Antisense Oligonucleotide Group Research Articles

The role of voltage-gated calcium channel α2δ-1 in the occurrence and development in myofascial orofacial pain

Patients who suffer from myofascial orofacial pain could affect their quality of life deeply. The pathogenesis of pain is still unclear. Our objective was to assess Whether Voltage-gated calcium channel α2δ-1(Cavα2δ-1) is related to myofascial orofacial pain. Rats were divided into the masseter tendon ligation group and the sham group. Compared with the sham group, the mechanical pain threshold of the masseter tendon ligation group was reduced on the 4th, 7th, 10th and 14th day after operation(P < 0.05). On the 14th day after operation, Cavα2δ-1 mRNA expression levels in trigeminal ganglion (TG) and the trigeminal spinal subnucleus caudalis and C1-C2 spinal cervical dorsal horn (Vc/C2) of the masseter tendon ligation group were increased (PTG=0.021, PVc/C2=0.012). Rats were divided into three groups. On the 4th day after ligating the superficial tendon of the left masseter muscle of the rats, 10 ul Cavα2δ-1 antisense oligonucleotide, 10 ul Cavα2δ-1 mismatched oligonucleotides and 10 ul normal saline was separately injected into the left masseter muscle of rats in Cavα2δ-1 antisense oligonucleotide group, Cavα2δ-1 mismatched oligonucleotides group and normal saline control group twice a day for 4 days. The mechanical pain threshold of the Cavα2δ-1 antisense oligonucleotides group was higher than Cavα2δ-1 mismatched oligonucleotides group on the 7th and 10th day after operation (P < 0.01). After PC12 cells were treated with lipopolysaccharide, Cavα2δ-1 mRNA expression level increased (P < 0.001). Cavα2δ-1 may be involved in the occurrence and development in myofascial orofacial pain.

Abstract P101: Suppression Of Serum Amyloid A Limits Progression Of Obesity Associated Abdominal Aortic Aneurysms

Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans, and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. Obesity is also associated with increases in serum amyloid A (SAA). We previously reported that deficiency of SAA significantly reduces AngII-induced inflammation and AAA in apoE-deficient mice. In this study we investigated whether SAA plays a role in progression of an established AAA in obese C57BL/6 mice. Approach and Results: Male C57BL/6 mice were fed a high fat diet (60% kcal as fat) throughout the study. After 4 months of diet the mice were infused with angiotensin II (AngII) at 1000ng/kg/min until the end of the study. Ultrasound (US) was performed in all mice before and after 28 days of AngII infusion, and mice that had at least a 25% increase in the luminal diameter of the abdominal aorta were stratified by luminal diameter into 3 groups. Group 1 was killed to establish baseline AAA. Groups 2 and 3 continued to receive AngII for a further 8 weeks along with an antisense oligonucleotide (ASO) that suppresses all 3 acute phase SAA isoforms (SAA-ASO), or a control ASO (5 mg/kg/wk). US was repeated at study end to assess AAA progression. Plasma SAA at the end of the experiment was 89.2±83.2 mg/L in the control ASO group, and 18.6±0.7 mg/L in the SAA-ASO group (mean±SD , p=0.008). There was no impact of SAA suppression on body weight, body fat, or blood pressure. After the first 4 weeks of AngII infusion, the average luminal diameter in all mice was 1.81±0.40 mm (mean±SD). Mice that received the control ASO had continued aortic dilation (average luminal aortic diameter 2.06±0.42 mm), whereas the mice that received the SAA-ASO had a significant reduction in progression of aortic dilation (average luminal diameter 1.64±0.43 mm, p=0.0015 for interaction between time and group). Conclusions: We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

Abstract B09: Selective depletion of YAP1 with next-generation (constrained ethyl-cEt) antisense oligonucleotides results in tumor regression in mouse models of HCC with YAP1 activation

Abstract The Hippo pathway plays important roles in tissue homeostasis. When dysregulated in adult tissues, however, it contributes to the development of a variety of cancers including hepatocellular carcinoma (HCC). YAP1 is the final regulator of the Hippo pathway and is associated with poor prognosis of HCC patients, and more recently has been suggested to be involved in tumor immune evasion. Here, we employed next-generation constrained ethyl (cEt) antisense oligonucleotides (ASOs) to selectively target YAP1 in the genetically engineered mouse (GEM) model of HCC (Salvador KO mice) as well as in a spontaneous carcinogen-induced model of HCC (diethylnitrosamine, DEN-induced HCC). In YAP1-activated Salvador KO mice with established HCC tumors, systemic treatment with YAP1 ASO at 25 mg/kg resulted in a marked reduction in YAP1 protein level in tumor (&amp;gt;90%) and regression of existing tumors, in contrast to the tumors in vehicle or control ASO-treated animals that significantly increased in size. In the DEN-induced HCC model, YAP1 was strongly activated in tumors as demonstrated by nuclear localization of YAP1 protein and the gene signature we previously identified (SOH). YAP1 ASOs produced either complete tumor stasis when DEN tumors were grown in immune-deficient mice, or complete regressions when tumors were grown in immune-competent animals. In accordance with this, YAP1 ASO treatment resulted in significant infiltration of T cells and myeloid lineage cells in the tumors in immune-competent mice. Importantly, when tumor-bearing animals were taken off the treatment after tumor regression was observed with YAP1 ASO, the antitumor response was sustained for more than 1 year, with 4 out of 16 animals remaining tumor-free in YAP1 ASO group. Furthermore, YAP1 ASO in combination with anti-PD1 antibody led to enhanced antitumor activity in the same tumor model. Collectively, these results suggest a potential therapeutic use of YAP1 ASO in the treatment of HCC with YAP1 activation as a single agent or in combination with immuno-oncology drugs. Broad screens have identified the human YAP1 ASO clinical development compound YAP1RX. Citation Format: Youngsoo Kim, Joanna Schmidt, Minji Jo, Jing Yi, Xiaolin Luo, Rachel Fleming, Sun Young Yim, Ju-Seog Lee, Brett P. Mornia, Randy L. Johnson, Gordon Mills, A. Robert MacLeod. Selective depletion of YAP1 with next-generation (constrained ethyl-cEt) antisense oligonucleotides results in tumor regression in mouse models of HCC with YAP1 activation [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B09.

The effect of miRNA-34a antisense oligonucleotide on non-small cell lung cancer cell line HCC827

Objective To investigate the effect of antisense oligonucleotides of miRNA-34a on non-small cell lung cancer (NSCLC) and its molecular mechanism. Methods The expression of miRNA-34a in human non-small cell lung cancer cell line HCC827 and human normal lung cell MRC-5 was detected by real time fluorescence quantitative polymerase chain reaction (qRT-PCR). HCC827 cells were divided into three groups: blank control group, negative control group, anti-sense oligonucleotide group(liposome 2000 transfected anti-sense oligonucleotide miRNA-34a); cell counting kit-8 (CCK-8) method was used to detect cell proliferation, Jimsa staining was used to detect cell cloning ability, Transwell test was used to detect cell migration and invasion ability; RT-PCR and Western blot were used to detect phosphatase and tensin homolog (PTEN), phosphorylation-protein kinase B (p-Akt), phosphatidylinositol-3-kinase (PI3K) mRNA and protein expression. Results The relative expression of miRNA34a in HCC827 cells was significantly higher than that in human normal lung cells (P 0.05). At 48 h, 72 h and 96 h, the proliferation level of HCC827 cells in anti-sense oligonucleotide miRNA-34a group was significantly lower than that in negative control group and blank control group (P<0.05). The cell cloning rate of antisense oligonucleotide miRNA-34a group was significantly lower than that of negative control group and blank control group (P<0.01). The number of migration and invasion of HCC827 cells in antisense oligonucleotide RNA-34a group was significantly lower than that in negative control group and blank control group (P<0.01). The relative expression of PTEN mRNA and protein in antisense oligonucleotide miRNA-34a group was significantly higher than that in negative control group and blank control group (P<0.05); the relative expression of p-Akt, PI3K mRNA and protein in antisense oligonucleotide miRNA-34a group were significantly lower than that in negative control group and blank control group (P<0.05). Conclusions The expression level of miRNA-34a in human non-small cell lung cancer cells is significantly higher than that in human normal lung cells. Antisense oligonucleotides of miRNA-34a can inhibit the proliferation, cloning, migration and invasion of human non-small cell lung cancer cells. The mechanism may be related to the negative regulation of PTEN/p-Akt/PI3K signaling pathway. Key words: Oligonucleotides, antisense; miRNA-34a; Carcinoma, non-small-cell lung; Cell line, tumor

Inhibition of Transcriptional Regulator Taz (Wwtr1) by Antisense Oligonucleotide (ASO) Treatment Improves Obesity and Hepatic Steatosis in a Mouse Model of NASH

Nonalcoholic fatty liver disease (NAFLD) is a common disorder that affects millions of people around the world. People with fatty liver disease alone are usually asymptomatic until a second insult to the liver, such as oxidative stress, triggers more severe steatosis, inflammation and fibrosis resulting in nonalcoholic steatohepatitis (NASH). The additional stress and damage to the hepatocytes results in the release of signals, such as hedgehog (Hh), to induce the wound-healing response. Data from animal models and human NASH have demonstrated Hh pathway activation correlates with the severity of liver disease. (1). In addition, the transcriptional coactivator, Taz/Wwtr1, was shown to be increased in hepatocytes from human and mouse NASH livers. (2). Based on these findings we performed DGE and qRT-PCR analysis on mice fed a high fat, fructose, and cholesterol (AMLN) diet for 24 weeks and found elevated mRNA expression of Taz in the livers compared to the lean mice. We treated C57B/6 mice fed the AMLN diet for 24 weeks with ASOs targeting Taz RNA. After 12 weeks of Taz ASO treatment, we observed a 95% knockdown in Taz mRNA expression in the liver compared to the control ASO group. This was accompanied by a 38% loss in whole-body fat mass (EchoMRI) and a 69% decrease in epidydimal white adipose tissue weight. Plasma analysis demonstrated a significant decrease in glucose and cholesterol compared to the control ASO levels. In addition, we observed reductions in liver triglyceride content resulting in levels similar to a lean mouse. No improvements in liver fibrosis were observed with Taz ASO treatment. These positive effects on obesity, plasma cholesterol, and liver steatosis suggest that Taz is a potential therapeutic target for preventing the progression of NASH into a more dangerous and irreversible liver disease. Disclosure R. Peralta: None. A.K. Low: Employee; Self; Ionis Pharmaceuticals, Inc.. Other Relationship; Spouse/Partner; Abbott. J. Schmidt: Employee; Self; Ionis Pharmaceuticals, Inc.. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.. A. Ferng: None. S. Murray: Employee; Self; Ionis Pharmaceuticals, Inc. S. Guo: Employee; Self; Ionis Pharmaceuticals, Inc..

Neprilysin Inhibition by an Antisense Oligonucleotide (ASO) Ameliorates Kidney Damage in a Salt‐induced Rat Chronic Kidney Disease Model

Neprilysin (NEP) is the key enzyme that degrades natriuretic peptides (NPs), which are the neurohormones contributing to natriuresis, diuresis and blood pressure regulation. NEP inhibition results in enhanced natriuresis and vasodilation by augmenting NP levels, which lead to the reduction of intra‐glomerular pressure and proteinuria in the damaged kidneys. We evaluated the renoprotective effects of a Generation 2.5 NEP antisense oligonucleotide (ASO) in a hypertensive and salt‐induced severe chronic kidney disease (CKD) model. Male, uninephrectomized (UNX) spontaneously hypertensive rats (SHR) were randomized to concurrently receive seven weeks of high salt intake (1% NaCl in drinking water for 5 weeks and then 1.5% for remaining two weeks) and drug treatments (vehicle control, NEP ASO at 20 mg/kg SQ weekly or the small molecule NEP inhibitor AHU377 at 50 mg/kg PO daily). AHU377 (Sacubitril) is the main ingredient of the recently approved heart failure drug Entresto®. Age‐matched SHR naïve (SHR control, normal salt intake) and SHRs with UNX (UnX control, normal salt intake + UnX) served as negative controls. NEP ASO treated rats demonstrated improvements in proteinuria compared to vehicle control or AHU377 treatments1. There were no differences between the treatment groups in blood BUN, creatinine level and creatinine clearance. In the NEP ASO group, kidney qPCR analysis demonstrated marked reductions of NEP expression (&gt;90%) and significantly less inflammation (MCP1, KIM1, NGAL) and fibrosis (ColIa1) relative to control or AHU377 treatments2. Western blot and NEP activity assays of kidney homogenates corroborated the qPCR findings. Meanwhile, NEP ASO and AHU377 treatments restored urinary cGMP/creatinine ratio3 suppressed by salt‐induced kidney damage. Histological examination of the kidney revealed less interstitial fibrosis, eosinophilic casts and glomerulosclerosis with NEP ASO treatment. In summary, in a severe rat CKD model, NEP ASO treatment was effective in blunting disease progression (i.e. less proteinuria, inflammation and fibrosis), which was not observed with the treatment by a small molecular NEP inhibitor AHU377. SHR control UnX control UnX, salt + vehicle UnX, salt + NEP ASO UnX, salt + AHU377 1 MicroAlb/CRE (mg/ml/mg/dL) 0.22 ± 0.04 1.25 ± 0.23 16.2 ± 3.3 2.40 ± 0.80** 26.3 ± 10.1 2 MCP1 gene expression (%) 100 ± 8 121 ± 8 347 ± 48 239 ± 18* 351 ± 31 KIM1 gene expression (%) 100 ± 28 248 ± 47 10,189 ± 2,382 1,430 ± 307* 18,970 ± 12,266 NGAL gene expression (%) 100 ± 7 157 ± 7 1,870 ± 387 463 ± 36** 1,987 ± 794 ColIa1 gene expression (%) 100 ± 10 139 ± 13 753 ± 122 474 ± 28* 752 ± 95 3 Urinary cGMP/CRE (pmol/ml/mg/dL) 33.6 ± 4.6 26.6 ± 2.4 19.5 ± 1.6 26.4 ± 2.2* 34.4 ± 4.2 1(UnX, salt + vehicle) vs. (UnX, salt + NEP ASO): ** p &lt; 0.01 2(UnX, salt + vehicle) vs. (UnX, salt + NEP ASO): * p &lt; 0.05, ** p &lt; 0.01 3(UnX, salt + vehicle) vs. (UnX, salt + NEP ASO): * p &lt; 0.05 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract B197: Selective depletion of YAP1 with next-generation antisense oligonucleotides leads to immune cell infiltration and tumor regression in mouse models of HCC

Abstract The Hippo pathway plays important roles in maintaining tissue homeostasis. When dysregulated, however, it contributes to the development of a variety of cancers including hepatocellular carcinoma (HCC). YAP1 is the final regulator of the pathway and its causal role in promoting HCC growth has been demonstrated previously in multiple animal models. YAP1 is therefore considered an attractive drug target for HCC but as a transcription coactivator is difficult to inhibit by conventional approaches. We investigated how selective downregulation of YAP1 would affect the growth of established HCC using optimized Gen2.5 antisense oligonucleotides (ASOs) for mouse YAP1. Here, we describe the antitumor activity of YAP1 ASOs in 2 mouse models of HCC, which appears to be mediated by both tumor autonomous and immune modulatory functions of YAP1. In YAP1-activated Salvador KO mice with established HCC, systemic treatment of YAP1 ASO for 2 months at 75 mg/kg/week resulted in a marked reduction (&amp;gt;90%) in tumor YAP1 levels and regression of HCC tumors, which is in contrast to the tumors in the control ASO-treated animals that significantly increased in size. Proliferation of tumor cells was drastically reduced in YAP1 ASO-treated animals as demonstrated by low Ki-67 staining along with a decrease in Cyr61, a downstream YAP1 target. YAP1 ASOs were also tested in the diethylnitrosamine-induced HCC model. Although the model has been widely used in HCC research, lack of reliable biomarkers and high variability in tumor take between animals make it less ideal for an efficacy study. To overcome these limitations, we established a subcutaneous model by in vivo passaging tumors excised from the liver in either nude or immune-competent C57BL/6 mice. Activation of YAP1 in this model was demonstrated by strong nuclear localization of YAP1 protein in tumors. While the effect of YAP1 ASO on tumor was primarily delaying its growth in nude mice, the same YAP1 ASO induced tumor regression when DEN tumors were grown in immune competent mice, suggesting an immune component for activity. Strong infiltration of myeloid lineage cells was observed in tumors of YAP1 ASO-treated animals at the end of the 6 week study, suggesting potential activation of immune cells as a result of YAP1 depletion. Significant infiltration of T cells and myeloid lineage cells into YAP1 ASO-treated tumors was also observed at an early time point as demonstrated by increases in CD3 and F4/80 signals by IHC. Immune cell infiltration into tumors was more pronounced in the combination of ASOs for YAP1 and its paralog mouse TAZ than YAP1 ASO alone; however, the combination group exhibited notable signs of inflammation on the skin and in the liver. Importantly, when tumor-bearing animals were taken off the treatment after tumor regression was observed with YAP1 ASO, the antitumor response was sustained for more than 6 weeks with 2 out of 8 animals remaining tumor free in YAP1 ASO group. Further, safety of ASO-mediated YAP1 inhibition was confirmed in both normal mice with no notable toxicity and tumor-bearing mice with improvement in liver transaminases despite near complete depletion of YAP1 in the liver. Collectively, these results provide evidence for immune activation as a result of YAP1 downregulation and also suggest a potential therapeutic use of YAP1 ASO in the treatment of HCC with YAP1 activation. Citation Format: Youngsoo Kim, Joanna Schmidt, Minji Jo, Jing Yi, Tianyuan Zhou, Xiaolin Luo, Sun Young Yim, Ju-Seog Lee, Randy L. Johnson, Brett P. Monia, Gordon Mills, A. Robert MacLeod. Selective depletion of YAP1 with next-generation antisense oligonucleotides leads to immune cell infiltration and tumor regression in mouse models of HCC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B197.

Effects of heat shock protein 70 regulating endoplasmic reticulum calcium channel related protein on cardiomyocyte apoptosis

Objective To observe the role of heat shock protein 70 (HSP70) in endoplasmic reticulum (ER) stress-induced cell apoptosis, and to investigate the molecular mechanism underlying the HSP70 regulating ER stress induced-apoptosis, secretion and calcium signal transduction. The fidings provide new intervention targets and theoretical basis of subcellular molecular application for myocardial protection. Methods We used SD neonatal rat cardiomyocytes to establish a hypoxia cell model, and then we further detected the role of HSP70 in cell apoptosis, production of reactive oxygen species (ROS), expression of calcium sensing receptor (CaSR) and sarco endoplasmic reticulum Ca2+ -ATPase (SERCA) in ER caused by ER stress, by overexpression or downexpression of HSP70. Results The early apoptosis rate of the blank group [(9.28±0.83)%], late apoptosis rate [(28.60±1.83)%], ROS formation rate is 80.0%, HSP70 gene transfection group early apoptosis rate [(4.30±0.72)%], late apoptosis rate [(11.6±1.66)%], ROS formation rate is 38.5%, HSP70 treated with antisense oligonucleotides early apoptosis rate [(29.3±2.54)%], late apoptosis rate [(34.4±2.43)%], ROS formation rate of 77.0%, respectively, suggesting that HSP70 significantly inhibited the cell apoptosis and the production of ROS. The expression of CaSR in HSP70 gene transfection group was significantly lower than that in HSP70 antisense oligonucleotide group, but SERCA showed no significant difference, indicating that HSP70 significantly inhibited the expression of CaSR, but it had no influence on the expression of SERCA. Conclusion HSP70 inhibited the Ca2+ current via inhibiting the expression of CaSR, and the JNK pathway might play important roles in cell apoptosis and ROS-induced Ca2+ current. Key words: Heat shock protein 70; Endoplasmic reticulum stress; Myocardial protection; Calmodulin; Calcium sensitive receptors; Apoptosis

Inhibitory effect of bcl-XL antisense oligodeoxynucleotides on the proliferation of colon cancer cells and the growth of human colon cancer xenografts in nude mice

Objective To investigate the effect of bcl-XL antisense oligonucleotide transfection on the proliferation of colon cancer cells and the growth of human colon cancer xenografts in nude mice. Methods A long-term SW620 single cell suspension was prepared for subcutaneous injection in nude mice. Nude mice were randomly divided into 3 groups: control group, N-ODN group and antisense oligonucleotide group (n=5), with an average of 50 mm3. The antisense oligonucleotide group and N-ODN group were injected with 2 ml/kg antisense oligonucleotide or N-ODN, and the control group was injected with equal amount of normal saline. At 16th day after the operation, the tumor cells were taken out, and some tumor tissues were extracted. RNA and protein were extracted. The cationic liposome mediated transfection of antisense oligonucleotide, real-time quantitative polymerase chain reaction (Real-time PCR), Western blotting, methyl thiazol tetrazolium (MTT) method and TdT-mediated dUTP nick end labeling (TUNEL) method were applied to observe the effects of bcl-XL antisense oligonucleotide on the proliferation and apoptosis of colorectal cancer cells in vivo and the growth of human colon cancer xenografts. Results (1) The inhibition rate of cell proliferation activity was gradually increased with the increase of antisense oligonucleotide concentration. When the concentration was over 200 nmol/L, the inhibition rate of cell proliferation was significantly decreased with the difference being statistically significant (t=2.418, 1.531 and 1.838, P=0.003, 0.016 and 0.011). (2) The relative expression levels in the control group, blank control group, N-ODN group and mRNA antisense oligonucleotides group were 0.96±0.25, 0.97±0.48, 0.78±0.32 and 0.41±0.13, the inhibition rate was 0.00%, -6.25%, 18.75% and 57.29%, respectively. There was significant difference between antisense oligonucleotide group and control group, blank control group or N-ODN group (P=0.025). (3) Compared with the control group, there was no significant change in the blank control group; the N-ODN group was slightly lower than that in the control group; the antisense oligonucleotide group was significantly lower (P=0.017). (4) The positive rates of cells in control group, blank control group, N-ODN group and antisense oligonucleotide group were 3.7%, 5.0%, 7.0% and 35.0%, respectively. By statistical analysis, antisense oligonucleotide group and cell control group, blank control group and N-ODN group, the difference was statistically significant (χ2=181.880, P=0.000); cells in the control group, N-ODN group and control group comparison between 22 blank, there was no statistically significant difference (χ2=1.410, P=0.084). (5) There was no significant difference in tumor volume between first groups (P=0.162). On the sixth day, there was significant difference between the antisense oligonucleotide group and the control group (t=2.410, P=0.027). On the eleventh and the 16 day, there was significant difference between the two groups in (P=0.028, 0.016). (6) The expression of bcl-XL gene in the tumor tissues of the 3 groups of nude mice was significantly decreased after antisense oligonucleotide treatment, but the expression of antisense oligonucleotide group was significantly decreased, while the expression of N-ODN in the control group and the control group did not change significantly. Conclusion bcl-XL antisense oligonucleotides can effectively inhibit the proliferation of colon cancer cells and the growth of human colon cancer xenografts in nude mice. To reduce the expression of bcl-XL by antisense oligonucleotides, and to provide the experimental basis for gene therapy of colon cancer. Key words: Colon cancer; Oligonucleotides; Bcl-XL; Proliferation

Abstract 625: Targeting of Heparin Binding EGF-like Growth Factor (HBEGF) Suppresses Hyperlipidemia and Atherosclerosis in LDL Receptor Deficient Mice

Objective: Elevation of apoB-containing lipoproteins is a well-established risk factor for the development of atherosclerosis. Previous reports showed that expression of heparin-binding EGF-like growth factor (HBEGF), a ligand of epidermal growth factor receptor (EGFR), is associated with atherosclerosis development. In this study, we examined in vivo effects of HBEGF targeting on hyperlipidemia-induced atherosclerosis by suppressing HBEGF expression using antisense oligonucleotide (ASO). Methods and Results: Female and male LDLR deficient mice were fed a high fat diet (HFD; 21% fat, 0.2% cholesterol) throughout the study. After 8 weeks of HFD feeding, mice were injected intraperitoneally with either control or HBEGF ASOs weekly for 12 weeks. At termination, we measured circulating lipid concentrations and atherosclerotic lesion size in the aorta. Compared to control ASO group, HBEGF ASO group had a significant reduction of circulating total cholesterol, triglyceride, and apoB-containing lipoprotein concentrations but no change of high-density lipoprotein (HDL) concentration. Importantly, HBEGF ASO injection significantly suppressed atherosclerosis in the aortic arch, thoracic, and abdominal aorta. HBEGF ASO suppressed sterol synthetic gene expression in the liver but elevated lipid contents in the liver. HBEGF gene silencing in a liver cell system induced downregulation of sterol regulatory element binding protein (SREBP) target genes including LDLR and Insig1. Conclusion: Targeting HBEGF using ASOs is an efficient approach to suppress dyslipidemia and hyperlipidemia-induced atherosclerosis. The differential gene expression analysis suggests that HBEGF ASO administration suppresses SREBP-regulated gene expression in the liver leading to downregulation of circulating cholesterol and TG concentrations.

Abstract A150: Anti-BIRC6 antisense oligonucleotide inhibits enzalutamide-resistant castration-resistant prostate cancer growth in patient-derived xenograft model by suppressing multiple signaling pathways

Abstract Background: Enzalutamide (ENZ) is the latest androgen receptor antagonist approved for treating castration-resistant prostate cancer (CRPC) and improves patient survival. However, increasing reports showing the development of ENZ-resistant tumor in patients and no known therapies were shown to be effective to-date. Our group has identified that BIRC6, an Inhibitor of Apoptosis (IAP) member, was elevated in CRPC. We developed antisense oligonucleotides (ASOs) that specifically target BIRC6 and demonstrated a significant inhibitory effect towards various CRPC models in vitro and in vivo. Most recently, we observed that BIRC6 expression was also elevated in an ENZ-resistant CRPC patient-derived xenograft (PDX) model. Thus, we hypothesize that anti-BIRC6 ASO may inhibit Enz-resistant CRPC. Materials and Methods: PDX model LTL313BR was a castration relapsed tumor line developed after androgen ablation in animal bearing LTL313B, a patient derived prostate cancer tumor line (androgen sensitive, AR+). LTL313BR was a typical CRPC (adenocarcinoma, AR+) and was resistant to Bicalutamide and Enzalutamide. The model was used to study the effect of anti-BIRC6 ASO on the growth of ENZ-resistant CRPC. Mice bearing LTL313BR tumors were randomized into scrambled control (Scrb) ASO or anti-BIRC6 ASO groups for 21-day treatment (n = 30 per group). Tumors were harvested 1 week after the end of treatment. Tumor volume and serum PSA was measured and the effect on tumor apoptosis was examined. Gene expression profiling was performed to investigate the mechanism of action of anti-BIRC6 ASO. Results: We demonstrated that anti-BIRC6 ASO significantly impeded the growth of Enz- resistant CRPC LTL313BR. Anti-BIRC6 ASO treated group showed median tumor volume of 385mm3 comparing to 521 mm3 in control ASO group, i.e. a 37% reduction. The marked tumor suppression was also coupled with significant reduction in serum PSA and induction of tumor apoptosis. Pathway enrichment analysis of gene expression profile indicates that anti-BIRC6 ASO altered gene expressions that inhibit pathways in mitogenic signalling, proliferation, cell migration, neutrophils chemotaxis and increased T-cell recruitment. Conclusion: Current study provides proof-of-principle data that anti-BIRC6 ASO may represent as novel therapeutic agent against ENZ-resistant CRPC. Citation Format: Iris Sze Ue Luk, Raunak Shrestha, Hui Xue, Yuwei Wang, Peter Gout, Colin Collins, Martin Gleave, Yuzhuo Wang. Anti-BIRC6 antisense oligonucleotide inhibits enzalutamide-resistant castration-resistant prostate cancer growth in patient-derived xenograft model by suppressing multiple signaling pathways. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A150.

Impact of microRNA- 21 antisense oligonucleotide on cisplatin resistance of EC9706/DDP cells

Objective To investigate the impact of microRNA(miRNA)- 21 antisense oligonucleotide on cisplatin(DDP) resistance of EC9706/DDP cells. Methods miRNA- 21 antisense oligonucleotides were designed and transfected into EC9706/DDP- resistant cells.The miRNA- 21 expression levels were detected by real- time fluorescent quantitative polymerase chain reaction(FQ- PCR).Methyl thiazol tetrazolium(MTT) method was used to measure the sensitivity of cells to DDP drugs.Colony formation assay was used to test the cell proliferation. The change of apoptosis was examined by flow cytometry. Western blotting was performed to detect the expression of B cell lymphoma/leukemia- 2(bcl- 2), and bcl- 2 associated X protein(bax) proteins. Results As compared with miRNA- NC negative control group and blank control group, the miRNA- 21 relative expression was decreased to(0.21±0.89),50% inhibitory dose(IC50)decreased to(16.22±1.85)mg/L, the number of clonal colonies was reduced, the apoptosis rate significantly increased to(30.5±2.9)%, bcl- 2 protein expression decreased,and bax protein expression increased in miRNA- 21 antisense oligonucleotide group(P< 0.05). Conclusion miRNA - 21 decreased the resistance of EC9706/DDP cells to DDP possibly through reducing the bcl- 2 protein expression, and increasing the bax protein expression. Key words: MicroRNA-21; Esophageal cancer; Cisplatin

MicroRNA-21 antisense oligonucleotides to inhibit the growth of EC9706 cells

Objective To observe microRNA(miRNA)-21 antisense oligonucleotide inhibition of the growth of EC9706 cells. Methods Antisense oligonucleotides of EC9706 cells, tetrazolium blue(MTT)assay of cell growth inhibition rate, real-time fluorescent quantitative polymerase chain reaction(FQ-PCR)miRNA-21 level detection cell apoptosis by flow cytometry, FQ-PCR programmed cell death 4(PDCD4)mRNA expression levels, immunofluorescence PDCD4 protein expression. Results The optimum concentration of antisense oligonucleotide inhibits cell activity was 0.5μmol/L, after transfection of antisense oligonucleotide expression levels of intracellular miRNA-2l significantly lower for non- defined group of 0.045±0.021,apoptosis a significant increase of 12.95%, the survival rate was significantly lower(48.21±9.56)%, the difference was statistically significant(P< 0.05).Antisense oligonucleotides group PDCD4 mRNA expression increased relative to 0.452±0.002, and increased protein expression levels, and the difference was statistically significant(P< 0.05). Conclusion miRNA-2l antisense oligonucleotides can increase PDCD4 of EC9706 effectively inhibit cell proliferation and induce apoptosis. Key words: MicroRNA-21; Esophageal cancer; Antisense oligonucleotide; Programmed cell death 4

Effect of peroxisome proliferator-activated receptor γ on the cholesterol efflux of peritoneal macrophages in inflammation

Atherosclerosis, a chronic inflammatory disorder characterized by lipid and cholesterol accumulation, is the principal contributing factor to the pathology of cardiovascular disease. Macrophages contribute to plaque development by internalizing native and modified lipoproteins that convert them into cholesterol-rich foam cells. With multiple factors, including hypercholesterolemia and inflammation, promoting atherosclerosis, it is of great significance to elucidate how the mechanism of cholesterol efflux from the macrophages changes and the role of peroxisome proliferator-activated receptor γ (PPARγ) in these situations. Following isolation and culture of peritoneal macrophages from C57BL/6 mice in the present study, the cells were divided into three groups: The control group, the ciglitazone group and the PPARγ antisense oligonucleotide group. The expression of PPARγ and nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitorα (IκBα) in each group was observed through the levels of protein and mRNA, and then the cholesterol efflux of each group was investigated. In addition, the same experiments were repeated following stimulation of each group with lipopolysaccharide (LPS). No significant difference in the expression levels of PPARγ between the control group and ciglitazone group was observed. The expression levels of PPARγ in the PPARγ antisense oligonucleotide group were evidently lower than those in the control group. Subsequent to stimulation with LPS, the expression levels of PPARγ in the three groups were higher than those of each group prior to stimulation. The cholesterol efflux of the PPARγ antisense oligonucleotide group was clearly suppressed following stimulation with LPS in comparison with that of the other groups. PPARγ contributes to anti-inflammation by protecting IκBα from being phosphorylated and degraded and promoting cholesterol efflux from peritoneal macrophages in inflammation.

Role of neuron-restrictive silencer factor in spinal cord in remifentanil-induced hyperalgesia in a mouse model of incisional pain

Objective To evaluate the role of neuron-restrictive silencer factor （NRSF） in the spinal cord in remifentanil-induced hyperalgesia in a mouse model of incisional pain （IP） .Methods Fifty-six male Kunming mice were randomly divided into 7 groups （n=8 each）：control group （group C） ,IP group （group I） ,IP ＋remifentanil group （group IR ） , NRSF antisense oligonucleotide group （NAS group ） , IP ＋ NRSF antisense oligonucleotide group （I＋NAS group ） ,IP ＋ remifentanil ＋ NRSF mismatch oligonucleotide group （IR＋NMS group） , and IP ＋ remifentanil ＋ NRSF antisense oligonucleotide group （IR ＋ NAS group ） . Artificial cerebrospinal fluid 5 μl was injected intrathecally once a day for 3 consecutive days in C ,I and IR groups .NRSF antisense oligonucleotide NAS 10μg was injected intrathecally once a day for 3 consecutive days in NAS ,I＋NAS and IR ＋ NAS groups . NRSF mismatch oligonucleotide 10 μg was injected intrathecally once a day for 3 consecutive days in IR＋NMS group .A 1-cm longitudinal incision was made through skin ,fascia and muscle of the plantar aspect of the right hindpaw to establish the model of incisional pain in sevoflurane-anesthetized rats .At 30 min after the last injection ,normal saline 0.4 ml was infused subcutaneously in C and NAS groups ,the model＆nbsp;was established and normal saline 0.4 ml was subcutaneously infused simultaneously in I and I＋NAS groups ,and the model was established and remifentanil 0.04 mg/kg was subcutaneously infused simultaneously in IR ,IR＋NMS and IR＋NAS groups .At 3 days before operation （T0 ） ,4 h before operation （T1 ） and 4 ,12 ,24 and 48 h after operation （T1-5 ） ,mechanical paw withdrawal threshold to von Frey stimuli （PMWT ） and paw withdrawal latency to thermal nociceptive stimulus （PTWL ） were measured .Results Compared with C group ,the PWMT and PWTL were significantly decreased at T2-5 in I ,IR ,I＋NAS ,IR＋NMS and IR＋NAS groups （ P0.05 ） .Compared with I group ,the PWMT and PWTL were significantly decreased at T2-5 in IR and IR＋NMS groups （ P0.05） . Compared with IR group ,no significant change was found in the PWMT and PWTL at each time point in IR＋NMS group （ P〉0.05） ,and the PWMT and PWTL were significantly increased at T2-5 in IR＋NAS group （ P〈0.05） . Conclusion NRSF in the spinal cord is involved in the development and maintenance of hyperalgesia induced by remifentanil in a mouse model of IP . Key words: Repressor proteins; Piperidines; Hyperalgesia; Spinal cord

Effect of antisense miR-224 on gastric cancer cell proliferation and apoptosis

To observe the effects of miR-224 antisense oligonucleotide (ASO) on the proliferation and apoptosis of gastric cancer cells in vitro and vivo. The expression of miR-224 in the cancer tissues and their adjacent tissues in 120 gastric cancer patients were detected by real-time quantitative PCR. The biological effects of miR-224 ASO on human gastric cancer SGC7901 cells was assessed by MTT assay, clone formation assay, flow cytometry and in vivo experiment in nude mice. Compared with the control group (0.50 ± 0.07), miR-224 ASO significantly reduced the miR-224 mRNA expression in the cancer patients (0.09 ± 0.01, P < 0.05). MTT assay results showed that the survival rate of gastric cells at 24 h, 48 h and 72 h was 53.6%, 59.1% and 70.1% in the miR-224 ASO group, and 12.3%, 17.4% and 24.7%, respectively, in the control group (P < 0.05 for all). Clone formation assay revealed that clone formation rate in the miR-224 ASO group was (5.33 ± 0.74)%, significantly lower than the (33.33 ± 8.38)% in the control group (P < 0.05). Flow cytometry indicated that the apoptotic index was (15.68 ± 1.46)% in the miR-224 ASO group and (3.36 ± 0.88)% in the control group (P < 0.01). In addition, the expressions of Bcl2 mRNA and protein were 1.05 ± 0.04 and 0.21 ± 0.03 in the miR-224 ASO group, significantly lower than that in the control group (4.87 ± 0.96 and 0.88 ± 0.09, P < 0.01). The in vivo study further showed that the tumor volume in the experimental group is significantly smaller than that in the control group (P = 0.01). MiR-224 is overexpressed in human gastric cancer. Reducing the expression of miR-224 can effectively inhibit the growth and promote apoptosis of gastric cancer cells. miR-224 may become a new target for the regulation of gene expression in gastric cancer.

Regulatory effect of iron regulatory protein-2 on iron metabolism in lung cancer.

Iron metabolism plays an important role in the pathogenesis of lung cancer. This study aimed to investigate the effect of gene silencing of iron regulatory protein-2 (IRP2) on mRNA and protein expression of transferrin (Tf), transferrin receptor (TfR), and ferritin (Fn) in A549 lung cancer cells. A549 cells were cultured and divided into a liposome control group, a liposome + oligonucleotide (SCODN) control group, and a Lipofectamine + antisense oligonucleotide (ASODN) group. RT-PCR and Western blotting were used to detect mRNA and protein expression of Tf, TfR, and Fn. We found no significant change in Tf mRNA expression among the 3 groups (P = 0.078). TfR and Fn mRNA expressions in the ASODN group notably decreased compared to the liposome and SCODN groups (P < 0.01). IRP2 and TfR protein expressions in the ASODN group were significantly lower than in the liposome or SCODN groups (P < 0.05), whereas no significant change in Tf protein expression was observed between the 3 groups (P = 0.088). Fn protein expression in the ASODN group was significantly higher than in the liposome or SCODN group (P < 0.05). IRP2 can regulate the expression of TfR and Fn by changing its own protein expression and thereby regulate iron metabolism.

GW24-e2991 Protective effect of cardiotrophin-1 in preventing acute cardiomyocyte hypoxia- reoxygeonation injury mediated by supressor of cytokine signalling 1--SOCS1/stat3 pathway

ObjectivesTo investigate the signalling pathways supressor of cytokine signalling 1 (SOCS1) how to involve in the protective effect of CT-1 in preventing cardiomyocyte I/R injury from cellular and molecular aspects.MethodsCardiomyocytes...

Metabolic Consequences of High-Fat Diet Are Attenuated by Suppression of HIF-1α

Obesity is associated with tissue hypoxia and the up-regulation of hypoxia inducible factor 1 alpha (HIF-1α). Prior studies in transgenic mice have shown that HIF-1α plays a role in the metabolic dysfunction associated with obesity. Therefore, we hypothesized that, after the development of diet-induced obesity (DIO), metabolic function could be improved by administration of HIF-1α antisense oligonucleotides (ASO). DIO mice were treated with HIF-1α ASO or with control ASO for 8 weeks and compared with an untreated group. We found that HIF-1α ASO markedly suppressed Hif-1α gene expression in adipose tissue and the liver. HIF-1α ASO administration induced weight loss. Final body weight was 41.6±1.4 g in the HIF-1α ASO group vs 46.7±0.9 g in the control ASO group and 47.9±0.8 g in untreated mice (p<0.001). HIF-1α ASO increased energy expenditure (13.3±0.6 vs 12±0.1 and 11.9±0.4 kcal/kg/hr, respectively, p<0.001) and decreased the respiratory exchange ratio (0.71±0.01 vs 0.75±0.01 and 0.76±0.01, respectively, p<0.001), which suggested switching metabolism to fat oxidation. In contrast, HIF-1a ASO had no effect on food intake or activity. HIF-1α ASO treatment decreased fasting blood glucose (195.5±8.4 mg/dl vs 239±7.8 mg/dl in the control ASO group and 222±8.2 mg/dl in untreated mice, p<0.01), plasma insulin, hepatic glucose output, and liver fat content. These findings demonstrate that the metabolic consequences of DIO are attenuated by HIF-1α ASO treatment.

Regulating action of iron regulatory protein-2 in iron metabolism of lung cancer

To discuss the regulating mechanism of iron regulatory protein-2 (IRP2) in the iron metabolism of lung cancer. The cultured A549 cells were divided into 3 groups: liposome group (including liposomes 20 mg/L), random oligonucleotide group (SCODN group) and antisense oligonucleotide group (ASODN group). And the liposome-mediated transfection was employed with the liposome and SCODN groups as controls. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to examine the mRNA and protein expressions of iron metabolism-related transferring (Tf), transferrin receptor (TfR) and ferritin (Fn) genes, etc. After a 48-hour transfection, the mRNA expression of Tf had no statistically significant difference among three groups (F = 2.18, P = 0.078); the mRNA expression of TfR in the ASODN group was significantly lower than that in the liposome and SCODN groups (P < 0.05). The expression of Fn mRNA in the ASODN group (0.56 ± 0.06) was higher than that in the liposome (0.36 ± 0.05) and SCODN groups (0.39 ± 0.03) (P < 0.05). After a 48-hour transfection, the IRP2 protein expression of the ASODN group was significantly lower than those of the liposome and SCODN groups (P < 0.05). The Tf protein expression was not statistically different in three groups (F = 2.67, P = 0.088). The TfR protein expression of the ASODN group was lower than those of the liposome and SCODN groups (P < 0.05). And the Fn protein expression of the ASODN group was higher than those of the liposome and SCODN groups (P < 0.05). IRP2 may affect the expressions of TfR and Fn in lung adenocarcinoma A549 cells by changing the amount of protein and regulating the iron metabolism.