controls. PBL ADIPOR1 and -R2 mRNA expression was also significantly higher in obese (R1=333 mean fold higher, R2=64 mean fold higher) and ID dogs pre- insulin treatment (R1 =48 mean fold higher, R2=16 mean fold higher) as compared to normal PBL. Insulin treatment reduced ADIPOR1 (3 mean fold higher) and -R2 (1.5 mean fold higher) expression back down closer to control PBL levels in ID animals. The upregulation of adiponectin receptor expression might reflect an increased need for adiponectin signaling; however, the increase may have a different implication between obese and ID dogs. This may be due to adiponectin's contradictory paradoxical dual role, having both anti-inflammatory and pro-inflammatory properties on peripheral leukocytes, especially on monocytes depending on the biological context and adiponectin isoform.

A cis-acting region in the N-methyl-d-aspartate R1 3′-untranslated region interacts with the novel RNA-binding proteins beta subunit of alpha glucosidase II and annexin A2 - effect of chronic ethanol exposure in vivo

A cis-acting region, Δ4, located in the 3'-untranslated region of N-methyl-d-aspartate R1(NR1) mRNA interacts with several trans-acting proteins present in polysomes purified from fetal cortical neurons. Chronic ethanol exposure of fetal cortical neurons increases Δ4 RNA-protein interactions. This increased interaction is due to an increase in one of the Δ4-binding trans-acting proteins identified as beta subunit of alpha glucosidase II (GIIβ). In this study, we examined whether ethanol-mediated regulation of NR1 mRNA in vivo is similar to that in vitro and whether Δ4-trans interactions are important for ethanol-mediated NR1 mRNA stability. Our data show that polysomal proteins from adult mouse cerebral cortex (CC) formed a complex with Δ4 RNA, suggesting the presence of NR1 mRNA-binding trans-acting proteins in CC polysomes. The intensity of the Δ4 RNA-protein complex was increased with polysomes from chronic ethanol-exposed CC. The Δ4 RNA-protein complex harbored GIIβ and a second trans-acting protein identified as annexin A2 (AnxA2). Ethanol-sensitive GIIβ was upregulated by 70% in ethanol-exposed CC. Heparin, a known binding partner of AnxA2, inhibited Δ4 RNA-protein complex formation. Transient transfection studies using chimeric constructs with and without the Δ4 region revealed that cis-trans interactions are important for ethanol-mediated stability of NR1 mRNA. Furthermore, our data highlight, for the first time, the presence of a binding site on the 3'-untranslated region of NR1 mRNA for AnxA2 and demonstrate the regulation of NR1 mRNA by AnxA2, GIIβ and a third NR1 mRNA-binding protein, which is yet to be identified.

IL-22 inhibits ox-LDL-induced apoptosis and increases Bcl-2 expression in CRL-1730 cells

Objective To investigate the effect of interleukin-22(IL-22) on apoptosis of CRL-1730 cells(human umbilical vein endothelial cell line) induced by ox-LDL and the possible mechanism.Methods Expression of IL-22 receptor R1(IL-22R1) mRNA in CRL-1730 cells stimulated with ox-LDL(100 μg/ml) was detected by fluorescence quantitative RT-PCR.Flow cytometry was applied to examine the effect of exogenous IL-22(20 ng/ml) on the ox-LDL-induced apoptosis of CRL-1730 cells.The expression of Bcl-2,Bcl-xl,Bcl-w,caspase 3,and STAT3 mRNA in CRL-1730 cells treated or untreated with IL-22 and ox-LDL were measured by fluorescence quantitative RT-PCR;the phosphorylation status of STAT3 and the Bcl-2 protein level were examined by Western blotting analysis.Results The expression of IL-22R1 mRNA increased in a time-dependent manner(P0.05) and peaked at 12 h after ox-LDL stimulation.Treatment with IL-22 significantly inhibited the apoptosis of CRL-1730 cells induced by ox-LDL(P0.01),increased the expression of anti-apoptosis genes Bcl-2,Bcl-xl,and Bcl-w,and decreased expression of caspase 3 mRNA(P0.01).STAT3 phosphorylation occurred 1-2 h after IL-22 treatment and Bcl-2 protein expression increased 4 h after IL-22 treatment in ox-LDL stimulated CRL-1730 cells.Conclusion IL-22 can inhibit the pro-apoptosis effect of ox-LDL in the CRL-1730 cells,which might be associated with the increased expression of Bcl-2,Bcl-xl,and Bcl-w mRNA,decreased expression of caspase 3,and induced STAT3 phosphorylation.