Objective To investigate the mechanism of dasatinib,tyrosine kinase inhibitor,inhibiting androgen receptor (AR) phosphorylation in prostate cancer cells.Methods HEK-293T and COS7 cell lines were cotransfected by wild-type (WT)-AR,ARY267F or ARY534F with Ack1 or Src,respectively,and Western blot was used to detect the AR phosphorylation sites.LNCaP cells were treated by EGF or heregulin without androgen,then Western blot was used to detect AR phosphorylation.After these LNCaP cells were treated by dasatinib or transfection with siRNA to silence Ack1 or Src gene,Western blot was used to observe the effect on AR phosphorylation,and quantitative real-time reverse transcription polymerase chain (RT-PCR)was applied to detect PSA mRNA and hk2 mRNA.Results After transfection,Ack1 kinase mediated the phosphorylation of AR Tyr267 in HEK-293T cells,and Src mediated AR Tyr534 phosphorylation in COS7cells.When LNCaP cells were treated by heregulin,AR Tyr267 was phosphorylated,but its phosphorylation was inhibited after these cells were treated by dasatinib or ack1 gene was silenced.When LNCaP cells were treated by EGF,AR Tyr534 was phosphorylated,but its phosphorylation was inhibited after these cells were treated by dasatinib or Src gene was silenced.EGF or heregulin raised endogenous AR target gene,PSA and hK2,mRNA levels in LNCaP cells (P ＜ 0.05).However,after these cells were treated by dasatinib,PSA and hK2 mRNA levels induced by heregulin were decreased (P ＜ 0.05),but those induced by EGF PSA were no significant changes (P ＞ 0.05).Conclusion Dasatinib can inhibit AR Tyr267 and AR Tyr 534phosphorylation,and it may play a significant role in anti-prostate cancer cells by inhibiting Ack1-mediated AR Tyr-267 phosphorylation and the expression of PSA mRNA and hk2 mRNA induced by heregulin.

Objective To evaluate the relationship between the effect of peroxisome proliferator-activated receptor-γ (PPARγ) in pulmonary vascular remodeling and NADPH oxidase 4 (NOX-4) in rats with pulmonary hypertension.Methods Thirty-two healthy adult male Sprague-Dawley rats,aged 2 months,weighing 200-250 g,were divided into 4 groups (n =8 each) using a random number table method:control group (group C),pulmonary arterial hypertension group (group PH),PPARγ agonist rosiglitazone treatment group (group R),and PPARγ antagonist GW9662 treatment group (group G).In group PH,monocrotaline 60 mg/kg was injected subcutaneously in the neck and back to induce pulmonary hypertension.The suspension of rosiglitazone and normal saline 5 mg · kg-1 · d-1 and GW9662 solution 2 mg · kg-1 · d-1 were administered by intragastric gavage after injecting monocrotaline,in group R and group G,respectively,for 4 consecutive weeks.The mean pulmonary arterial pressure (mPAP) was measured at 4 weeks after establishing the model.The animals were then sacrificed,and the lungs were removed for microscopic examination of the pathological changes (with a light microscope) and for determination of the expression of PPARγ and NOX-4 protein and mRNA in lung tissues (by real-time polymerase chain reaction or Western blot).The percentage of media thickness of pulmonary arterioles was calculated.Results Compared with group C,the mPAP and percentage of media thickness of pulmonary arterioles were significantly increased,the expression of PPARγ protein and mRNA was down-regulated,and the expression of NOX-4 protein and mRNA was up-regulated in PH,R and G groups (P＜0.05).Compared with group PH,the mPAP and percentage of media thickness of pulmonary arterioles were significantly decreased,the expression of PPARγ protein and mRNA was up-regulated,and the expression of NOX-4 protein and mRNA was down-regulated in group R,and the mPAP and percentage of media thickness of pulmonary arterioles were significantly increased,the expression of PPARγ protein and mRNA was down-regulated,and the expression of NOX-4 protein and mRNA was up-regulated in group G (P＜0.05).Conclusion The mechanism of endogenous protective effect of PPARγ in the development of pulmonary hypertension and pulmonary vascular remodeling may be related to down-regulating the expression of NOX-4 in rats.

Objective:To investigate the influence of long non-coding RNA (lncRNA) DIO3OS in ketamine-induced neurotoxicity and its mechanism in mouse hippocampal neurons.Methods:(1) Primary mouse hippocampal neurons were isolated and cultured; CCK-8 assay was used to detect the viability of cells treated with different concentrations of ketamine (0, 25, 50, 100, 200 μmol/L), and qPCR was used to detect DIO3OS mRNA expression. (2) Hippocampal neurons were divided into 4 groups: control group, ketamine group (cultured with 50 μmol/L ketamine for 24 h), ketamine+pc group (transfected with pcDNA3.1 plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h), and ketamine+DIO3OS group (transfected with pcDNA3.1-DIO3OS plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h); cell viability was detected by CCK-8 assay; lactic dehydrogenase (LDH) release was determined by LDH cytotoxicity assay kit; cell apoptosis was detected by flow cytometry; mRNA expressions of DIO3OS and brain-derived neurotrophic factor ( BDNF) were examined by qPCR; protein expressions of polypyrimidine tract-binding protein 1 (PTBP1) and BDNF were detected by Western blotting. (3) Total proteins of routinely cultured neurons were extracted, and RNA Pull-Down assay was used to detect whether DIO3OS mRNA and BDNF mRNA could directly bind to PTBP1 protein. (4) Hippocampal neurons were divided into ketamine+DIO3OS+si-NC group (co-transfected with pcDNA3.1-DIO3OS plasmid and si-NC plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h) and ketamine+DIO3OS+si-PTBP1 group (co-transfected with pcDNA3.1-DIO3OS plasmid and si-PTBP1 plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h); qPCR was used to examine the mRNA expressions of DIO3OS and BDNF; Western blotting was used to detect the protein levels of PTBP1 and BDNF. (5) Hippocampal neurons were divided into ketamine group (cultured with 50 μmol/L ketamine for 24 h), ketamine+si-DIO3OS group (transfected with si-DIO3OS plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h), ketamine+si-PTBP1 group (transfected with si-PTBP1 plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h), and ketamine+si-DIO3OS+si-PTBP1 group (co-transfected with si-DIO3OS plasmid and si-PTBP1 plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h); qPCR was used to examine the mRNA expressions of DIO3OS and BDNF; Western blotting was used to detect the BDNF protein expression. (6) Hippocampal neurons were divided into ketamine+DIO3OS+si-NC group (co-transfected with pcDNA3.1-DIO3OS plasmid and si-NC plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h), ketamine+DIO3OS+si-BDNF group (co-transfected with pcDNA3.1-DIO3OS plasmid and si-BDNF plasmid for 48 h, and then cultured with 50 μmol/L ketamine for 24 h); cell viability was detected by CCK-8 assay; LDH release was determined by LDH cytotoxicity assay kit; cell apoptosis was detected by flow cytometry. Results:(1) Compared with 0 μmol/L ketamine, 25, 50, 100 and 200 μmol/L ketamine could significantly inhibit the cell viability and DIO3OS mRNA expression ( P<0.05). (2) Compared with control group, ketamine group had significantly decreased DIO3OS mRNA expression and cell viability, significantly increased LDH release and apoptotic rate, and statistically inhibited BDNF mRNA and protein expressions and PTBP1 protein expression ( P<0.05); compared with ketamine+pc group, ketamine+DIO3OS group had significantly increased DIO3OS mRNA expression and cell viability, significantly decreased LDH release and apoptotic rate, significantly elevated BDNF mRNA and protein expressions ( P<0.05). (3) RNA Pull-Down assay showed that Bio-labeled DIO3OS (Bio-DIO3OS) and Bio-labeled BDNF (Bio-BDNF) could adsorb PTBP1 protein, while Bio-labeled antisense strands of DIO3OS or BDNF (Bio-DIO3OS-AS and Bio-BDNF-AS) could not adsorb PTBP1 protein. (4) Compared with ketamine+DIO3OS+si-NC group, ketamine+DIO3OS+si-PTBP1 group had significantly inhibited BDNF mRNA and protein expressions and PTBP1 protein expression ( P<0.05); no significant difference was noted in DIO3OS mRNA expression ( P>0.05). (5) Compared with ketamine group, ketamine+si-DIO3OS and ketamine+si-DIO3OS+si-PTBP1 groups had significantly decreased DIO3OS mRNA expression ( P<0.05); compared with ketamine group, ketamine+si-DIO3OS and ketamine+si-PTBP1 groups had significantly decreased BDNF mRNA and protein expressions ( P<0.05); compared with ketamine+si-DIO3OS and ketamine+si-PTBP1 groups, ketamine+si-DIO3OS+si-PTBP1 group had significantly elevated BDNF mRNA and protein expressions ( P<0.05). (6) Compared with ketamine+DIO3OS+si-NC group, ketamine+DIO3OS+si-BDNF group had significantly reduced cell viability, and significantly increased LDH release and apoptotic rate ( P<0.05). Conclusion:LncRNA DIO3OS expression is decreased in ketamine-induced primary mouse hippocampal neurons; DIO3OS overexpression can alleviate ketamine-induced neurotoxicity in mouse hippocampal neurons by regulating BDNF expression via binding to PTBP1.

Sensory modalities are important for survival but the molecular mechanisms remain challenging due to the polymodal functionality of sensory neurons. Here, we report the C. elegans outer labial lateral (OLL) sensilla sensory neurons respond to touch and cold. Mechanosensation of OLL neurons resulted in cell-autonomous mechanically-evoked Ca