Urinary Extracellular Vesicles Carrying Klotho Improve the Recovery of Renal Function in an Acute Tubular Injury Model

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Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex

Background

The human hemoglobin switch (HbF→HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor.

Design and Methods

We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34⁺ cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production.

Results

In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80–90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects.

Conclusions

Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221–222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.     

负载hTERT调控相关miR-138慢病毒的包装及鉴定

目的:构建hTERT(人端粒酶逆转录酶)调控相关miR-138慢病毒表达载体.方法:化学合成miR-138前体序列,连接到包含U6启动子及GFP(绿色荧光蛋白)报告基因的慢病毒载体pGCL-GFP中,获得重组质粒,经双酶切和测序鉴定后,与慢病毒包装质粒pHelper 1.0及pHelper 2.0共转染至HEK 293T细胞包装病毒,并对病毒进行鉴定.结果:双酶切和测序结果证实miR-138前体核苷酸链序列捕入正确,包装的慢病毒滴度达7×107TU/ml;包装成功的慢病毒可有效增加感染细胞内miR-138的表达丰度,并可下调hTERT表达水平.结论:成功包装并鉴定人miR-138慢病毒表达载体.     

大鼠脑皮质NF-κB和miR-146a在轻型脑创伤后炎症反应过程中的协同作用

目的探讨轻型脑创伤后大鼠皮质区核因子κB(NF-κB)活化及其microRNA(miRNA)-146a转录调控模式在炎症反应过程中的作用机制。方法选择健康雄性SD大鼠60只,随机分为对照组(n=10)、轻型脑创伤组(n=50)。轻型脑创伤组分为创伤后1、3、6、12和24h5个时间点,10%水合氯醛(0.3mL/100g)腹腔内注射,采用Marmarou’s方法,用质量450g、直径18mm的铜棒由1.0m高度自由落下,造成大鼠轻型脑创伤。对照组仅给予相应麻醉、头皮切开及缝合处理。采用实时定量PCR及Western blot方法比较两组大鼠脑皮质NF-κB和miR-146a表达水平的变化。结果脑创伤1、3、6、12、24h后miR-146a表达水平显著上调,分别为对照组的1.21±0.15,1.73±0.29,2.36±0.24,3.60±0.37,1.97±0.26倍。伤后6～12h为表达高峰,24h其转录水平有所下调。NF-κB的活化与核转位与miR-146a的表达模式基本一致。结论轻型脑创伤后皮质区miR-146a表达水平显著上调,NF-κB通过miR-146a上游调控元件增强了miR-146a的转录水平,NF-κB-miR-146a途径可能在促进脑创伤后炎症反应过程中发挥重要作用。     

miR-1、miR-16和miR-208前体在乳鼠心肌细胞和心肌成纤维细胞中的表达

目的检测心肌特异的miR-1、miR-16和miR-208前体在乳小鼠、乳大鼠心肌细胞及心肌成纤维细胞中的表达。方法利用出生1～3d的Sprague-Dawley(SD)大鼠和C57BL/6小鼠,采用2.5%胰酶消化和差速贴壁法分离心肌细胞和心肌成纤维细胞。提取原代的乳小鼠、乳大鼠心肌细胞及P1代的心肌成纤维细胞总RNA,以β-actin为内参照,逆转录酶-聚合酶链式反应(RT-PCR)分别检测miR-1、miR-16和miR-208的前体表达水平。用FluorChem8900软件分析琼脂糖凝胶电泳后PCR产物条带的灰度值,计算出表达的目的miRNA前体与β-actin的比值。结果有效分离并培养了乳C57BL/6小鼠、乳SD大鼠心肌细胞及心肌成纤维细胞。RT-PCR结果显示,在乳SD大鼠心肌细胞及心肌成纤维细胞中miR-1和miR-16均有相近水平的表达;心肌细胞中miR-208的表达显著低于miR-208b(P0.01),miR-208b在心肌细胞中的表达显著高于其在心肌成纤维细胞中的表达(P0.05)。乳C57BL/6小鼠心肌细胞中miR-1a-1的表达显著低于miR-1a-2(P0.01),而miR-1a-2在心肌细胞和心肌成纤维细胞中的表达水平一致;miR-16-1在心肌细胞和心肌成纤维细胞中表达水平相近,而miR-16-2在两种细胞中均不表达;心肌细胞中miR-208a和miR-208b表达水平差别不明显,心肌成纤维细胞中只有miR-208a表达,并且在水平上低于其在心肌细胞中的表达(P0.05)。结论 miR-1、miR-16和miR-208前体在乳小鼠、乳大鼠心肌细胞及心肌成纤维细胞中呈细胞特异性的表达。     

The miR-124 regulates the expression of BACE1/β-secretase correlated with cell death in Alzheimer's disease

The role of miR-124 on the expression of β-site APP cleaving enzyme 1 (BACE1), an important cleavager of amyloid precursor protein that plays a pivotal role in the β-amyloid production, was studied in this paper using cellular models for Alzheimer’ disease (AD) of cultured PC12 cell lines and primary cultured hippocampal neurons. The aim of the present study was to uncover novel potential miR-124 targets and shed light on its function in the cellular AD model. MiR-124 expression was steadily altered when its mimic and inhibitor were transfected in vitro. The results showed the expression of BACE1, one of the potential functional downstream targets of miR-124, was well correlated with cell death induced by Aβ neurotoxicity, and its expression level could be up- and down-regulated by suppression or over expression of miR-124 level respectively. These findings suggest that miR-124 may work as a basilic regulating factor to alleviate cell death in the process of AD by targeting BACE1, play an essential role in the control of BACE1 gene expression, and might be considered as a novel therapeutic target in treating AD.     

microRNAs调控骨骼肌分化的分子机制

microRNA(miRNA)是一大类广泛存在于真核细胞当中的长度约22 nt的内源性单链非编码RNA,通过与靶基因mRNA的3'非翻译区(3'untranslated region,3'UTR)结合在转录后水平调控靶基因的表达.miRNA作为调控基因表达的重要分子在骨骼肌分化调控中的作用越来越受到关注,阐明miRNA在骨骼肌增殖与分化中的作用机制具有重要的理论意义,同时也可为骨骼肌相关疾病的治疗提供新的思路.文章总结了miRNA,尤其是miR-1、miR-133和miR-206等肌肉特异性miRNA,在调控骨骼肌分化过程中作用机制的研究进展,以便于进一步工作的开展.     

Inhibition of hypoxia-induced miR-155 radiosensitizes hypoxic lung cancer cells

miR-155 is a prominent microRNA (miRNA) that regulates genes involved in immunity and cancer-related pathways. miR-155 is overexpressed in lung cancer, which correlates with poor patient prognosis. It is unclear how miR-155 becomes increased in lung cancers and how this increase contributes to reduced patient survival. Here, we show that hypoxic conditions induce miR-155 expression in lung cancer cells and trigger a corresponding decrease in a validated target, FOXO3A. Furthermore, we find that increased levels of miR-155 radioprotects lung cancer cells, while inhibition of miR-155 radiosensitizes these cells. Moreover, we reveal a therapeutically important link between miR-155 expression, hypoxia, and irradiation by demonstrating that anti-miR-155 molecules also sensitize hypoxic lung cancer cells to irradiation. Our study helps explain how miR-155 becomes elevated in lung cancers, which contain extensive hypoxic microenvironments, and demonstrates that inhibition of miR-155 may have important therapeutic potential as a means to radiosensitize hypoxic lung cancer cells.