Down-Regulation of MicroRNA-223 Promotes Degranulation via the PI3K/Akt Pathway by Targeting IGF-1R in Mast Cells

Background

Mast cells play a central role in allergic and inflammatory disorders by inducing degranulation and inflammatory mediator release. Recent reports have shown that miRNAs play an important role in inflammatory response regulation. Therefore, the role of miR-223 in mast cells was investigated.

Methods

The expression of miR-223 was quantified by quantitative real-time polymerase chain reaction (qRT-PCR) in immunoglobulin E (IgE)-mediated mast cells. After successful miR-223 inhibition by transfection, degranulation was detected in IgE-mediated mast cells. The phosphorylation of IκB-α and Akt were examined using western blotting. NF-κB was tested using electrophoretic mobility shift assay. PI3K-inhibitor (LY294002) was used to investigate whether the PI3K/Akt pathway was essential for mast cell activation. The TargetScan database and a luciferase reporter system were used to identify whether insulin-like growth factor 1 receptor (IGF-1R) is a direct target of miR-223.

Results

MiR-223 expression was up-regulated in IgE-mediated mast cells, whereas its down-regulation promoted mast cell degranulation. Levels of IκB-α and Akt phosphorylation as well as NF-κB were increased in miR-223 inhibitor cells. LY294002 could block the PI3K/Akt signaling pathway and rescue the promotion caused by suppressing miR-223 in mast cells. IGF-1R was identified as a direct target of miR-223.

Conclusions

These findings suggest that down-regulation of miR-223 promotes degranulation via the PI3K/Akt pathway by targeting IGF-1R in mast cells.     

MicroRNA-576-3p Inhibits Proliferation in Bladder Cancer Cells by Targeting Cyclin D1

MicroRNAs (miRNAs) are small, endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3′-UTR of mRNAs and directing their gene expression. Here, we first discovered that miR-576-3p was down-regulated in human bladder cancer cell lines compared with the non-malignant cell line. To better characterize the role of miR-576-3p in bladder cancer cells, we over-expressed or down-regulated miR-576-3p in bladder cancer cells by transfecting with chemically synthesized mimic or inhibitor. The overexpression of miR-576-3p remarkably inhibited cell proliferation via G1-phase arrest, and decreased both mRNA and protein levels of cyclin D1 which played a key role in G1/S phase transition. The knock-down of miR-576-3p significantly promoted the proliferation of bladder cancer cells by accelerating the progression of cell cycle and increased the expression of cyclin D1. Moreover, the dual-luciferase reporter assays indicated that miR-576-3p could directly target cyclin D1 through binding its 3′-UTR. All the results demonstrated that miR-576-3p might be a novel suppressor of bladder cancer cell proliferation through targeting cyclin D1.     

MicroRNA-217 Promotes Angiogenesis of Human Cytomegalovirus-Infected Endothelial Cells through Downregulation of SIRT1 and FOXO3A

Human cytomegalovirus(HCMV) infection has been shown to contribute to vascular disease through the induction of angiogenesis. However, the role of microRNA in angiogenesis induced by HCMV infection remains unclear. The present study was thus designed to explore the potential effect of miR-1217 on angiogenesis and to disclose the underlying mechanism in endothelial cells. We found that HCMV infection of endothelial cells(ECs) enhanced expression of miR-217 and reduced SIRT1 and FOXO3A protein level in 24 hours post infection(hpi). Transfection of miR-217 inhibitor not only depressed cellular migration and tube formation induced by HCMV infection, but also enhanced SIRT1 and FOXO3A protein expression. Additionally, luciferase assay confirmed that miR-217 directly targeted FOXO3A mRNA 3`UTR. Furthermore, pretreatment with resveratrol depressed motility and tube formation of HCMV-infected ECs, which could be reversed by SIRT1 siRNA. Similarly, delivery of FOXO3A overexpression lentivirus suppressed proliferative rate, migration and tube formation of HCMV-infected ECs, which reversed by transfection of FOXO3A siRNA. In summary, HCMV infection of endothelial cells induces angiogenesis by both of miR-217/SIRT1 and miR-217/FOXO3A axis.     

Trx通过Sirt3-P53通路促进自噬并改善高糖诱发的CMECs损伤

目的:明确硫氧还蛋白(Thioredoxin,Trx)通过自噬调节对大鼠心脏微血管内皮细胞损伤的保护作用及相关机制。方法:分离成年大鼠心脏微血管内皮细胞并分为:(1)正常对照组;(2)高糖组;(3)高糖+Trx组;(4)高糖+Trx+Ad-sh Sirt3组;(5)高糖+Trx+Ad-sh P53组;(6)高糖+DMSO空载组。通过In Vitro Vascular Permeability Assay Kit检测单层心脏微血管内皮细胞通透性,TUNEL染色检测细胞凋亡,Western blot法检测Sirt3、P53、Atg5、LC3BI/II等相关自噬相关信号通路关键蛋白的表达水平。结果:与正常对照组相比,高糖引起单层心脏微血管内皮细胞通透功能损伤,增加细胞凋亡,抑制自噬,且Sirt3、Atg5、LC3BI/II表达下降而P53表达上升;给予Trx可以上调Sirt3、Atg5、LC3BI/II蛋白表达水平,抑制P53表达,并显著减轻上述高糖引起的细胞损伤;但是,分别干扰Sirt3和P53表达后,Trx的作用明显减弱。结论:Trx通过Sirt3-P53信号通路促进心脏微血管内皮细胞自噬,降低细胞凋亡,改善高糖诱发的大鼠心脏微血管内皮细胞损伤。     

microRNA-151-5p在肾血管性高血压大鼠血管内皮细胞中的表达及意义

目的:研究microRNA-151-5p(miR-151-5p)在两肾一夹(2K1C)肾血管性高血压大鼠中的表达,并为miR-151-5p参与调节血管内皮细胞功能提供理论依据。方法:建立2K1C大鼠模型,获得胸主动脉血管内皮,采用荧光定量PCR技术检测血管内皮细胞中miR-151-5p的表达,通过数据库及生物信息学软件预测miR-151-5p的靶基因,并对靶基因进行GO富集和KEGG pathway分析。结果:与假手术组大鼠相比较,实验组大鼠胸主动脉血管内皮细胞miR-151-5p的表达量显著升高(P0.05)。GO分析显示miR-151-5p的靶基因参与蛋白加工水解、Notch受体加工等多种生物学功能(P0.01);KEGG pathway分析显示miR-151-5p的靶基因参与Notch信号通路、血管平滑肌收缩、代谢途径、细菌感染和RNA转运等信号通路。结论:2K1C大鼠血管内皮细胞中miR-151-5p表达升高,可能通过对其靶基因APH1A的调控参与血管内皮细胞功能调节。     

高浓度葡萄糖通过p38MAPK-TXNIP/TRX-ROS通路抑制MC3T3-E1 细胞成骨分化

目的：研究高浓度葡萄糖抑制MC3T3-E1细胞成骨分化的机理。方法：建立MC3T3-E1 细胞成骨分化诱导体系,观察不同浓 度葡萄糖（5.5mM 和22mM）对MC3T3-E1 细胞成骨分化的影响;用不同浓度的p38 MAPK 抑制剂Fr167653（0.1 滋M、1.0 滋M 和 10 滋M）进行药物干预,观察MC3T3-E1 细胞在22mM葡萄糖浓度下成骨分化的变化情况。通过钙含量检测、Real time PCR 检测 相关分化的变化;用Western Blot 方法检测MC3T3-E1 细胞分化过程中p38 MAPK 磷酸化状态、TXNIP 表达水平的变化;使用胰 岛素二硫键还原法检测细胞内TRX活性水平;使用活性氧检测试剂盒检测细胞内自由氧生成水平。结果：体外诱导条件下,高浓 度（22mM）葡萄糖通过升高p38 MAPK 磷酸化水平,上调TXNIP 表达水平,同时降低TRX 活性,使细胞内自由氧生成增加,抑制 MC3T3-E1 细胞的成骨分化;Fr167653通过抑制p38 MAPK 磷酸化,下调TXNIP 表达同时升高TRX活性,抑制细胞内自由氧生 成,解除高浓度葡萄糖对细胞成骨分化的抑制作用。结论：高浓度葡萄糖通过p38 MAPK-TXNIP/TRX-ROS 信号通路抑制 MC3T3-E1细胞成骨分化。     

高浓度葡萄糖通过p38MAPK-TXNIP/TRX-ROS通路抑制MC3T3-E1细胞成骨分化

目的：研究高浓度葡萄糖抑制MC3T3-E1细胞成骨分化的机理。方法：建立MC3T3-E1细胞成骨分化诱导体系,观察不同浓度葡萄糖（5.5mM和22mM）对MC3T3-E1细胞成骨分化的影响;用不同浓度的p38 MAPK抑制剂Fr167653（0.1μM、1.0μM和10μM）进行药物干预,观察MC3T3-E1细胞在22mM葡萄糖浓度下成骨分化的变化情况。通过钙含量检测、Real time PCR检测相关分化的变化;用Western Blot方法检测MC3T3-E1细胞分化过程中p38 MAPK磷酸化状态、TXNIP表达水平的变化;使用胰岛素二硫键还原法检测细胞内TRX活性水平;使用活性氧检测试剂盒检测细胞内自由氧生成水平。结果：体外诱导条件下,高浓度（22mM）葡萄糖通过升高p38 MAPK磷酸化水平,上调TXNIP表达水平,同时降低TRX活性,使细胞内自由氧生成增加,抑制MC3T3-E1细胞的成骨分化;Fr167653通过抑制p38 MAPK磷酸化,下调TXNIP表达同时升高TRX活性,抑制细胞内自由氧生成,解除高浓度葡萄糖对细胞成骨分化的抑制作用。结论：高浓度葡萄糖通过p38 MAPK-TXNIP/TRX-ROS信号通路抑制MC3T3-E1细胞成骨分化。     

MiR-210-3p Inhibits Proliferation and Migration of C6 Cells by Targeting Iscu

Neurochemical Research - Glioma is the most common primary brain tumor and the most malignant type of glioma is glioblastoma with the character of high mortality, high recurrence rate and poor...     

Sphingosine-1-Phosphate Induces the Migration of Thyroid Follicular Carcinoma Cells through the MicroRNA-17/PTK6/ERK1/2 Pathway

Sphingosine-1-phosphate (S1P) is a bioactive lipid known to play a role in tumorigenesis and cancer progression. However, the molecular mechanisms of S1P regulated migration of papillary thyroid cancer cells are still unknown. In this study, we showed that S1P induced PTK6 mRNA and protein expression in two thyroid follicular cancer cell lines (ML-1 and FTC-133). Further studies demonstrated that induced PTK6 and its downstream signal component (ERK1/2) are involved in S1P-induced migration. Upon investigating the mechanisms behind this event, we found that miR-17 inhibited the expression of PTK6 through direct binding to its 3’-UTR. Through overexpression and knockdown studies, we found that miR-17 can significantly inhibit S1P-induced migration in thyroid follicular cancer cells. Interestingly, overexpression or knockdown of PTK6 or ERK1/2 effectively removed the inhibition of S1P-induced migration by miR-17. Furthermore, we showed that S1P decreased miR-17 expression levels. Meanwhile, in papillary thyroid cancers, miR-17 is downregulated and negatively associated with clinical staging, whereas PTK6 is upregulated and positively associated with clinical stages. Collectively, our work defines a novel signaling pathway implicated in the control of thyroid cancer migration.     

LncRNA MALAT1 Promotes OGD-Induced Apoptosis of Brain Microvascular Endothelial Cells by Sponging miR-126 to Repress PI3K/Akt Signaling Pathway

Neurochemical Research - Ischemic stroke (IS) is a common disease that seriously endangers human health. Patients with IS present with increased death of brain microvascular endothelial cells...     

LncRNA Kcnq1ot1/miR-214-3p/caspase-1通路调控高糖处理的心脏成纤维细胞焦亡

摘要 目的：探讨长链非编码RNA Kcnq1ot1在高糖处理的心脏成纤维细胞中调控焦亡的作用及具体机制。方法：培养C57BL/6乳鼠原代心脏成纤维细胞,分别用5.5 mM和30 mM葡萄糖培养,用免疫荧光、qRT-PCR和western blot方法检测NLRP3、caspase-1和IL-1β的表达。高糖处理的成纤维细胞抑制Kcnq1ot1,检测caspase-1的表达。生物信息学和荧光素酶报告基因检测验证与Kcnq1ot1和caspase-1存在共同互补结合位点的microRNA。应用qRT-PCR和western blot方法检测高糖诱导的心脏成纤维细胞干扰Kcnq1ot1后miR-214-3p的表达,以及过表达或干扰miR-214-3p后caspase-1的表达水平。高糖诱导的细胞单独干扰Kcnq1ot1或同时抑制Kcnq1ot1和miR-214-3p,检测caspase-1、NLRP3和IL-1?茁的表达水平。结果：高糖诱导的成纤维细胞中焦亡激活,Kcnq1ot1表达明显升高;抑制Kcnq1ot1后caspase-1表达显著下调。生物信息学和荧光素酶报告基因检测发现miR-214-3p与Kcnq1ot1和caspase-1存在共同互补结合位点。高糖诱导的心脏成纤维细胞干扰Kcnq1ot1后miR-214-3p表达升高;过表达 miR-214-3p 后caspase-1表达降低,抑制miR-214-3p后caspase-1表达升高。同时抑制Kcnq1ot1和miR-214-3p可逆转干扰Kcnq1ot1对caspase-1的降低作用。结论：干扰Kcnq1ot1能够通过抑制miR-214-3p/caspase-1信号转导通路,抑制高糖诱导的心脏成纤维细胞焦亡。     

MicroRNA-449a/b抑制人结肠癌细胞内Fra-1的表达

MicroRNAs (miRNAs) 是一类长度约为22 nt的非编码的调控性小RNA,它们在诸多的生命活动中发挥重要作用,如参与调控细胞的增殖、分化、凋亡以及肿瘤的发生发展. MicroRNA-449a/b (miR 449a/b) 是脊椎动物中进化保守的miRNA,作为抑癌基因,参与了许多癌症的发生过程,但其在结肠癌中的作用尚不清楚. 本文利用实时荧光定量技术研究了miR-449a/b在结肠癌组织中的表达. 利用双荧光素酶报告基因检测系统及Western印迹鉴定miR-449a/b的靶基因. 应用MTS法和Transwell分别检测miR-449a/b对结肠癌细胞增殖和迁移的影响. 检测组蛋白乙酰化酶抑制剂曲古菌素A (trichostatin A, TSA) 对结肠癌细胞中miR-449a/b表达的影响. 研究结果表明：与正常结肠组织相比,miR-449a/b在结肠癌组织中低表达;miR 449a/b能够结合到FRA-1 mRNA 3′-非翻译区 (3′-untranslated region, 3′-UTR),从而抑制结肠癌细胞HCT116内源Fra 1的表达;外源转染miR-449a/b明显抑制结肠癌细胞HCT116的增殖和迁移;并且TSA处理能够诱导结肠癌细胞HCT116中miR-449a/b的表达. 以上结果提示: miR-449a/b可能通过抑制靶基因Fra-1的表达,进而抑制结肠癌细胞的增殖和迁移．     

BJ-1108, a 6-Amino-2,4,5-Trimethylpyridin-3-ol Analog,Inhibits Serotonin-Induced Angiogenesis and Tumor Growth through PI3K/NOX Pathway

5-Hydroxytryptamine (5-HT) induces proliferation of cancer cells and vascular cells. In addition to 5-HT production by several cancer cells including gastrointestinal and breast cancer, a significant level of 5-HT is released from activated platelets in the thrombotic environment of tumors, suggesting that inhibition of 5-HT signaling may constitute a new target for antiangiogenic anticancer drug discovery. In the current study we clearly demonstrate that 5-HT-induced angiogenesis was mediated through the 5-HT₁ receptor-linked Gβγ/Src/PI3K pathway, but not through the MAPK/ERK/p38 pathway. In addition, 5-HT induced production of NADPH oxidase (NOX)-derived reactive oxygen species (ROS). In an effort to develop new molecularly targeted anticancer agents against 5-HT action in tumor growth, we demonstrate that BJ-1108, a derivative of 6-amino-2,4,5-trimethylpyridin-3-ol, significantly inhibited 5-HT-induced angiogenesis. In addition, BJ-1108 induced a significant reduction in the size and weight of excised tumors in breast cancer cell-inoculated CAM assay, showing proportionate suppression of tumor growth along with inhibition of angiogenesis. In human umbilical vein endothelial cells (HUVECs), BJ-1108 significantly suppressed 5-HT-induced ROS generation and phosphorylation of PI3K/Akt but not of Src. Unlike NOX inhibitors, BJ-1108, which showed better antioxidant activity than vitamin C, barely suppressed superoxide anion induced by mevalonate or geranylgeranyl pyrophosphate which directly activates NOX without help from other signaling molecules in HUVECs, implying that the anti-angiogenic action of BJ-1108 was not mediated through direct action on NOX activation, or free radical scavenging activity. In conclusion, BJ-1108 inhibited 5-HT-induced angiogenesis through PI3K/NOX signaling but not through Src, ERK, or p38.     

Circ_0003423 Alleviates ox-LDL-Induced Human Brain Microvascular Endothelial Cell Injury via the miR-589-5p/TET2 Network

Brain microvascular endothelial cells (BMECs) injury is one of the main causes of cerebrovascular diseases. Circular RNA (circRNA) has been found to be involved in the regulation of cerebrovascular diseases progression. However, the role and mechanism of circ_0003423 in cerebrovascular diseases is still unclear. In our study, oxidized low density lipoprotein (ox-LDL)-induced HBMEC-IM cells were used to construct cerebrovascular cell injury model in vitro. Quantitative real-time PCR was used to determine the expression levels of circ_0003423, miR-589-5p and Ten-eleven translocation 2 (TET2). The interactions between miR-589-5p and circ_0003423 or TET2 were confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Cell viability, angiogenesis and apoptosis were measured using cell counting kit 8 assay, tube formation assay and flow cytometry. Cell oxidative stress was evaluated by detecting the levels of reactive oxygen species and lactate dehydrogenase. The protein levels were examined by western blot analysis. Our results showed that circ_0003423 was a downregulated circRNA in ox-LDL-induced HBMEC-IM cells. In the terms of mechanism, circ_0003423 was found to be a sponge of miR-589-5p. Function analysis showed that circ_0003423 overexpression could relieve ox-LDL-induced HBMEC-IM cell injury, and this effect could be reversed by miR-589-5p mimic. In addition, TET2 was confirmed to be a target of miR-589-5p, and its overexpression could alleviate ox-LDL-induced HBMEC-IM cell injury. Moreover, the rescue experiments also confirmed that TET2 silencing could abolish the inhibition effect of anti-miR-589-5p on ox-LDL-induced HBMEC-IM cell injury. In summary, our data showed that circ_0003423 alleviated ox-LDL-induced HBMEC-IM cells injury through regulating the miR-589-5p/TET2 axis.