miR-21 mediates fibrogenic activation of pulmonary fibroblasts and lung fibrosis

Uncontrolled extracellular matrix production by fibroblasts in response to tissue injury contributes to fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), a progressive and ultimately fatal process that currently has no cure. Although dysregulation of miRNAs is known to be involved in a variety of pathophysiologic processes, the role of miRNAs in fibrotic lung diseases is unclear. In this study, we found up-regulation of miR-21 in the lungs of mice with bleomycin-induced fibrosis and also in the lungs of patients with IPF. Increased miR-21 expression was primarily localized to myofibroblasts. Administration of miR-21 antisense probes diminished the severity of experimental lung fibrosis in mice, even when treatment was started 5–7 d after initiation of pulmonary injury. TGF-β1, a central pathological mediator of fibrotic diseases, enhanced miR-21 expression in primary pulmonary fibroblasts. Increasing miR-21 levels promoted, whereas knocking down miR-21 attenuated, the pro-fibrogenic activity of TGF-β1 in fibroblasts. A potential mechanism for the role of miR-21 in fibrosis is through regulating the expression of an inhibitory Smad, Smad7. These experiments demonstrate an important role for miR-21 in fibrotic lung diseases and also suggest a novel approach using miRNA therapeutics in treating clinically refractory fibrotic diseases, such as IPF.Fibroblast proliferation and generation of provisional extracellular matrix (ECM) are primary tissue responses to injury (Tomasek et al., 2002). Successful wound repair processes are tightly regulated, with a balance of ECM synthesis and resolution as well as reepithelization (Tomasek et al., 2002). Uncontrolled ECM production can lead to clinically important fibrotic diseases, including idiopathic pulmonary fibrosis (IPF) (Tomasek et al., 2002; Thannickal et al., 2004). An important pathological feature of IPF is the presence of fibroblastic foci in the lungs, and the presence and extent of such fibroblastic foci is one of the most reliable markers of poor prognosis in patients with IPF (Wynn, 2007; Hardie et al., 2009). Fibroblastic foci are aggregations of fibroblasts/myofibroblasts that produce excessive ECM components (Wynn, 2007; Hardie et al., 2009). TGF-β1 has been shown to be an important mediator of lung fibrosis and can induce differentiation of pulmonary fibroblasts into myofibroblasts characterized by α-smooth muscle actin (α-SMA) expression and active synthesis of ECM proteins (Lee et al., 2006; Cutroneo et al., 2007).microRNAs (miRNAs) are a class of noncoding small RNAs, 22 nt in length, which bind to the 3′ UTR of target genes and thereby repress translation of target genes and/or induce degradation of target gene mRNA (Stefani and Slack, 2008). miRNAs play essential roles in numerous cellular and developmental processes, including intracellular signaling pathways and organ morphogenesis (Stefani and Slack, 2008). Aberrant expression of miRNAs is closely associated with initiation and progression of pathophysiologic processes including diabetes, cancer, and cardiovascular disease (Thum et al., 2008; Croce, 2009; Latronico and Condorelli, 2009; Pandey et al., 2009). However, the role of miRNAs in lung fibrosis is just beginning to unravel (Pandit et al., 2010). Therefore, determining the roles of specific miRNAs involved in the pathogenesis of lung fibrosis is likely to suggest important new directions for the treatment of IPF and other interstitial lung diseases.In the present study, we explored the role of miRNA in the pathogenesis and progression of lung fibrosis. We found that miR-21 is highly up-regulated in the lungs of mice with bleomycin-induced lung fibrosis and in the lungs of patients with IPF. The enhanced expression of miR-21 is primarily located to myofibroblasts in the fibrotic lungs. In addition, miR-21 is up-regulated by TGF-β1 and functions in an amplifying circuit to enhance the fibrogenic activity of TGF-β1 in human primary fibroblasts. More importantly, we found that miR-21 sequestration in mouse lungs attenuates bleomycin-induced lung fibrosis. Overall, these data suggest that miR-21 is a central mediator in the pathogenesis of lung fibrosis and a potential target for developing novel therapeutics in treating fibrotic diseases, including IPF.     

Cigarette smoke induces aberrant EGF receptor activation that mediates lung cancer development and resistance to tyrosine kinase inhibitors

The EGF receptor (EGFR) and its downstream signaling are implicated in lung cancer development. Therefore, much effort was spent in developing specific tyrosine kinase inhibitors (TKI) that bind to the EGFR ATP-pocket, blocking EGFR phosphorylation/signaling. Clinical use of TKIs is effective in a subset of lung cancers with mutations in the EGFR kinase domain, rendering the receptor highly susceptible to TKIs. However, these benefits are limited, and emergence of additional EGFR mutations usually results in TKI resistance and disease progression. Previously, we showed one mechanism linking cigarette smoke to EGFR-driven lung cancer. Specifically, exposure of lung epithelial cells to cigarette smoke-induced oxidative stress stimulates aberrant EGFR phosphorylation/activation with impaired receptor ubiquitination/degradation. The abnormal stabilization of the activated receptor leads to uncontrolled cell growth and tumorigenesis. Here, we describe for the first time a novel posttranslational mechanism of EGFR resistance to TKIs. Exposure of airway epithelial cells to cigarette smoke causes aberrant phosphorylation/activation of EGFR, resulting in a conformation that is different from that induced by the ligand EGF. Unlike EGF-activated EGFR, cigarette smoke-activated EGFR binds c-Src and caveolin-1 and does not undergo canonical dimerization. Importantly, the cigarette smoke-activated EGFR is not inhibited by TKIs (AG1478; erlotinib; gefitinib); in fact, the cigarette smoke exposure induces TKI-resistance even in the TKI-sensitive EGFR mutants. Our findings show that cigarette smoke exposure stimulates not only aberrant EGFR phosphorylation impairing receptor degradation, but also induces a different EGFR conformation and signaling that are resistant to TKIs. Together, these findings offer new insights into cigarette smoke-induced lung cancer development and TKI resistance.     

Periostin mediates cigarette smoke extract-induced proliferation and migration in pulmonary arterial smooth muscle cells

Cigarette smoking is an important risk factor for pulmonary arterial hypertension (PAH). Pulmonary arterial smooth muscle cells (PASMCs) play a critical role in the pathogenesis of PAH-associated arterial remodeling. This study was done to explore the expression and biological roles of periostin in PASMCs following exposure to cigarette smoke extract (CSE). PASMCs were exposed to different concentrations of CSE and tested for gene expression and reactive oxygen species (ROS) production. PASMCs were incubated with recombinant periostin protein or transfected with small interfering RNA targeting periostin before CSE exposure and then examined for cell proliferation and migration. Compared to control cells, exposure to CSE led to a significant upregulation of periostin. Pretreatment with 5 mM N-acetyl-l-cysteine (an inhibitor of ROS formation) or 10 μM U0126 (an inhibitor of ERK1/2) significantly prevented the induction of periostin in CSE-treated PASMCs. The addition of recombinant periostin protein significantly enhanced the proliferation and migration of PASMCs. In contrast, knockdown of endogenous periostin counteracted the proliferation and migration of PASMCs induced by CSE treatment. In conclusion, CSE induces the expression of periostin in PASMCs via promotion of ROS and activation of ERK1/2. Periostin mediates the effects of CSE on PASMC proliferation and migration. These findings warrant further exploration of the roles of periostin in cigarette smoking-associated pulmonary arterial remodeling.     

Cigarette smoke selectively enhances viral PAMP- and virus-induced pulmonary innate immune and remodeling responses in mice

Viral infections have more severe consequences in patients who have been exposed to cigarette smoke (CS) than in those not exposed to CS. For example, in chronic obstructive pulmonary disease (COPD), viruses cause more severe disease exacerbation, heightened inflammation, and accelerated loss of lung function compared with other causes of disease exacerbation. Symptomatology and mortality in influenza-infected smokers is also enhanced. To test the hypothesis that these outcomes are caused by CS-induced alterations in innate immunity, we defined the effects of CS on pathogen-associated molecular pattern-induced (PAMP-induced) pulmonary inflammation and remodeling in mice. CS was found to enhance parenchymal and airway inflammation and apoptosis induced by the viral PAMP poly(I:C). CS and poly(I:C) also induced accelerated emphysema and airway fibrosis. The effects of a combination of CS and poly(I:C) were associated with early induction of type I IFN and IL-18, later induction of IL-12/IL-23 p40 and IFN-gamma, and the activation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF2alpha). Further analysis using mice lacking specific proteins indicated a role for TLR3-dependent and -independent pathways as well as a pathway or pathways that are dependent on mitochondrial antiviral signaling protein (MAVS), IL-18Ralpha, IFN-gamma, and PKR. Importantly, CS enhanced the effects of influenza but not other agonists of innate immunity in a similar fashion. These studies demonstrate that CS selectively augments the airway and alveolar inflammatory and remodeling responses induced in the murine lung by viral PAMPs and viruses.     

Cigarette smoke stimulates cathepsin B activity in alveolar macrophages of rats

Cathepsin B activity was determined in alveolar macrophages and cell-free bronchoalveolar lavage fluid from Sprague-Dawley rats exposed only through the nose to fresh mainstream smoke from University of Kentucky high-tar, high-nicotine reference cigarettes, and in cells and fluid from room control and sham control animals. Increased levels of blood carboxyhemoglobin and pulmonary aryl hydrocarbon hydroxylase activity in smoke-exposed animals indicated effective exposure of animals to cigarette smoke. Cathepsin B activity was quantitated with alpha-N-benzyloxycarbonyl-leucine-leucine-arginine-2-naphthylamide as substrate. Specific activity (nanomoles of substrate cleaved per milligram of protein per hour) in alveolar macrophages was increased by 43% at both 4- and 10-week exposure points in animals exposed twice daily to 10 puffs of cigarette smoke. These data indicate that maximal stimulation of the enzyme occurs within 4 weeks of the initiation of smoke exposure. When the activity was expressed on a per-cell basis, cathepsin B activity was also increased in the smoke-exposed group at both exposure points. Activity in bronchoalveolar lavage fluid of smoke-exposed animals was increased by approximately 50% at 4 and 10 weeks, but the differences were not statistically significant. These findings demonstrate that cigarette smoke is a potent inducer of cathepsin B activity in alveolar macrophages of rats.     

Cigarette smoke inhibition of ion transport in canine tracheal epithelium.

Inhalation of cigarette smoke is known to impair pulmonary mucociliary clearance. Active ion transport by airway epithelium plays an important role in maintaining effective mucociliary clearance by regulating the volume and composition of the airway secretions. To determine the effect of cigarette smoke on airway epithelial ion transport, the electrical properties and transepithelial Na and Cl fluxes were measured in canine tracheal epithelium. In vivo, the inhalation of the smoke from one cigarette acutely and reversibly decreased the electrical potential difference across the tracheal epithelium. In vitro, exposure of the mucosal surface of the epithelium to cigarette smoke decreased the short circuit current and transepithelial resistance. The decrease in short circuit current was due to an inhibition of the rate of Cl secretion with minimal effect on the rate of Na absorption. The effect of cigarette smoke was reversible, was not observed upon exposure of the submucosal surface to smoke, and was most pronounced when secretion was stimulated. The particulate phase of smoke was largely responsible for the inhibitory effect, since filtering the smoke minimized the effect. The effect of cigarette smoke was not prevented by addition of antioxidants to the bathing solutions, suggesting that the inhibition of Cl secretion cannot be entirely attributed to an oxidant mechanism. These results indicate that cigarette smoke acutely inhibits active ion transport by tracheal epithelium, both in vivo and in vitro. This effect may explain, in part, both the abnormal mucociliary clearance and the airway disease observed in cigarette smokers.     

miR-138在胃黏膜癌变过程中的表达及意义

摘要：目的：探讨人端粒酶逆转录酶（hTERT）蛋白和hTERT调控相关miR-138在正常胃黏膜（NM）、肠上皮化生(IM)、异型增生(DYS)、早期胃癌（EGC）和进展期胃癌(AGC)中的表达及其意义。 方法：选取经病理证实的胃黏膜病变组织96例,用免疫组化SP染色法检测hTERT表达水平,real-time PCR检测miR-138表达丰度。 结果：在NM、IM、DYS、EGC和AGC 5组标本中,hTERT阳性表达率逐渐升高,分别为0.0%、26.1%、33.3%、75.0%和77.4%;miR138表达水平呈逐步下降趋势,分别为2.93±1.24、2.12±0.98、1.90±0.58、1.27±0.89和1.68±1.02;低表达miR-138组织的百分率在低、未分化组中显著高于高、中分化组（83.3% vs 36.5%,P<0.01）,而与性别、年龄、有无淋巴结转移及TNM分期等无关。 结论：miR-138表达丰度的下降,是胃黏膜癌变过程中的早期事件;miR-138有望成为新的胃癌早期诊断指标及治疗靶标。     

烟草烟雾提取物影响主动脉平滑肌细胞GATA-2的表达

背景：吸烟是动脉粥样硬化形成的主要危险因素之一。目的：观察烟草烟雾提取物对大鼠血管平滑肌细胞中GATA-2浓度的影响及早期生长反应因子1在其中的作用。方法：体外培养大鼠血管平滑肌细胞,用不同浓度(0,5%,10%,20%)烟草烟雾提取物刺激,采用RT-PCR的方法检测烟草烟雾提取物对GATA-2的影响。用筛选出的最适合的烟草烟雾提取物浓度处理大鼠血管平滑肌细胞不同时间(0,4,8,12,24 h)后,检测GATA-2 mRNA的变化,以及加入生长反应因子1抑制剂后GATA-2 mRNA的变化。结果与结论：与0浓度烟草烟雾提取物相比,5%低浓度烟草烟雾提取物刺激后,GATA-2 mRNA表达较10%中浓度和20%高浓度增加的更显著。不加烟草烟雾提取物组即0 h 组血管平滑肌细胞中有少量 GATA-2的mRNA,5%烟草烟雾提取物刺激后于4 h内开始增加,8 h达高峰。加入生长反应因子1抑制剂后5%烟草烟雾提取物诱导的血管平滑肌细胞GATA-2 mRNA表达明显降低。提示烟草烟雾提取物可通过生长反应因子1促进GATA-2增加,抑制生长反应因子1后,GATA-2的表达减少。     

氨基胍对烟雾吸入性肺损伤的影响

目的探讨诱导型一氧化氮合酶(iNOS)抑制剂氨基胍(aminoguanidine,AG)对大鼠烟雾吸入性肺损伤的影响。方法40只SD雄性大鼠随机分为正常对照组(n=8)、烟雾吸入组(n=16)和AG治疗组(n=16)。建立烟雾吸入性肺损伤模型,AG治疗组于致伤后立即腹腔注射AG50mg/kg。正常对照组及烟雾吸入组腹腔注射等容量生理盐水。分别于2、6、12、24h时相点进行动脉血气分析,并分批处死大鼠,取肺组织测肺含水量,制备肺组织匀浆检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、各型一氧化氮合酶(NOS)活性及一氧化氮(NO)含量。制作肺组织病理切片,光镜下观察病理变化。结果与烟雾吸入组相比,AG治疗组动脉血氧分压显著升高(P<0.05),肺组织含水量降低(P<0.05),肺组织中SOD活性及CAT活性升高(P<0.05),iNOS活性及NO含量降低(P<0.05)。结论AG能够升高动脉血氧分压,提高烟雾吸入性肺损伤肺组织抗氧化能力,减轻肺组织氧化性损伤,减轻肺水肿,并减轻组织炎性浸润,对烟雾吸入性肺损伤有较好的保护作用。     

血清外泌体miR-125b,miR-21及miR-122对肝细胞癌诊断价值

目的 评估血清外泌体中miR-125b、miR-21、miR-122及miR-375诊断肝细胞癌（HCC）的效能，预测miR-125b潜在调控的靶基因。方法 以体检健康者（健康人对照组）50例、HCC患者组46例和慢性乙型肝炎（CHB）患者组11例为研究对象。收集各研究对象的临床资料，包括年龄、性别、甲胎蛋白（AFP）、α-L-岩藻糖苷酶（AFU）等指标。采集各组血液标本，分离纯化血清外泌体并鉴定，应用实时荧光定量聚合酶链反应（RT-qPCR）检测各组血清外泌体miR-125b、miR-21、miR-122及miR-375的表达水平。ROC曲线评估各指标单独及联合检测对肝癌的诊断效能。基于miRTarBase.V8、TarBase.V8、miRecords数据库预测miR-125b潜在靶基因，通过clusterProfiler软件分析，间接推测其可能参与的信号转导通路。结果 提取纯化的血清样本中含有大量外泌体颗粒。RT-qPCR检测结果表明，与健康人对照组及CHB组相比，HCC组miR-125b、miR-21和miR-122的表达水平显著上调（P<0.05）。ROC曲线分析结果表...     

Identification of miR-130a, miR-27b and miR-210 as serum biomarkers for atherosclerosis obliterans

BackgroundArteriosclerosis obliterans (ASO) is a kind of peripheral arterial disease. Most patients with ASO have no apparent clinical symptoms early on, but a diagnosis at the early stage is essential to prevent progression. Unfortunately, the specific and sensitive markers for ASO are still lacking currently. In this study, using both tissue samples and blood samples obtained from ASO patients, we aim to find a cluster of miRNAs that can be used as new risk-markers for the diagnosis of ASO in the earlier stages.MethodsWe enrolled 104 patients diagnosed with ASO and 105 age-matched controls. We examined the expression levels of a series of miRNAs in both intima samples and serum samples from these patients.ResultsLevels of miR-21, miR-130a, miR-27b, let-7f and miR-210 significantly increased, while levels of miR-221 and miR-222 significantly decreased in the sclerotic samples compared with normal samples. Significant increase of miR-130a, miR-27b and miR-210 expression was observed in the serum samples of ASO patients. Moreover, the expression of miR-130a and miR-27b in sera of ASO patients was positively correlated with Fontaine stages.ConclusionsThe serum levels of miR-130a, miR-27b and miR-210 may serve as potential biomarkers for early stage ASO.     

Lack of hematogenous mediated pulmonary injury with smoke inhalation

Inhalation injury was studied in chronically prepared sheep (n = 12) by insufflating one lung with cotton smoke from burning cotton cloth. The contralateral lung was insufflated with air. There was also a sham group in which both lungs were insufflated with air (n = 6). The pulmonary status of the smoked animals gradually deteriorated; by 24 hours shunt blood flow had increased to 32 +/- 3% and the animals were sacrificed. Wet-weight/dry-weight ratios were elevated only in the smoke-exposed lungs. They likewise showed histologic evidence of injury. Lavage materials from the injured lungs had higher percentages of neutrophils than the others. The lung lesion produced by the inhalation of cotton smoke appears to be localized to the area of injury, rather than being a generalized pulmonary response.     

7-硝基吲唑在烟雾吸入性肺损伤中的作用

目的 探讨神经型一氧化氮合酶（nNOS）抑制剂7-硝基吲唑（7-NI）在烟雾吸入性肺损伤中的作用。方法 40只SD雄性大鼠被随机分为正常对照组（n=8）、烟雾吸入性肺损伤模型组（n=16）和7-NI治疗组（n=16），建立烟雾吸入性肺损伤模型。7-NI治疗组在致伤后立即腹腔注射7-NI 20mg／kg（溶于2ml花生油中）；正常对照组及模型组腹腔注射2ml花生油。分别于伤后2、6、12和24h时间点监测动脉血气分析；并分批处死大鼠，取肺组织测肺含水量，制备肺组织匀浆检测超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、各型一氧化氮合酶（NOS）活性及肿瘤坏死因子-α（TNF—α）和一氧化氮（NO）含量。光镜下观察肺组织病理学变化。结果 与模型组比较，7～NI治疗组各时间点动脉血氧分压均显著升高（P均〈0．05），肺组织含水量显著降低（P〈0．05），肺组织中SOD及CAT活性均明显升高（P均〈0．05），nNOS活性及NO含量均明显降低（P均〈0．05）。治疗组2h和6h肺组织中TNF—α含量均较模型组降低（P均〈0．05）。光镜下7-NI治疗组较模型组损伤程度减轻，炎性细胞浸润减少，肺间质内未见点状出血。结论 7-NI对烟雾吸人性肺损伤有较好的保护作用，可提高动脉血氧分压，减轻肺水肿程度，增加组织抗氧化能力，并减轻组织炎性细胞浸润。     

Regulation of circadian behavioural output via clock-responsive miR-276b

Growing evidence indicates that microRNAs play numerous important roles. However, the roles of some microRNAs involved in regulation of circadian rhythm and sleep are still not well understood. In this study, we show that the miR-276b is essential for maintaining both sleep and circadian rhythm by targeting tim, npfr1 and DopR1 genes, with miR-276b deleted mutant flies sleeping more, and vice versa in miR-276b overexpressing flies. Through analysing its promoter, we found that mir-276b is responsive to CLOCK and regulates circadian rhythm through the negative feedback loop of the CLK/CYC-TIM/PER. Furthermore, miR-276b is broadly expressed in the clock neurons and the central complexes such as the mushroom body and the fan-shape body of Drosophila brain, in which up-regulation of miR-276b in tim, npfr1 and DopR1 expressing tissues significantly causes sleep decreases. This study clarifies that the mir-276b is very important for participating in regulation of circadian rhythm and sleep.     

人胶质瘤组织中miR-125b表达研究

目的:探讨miR-125b在人胶质瘤组织中的表达。方法:选取不同恶性程度胶质瘤手术患者20例为胶质瘤组(Ⅰ级5例、Ⅱ级5例、Ⅲ级5例、Ⅳ级5例),另选择实施脑外伤内减压手术的患者5例为正常对照组。术中取下胶质瘤组胶质瘤组织和正常对照组的正常脑组织,采用荧光定量PCR方法检测miR-125b的表达。结果:正常对照组的miR-125b相对表达量为(0.28±0.12),胶质瘤组Ⅰ、Ⅱ、Ⅲ、Ⅳ级的miR-125b相对表达量分别为(0.43±0.07)、(0.69±0.08)、(0.90±0.05)、(1.12±0.04),差异有统计学意义(P0.05)。肿瘤病理恶性程度越高,生存期越短(P0.05)。结论:miR-125b异常表达可能与胶质瘤的发生和发展密切相关。