高车前素对大肠癌细胞生长和转移的影响

高车前素是一种分离自菊科植物毛莲蒿的黄酮类化合物,具有多种生物活性。原癌基因PIM1可调节癌细胞存活、分化、增殖、凋亡和肿瘤发生。为了揭示高车前素对大肠癌细胞生长和转移的影响,及其对PIM1表达的影响,本研究应用不同浓度的高车前素处理人大肠癌细胞系SW620,并通过转染PIM1 siRNA来敲低大肠癌细胞中PIM1的表达,通过转染高表达PIM1的质粒载体再上调PIM1的表达。研究发现敲低PIM1可显著抑制大肠癌细胞的增殖和侵袭,并促进细胞凋亡;高车前素处理以浓度依赖性方式抑制大肠癌细胞系SW620的增殖和侵袭,并增加细胞凋亡。此外,高车前素处理以浓度依赖性方式抑制PIM1的表达,而上调PIM1的表达可显著抑制高车前素对大肠癌细胞系SW620的增殖、凋亡和侵袭能力的影响;高车前素以浓度依赖性方式抑制JAK2/STAT3的活化,并上调癌细胞内ROS水平;应用ROS清除剂N-乙酰半胱氨酸(NAC)预处理可恢复JAK2/STAT3信号传导的激活及PIM1的表达。本研究证实原癌基因PIM1在大肠癌中起着致癌基因的作用,而高车前素通过ROS/JAK2/STAT3信号通路来调节PIM1的表达,进而发挥其抗肿瘤作用。     

microRNA-21在食管癌细胞增殖、迁移和凋亡中的作用

本研究检测了40例食管癌组织和40例癌旁组织中的miR-21、PTEN、PI3K和AKT表达,并通过转染miR-21抑制剂来敲低人食管癌细胞系EC9706的miR-21表达,考察了miR-21对食管癌细胞生长的影响。研究发现,食管癌组织中PTEN蛋白的阳性染色评分低于癌旁组织(p<0.05),而PI3K和AKT蛋白的阳性染色评分高于癌旁组织(p<0.05)。miR-21在人食管癌组织中被上调(3.56 vs 1.21,p<0.05)。转染miR-21抑制剂导致PTEN蛋白表达升高,而PI3K和AKT蛋白表达降低(p<0.05)。转染miR-21抑制剂抑制了EC9706细胞的增殖和迁移,但促进了细胞凋亡(p<0.05)。miR-21的上调可通过激活PTEN/PI3K/AKT信号通路来促进食道癌细胞的增殖和迁移,并抑制细胞凋亡。     

鸦胆子油乳注射液可能通过调节JAK2/STAT3信号通路抑制胰腺癌AsPC-1细胞上皮–间质转化

该文旨在探讨鸦胆子油乳注射液(简称鸦胆子油乳)调节JAK2/STAT3信号通路对胰腺癌细胞上皮–间质转化(EMT)的影响。将胰腺癌AsPC-1细胞分为对照组、鸦胆子油乳低剂量组、鸦胆子油乳中剂量组、鸦胆子油乳高剂量组、鸦胆子油乳高剂量+Colivelin(JAK2/STAT3激活剂)组。该研究采用细胞划痕实验检测细胞迁移; Transwell实验检测细胞侵袭; MTT、EdU检测细胞增殖;流式细胞术检测细胞凋亡率; qRT-PCR检测细胞JAK2 mRNA、STAT3 mRNA表达; Western blot检测细胞JAK2/STAT3通路、E-cadherin、N-cadherin、Vimentin蛋白表达水平。结果显示,与对照组相比,鸦胆子油乳低、中、高剂量组细胞活力、增殖率、细胞划痕愈合率、侵袭细胞个数、JAK2mRNA、STAT3 mRNA、N-cadherin、Vimentin、p-JAK2/JAK2、p-STAT3/STAT3表达水平显著降低,细胞凋亡率和细胞E-cadherin表达水平显著升高(P<0.05)。与鸦胆子油乳高剂量组相比,鸦胆子油乳高剂量+Colive...     

miR-138对小鼠乳腺上皮细胞的作用及其调控的靶序列

miRNA是一类长约20～25 nt的内源性非编码蛋白的小RNA.miRNAs的作用遍及生命体的发生、生长、发育、分化和死亡等过程,但目前对miRNA在细胞中的确切功能和其如何发挥作用知之甚少.应用miRNA基因沉默技术,抑制小鼠乳腺上皮细胞miR-138的表达,应用荧光定量PCR、Western blot、细胞活力分析技术等分析mi-138的表达及mLR-138对小鼠乳腺上皮细胞的影响,结果显示,miR-138抑制后细胞活性增强(P＜0.05),PRL-R蛋白表达增强(P＜0.05),信号转导通路分子STAT5、MAPK表达加强.研究认为,miR-138抑制小鼠乳腺上皮细胞的增殖.miR-138在乳腺上皮细胞调控的靶序列是PRL-R,抑制其表达而发挥作用.miR-138通过调控STAT5、MAPK信号转导通路而调控乳腺上皮细胞增殖.     

RNAi沉默STAT3基因对人大细胞肺癌细胞增殖的影响

旨在研究RNAi沉默STAT3基因对人大细胞肺癌NCI-H460细胞增殖的影响。针对STAT3基因mRNA设计合成5条短发夹DNA,构建重组SiRNA-ST3质粒(命名为SiRNA-ST3-1,2,3,4,N)。用重组质粒分别转染NCI-H460细胞,RT-PCR法检测转染24 h后STAT3 mRNA的表达;Western blotting法检测转染24 h和48 h后STAT3、pSTAT3蛋白表达;MTT法检测转染24 h、48 h、72 h后NCI-H460细胞增殖情况。结果显示,SiRNA-ST3载体构建成功。RT-PCR和Western blotting检测结果表明,NCI-H460细胞转染重组质粒SiRNA-ST3-2和SiRNA-ST3-3后STAT3基因mRNA转录和STAT3、pSTAT3蛋白表达都明显下降(P<0.05)。与未转染组比,SiRNA-ST3-2组和SiRNA-ST3-3组NCI-H460增殖能力24 h、48 h降低明显(P<0.05);与SiRNA-ST3-N组比,SiRNA-ST3-2组和SiRNA-ST3-3组NCI-H460增殖能力48 h降低明显(P<0.05)。由此证实,构建的重组质粒SiRNA-ST3-2、SiRNA-ST3-3能有效靶向沉默STAT3基因,并抑制人大细胞肺癌NCI-H460细胞的增殖能力。     

糖皮质激素下调JAK/STAT抑制嗜酸粒细胞哮喘

摘要 目的：探究糖皮质激素对嗜酸粒细胞哮喘(Eosinophilic asthma, EA) 2 型固有免疫细胞(Type 2 innate lymphoid cells, ILC2s)的影响及相关机制。方法：研究对象来自我院 2021年6月至 2022年6月的EA患者和健康对照（Healthy control, HC）,收集相应临床基线资料并评估病情、进行血常规、肺功能等检查;应用流式细胞术检测外周血单个核细胞（Peripheral blood mononuclear cell, PBMC） ILC2s(CD45+Lin-CD127+CD294+);ELISA检测外周血IL-5、IL-13浓度。糖皮质激素治疗EA 患者3月后,观察PBMC中ILC2s及IL-5、IL-13浓度。C57BL/6J小鼠给予鸡卵清蛋白(Ovalbumin,OVA) 20 μg 腹腔注射致敏后用1%OVA雾化吸入激发哮喘EA模型,阴性对照（Negative control, NC）组小鼠用同等体积PBS作为对照。EA造模成功的小鼠通过流式细胞术检测血液及肺泡灌洗液中ILC2s,HE染色检测小鼠肺泡灌洗液中嗜酸性粒细胞(Eosinophil, EOS)及肺部炎症。EA小鼠经糖皮质激素处理后,检测肺部炎症情况;流式细胞术检测PBMC、肺泡灌洗液（Bronchoalveolar lavage fluid, BALF）中 ILC2s;分离肺组织ILC2s,western blot检测相关蛋白表达情况。结果：EA组的ILC2s比例升高, EOS升高,2型细胞因子IL-5、IL-13增加,糖皮质激素治疗1月及3月后ILC2s比例下降,2型细胞因子IL-5、IL-13下降。与NC组小鼠比较,EA组小鼠PBMC及BALF中ILC2s升高,BALF中EOS升高,血清中2型细胞因子IL-5、IL-13升高,肺部炎症加重。糖皮质激素治疗后,肺部炎症减轻,EOS下降,ILC2s减少,2型细胞因子IL-5、IL-13下降,下调JAK/STAT蛋白。结论：在EA中,糖皮质激素通过下调JAK/STAT蛋白抑制ILC2s的功能减轻肺部炎症,为激素治疗嗜酸性粒细胞哮喘的机制提供了新方向。     

STAT3小干扰RNA的构建及对缺氧复氧人肾小管上皮细胞凋亡的影响

目的:构建信号转导与转录因子3(STAT3)小干扰RNA(siRNA)表达载体,并观察其对缺氧复氧后人肾小管上皮细胞(HKC)凋亡的影响。方法:设计3对人STAT3 siRNA靶序列,用DNA重组技术克隆至质粒pRNAT-U6.1/neo中,构建重组质粒pRNAT-U6.1-STAT3 siRNA,检测并筛选出最佳抑制效率的siRNA质粒载体。重组质粒转染至缺氧复氧后HKC细胞,Western blotting和Real Time-PCR测定STAT3蛋白和mRNA表达量,流式细胞仪测定细胞凋亡,间接荧光法测定Bcl-2和Bax表达的变化。结果:靶向STAT3基因表达的质粒载体构建成功,并筛选出抑制效率最佳的重组质粒。缺氧复氧后HKC细胞STAT3表达、凋亡率和Bax/Bcl-2比值增加;缺氧复氧后HKC细胞转染重组质粒后STAT3表达、凋亡率和Bax/Bcl-2比值明显降低。结论:成功构建并筛选最佳抑制效率的靶向STAT3的重组质粒载体。该载体可有效抑制缺氧复氧后HKC细胞中STAT3信号转导通路的活化,并进一步通过上调Bcl-2、下调Bax蛋白的表达,从而抑制细胞凋亡。     

Galectin-7在哮喘儿童支气管黏膜中的表达及对支气管上皮细胞凋亡的影响

目的:探讨半乳糖凝集素-7(Galectin-7)在哮喘儿童支气管黏膜中的表达及对支气管上皮细胞凋亡的影响。方法:收集哮喘儿童支气管黏膜及支气管扩张非哮喘儿童支气管黏膜,Western blot检测其Galectin-7的表达。体外培养人支气管上皮细胞,分为正常组、对照组、感染组和实验组,正常组用正常的人支气管上皮细胞,对照组细胞用转染siRNA control后的人支气管上皮细胞,感染组细胞用RSV感染后的人支气管上皮细胞,实验组细胞为RSV感染后并转染siRNA Galectin-7的人支气管上皮细胞。培养24 h后,检测各组细胞中Galectin-7蛋白表达,并采用流式细胞术检测各组细胞的凋亡情况,Western blot检测细胞中Bcl-2、Bax、STAT3、p-STAT3蛋白的表达。结果:哮喘儿童支气管黏膜中Galectin-7的表达明显高于非哮喘儿童支气管黏膜组织(P0.01)。正常组和对照组Galectin-7水平比较差异无统计学意义(P0.05),感染组Galectin-7、Bax表达和细胞凋亡率均明显高于正常组,而Bcl-2、p-STAT3的表达均明显低于正常组(P0.01),实验组Galectin-7、Bax表达和细胞凋亡率明显低于感染组,而Bcl-2、p-STAT3的表达均明显高于感染组(P0.01)。结论:Galectin-7在哮喘儿童支气管黏膜中表达上调,可能通过活化STAT3,促进支气管上皮细胞凋亡。     

Enhancement of placenta growth factor expression by oncostatin M in human rheumatoid arthritis synovial fibroblasts

Oncostatin M (OSM) belongs to IL‐6 subfamily and is mostly produced by T lymphocytes. High levels of OSM are detected in the pannus of rheumatoid arthritis (RA) patients and it may arouse the inflammation responses in joints and eventually leads to bone erosion. Placenta growth factor (PLGF) is an angiogenic factor and highly homologous with vascular endothelial growth factor (VEGF). It has been recently reported that PLGF is highly expressed in synovial tissue and enhances the production of proinflammatory cytokines including TNF‐α and IL‐6. Here, we demonstrated that OSM increased mRNA and protein levels of PLGF in a time‐ and concentration‐dependent manner in RA synovial fibroblasts. Inhibitors of JAK3 and PI3K antagonized OSM‐induced production of PLGF. OSM enhanced the phosphorylation of Tyr705‐STAT3, Ser727‐STAT3, Ser473‐Akt, and increased the nuclear translocation of phosphorylated STAT3 time‐dependently. Transfection of dominant negative Akt or application of PI3K inhibitorLY294002 significantly inhibited p‐Tyr705‐STAT3, p‐Ser727‐STAT3, and PLGF expression, indicating that Akt is involved in JAK3/STAT3/PLGF signaling cascade. To further examine whether STAT3 binds to the promoter region of PLGF, Chip assay was used and it was found that OSM could bind with PLGF promoter, which was inhibited by JAK3 and PI3K inhibitors. Accumulation of PLGF in the pannus may contribute to the inflammation, angiogenesis and joints destruction in RA patients. These findings demonstrated the important role of OSM in the pathology network of RA and provided novel therapeutic drug targets for RA treatment. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Sodium orthovanadate suppresses palmitate-induced cardiomyocyte apoptosis by regulation of the JAK2/STAT3 signaling pathway

Elevated circulatory free fatty acids (FFAs) especially saturated FFAs, such as palmitate (PA), are detrimental to the heart. However, mechanisms responsible for this phenomenon remain unknown. Here, the role of JAK2/STAT3 in PA-induced cytotoxicity was investigated in cardiomyocytes. We demonstrate that PA suppressed the JAK2/STAT3 pathway by dephosphorylation of JAK2 (Y1007/1008) and STAT3 (Y705), and thus blocked the translocation of STAT3 into the nucleus. Conversely, phosphorylation of S727, another phosphorylated site of STAT3, was increased in response to PA treatment. Pretreatment of JNK inhibitor, but not p38 MAPK inhibitor, inhibited STAT3 (S727) activation induced by PA and rescued the phosphorylation of STAT3 (Y705). The data suggested that JNK may be another upstream factor regulating STAT3, and verified the important function of P-STAT3 (Y705) in PA-induced cardiomyocyte apoptosis. Sodium orthovanadate (SOV), a protein tyrosine phosphatase inhibitor, obviously inhibited PA-induced apoptosis by restoring JAK2/STAT3 pathways. This effect was diminished by STAT3 inhibitor Stattic. Collectively, our data suggested a novel mechanism that the inhibition of JAK2/STAT3 activation was responsible for palmitic lipotoxicity and SOV may act as a potential therapeutic agent by targeting JAK2/STAT3 in lipotoxic cardiomyopathy treatment.     

Coexpression of oncostatin M and its receptors and evidence for STAT3 activation in human ovarian carcinomas

The expression of oncostatin M and leukemia inhibitory factor (LIF), JAK-STAT activators and members of the interleukin-6 family of cytokines, were examined in a series of primary ovarian carcinomas using immunohistochemistry. The malignant epithelial cells of all 29 ovarian carcinomas examined expressed oncostatin M; none expressed LIF. Oncostatin M can activate two related receptors, one consisting of a low-affinity LIF receptor subunit, LIFR beta, which forms a heterocomplex with the gp130 signal transducing protein and can recognize both oncostatin M and LIF, and a second heterocomplex consisting of a subunit that specifically recognizes oncostatin M, OSMR beta, and the gp130 protein. By immunohistochemistry, 25 of 25 ovarian carcinomas examined expressed the LIFR beta subunit in the malignant epithelial cells (all samples express gp130), and two-thirds the ovarian carcinomas studied expressed OSMR beta mRNA as determined by RT-PCR. Thus oncostatin M and its receptors are commonly coexpressed in malignant ovarian epithelial cells, and represent a potential autocrine loop in this tumor type. STAT3, of one the signaling proteins downstream of the oncostatin M/LIF receptors, was found in its phosphorylated, activated form (phosphotyrosine 705 STAT3) in the malignant epithelial cells of 17 of 23 ovarian carcinomas examined (74%) as determined by immunohistochemistry; this suggests that this protein is constitutively activated in most ovarian carcinomas, as it is in many other human malignancies. Recombinant human Oncostatin M (rhOSM) can induce the transient tyrosine 705 phosphorylation of STAT3 in serum-starved LIFR beta/OSMR beta expressing ovarian carcinoma cell lines, but does not alter cell growth and effects only a modest increase in the apoptotic rate in these cultured cells. Oncostatin M and its receptors may be part of a network of cytokine systems within ovarian carcinomas that may act to maintain STAT3 in its activated form, a phenomenon associated with the malignant phenotype.     

STAT3 activation inhibits human bronchial epithelial cell apoptosis in response to cigarette smoke exposure

We have previously reported that cigarette smoke can induce DNA damage in human lung cells without leading to apoptosis or necrosis. In this study, we report that STAT3 is required for the survival of human bronchial epithelial cells (HBECs) following cigarette smoke-induced DNA damage. Cigarette smoke extract (CSE) exposure increases STAT3 phosphorylation (Tyr 705) and DNA binding activity in HBECs. CSE also stimulates IL-6 release and mRNA expression. Anti-IL-6 neutralizing antibody partially blocks STAT3 activation and renders the cells sensitive to CSE-induced DNA damage. Suppression of STAT3 by siRNA results in severe DNA damage and cell death in response to CSE exposure. These findings suggest that STAT3 mediates HBEC survival in response to CSE-induced DNA damage, at least in part, through the IL-6/STAT3 signaling pathway.