miR-205-5p、PDCD5、Beclin1在子宫内膜癌组织中的表达及相关性研究

目的 研究子宫内膜癌组织中微小RNA(microRNA miR-205-5p)、程序性细胞坏死因子5（programmed cell necrosis factor 5,PDCD5）、Beclin1的表达情况及三者之间的相关性。 方法 选择75例子宫内膜癌组织和距离癌组织边缘5 cm癌旁组织。通过免疫组织化学、实时荧光定量法检测miR-205-5p、PDCD5、Beclin1的表达情况,分析miR-205-5p、PDCD5、Beclin1的相关性及对子宫内膜癌的诊断价值。 结果 癌组织中miR-205-5p表达高于癌旁组织,PDCD5、Beclin1表达低于癌旁组织（P＜0.05）。不同组织学分级、临床分期、淋巴结转移、肌层浸润子宫内膜癌患者miR-205-5p、PDCD5、Beclin1表达差异有统计学意义（P＜0.05）,Ⅲ~Ⅳ期、中低分化、有淋巴结转移、≥1/2肌层浸润子宫内膜癌患者miR-205-5p表达升高,PDCD5、Beclin1表达降低,差异有统计学意义（P＜0.05）。不同年龄、病理类型子宫内膜癌患者miR-205-5p、PDCD5、Beclin1表达差异无统计学意义（P＞0.05）。miR-205-5p、PDCD5之间呈负相关,miR-205-5p、Beclin1之间呈负相关,PDCD5、Beclin1之间呈正相关（P＜0.05）。三项联合诊断子宫内膜癌的价值高于miR-205-5p、PDCD5、Beclin1单项诊断（P＜0.05）。 结论 miR-205-5p表达升高,PDCD5、Beclin1表达降低对子宫内膜癌的发展起到了促进作用,参与子宫内膜癌的演变进程,临床可根据上述指标对子宫内膜癌进行预测。     

MicroRNA-132通过诱导线粒体氧化应激障碍-铁死亡进程促进动脉粥样硬化

目的 探讨MicroRNA-132（miR-132）在动脉粥样硬化斑块中的表达及生物学意义。方法 收集在本医院行外周血管造瘘手术的动脉粥样硬化患者的斑块样本及周围正常血管样本各30例,分为实验组（n=30）与对照组（n=30）;利用RT-qPCR验证miR-132在30例组织标本中的表达水平;采用脂质体转染技术上调人脐静脉内皮细胞（HUVEC）中miR-132的表达,继而通过流式细胞及激光共聚焦技术分析过表达miR-132的HUVEC内活性氧（ROS）、ROS与线粒体的定位关系、线粒体活性氧超氧化物（mtROS）、线粒体膜电位状态（MMP）以及线粒体膜转换孔通透性（mPTP）的功能变化;通过ELISA检测HUVEC内线粒体氧化还原呼吸链复合体（Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ型）活性的状态;通过Western blot检测铁死亡关键蛋白的表达水平。结果 与正常血管样本（对照组）相比,miR-132在动脉粥样硬化斑块的表达水平显著上调（P<0.001）;相比于正常HUVEC,脂质体转染的HUVEC内miR-132表达量显著上升（P<0.001）,细胞内ROS明显增加（P<0.001）,且大部分ROS与线粒体存在共定位关系;同时于正常HUVEC,miR-132过表达的HUVEC细胞内MMP下降（P<0.001）、mtROS升高（P<0.001）、线粒体活性氧mPTP更多开放（P< 0.001）,继而引起线粒体氧化还原呼吸链应激障碍,铁死亡关键蛋白GPX4显著下调（P<0.001）、氧化蛋白NOX4显著增多（P< 0.001）。结论 MiR-132可通过诱导线粒体氧化应激障碍-铁死亡进程促进动脉粥样硬化,有望成为动脉粥样硬化的治疗靶点。     

MicroRNA-21与骨质疏松症相关性的研究进展

骨质疏松症(osteoporosis,OP)是一种以易骨折为特点的全身代谢性骨骼疾病。在老龄化程度不断加剧的当今社会,其发病率逐年呈现显著上升趋势,尤其是骨质疏松性骨折导致的后遗症、副损伤给社会及患者带来极大的经济和生活负担。近年来诸多研究发现microRNA具有明确的抗骨质疏松作用,随着基因疗法应用的推广,microRNA在临床治疗骨质疏松起到了很好的靶向作用,同时相关文献阐释,尤其是其家族成员microRNA-21可通过调控成骨细胞和破骨细胞的分化与功能,在OP等骨疾病的发生发展过程中起着重要作用。本文将通过对microRNA-21在骨质疏松中的相关作用机制进行综述,旨在为OP靶向治疗及相关分子机制研究提供理论依据和新的思路。     

MicroRNA-34a通过上调Cdc42和Rac1促进人晶状体上皮细胞衰老和凋亡

目的:观察MicroRNA-34a对人晶状体上皮细胞系SRA01/04衰老和凋亡的影响及作用机制。方法:qRT-PCR检测年龄相关性白内障(ARC)晶状体和透明晶状体上皮细胞中MicroRNA-34a表达水平,采用脂质体转染试剂盒将MicroRNA-34a mimics(过表达组)、MicroRNA-34a inhibitors(抑制组)和空脂质体(对照组)转染至SRA01/04细胞,qRT-PCR检测MicroRNA-34a的表达量;采用β-半乳糖苷酸(SA-β-gal)染色检测转染后细胞的衰老情况。Annexin V-FITC/PI双染色流式细胞仪检测MicroRNA-34a对人晶状体细胞系SRA01/04细胞凋亡的影响;蛋白免疫印迹检测Cdc42、Rac1蛋白的表达。结果:透明晶状体前囊膜组织中MicroRNA-34a的表达量显著低于ARC晶状体前囊膜组织(P<0.05);MicroRNA-34a过表达组、对照组、MicroRNA-34a抑制组SA-β-gal阳性率分别为(87.56±2.34)%、(12.22±2.74)%、(3.45±0.45)%。MicroRNA-34a过表达组明显高于对照组,而MicroRNA-34a抑制组SA-β-gal阳性率明显低于对照组(P<0.05);MicroRNA-34a抑制组、对照组和MicroRNA-34a过表达组的细胞凋亡率分别为(5.87±1.22)%、(12.26±2.14)%、(29.45±3.12)%,MicroRNA-34a抑制组细胞凋亡率明显低于对照组,而MicroRNA-34a过表达组细胞凋亡率明显高于对照组(P<0.05);MicroRNA-34a过表达组中Cdc42和Rac1的表达明显高于对照组(P<0.05),MicroRNA-34a抑制组中Cdc42和Rac1的表达明显低于对照组(P<0.05)。结论:MicroRNA-34a可能通过上调Cdc42和Rac1促进人晶状体上皮细胞衰老和凋亡。     

MicroRNA-96对人视网膜色素上皮细胞增殖与迁移的影响

目的：探讨MicroRNA-96（miR-96）对人视网膜色素上皮（RPE）细胞增殖和迁移的影响。方法：实验研究。通过RNA原位杂交检测人胚胎眼（20周）石蜡切片中RPE层miR-96的表达情况。将miR-96和阴性对照（NC）通过阳离子脂质体介导转染人眼RPE细胞,采用细胞增殖实验（MTS）、流式细胞术和Transwell实验分别检测细胞增殖、细胞周期以及细胞迁移能力。通过生物信息学及Western blot法确定miR-96作用的靶基因。应用Western blot检测miR-96对细胞增殖迁移相关信号通路蛋白（Akt、ERK）及细胞周期相关蛋白（p-Cdc2、CyclinD2、p-Rb）表达的影响。组间数据比较采用独立样本t检 验。结果：MiR-96在人眼RPE细胞中有表达。MTS结果显示,转染NC、miR-96后,人眼RPE细胞的相对增殖速率分别为100%、74%±2%,差异有统计学意义（t=42.174,P=0.002）。流式细胞术检测结果显示,转染miR-96后阻滞在G1期的RPE细胞显著多于转染NC后,且差异有统计学意义（t= -18.444,P=0.003）。Transwell实验结果显示,与转染NC相比,转染miR-96能显著抑制RPE细胞的迁移,差异具有统计学意义（t=6.754,P=0.002）。进而,明确了MITF是miR-96作用的靶基因。Western blot检测结果显示,转染miR-96后细胞中细胞周期相关蛋白p-Rb（t=11.211,P=0.002）、p-Cdc2（t=9.133, P=0.003）、CyclinD2（t=7.542,P=0.005）以及迁移相关信号通路蛋白p-ERK（t=16.699,P<0.001）,p-Akt （t=23.552,P<0.001）的表达水平均降低。结论：miR-96通过作用于靶基因MITF,并调控细胞周期和迁移相关蛋白的表达从而抑制人RPE细胞的增殖和迁移。     

Overexpression of microRNA-124 promotes the neuronal differentiation of bone marrow-derived mesenchymal stem cells

microRNAs(miRNAs) play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124(miR-124) overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesenchymal stem cells, neural stem cells and neurons. miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We constructed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers β-III tubulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These results suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.     

雌二醇联合孕激素对卵巢癌细胞凋亡及microRNA-15a表达的影响

目的 探讨雌二醇（E2）联合孕激素（P4）对microRNA-15a （miR-15a）表达的影响。方法 采集手术中切除的卵巢癌组织标本进行细胞培养,分3个处理组：E2组,P4组和雌二醇联合孕激素（E2+P4）组。激素处理后,MTT法检测细胞存活率;流式细胞技术检测细胞凋亡与细胞周期;qRT-PCR法检测Bcl-2,Bax和miR-15a的相对表达量。结果 高浓度（浓度均为10^-4 mol·L^-1）的E2,P4,E2+P4能够降低卵巢癌细胞的存活率,并表现为时间依赖性;低浓度（≤ 10^-8 mol·L^-1） E2能够提高卵巢癌细胞的存活率。与对照组相比,高浓度E2,P4和E2+P4能够增加卵巢癌细胞的凋亡率（P<0.001）,并且E2+P4的作用最明显;低浓度E2能够抑制肿瘤细胞的凋亡（P<0.001）。E2,P4和E2+P4对细胞周期的影响没有统计学差异。高浓度的E2,P4和E2+P4能够下调Bcl-2的表达（P<0.05或P<0.001）,上调Bax的表达（P<0.001）;但是低浓度E2作用却相反。高浓度的E2+P4能够促进miR-15a的表达（P<0.001）。结论 高浓度的E2,P4和E2+P4能够降低细胞的存活率促进细胞凋亡,下调Bcl-2的表达,上调Bax的表达;低浓度E2的作用则相反;高浓度的E2+P4能够促进miR-15a的表达。     

Inhibition of microRNA-17-5p reduces the inflammation and lipid accumulation,and up-regulates ATP-binding cassette transporterA1 in atherosclerosis

Atherosclerosis (AS) is a chronic inflammatory disease of the arterial wall. Macrophages are considered to be closely associated with the development and progression of AS. However, the precise mechanism of miR-17-5p in the macrophages under AS remains incompletely clarified. This study investigated the regulatory effect of miR-17-5p on the inflammation and lipid accumulation in mouse macrophages both in vivo and in vitro. It was found that miR-17-5p was highly expressed with lowered ATP-binding cassette transporterA1 (ABCA1) level in the peripheral blood leucocytes (PBLs) of AS patients. Moreover, the level of miR-17-5p was up-regulated in the macrophages of ApoE^?/? mice fed with a high-cholesterol diet. Furthermore, we injected miR-17-5p antagomir into AS mice or transfected miR-17-5p inhibitors into mouse macrophage RAW264.7 cells. Results showed that downregulation of miR-17-5p significantly reduced the production of inflammatory cytokines, inhibited the lipid accumulation and up-regulated ABCA1, and activated peroxisome proliferator-activated receptor (PPAR) γ/Liver X receptor (LXR) α signaling pathway. Additionally, ABCA1 was found to be a target of miR-17-5p by directly binding to 3′-untranslated region (3′-UTR) of its mRNA. Our study indicates a novel regulatory mechanism for miR-17-5p by interacting with ABCA1, which could be a therapy-target for the treatment of AS.