microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization

Non-small cell lung cancer (NSCLC) accounts for approximately 70% of all lung cancer-related deaths worldwide. Prognostic markers are essential for the early detection of lung cancer in patients. In this study, we first identified microRNA146 (miR-146) expression in cancer cell lines using miRNA in situ hybridization (MISH) and confirmed the accuracy of MISH using q-RT-PCR. In addition, two different systems, BCIP/NBT and ELF, were used to detect the signal for a comparative analysis of the specificity of MISH. Compared to the BCIP/NBT system, the ELF detection system was more effective for MISH. Furthermore we detected the expression of miR-146 in NSCLC tissues (43 cases) and normal tissues (32 cases). Based on our results, we can conclude that miR-146 is more highly expressed in cancer tissue than normal tissue (t-test, P < 0.05) and that miR-146 can predict the prognosis of NSCLC by MISH. Our findings preliminary demonstrate that MISH can be applied as a molecular diagnostic tool to determine the expression and localization of miRNAs in cancer tissues and that miR-146, determined by MISH, predicts the prognosis of NSCLC patients.     

MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells

Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3′-untranslated regions (3′UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.     

Human Prostate DU145 Carcinoma Cells Implanted in Nude Mice Remove the Peritoneal Mesothelium to Invade and Grow as Carcinomas

Implanted human, androgen‐independent prostatic carcinoma cells (DU145) into athymic (NCr nu/nu) mice produce diverse tumors on the peritoneal surfaces of many organs. Light and ultrastructural observations show that the mesothelial covering these surfaces are typically microvilli‐coated, squamous cells or secretory cuboidal cells. The peritoneal regions colonized by tumors lack mesothelial cells and are covered by actively replicating carcinoma cells that grow as poorly differentiated cell clusters made of cell aggregates to somewhat compact spheroids covered with pleiomorphic microvilli and containing an undifferentiated vascular supply. These xenografts clusters invade the diaphragm and develop into tumors with both a basal solid aspect and an upper region of cribriform morphology. Furthermore, each tumor contains two cell types: (1) a poorly differentiated clear cell type, which grows into intraperitoneal tumors and (2) a large, basophilic cell type, which invades the peritoneal stroma of organs, including of the diaphragm. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

Relationships between microRNA-20a and microRNA-125b expression and apoptosis and inflammation in experimental spinal cord injury

ABSTRACT

Objectives: The aim of the study was to determine the relationships between microRNA-20a and microRNA-125b expression and apoptosis and inflammation in a rat model of spinal cord injury (SCI) using microscopy, immunohistochemistry, and molecular biology.

Methods: Sixty-one rats were divided into three groups: a control group that was not subjected to any operation; a sham-operated group; and an experimental group that was subjected to spinal cord compression. The experimental group was further subdivided into two subgroups: the experimental control group, which did not receive any drug treatment; and the methylprednisolone treatment group, which received 30 mg/kg methylprednisolone on day 0 followed by 10 mg/kg/day methylprednisolone from days 1–14.

Results: Tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 levels increased in the experimental control group on days 1 and 3, and decreased in the experimental control group and methylprednisolone treatment group on days 7 and 14. Caspase-3 levels increased in the experimental control group on day 1, and decreased in the experimental control group and methylprednisolone treatment group on days 3, 7, and 14. MicroRNA-20a expression was upregulated in the experimental control group on days 1 and 3, and microRNA-125b expression was downregulated on days 3 and 7.

Conclusions: After SCI, upregulated microRNA-20a expression and increased proinflammatory cytokines may lead to an increase in inflammation. MicroRNA-125b may be associated with caspase-3, and microRNA-125b downregulation may inhibit apoptosis. Although the results of this study suggest potential relationships between microRNA-20a and microRNA-125b expression and apoptosis and inflammation in SCI, further studies are needed to confirm microRNA-20a and microRNA-125b as biomarkers in SCI and to develop new strategies for the treatment of SCI.     

microRNA-21参与调控膀胱癌细胞增殖及多柔比星敏感性的研究

目的膀胱移行细胞癌是一种常见的恶性肿瘤,且复发率很高。由于microRNA-21(miR-21)在多种类型肿瘤中参与肿瘤发生及耐药,故通过此次研究探索其在膀胱移行细胞癌中的功能。方法肿瘤组织及癌旁正常组织中的miR-21及目的蛋白PTEN表达水平分别采用实时qRT-PCR及Western blot方法进行测量。采用MTT法评估miR-21对于癌细胞增殖以及多柔比星敏感性的影响。采用流式细胞术探究T24细胞系中多柔比星引起的细胞凋亡。结果与癌旁正常膀胱组织相比,miR-21在膀胱肿瘤组织中的表达水平显著上调,而PTEN蛋白表达低水平。活体内研究发现miR-21与PTEN表达呈负相关。miR-21过表达可显著促进T24细胞系的细胞增殖与多柔比星敏感性。结论 miR-21可能是膀胱移行细胞癌的致癌因素,有望成为膀胱移行细胞癌的治疗新靶点。     

microRNA-21对人甲状腺乳头状癌细胞增殖和凋亡的影响

目的:探讨microRNA-21(miR-21)在人甲状腺乳头状癌中的表达及其对人甲状腺乳头状癌K1细胞增殖和凋亡能力的影响?方法:通过qRT-PCR检测12对甲状腺乳头状癌组织(PTC)与癌旁正常组织中miR-21的表达差异;将miR-21抑制试剂(Anti-miR-21)?过表达试剂(miR-21 mimic)分别转染入K1细胞,并各自设立阴性对照组,运用qRT-PCR验证K1细胞中miR-21的表达;MTT实验检测miR-21对K1细胞增殖能力的影响;流式细胞术检测转染后K1细胞凋亡的变化,Western blot检测细胞凋亡增殖相关蛋白的表达情况?结果:miR-21在PTC组织中的表达明显高于癌旁正常甲状腺组织(P < 0.05);与阴性对照组相比,瞬时转染Anti-miR-21的K1细胞miR-21的表达明显减弱(P < 0.01);MTT实验结果显示抑制miR-21表达后,K1细胞的增殖能力明显下降(P < 0.05);流式细胞仪检测显示转染Anti-miR-21后的K1细胞凋亡明显增加,凋亡率为19.5%,阴性对照组的凋亡率为9.4%;Western blot结果显示转染Anti-miR-21组K1细胞Bcl-2表达降低,Bax表达升高?转染miR-21mimic的K1细胞增殖和凋亡无明显改动?结论:miR-21在人甲状腺乳头状癌中呈高表达,抑制miR-21的表达能显著降低K1细胞的增殖能力,增加K1细胞凋亡率?     

不同起源胶质瘤中microRNA-210表达差异及其与星形胶质细胞肿瘤级别的相关性

目的:探讨microRNA-210(miR-210)在星形胶质细胞肿瘤和少突胶质细胞肿瘤中的表达差异及意义?方法:将组织标本分为正常脑组织?少突胶质细胞肿瘤?星形胶质细胞肿瘤3组,星形胶质细胞肿瘤再分为A[毛细胞型星形细胞瘤(WHO Ⅰ级)]?B[弥漫性星形细胞瘤(WHO Ⅱ级)]?C[间变性星形细胞瘤(WHO Ⅲ级)]?D[胶质母细胞瘤(GBM,WHO Ⅳ级)]4个亚组?采用实时定量PCR方法检测各组中miR-210的表达水平?统计学分析miR-210的表达和星形胶质细胞肿瘤级别的相关性?结果:miR-210在各级别星形胶质细胞肿瘤中的表达水平依次为D> C> A和B,除A?B组间外,其他组间的差异都具有统计学意义(P < 0.001),星形胶质细胞肿瘤级别越高,miR-210表达水平越高;然而miR-210在少突胶质细胞肿瘤中呈低表达(P < 0.05),且间变性少突胶质细胞瘤中表达量低于少突胶质细胞瘤中的表达量?结论:miR-210在不同起源?不同级别胶质瘤中的表达水平不同,可以作为不同起源胶质瘤病理检查的辅助鉴别手段,也可作为星形胶质细胞肿瘤恶性进展的生物学标志物?     

miR⁃145⁃5p靶向调控LOX基因对肝癌细胞侵袭和迁移的影响

目的 探讨miR-145-5p靶向调控LOX对肝癌细胞侵袭和迁移的影响及其作用机制。方法 收集2017年9月至2019年9月广西医科大学附属肿瘤医院肝胆外科手术切除的73例肝癌患者肿瘤组织及其配对癌旁正常组织,以及肝癌细胞系SMMC-7721、SK-Hep1,采用RT-PCR法检测组织中miR-145-5p和LOX的表达。分别将miR-145-5p慢病毒载体、LOX过表达质粒及LOX慢病毒沉默载体转染至肝癌细胞SMMC-7721或SK-Hep1,同时用LOX过表达质粒和过表达miR-145-5p慢病毒载体共转染SMMC-7721细胞,各转染组均设置相应阴性对照组及空白对照组;然后采用Transwell实验检测肝癌细胞迁移和侵袭能力;Western blot法检测LOX蛋白的表达水平;通过TargetScan数据库预测miR-145-5p的靶点,并利用Cluster Profiler包进行GO和KEGG富集分析。结果 qRT-PCR检测结果显示,miR-145-5p在癌旁正常组织中的表达高于肝癌组织（4.196±2.288 vs 2.835±1.817,P<0.0001）;LOX在肝癌组织中的表达高于癌旁正常组织（12.17±1.369 vs 11.26±1.556,P<0.001）。Transwell检测结果显示,过表达LOX能促进肝癌细胞侵袭、迁移,敲低LOX则抑制肝癌细胞侵袭、迁移（均P<0.05）;过表达miR-145-5p能抑制肝癌细胞的侵袭、迁移能力,LOX过表达能逆转miR-145-5p对SMMC-7721细胞迁移和侵袭的抑制能力。TargetScan数据库预测显示,LOX是miR-145-5p的靶基因。Western blot检测结果显示,miR-145-5p能抑制LOX蛋白表达水平（P<0.001）。结论 miR-145-5p在肝癌组织中低表达,LOX则高表达,miR-145-5p可能通过负向调控LOX抑制肝癌细胞侵袭和迁移。     

未足月胎膜早破合并绒毛膜羊膜炎孕妇外周血miR-145表达及与Th1/Th2平衡的关系

目的探讨miR-145在未足月胎膜早破(preterm prelabor rupture of membranes,pPROM)合并绒毛膜羊膜炎(histological chorioamnionitis,HCA)孕妇外周血中表达情况,并分析其与Th1/Th2平衡的关系。方法选取2018年3月至2019年4月河北省儿童医院因胎膜早破入院的pPROM孕妇50例作为研究组,根据是否发生HCA分为HCA组(17例)与无HCA组(33例),另选取正常妊娠孕妇34例作为对照组,采集静脉血,分离血浆及外周血单个核细胞(peripheral blood mononuclear cells,PBMC),通过qRT-PCR法检测外周血血浆中miR-145的表达情况,利用流式细胞术检测PBMC中Th1细胞、Th2细胞占PBMC的比例,采用酶联免疫吸附法检测Th 1细胞分泌的细胞因子IL-2、IFN-γ、TNF-α以及Th 2细胞分泌的细胞因子IL-4、IL-6水平,Pearson法对miR-145与Th1、Th2细胞以及细胞分泌因子相关性进行分析,并通过ROC曲线分析外周血血浆miR-145对pPROM患者HCA发生的预测价值。结果与对照组、无HCA组相比,HCA组外周血血浆miR-145水平显著降低(P<0.05);与对照组、无HCA组相比,HCA组外周血Th1细胞比例、Th1/Th2比值显著升高(P<0.05),Th2细胞比例显著降低(P<0.05);与对照组、无HCA组相比,HCA组外周血血浆IL-2、IFN-γ、TNF-α水平显著升高(P<0.05),IL-4、IL-6水平显著下降(P<0.05);Pearson分析显示:外周血血浆miR-145表达与Th1细胞比例、Th1/Th2比值、IFN-γ、TNF-α、IL-2水平呈负相关(P<0.05),与Th2细胞比例、IL-4、IL-6水平呈正相关(P<0.05);ROC曲线分析显示miR-145水平预测pPROM患者HCA发生的截断值为0.56(曲线下面积0.859,敏感度89.3%,特异性68.2%)。结论pPROM合并HCA孕妇外周血血浆miR-145低表达,PBMC呈Th1优势,miR-145可能通过调控Th1/Th2平衡影响HCA的发生。