microRNA分子表达与调控研究的最新进展

microRNA(miRNA)是一类分子长度为19~24nt的微小RNA,通常在转录后水平调控靶基因的降解或抑制翻译。miRNA分子在进化上高度保守,已经发现越来越多的miRNA分子参与真核生物的生长发育、生理活性、细胞增殖、组织分化、细胞凋亡、复杂疾病调控等功能。通过介绍miRNA的起源、合成、修饰、细胞表达特点,以及对真核细胞调控等的最新进展与研究方法,阐述miRNA在基因表达调节中的重要地位及应用前景。     

Regulation of Cardiac Expression of the Diabetic Marker MicroRNA miR-29

Diabetes mellitus (DM) is an independent risk factor for heart disease and its underlying mechanisms are unclear. Increased expression of diabetic marker miR-29 family miRNAs (miR-29a, b and c) that suppress the pro-survival protein Myeloid Cell Leukemia 1(MCL-1) is reported in pancreatic β-cells in Type 1 DM. Whether an up-regulation of miR-29 family miRNAs and suppression of MCL-1 (dysregulation of miR-29-MCL-1 axis) occurs in diabetic heart is not known. This study tested the hypothesis that insulin regulates cardiac miR-29-MCL-1 axis and its dysregulation correlates with DM progression. In vitro studies with mouse cardiomyocyte HL-1 cells showed that insulin suppressed the expression of miR-29a, b and c and increased MCL-1 mRNA. Conversely, Rapamycin (Rap), a drug implicated in the new onset DM, increased the expression of miR-29a, b and c and suppressed MCL-1 and this effect was reversed by transfection with miR-29 inhibitors. Rap inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling in HL-1 cells. Moreover, inhibition of either mTORC1 substrate S6K1 by PF-4708671, or eIF4E-induced translation by 4E1RCat suppressed MCL-1. We used Zucker diabetic fatty (ZDF) rat, a rodent model for DM, to test whether dysregulation of cardiac miR-29-MCL-1 axis correlates with DM progression. 11-week old ZDF rats exhibited significantly increased body weight, plasma glucose, insulin, cholesterol, triglycerides, body fat, heart weight, and decreased lean muscle mass compared to age-matched lean rats. Rap treatment (1.2 mg/kg/day, from 9-weeks to 15-weeks) significantly reduced plasma insulin, body weight and heart weight, and severely dysregulated cardiac miR-29-MCL1 axis in ZDF rats. Importantly, dysregulation of cardiac miR-29-MCL-1 axis in ZDF rat heart correlated with cardiac structural damage (disorganization or loss of myofibril bundles). We conclude that insulin and mTORC1 regulate cardiac miR-29-MCL-1 axis and its dysregulation caused by reduced insulin and mTORC1 inhibition increases the vulnerability of a diabetic heart to structural damage.     

Targeted Expression of Suicide Gene by Tissue-Specific Promoter and MicroRNA Regulation for Cancer Gene Therapy

In order to realise the full potential of cancer suicide gene therapy that allows the precise expression of suicide gene in cancer cells, we used a tissue specific Epithelial cell adhesion molecule (EpCAM) promoter (EGP-2) that directs transgene Herpes simplex virus–thymidine kinase (HSV-TK) expression preferentially in EpCAM over expressing cancer cells. EpCAM levels are considerably higher in retinoblastoma (RB), a childhood eye cancer with limited expression in normal cells. Use of miRNA regulation, adjacent to the use of the tissue-specific promoter, would provide the second layer of control to the transgene expression only in the tumor cells while sparing the normal cells. To test this hypothesis we cloned let-7b miRNA targets in the 3’UTR region of HSV-TK suicide gene driven by EpCAM promoter because let-7 family miRNAs, including let-7b, were found to be down regulated in the RB tumors and cell lines. We used EpCAM over expressing and let-7 down regulated RB cell lines Y79, WERI-Rb1 (EpCAM ^+ve/let-7b^{down-regulated}), EpCAM down regulated, let-7 over expressing normal retinal Müller glial cell line MIO-M1(EpCAM ^−ve/let-7b^up-regulated), and EpCAM up regulated, let-7b up-regulated normal thyroid cell line N-Thy-Ori-3.1(EpCAM ^+ve/let-7b^up-regulated) in the study. The cell proliferation was measured by MTT assay, apoptosis was measured by probing cleaved Caspase3, EpCAM and TK expression were quantified by Western blot. Our results showed that the EGP2-promoter HSV-TK (EGP2-TK) construct with 2 or 4 copies of let-7b miRNA targets expressed TK gene only in Y79, WERI-Rb-1, while the TK gene did not express in MIO-M1. In summary, we have developed a tissue-specific, miRNA-regulated dual control vector, which selectively expresses the suicide gene in EpCAM over expressing cells.     

MicroRNA与神经系统重大疾病

目前有研究证实microRNA参与了神经系统生长发育和生理功能的调控,它也与可塑性障碍性疾病、神经系统退行性疾病、神经系统肿瘤、脑血管疾病等重大疾病的发生发展相关．随着microRNA研究领域的发展,一些重大神经系统疾病的相关发病机制将有可能被阐释．     

微RNA介导巨噬细胞极化的转录后调控

巨噬细胞极化是根据周围刺激环境做出表型调节的一个过程.一般极化为2个表型,分别为经典激活的M1巨噬细胞和替代激活的M2巨噬细胞.简而言之,M1巨噬细胞的特征是促炎和抗肿瘤;M2巨噬细胞是抗炎和促肿瘤.巨噬细胞极化被认为是人体生理和病理的关键调节器,其发挥作用的有效性依赖于关键因子的协调表达,而这些关键因子的表达在转录后...     

微小RNA与胃癌的关系

微小RNA（microRNA、miRNA）与胃癌的发生发展可通过调控其靶基因参与的信号传导通路,影响胃癌的发生、侵袭和转移等过程,发挥着类似于癌基因或抑癌基因的作用。目前,已发现多种microR—NA与胃癌关系密切,包括通过调节周期蛋白依赖性蛋白激酶（Cdk）表达影响胃癌细胞增殖的miR-106b-93～25家族、miR-222—221家族和抑制高迁移率族蛋白A2（HMGA2）基因表达抑制胃癌细胞转移的miR-129和let-一7miRNA家族等。另有研究表明,miR-d21和miR-31检测阳性率显著高于血清CEA,可能成为新的胃癌肿瘤标志物。miR-15b和miR-16与胃癌多药耐药的关系也说明microRNA可能成为胃癌治疗新的靶点。     

微小RNA正调控基因表达的分子机制

microRNAs（miRNAs）是近年来发现的普遍存在于动植物体内的一类非编码RNA,传统观点认为,它们通过其种子序列定位于靶mRNA的3′非编码区,并发挥抑制靶mRNA翻译的作用.最新的研究揭示少数miRNAs也可以活化翻译,且这些现象的发生均与miRNA和翻译调控元件的相互作用有关.这些发现进一步扩展了对微小RNA功能的认识领域.     

MicroRNA 340 Is Involved in UVB-Induced Dendrite Formation through the Regulation of RhoA Expression in Melanocytes

The influence of UV irradiation on pigmentation is well established, but the molecular and cellular mechanisms controlling dendrite formation remain incompletely understood. MicroRNAs (miRNAs) are a class of small RNAs that participate in various cellular processes by suppressing the expression of target mRNAs. In this study, we investigated the expression of miRNAs in response to UVB irradiation using a microarray screen and then identified potential mRNA targets for differentially expressed miRNAs among the genes governing dendrite formation. We subsequently determined the ability of miRNA 340 (miR-340) to suppress the expression of RhoA, which is a predicted miR-340 target gene that regulates dendrite formation. The overexpression of miR-340 promoted dendrite formation and melanosome transport, and the downregulation of miR-340 inhibited UVB-induced dendrite formation and melanosome transport. Moreover, a luciferase reporter assay demonstrated direct targeting of RhoA by miR-340 in the immortalized human melanocyte cell line Pig1. In conclusion, this study has established an miRNA associated with UVB irradiation. The significant downregulation of RhoA protein and mRNA expression after UVB irradiation and the modulation of miR-340 expression suggest a key role for miR-340 in regulating UVB-induced dendrite formation and melanosome transport.     

微RNA参与调控肿瘤血管生成

肿瘤血管生成是由多种因子参与调控的复杂过程,对肿瘤的生长、侵袭和转移起着关键作用.微 RNA(microRNA, miRNA)是一段长约22 nt的非编码RNA序列,参与调节细胞增殖、分化、凋亡,肿瘤发生、发展等多种生命活动.认识miRNA与肿瘤血管生成的关系不但能加深对血管生成分子机制的理解,同时对阐明 miRNA的功能具有重要意义.     

MicroRNA Regulation of the Synaptic Plasticity-Related Gene Arc

Expression of activity-regulated cytoskeleton associated protein (Arc) is crucial for diverse types of experience-dependent synaptic plasticity and long-term memory in mammals. However, the mechanisms governing Arc-specific translation are little understood. Here, we asked whether Arc translation is regulated by microRNAs. Bioinformatic analysis predicted numerous candidate miRNA binding sites within the Arc 3'-untranslated region (UTR). Transfection of the corresponding microRNAs in human embryonic kidney cells inhibited expression of an Arc 3'UTR luciferase reporter from between 10 to 70% across 16 microRNAs tested. Point mutation and deletion of the microRNA-binding seed-region for miR-34a, miR-326, and miR-19a partially or fully rescued reporter expression. In addition, expression of specific microRNA pairs synergistically modulated Arc reporter expression. In primary rat hippocampal neuronal cultures, ectopic expression of miR-34a, miR-193a, or miR-326, downregulated endogenous Arc protein expression in response to BDNF treatment. Conversely, treatment of neurons with cell-penetrating, peptide nucleic acid (PNA) inhibitors of miR-326 enhanced Arc mRNA expression. BDNF dramatically upregulated neuronal expression of Arc mRNA and miR-132, a known BDNF-induced miRNA, without affecting expression of Arc-targeting miRNAs. Developmentally, miR-132 was upregulated at day 10 in vitro whereas Arc-targeting miRNAs were downregulated. In the adult brain, LTP induction in the dentate gyrus triggered massive upregulation of Arc and upregulation of miR-132 without affecting levels of mature Arc-targeting miRNAs. Turning to examine miRNA localization, qPCR analysis of dentate gyrus synaptoneurosome and total lysates fractions demonstrated synaptic enrichment relative to small nucleolar RNA. In conclusion, we find that Arc is regulated by multiple miRNAs and modulated by specific miRNA pairs in vitro. Furthermore, we show that, in contrast to miR-132, steady state levels of Arc-targeting miRNAs do not change in response to activity-dependent expression of Arc in hippocampal neurons in vitro or during LTP in vivo.     

Regulation of HD-ZIP III Genes by MicroRNA 165

MicroRNAs (miRNAs) 165 and 166 are able to cleave their target mRNAs of HD-ZIP III genes, thus regulating the functions of these genes. Although it is generally accepted that both miR165 and miR166 perform the same functions in the regulation of HD-ZIP III genes in Arabidopsis, no experimental data are available to support this notion. Recent work has shown that overexpression of miR166 downregulates the expression of three HD-ZIP III genes, ATHB-9/PHV, ATHB-14/PHB and ATHB-15, which in turn recapitulates the phenotypes of simultaneous loss-of-function mutations of these genes. In the March issue of Plant & Cell Physiology, we have demonstrated that overexpression of miR165 leads to the down-regulation of all five HD-ZIP III genes, and concomitantly recapitulates the phenotypes of loss-of-function mutation of IFL1/REV and those of simultaneous loss-of-function mutations of IFL1/REV, ATHB-9/PHV and ATHB-14/PHB. These results indicate that miR165 and miR166 differentially regulate the functions of HD-ZIP III genes in Arabidopsis. In this addendum, we show that overexpression of the antisense form of the miR165a gene leads to formation of amphivasal vascular bundles, a phenotype reminiscent of that of the dominant mutation of IFL1/REV. This finding provides direct evidence for a role of miR165 in regulation of vascular patterning.Key Words: HD-ZIP III genes, miR165, miR166, organ polarity, vascular patterning     

Regulation of Transient Site-specific Copy Gain by MicroRNA

Intra-tumor copy number heterogeneity is commonly observed in cancer; however, the molecular mechanisms that contribute to heterogeneity remain poorly understood. Up-regulation of the histone demethylase KDM4A promotes transient site-specific copy gain (TSSG) in cells; therefore, uncovering how KDM4A levels are controlled is important for understanding the regulation of copy number heterogeneity. Here, we demonstrate that KDM4A is regulated by hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137. Altering expression of these microRNAs (miRNAs) regulates KDM4A-dependent TSSG. miRNA inhibition promoted copy gains and increased expression of the drug-resistant oncogene CKS1B, which was further substantiated in primary breast tumors. Consistent with increased CKS1B expression, miRNA inhibition reduced breast cancer cell sensitivity to cisplatin. Our data identify these miRNAs as regulators of TSSG and copy gains of a drug resistance gene.