miR-142-3p在MCF-7细胞中靶向调节AKT pathway活性机理

为探究miR-142-3p在MCF-7细胞中作用机理,采用RNA免疫共沉淀技术和双荧光素酶报告基因技术筛选及验证miR-142-3p作用靶基因;蛋白质免疫印迹技术验证靶基因及其介导的PI3K-AKT-mTOR信号通路蛋白表达量及通路活性;应用实时荧光定量PCR验证靶基因PTEN对miR-142-3p调节关系。结果表明,miR-142-3p靶向调节AKT和PTEN表达;miR-142-3p过表达组中PI3K-AKT-mTOR通路活性显著降低;miR-142-3p抑制组中PI3K-AKT-mTOR通路活性显著上升;抑制PTEN表达显著提高miR-142-3p表达量。因此miR-142-3p靶向调节AKT和PTEN表达继而抑制PI3K-AKT-mTOR信号通路活性,PTEN与miR-142-3p存在调节关系。     

Cepharanthine induces autophagy via PI3K/AKT/mTOR signaling pathway in ovarian cancer SKOV3 cells

AIM: To investigate the autophagy of human ovarian cancer SKOV3 cells induced by cepharanthine and to explore its mechanism. METHODS: The effect of cepharanthine on the viability of ovarian cancer SKOV3 cells was measured by CCK-8 assay. The SKOV3 cells were treated with cepharanthine, and then the formation of autophagosome was observed with acridine orange staining under fluorescence microscope. The protein levels of LC3, AKT, p-AKT, mTOR, p-mTOR and GAPDH in the SKOV3 cells treated with cepharanthine were determined by Western blot.RESULTS: Cepharanthine significantly inhibited the viability of ovarian cancer SKOV3 cells in a dose-dependent manner (P<0.05). The number of the intracellular acidic autophagosomes with bright red fluorescence was significantly increased after cepharanthine treatment in the SKOV3 cells. The expression of LC3-Ⅱ in SKOV3 cells was significantly enhanced after cepharanthine treatment. Furthermore, treatment with cepharanthine in the SKOV3 cells also resulted in a significant down-regulation of phosphorylated form of AKT and mTOR (P<0.01), while the total protein level was not changed. Combination of cepharanthine and 3-methyladenine resulted in a substantial decrease in the cell viability compared with using cepharanthine alone.CONCLUSION: Cepharanthine significantly inhibits the growth of human ovarian cancer SKOV3 cells and induces the autophagy, which may be correlated with down-regulation of PI3K/AKT/mTOR signaling pathway.     

外源ACC通过激活乙烯合成及信号转导诱导小豆抗锈性

大量研究表明, 乙烯可激发植物对死体营养型真菌的抗性, 但我们前期研究发现, 乙烯合成前体ACC可提高小豆对活体营养型真菌——锈菌的抗性, 为初步明确其机制, 本研究分析了ACC处理对小豆乙烯合成及信号转导的影响, 结果表明, ACC处理显著提高了乙烯合成基因VaACS1及信号通路关键基因VaEIN2?VaEIN3?VaERF5的表达水平?此外, ACC处理后再接种锈菌, 小豆锈病的发病程度显著降低?对接种锈菌后不同时间VaPR2和VaPR4的表达分析表明, 相比ACC处理后不接种对照, ACC处理后再接种锈菌的处理, 接种后1~5 d这两个基因表达量显著升高; 与水处理不接种锈菌相比, 水处理接种锈菌5~8 d后VaPR2和VaPR4的表达量虽显著上调, 但应答时间较ACC处理滞后, 且总体表达水平低, 表明ACC激活乙烯通路进而诱导防卫反应基因上调表达是其诱导小豆抗锈性的关键?     

Effects of naringenin on PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways in rats with myocardial ischemia/reperfusion injury

AIM To observe the effect of naringenin on cardiac injury in ischemia/reperfusion （I/R） rats, and to explore whether the role of naringenin is involved in PI3K/AKT signaling pathway and endoplasmic reticulum stress and its related apoptotic pathways. METHODS SD rats （n=48） were randomly divided into sham operation （sham） group, model （I/R） group, naringenin treatment （NAR） group and naringenin+LY294002 （NL） group. Myocardial I/R injury model was prepared by ligation of left anterior descending coronary artery of rats for 30 min followed by reperfusion for 120 min. After reperfusion, the serum levels of cardiac troponin I （cTnI） was measured by ELISA. HE staining, TTC staining and TUNEL staining were used to detect the myocardial histopathological changes, myocardial infarction area and myocardial cell apoptotic rate. The mRNA levels of endoplasmic reticulum stress-related indicators glucose-regulated protein 78 （GRP78）, C/EBP homologous protein （CHOP） and caspase-12 were detected by RT-qPCR. The protein levels of cleaved caspase-3, GRP78, CHOP, caspase-12, p-PI3K and p-AKT were determined by Western blot. RESULTS Compared with I/R group, the serum content of cTnI, myocardial pathological damage, myocardial infarction area and myocardial cell apoptotic rate were significantly reduced （P<0.05）, the protein levels of cleaved caspase-3, GRP78, CHOP and caspase-12 were decreased （P<0.05）, and the protein levels of p-PI3K and p-AKT were increased in NAR group （P<0.05）. LY294002 attenuated the protective effect of naringenin to some extent. CONCLUSION Naringenin reduces myocardial I/R injury in rats possibly by activating PI3K/AKT signaling pathway and subsequently regulating endoplasmic reticulum stress and its related apoptotic pathways.     

Ethanol extract from Cortex Albiziae attenuates mouse acute liver injury induced by carbon tetrachloride via modulation of NF-κB pathway

AIM:To investigate the protective effect of ethanol extract from Cortex Albiziae on acute liver injury, and to explore its possible mechanism. METHODS:Acute liver injury in mice was induced by single intraperitoneal injection of 25% carbon tetrachloride (olive oil solubilization). The effective parts of ethanol extract from Cortex Albizziae against acute liver injury were screened. The pathological changes of the liver tissues were examined by pathological sections with HE staining. The activity of total superoxide dismutase (T-SOD) and the content of malondialdehyde (MDA) of the liver tissues were detected, the serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were mea-sured by ELISA, and the protein expression levels of NF-κB p65, Bcl-2 and Bax in the liver cells of the mice in each group were determined by Western blot. RESULTS:Compared with model group, the serum levels of AST and ALT in low-dose n-butanol phase of ethanol extract from Cortex Albiziae (AB-L, 4 mg·kg^-1·d^-1) group and high-dose n-butanol phase of ethanol extract from Cortex Albiziae (AB-H, 8 mg·kg^-1·d^-1) group were significantly decreased. The necrosis extent and degree of the hepatocytes and infiltration of inflammatory cells were significantly lower than that in model group. Compared with model group, the serum levels of TNF-α and IL-6 in AB-H group and AB-L group were significantly decreased (P<0.05). The protein level of NF-κB p65 in the nuclei of mouse liver cells in AB-H group and AB-L group were also decreased significantly (P<0.05). Compared with model group, the protein expression of Bax was decreased, the protein expression of Bcl-2 was increased, and the Bcl-2/Bax ratio was increased in AB-L group and AB-H group. CONCLUSION:The n-butanol phase of ethanol extract from Cortex Albiziae may protect the liver by reducing the activation of NF-κB p65, inhibiting the excessive release of inflammatory cytokines IL-6 and TNF-α, and decreasing hepatocyte apoptosis via regulating Bcl-2 and Bax expression.     

Up-regulation of miR-181a attenuates releases of CSE-induced pro-inflammatory factors and expression of collagen IV,fibronectin and α-SMA in human bronchial epithelial cells

AIM: To investigate the effect and potential mechanism of microRNA-181a (miR-181a) on cigarette smoke extract (CSE)-induced the productions of pro-inflammatory factors and the expression of collagen IV, fibronectin and α-smooth muscle actin (α-SMA) in human bronchial epithelial cells (HBECs). METHODS: CSE-induced miR-181a expression was detected by RT-qPCR in the HBECs. After tansfected with miR-181a mimic, the releases of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6 and transforming growth factor-β1 (TGF-β1) were measured by ELISA, the protein expression of collagen IV, fibronectin and α-SMA was determined by Western blot. The activation of NF-κB/TGF-β1/Smad3 pathway was also evaluated by Western blot. RESULTS: CSE increased the levels of TNF-α, IL-1β, IL-6 and TGF-β1 and the expression of collagen IV, fibronectin and α-SMA, and decreased the expression of miR-181a in the HBECs (P<0.05). However, transfected with miR-181a mimic partially prevented the releases of TNF-α, IL-1β, IL-6 and TGF-β1, and inhibited the expression of collagen IV, fibronectin and α-SMA (P<0.05). Additionally, the activation of NF-κB/TGF-β1/Smad3 evoked by CSE was attenuated after transfected with miR-181a mimic. CONCLUSION: Up-regulation of miR-181a prevents the releases of CSE-induced pro-inflammatory factors and expression of collagen IV, fibronectin and α-SMA in the HBECs, and its mechanism may be related to the inhibition of NF-κB/TGF-β1/Smad3 pathway.     

拟南芥T1N6_22在抵抗Pst DC3000侵染过程中的功能分析

【目的】明确拟南芥抗灰霉病基因T1N6_22在抗Pst DC3000过程中的功能,分析T1N6_22影响拟南芥对Pst DC3000的抗性原因。【方法】对t1n6_22突变体和转基因回复突变体（t1n6_22/T1N6_22）接种Pst DC3000,检测其症状;采用间苯胺蓝染色法检测接种突变体中胼胝质的积累情况;测定接种叶片中Pst DC3000的生长量,明确T1N6_22在拟南芥抗Pst DC3000过程中的功能。利用RT-PCR技术,检测SA、JA和ET对T1N6_22表达的影响及T1N6_22对抗病防御相关基因表达的影响;利用quantitative real-time PCR技术,检测Pst DC3000对T1N6_22及抗病相关基因表达的影响,分析T1N6_22影响拟南芥对Pst DC3000的抗性原因。【结果】t1n6_22突变体接种Pst DC3000后表现明显的抗病症状,而回复突变体t1n6_22/T1N6_22和拟南芥野生型表现明显的感病症状。SA处理拟南芥野生型,T1N6_22的表达量明显增强,经JA和ACC处理,该基因的表达量无显著变化。t1n6_22突变体中,PAL、PR4、PPO、SOD和CAT的表达水平均明显高于野生型Col-0和转基因回复植株。接种Pst DC3000后,拟南芥野生型中T1N6_22及抗病相关基因PR1、PR3、PR5和PDF1.2的表达量明显增强。【结论】T1N6_22在拟南芥抗Pst DC3000过程中起负调控作用,T1N6_22的表达受SA诱导,可能主要通过调节植物的次生代谢产物的分泌影响拟南芥对Pst DC3000的抗性。     

干扰lnc23后牛骨骼肌卫星细胞转录组测序的生物信息学分析

试验旨在分析lnc23在调控牛骨骼肌卫星细胞分化过程中转录组水平的变化,筛选出可能参与调节骨骼肌卫星细胞生长的调控通路及相关mRNAs。采用第二代测序技术（next-generation sequencing,NGS）基于Illumia hiSeq测序平台对成功构建的lnc23抑制模型及相应对照组的牛骨骼肌卫星细胞进行转录组测序,将数据整理、过滤及评估后,通过生物信息学方法分析样品间基因转录差异表达,对这些差异基因进行GO和KEGG富集分析,并应用实时荧光定量PCR技术对转录组数据结果进行验证。结果显示,转录组测序共鉴定到19 358个基因,筛选到1 297个差异表达基因,其中上调856个,下调441个。差异基因的GO功能分析共包括细胞组分、分子功能和生物学过程3大类222个分支,其中有大量的差异基因与催化活性、分子功能调节、信号转导、生物黏附、新陈代谢相关。KEGG分析显示,差异基因共涉及190个通路,显著富集在PI3K-Akt、P53、TNF、RIG-Ⅰ样受体、神经活性配体受体互作、细胞因子互作等信号通路上,进一步从中筛选出可能参与细胞生长、肌肉发育过程的差异基因,如CCNA2、RRM2、IL-6、MYL2等。实时荧光定量PCR结果显示,所选的11个差异基因中有9个与转录组测序结果变化一致,表明了测序结果的可靠性。本试验完成了干扰lnc23及其对照后牛骨骼肌卫星细胞的转录组测序分析,获取差异基因的功能注释信息,初步揭示lnc23调控牛骨骼肌卫星细胞分化的潜在基因和通路,为深入探究lnc23调控牛骨骼肌卫星细胞的分化机制打下良好的基础。     

Testing assumptions of cost surface analysis—a tool for invasive species management

Applied ecology could benefit from new tools that identify potential movement pathways of invasive species, particularly where data are sparse. Cost surface analysis (CSA) estimates the permeability (friction) across a landscape and can be applied to dispersal modelling. Increasingly used in a diversity of applications, several fundamental assumptions that might influence the outputs of CSA (cost surfaces and least-cost pathways) have yet to be systematically examined. Thus, we explore two issues: the presumed relationship between habitat preferences and dispersal behaviour as well as the degree of landscape fragmentation through which an organism moves by modelling a total of 18 sensitivity and dispersal scenarios. We explored the effect of fragmentation by altering the friction values (generally assigned using expert opinion) associated with patch and linear features. We compared these sensitivity scenarios in two sites that differed in fragmentation. We also used eastern grey squirrels (Sciurus carolinensis) as an example invading species and compared diffusion models and two contrasting cost surface dispersal scenarios. The diffusion model underestimated spread because squirrels did not move randomly through the landscape. Despite contrasting assumptions regarding dispersal behaviour, the two cost surfaces were strikingly similar while the least-cost paths differed. Furthermore, while the cost surfaces were insensitive to changes in friction values for linear features, they were sensitive to assumptions made for patch features. Our results suggest that movement in fragmented landscapes may be more sensitive to assumptions regarding friction values than contiguous landscapes. Thus, the reliability of CSA may depend not only on the range of friction values used for patches but also the degree of contiguity in the landscape.     

When, why, and how to perform cardiac radiofrequency catheter ablation

Radiofrequency catheter ablative techniques provide a unique opportunity to cure dogs of certain supraventricular tachyarrhythmias, rather than simply controlling the rhythm with drug therapy. Accessory pathways, which can participate as one limb of a rapid, narrow complex tachyarrhythmia circuit, have a particularly high cure rate with radiofrequency catheter ablation. Focal atrial tachycardias also provide a substrate that can be amenable to ablation. Tachycardia-induced myocardial dysfunction resulting from frequent tachyarrhythmic episodes will resolve after a successful ablation. This report outlines the indications, equipment, and techniques used in dogs for performing catheter ablation with temperature-controlled radiofrequency energy delivery.