MiR-302/367在体细胞向多能性干细胞重编程中的研究

MicroRNA-302/367(miR-302/367)发现于2003年,是一类长度在21～22 nt的miRNA簇,与多能性干细胞自我更新及多向分化有重要关系.在体细胞向多能性干细胞重编程中具有重要作用. miR-302/367簇中各miRNA具有相对保守的种子区及靶基因,主要通过抑制靶基因蛋白质的翻译,从而促进间质-上皮转化（mesenchymal epithelial transition, MET）、抑制细胞周期、调控细胞分化相关基因及表观遗传水平等方式促进体细胞向多能性细胞重编程.本文对miR 302/367的发现、结构、miR 302/367在多能性细胞中的作用及在体细胞向多能性干细胞重编程中的作用及其机理等做一综述.     

体细胞重编程为多能干细胞的研究进展

体细胞通过重编程转变成其他类型的细胞,在再生医学方面具有重要的应用前景。细胞重编程的方法主要有体细胞核移植、细胞融合、细胞提取物诱导、限定因子诱导等,这些方法可以不同程度地改变细胞命运。最近,限定因子诱导的多能干细胞（induced pluripotent stem cell。iPS）为重编程提供了一种崭新的方法,不仅可以避免伦理争议,还提供了一种更为便利的技术,为再生医学开辟了新的天地;同时,iPS技术为研究基因表达调控、蛋白质互作、机体生长发育等提供了一个非常重要的研究手段。本文主要论述了体细胞重编程的方法及iPS细胞的进展、面临的问题和应用前景。     

哺乳动物体细胞异种核移植胚胎早期发育的研究进展

体细胞异种核移植是指将一个物种的体细胞移植到另一物种的去核卵母细胞中,移入的体细胞核在受体胞质中重编程并发育成新个体的实验方法.该方法为拯救濒危物种和获取灵长类胚胎干细胞提供了可能的途径.但这方面的研究目前还只获得初步的进展,核重编程不完全以及异种胚胎的囊胚率低仍是其面临的主要难点.本文从基因表达、表观重编程、线粒体异质性、核重塑和核移植体系优化等方面入手,介绍近年来哺乳动物体细胞异种核移植的研究进展,并探讨异种重构胚重编程所面临的关键问题和可能获得成功的方法.     

诱导多能干细胞的研究进展

体细胞诱导成为多能性干细胞(induced pluripotent stem cell,iPS cell)的研究成果被国际生命科学界誉为具有里程碑意义的创新之举.在短短3年多的时间里,这项研究已经在细胞重编程的机理研究、探索疾病的发生发展机制以及临床医学的应用等领域引发了很多突破性的进展,而且,这一非克隆干细胞技术的诞生,成功地避开了长期以来争论不休的伦理问题,极大地推动该领域和相关科学领域的发展.从iPS细胞的研究历程、iPS细胞的构建机理、iPS细胞研究的最新应用成果以及iPS细胞的发展前景和研究方向等方面进行了评.     

体细胞重编程为多潜能干细胞的研究进展

人类的胚胎干细胞（embryonic stem cells,ES cells）可以用来治疗很多疾病,但是如果通过核移植来获得与供体或者患者相匹配的ES细胞,就会受到人卵母细胞来源等条件的制约。这就促使了将体细胞重编程为多潜能细胞这样一种技术策略的发展,其中包括将分化细胞与ES细胞融合,在卵细胞、ES细胞或多潜能癌细胞的抽提物中孵育,强制多潜能因子过表达等具体的方法。通过这些途径引出了一些核功能的重编程以及相应的DNA甲基化修饰、组蛋白翻译后修饰,使体细胞表达特定的多潜能因子,转变为类似胚胎干细胞的多潜能细胞。     

牛诱导多能干细胞的建立及应用研究进展

诱导性多能干细胞（induced pluripotent stem cells,iPSCs）是指通过导入特定的转录因子将终末分化的体细胞重编程为可以无限增殖更新并具有分化为三胚层多种细胞类型的一类干细胞系。目前对人与小鼠的iPSCs研究已经取得了很多重要成果,但其他动物,如牛等经济型有蹄类家畜iPSCs的研究始终没有突破性的进展。如何将外源转录因子通过重编程载体高效安全地导入体细胞中并持续表达是生产牛诱导多能干细胞（bovine induced pluripotent stem cells,biPSCs）的主要瓶颈。本文就biPSCs建立中重编程系统的选择、诱导因子的选择、小分子化合物的添加等方面进行综述,以期为进一步完善biPSCs及牛胚胎干细胞系的建立提供参考。     

MicroRNA-214 在心肌缺血/再灌注的研究进展

MicroRNA是一种内源性的小核苷酸片段,已检测出700余种。大约30%的人类基因受miRNAs调节。其中miRNA-214在不同细胞有多种生物学作用,通过调控多种靶基因在诸多疾病中都发挥着重要作用。microRNA-214在心肌损伤及免疫方面也发挥积极的作用,通过抑制心肌缺血/再灌注的细胞凋亡、HIF1AN等机制参与心肌缺血/再灌注,其有可能成为预防和治疗治疗心肌缺血/再灌注损伤性疾病的新型靶向分子,为临床预防和治疗心肌缺血/再灌注损伤性疾病提供思路和方法。     

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．     

过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。     

神经干细胞/前体细胞分化的功能学鉴定研究进展

神经干细胞／前体细胞分化是神经生物学研究的热点．以往对神经干细胞／前体细胞分化水平的鉴定主要依赖于形态学指标,而随着膜片钳技术的应用,神经干细胞／前体细胞分化过程中膜电特性的改变、离子通道活动等功能学指标越来越受到重视．综述了利用膜片钳技术研究神经干细胞／前体细胞功能分化的最新进展,并对存在的问题做出思考和展望．     

The microRNA-302-367 cluster suppresses the proliferation of cervical carcinoma cells through the novel target AKT1

The miR-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. Here, we investigated the role of the miR-302-367 cluster in cervical carcinoma. The cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. However, ectopic expression of the miR-302-367 cluster in HeLa and SiHa cervical cancer cells inhibited cell proliferation and tumor formation by blocking the G1/S cell cycle transition. We identified a new cell cycle regulatory pathway in which the miR-302-367 cluster directly down-regulated both cyclin D1 and AKT1 and indirectly up-regulated p27^Kip1 and p21^Cip1, leading to the suppression of cervical cancer cell proliferation. Our findings suggest that the miR-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.     

Vitamin C counteracts miR-302/367-induced reprogramming of human breast cancer cells and restores their invasive and proliferative capacity

Epigenetic reprogramming by embryonic stem cell-specific miR-302/367 cluster has shown some tumor suppressive effects in cancer cells of different tissues such as skin, colon, and cervix. Vitamin C has been known as a reprogramming enhancer of human and mouse somatic cells. In this study, first we aimed to investigate whether exogenous induction of miR-302/367 in breast cancer cells shows the same tumor suppressive effects previously observed in other cancer cells lines, and whether vitamin C can enhance reprogramming of breast cancer cells and also improve the tumor suppressive function of miR-302/367 cluster. Overexpression of miR-302/367 cluster in MDA-MB-231 and SK-BR-3 breast cancer cells upregulated expression of miR-302/367 members and also some core pluripotency factors including OCT4A, SOX2 and NANOG, induced mesenchymal to epithelial transition, suppressed invasion, proliferation, and induced apoptosis in the both cell lines. However, treatment of the miR-302/367 transfected cells with vitamin C suppressed the expression of pluripotency factors and augmented the tumorigenicity of the breast cancer cells by restoring their proliferative and invasive capacity and compromising the apoptotic effect of miR-302/367. Supplementing the culture medium with vitamin C downregulated expression of TET1 gene which seems to be the reason behind the negative impact of vitamin C on the reprogramming efficiency of miR-302/367 cluster and its anti-tumor effects. Therefore application of vitamin C may not always serve as a reprogramming enhancer depending on its switching function on TET1. This phenomenon should be carefully considered when considering a reprogramming strategy for tumor suppression.     

miR-302通过下调靶基因RAB22A抑制膀胱癌进展

目的:探讨miR-302通过靶向调控RAB22A影响膀胱癌进展的分子机制。方法:采用RT-qPCR检测miR-302在HTB1和RT112膀胱癌细胞系和膀胱内皮细胞系HBdNEC中的表达;以miRNA-NC、miR-302 mimic、miR-302 inhibitor转染细胞,并分为以下几组:NC+control si RNA、miR-302 inhibitor+control si RNA、miR-302 inhibitor+RAB22A si RNA、NC+vector、miR-302 mimic+vector或miR-302 mimic+RAB22A,再通过MTT实验分析膀胱癌细胞的增殖情况,细胞侵袭实验检测细胞侵袭情况,双荧光素酶报告载体检测分析miR-302靶基因,Western blot检测RAB22A在膀胱癌细胞中的表达。结果:HTB1和RT112细胞中miR-302的表达明显低于HBdNEC细胞(P0.05)。miR-302高表达抑制膀胱癌细胞的增殖和侵袭;miR-302低表达时,膀胱癌细胞的增殖和侵袭能力上升。生物信息学和双荧光素酶报告结果显示RAB22A为miR-302的靶基因。miR-302过表达后,细胞荧光素酶活性显著下降(P0.05),RAB22A表达下调(P0.05);miR-302表达沉默后,细胞荧光素酶活性显著上升(P0.05),RAB22A表达上调(P0.01)。拯救实验显示RAB22A表达沉默可逆转miR-302表达沉默时对膀胱癌细胞增殖和侵袭能力上调的影响;而RAB22A过表达可逆转miR-302过表达对膀胱癌细胞增殖和侵袭的抑制。结论:miR-302可通过抑制靶基因RAB22A的表达,抑制膀胱癌细胞的增殖及侵袭。     

MicroRNA-302c-3p inhibits endothelial cell pyroptosis via directly targeting NOD-, LRR- and pyrin domain-containing protein 3 in atherosclerosis

Inflammation and endothelial dysfunction are important participants and drivers in atherosclerosis. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome activation and the resulting pyroptosis are involved in the initiation and vicious circle of chronic inflammation, thus playing an indispensable role in atherosclerosis. Accordingly, blocking the activation of NLRP3 inflammasome may be a promising treatment strategy to blunt the progression of atherosclerosis. In this study, it was demonstrated that miR-302c-3p exerted anti-pyroptosis effects by directly targeting NLRP3 in vivo and in vitro. In brief, the expression of miR-302c-3p was down-regulated whereas the expression of NLRP3 was up-regulated in human plaques and in vitro pyroptosis model of endothelial cells. Overexpression of miR-302c-3p suppressed endothelial cell pyroptosis by targeting specific sites of NLRP3. By comparison, down-regulation of endogenous miR-302c-3p led to the opposite results, which were reversed by silencing the expression of NLRP3. Finally, the up-regulation of miR-302c-3p inhibited the inflammation and pyroptosis of atherosclerosis mouse model. In conclusion, miR-302c-3p may be a powerful and attractive target for suppressing endothelial inflammation and pyroptosis, providing a novel strategy for preventing or alleviating the progression of atherosclerosis.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

环六亚甲基双乙酰胺对胃癌不同细胞周期两条信号系统的调节

环六亚甲基双乙酰胺(HMBA)对MGc80-3不同时相细胞内cAMP-PKA与DAG-PKC两大系统不仅具有正负调控作用,而且其作用具有周期特异性. 其中G1期是最敏感的调控时相,与对照组相比,cAMP水平上升102.3%,PKA活性升高348%,DAG含量下降51.4%,PKC活性降低32.3%;次敏感时相为G2期;M期基本没受影响;S期变化规律不同于其他时相.