在子宫内膜癌ECC-1中他莫西芬通过mir-585调节PAX2蛋白表达

目的研究在子宫内膜癌ECC-1细胞中他莫西芬（tamoxifen,TAM）对PAX2（pairedbox2）蛋白表达的调节作用,寻找在这-过程中起调节作用的microRNA。方法用他莫西芬刺激ECC-1细胞,Western印迹检测PAX2蛋白表达的变化。利用MicrocosmTargets（miRBaseSequencedatabase）预测了PAX2相关的microRNA。用实时定量的方法检测他莫西芬刺激后ECC-1中PAX2相关microRNA表达的变化,找出差异变化明显的microRNA,合成这些microRNA的mimics,转染人ECC-1细胞中,用Western印迹检测其对PAX2蛋白表达的影响。结果他莫西芬刺激ECC-1细胞系后,Western印迹显示PAX2蛋白表达水平较对照组中等程度上调。实时定量PCR结果显示他莫西芬刺激ECC-1细胞后mir-135b＊,mir-604,mir-585,mir-181c＊表达较对照组明显下调。合成mir-135b＊,mir-604,mir-585,mir-181c＊的mimics并转染人ECC-1细胞后,Western印迹结果显示转入mir-585mimics的ECC-1细胞中PAX2蛋白表达较对照组下调。结论他莫西芬刺激可以引起ECC—1细胞中PAX2蛋白表达水平中等程度上调,通过抑制mir-585的表达减少其对PAX2mRNA翻译的抑制可能是这-调节作用中的部分机制。     

Importance of DNA methylation in the inheritance of radiation-induced aberrant expression of microRNA

We studied microRNA gene expression in HeLa cells following exposure for 6 h and 8 days to Co⁶⁰ gamma rays at a dose of 4 Gy using an approach of large-scale parallel DNA sequencing. We identified 12 microRNAs with aberrant expression which were maintained in cell generations. The analysis of radiation-induced aberrant expression of pre-microRNAs made it possible to assess the importance of nuclear and cytoplasmic stages of microRNA biogenesis for preservation of its aberrant expression. On cell treatment by 5-azacytidine, aberrant expression was maintained only in two microRNAs: miR-21-3p and miR-422a, which demonstrated an increase in expression. Radiation-induced decrease in expression in ten examined microRNAs was dependent on DNA demethylation. At the same time, expression in a microRNA set, which demonstrated inheritable alteration of the expression after gamma-radiation exposure in the untreated cells, was not dependent or was weakly dependent on DNA methylation. The obtained results suggest that ionizing radiation induces aberrant DNA methylation, which affects inherited expression changes in microRNAs in cell generations after exposure to the mutagen.     

The let-7 MicroRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans

The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3' UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions.     

Simultaneous determination of O6-methyl-2'-deoxyguanosine, 8-oxo-7,8-dihydro-2'-deoxyguanosine, and 1,N6-etheno-2'-deoxyadenosine in DNA using on-line sample preparation by HPLC column switching coupled to ESI-MS/MS

O(6)-Methyl-2'-deoxyguanosine (O(6)-mdGuo), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), and 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo) are promutagenic DNA lesions originating from both endogenous and exogenous agents and actions (methylation, hydroxylation, lipid peroxidation products). A highly sensitive quantitative method was developed to measure these DNA adducts simultaneously, using liquid chromatography tandem mass spectrometry with column switching. Deuterated O(6)-[(2)H(3)]mdGuo was synthesized and used as internal standard. The limits of quantification for O(6)-mdGuo, 8-oxodGuo, and epsilondAdo were 24, 98, and 48 fmol on column, respectively. The method showed linearity in the range 0.24-125 pmol/ml, 0.98-125 pmol/ml, and 0.49-62.5 pmol/ml for the three adducts, respectively. The inter-day precision in the linear concentration range was between 1.7 and 9.3% for O(6)-mdGuo, 10.6 and 28.7% for 8-oxodGuo, and 6.2 and 10.4%, for epsilondAdo. In DNA isolated from liver of untreated 12-week-old female F344 rats, O(6)-mdGuo was above the limit of detection (37 adducts per 10(9) normal nucleosides) but could not be quantified. 8-oxodGuo and epsilondAdo showed background levels of 500 and 130 adducts per 10(9) normal nucleosides, respectively. DNA analyzed 1h after treatment of rats with dimethylnitrosamine by oral gavage of 50 microg/kg b.wt. did not affect the levels of 8-oxodGuo and epsilondAdo but resulted in 200 O(6)-mdGuo adducts per 10(9) normal nucleosides. The method developed will be of use to study the biological significance of exogenous DNA adducts as an increment to background DNA damage and the role of modulating factors, such as DNA repair.     

Exposure of mice to cigarette smoke and/or light causes DNA alterations in heart and aorta

Cigarette smoke (CS) is a major risk factor for cardiovascular diseases, cancer, and other chronic degenerative diseases. UV-containing light is the most ubiquitous DNA-damaging agent existing in nature, but its possible role in cardiovascular diseases had never been suspected before, although it is known that mortality for cardiovascular diseases is increased during periods with high temperature and solar irradiation. We evaluated whether exposure of Swiss CD-1 mice to environmental CS (ECS) and UV-C-covered halogen quartz lamps, either individually or in combination, can cause DNA damage in heart and aorta cells. Nucleotide alterations were evaluated by (32)P postlabeling methods and by HPLC-electrochemical detection. The whole-body exposure of mice to ECS considerably increased the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and of bulky DNA adducts in both heart and aorta. Surprisingly, even exposure to a light that simulated solar irradiation induced oxidatively generated damage in both tissues. The genotoxic effects of UV light in internal organs is tentatively amenable to formation of unidentified long-lived mutagenic products in the skin of irradiated mice. Nucleotide alterations were even more pronounced when the mice were exposed to smoke and/or light during the first 5weeks of life rather than during adulthood for an equivalent period of time. Although the pathogenetic meaning is uncertain, DNA damage in heart and aorta may tentatively be related to cardiomyopathies and to the atherogenesis process, respectively.     

Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA

Human A549 lung epithelial cells were challenged with 18O-labeled hydrogen peroxide ([18O]-H2O2), the total RNA and DNA extracted in parallel, and analyzed for 18O-labeled 8-oxo-7,8-dihydroguanosine ([18O]-8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine ([18O]-8-oxodGuo) respectively, using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-MS/MS). [18O]-H2O2 exposure resulted in dose-response formation of both [18O]-8-oxoGuo and [18O]-8-oxodGuo and 18O-labeling of guanine in RNA was 14-25 times more common than in DNA. Kinetics of formation and subsequent removal of oxidized nucleic acids adducts were also monitored up to 24 h. The A549 showed slow turnover rates of adducts in RNA and DNA giving half-lives of approximately 12.5 h for [18O]-8-oxoGuo in RNA and 20.7 h for [18O]-8-oxodGuo in DNA, respectively.     

Inhibition of SNAP25 expression by HIV-1 Tat involves the activity of mir-128a

MicroRNAs (miRs) are short endogenous RNAs that regulate gene expression by incomplete pairing with messenger RNAs. An increasing number of studies show that mammalian microRNAs play fundamental roles in various aspects of cellular function including differentiation, proliferation, and cell death. Recent findings demonstrating the presence of microRNAs in mature neuronal dendrites suggest their possible involvement in controlling local protein translation and synaptic function. HIV-1 Encephalopathy (HIVE) is a manifestation of HIV-1 infection that often results in neuronal damage and dysfunction. While neurons are rarely, if ever, infected by HIV-1, they are exposed to cytotoxic viral and cellular factors including the HIV-1 transactivating factor Tat. In this study, we show that Tat deregulates expression levels of selected microRNAs, including the neuronal mir-128, in primary cortical neurons. We further show that mir-128a inhibits expression of the pre-synaptic protein SNAP25, whereas the anti-mir-128a partially restores Tat/mir-128a-induced downregulation of SNAP25 expression. Altogether, our data provide a novel mechanism by which HIV-Tat perturbs neuronal activity.     

Kaposi's sarcoma herpes virus taps into a host microRNA regulatory network

Kaposi's sarcoma-associated herpes virus (KSHV), the causative agent of several neoplasms, has recently been shown to encode 12 microRNAs. mir-155 is a host-encoded microRNA associated with lymphomagenesis. Two new papers provide strong evidence that a KSHV-encoded microRNA and mir-155 share a common set of mRNA targets and binding sites, implying a possible link between viral- and non-viral-mediated tumorigenesis.     

Interplay between histopathological alterations, cigarette smoke and chemopreventive agents in defining microRNA profiles in mouse lung

We have investigated alterations of microRNA expression profiles in the apparently healthy lung of mice and rats as an early response to exposure to cigarette smoke, either mainstream (MCS) or environmental, and/or to treatment with chemopreventive agents. Further on, we evaluated microRNA alterations at a later stage, when lung tumors were detectable in MCS-exposed mice. Lung samples were available from previous studies, in which strain H mice had been exposed to MCS for 4 months, starting immediately after birth, and then kept in filtered air for an additional 3 months. Some samples were from MCS-exposed mice treated either with N-acetyl-l-cysteine during pregnancy or with phenethyl isothiocyanate after weaning. The analysis of 576 mouse microRNAs showed that MCS strongly dysregulated microRNA expression and that both chemopreventive agents efficiently attenuated this trend, especially in noncancer tissue. MicroRNA expression was affected by histopathology, with specific signatures related to occurrence of pneumonia, adenoma, or bronchoalveolar carcinoma. Within pairs of samples from individual mice, microRNA analysis discriminated adenomatous tissue and especially carcinomatous tissue from the surrounding normal appearing tissue. A series of microRNA alterations characterized the sequential stages of pulmonary carcinogenesis. The involved functions included oncogene activation, inhibition of oncosuppressor genes, recruitment of undifferentiated stem cells, inflammation, inhibition of gap-junctional intercellular communications, angiogenesis, invasiveness, and metastatization. Thus, microRNA expression profiles in lung are dysregulated by MCS along all steps of the carcinogenesis process and depend on the interplay among exposure to noxious agents, treatment with dietary and pharmacological agents, and occurrence of pulmonary diseases.     

Insertion of microRNA targets into the flavivirus genome alters its highly neurovirulent phenotype

Flaviviruses such as West Nile, Japanese encephalitis, and tick-borne encephalitis (TBEV) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. Here, we demonstrate that incorporation into the viral genome of a target sequence for cellular microRNAs expressed in the central nervous system (CNS) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. As a model virus for this type of modification, we used a neurovirulent chimeric tick-borne encephalitis/dengue virus (TBEV/DEN4) that contained the structural protein genes of a highly pathogenic TBEV. The inclusion of just a single target copy for a brain tissue-expressed mir-9, mir-124a, mir-128a, mir-218, or let-7c microRNA into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in mice infected intracerebrally with a large dose of virus. Viruses bearing a complementary target for mir-9 or mir-124a were highly restricted in replication in primary neuronal cells, had limited access into the CNS of immunodeficient mice, and retained the ability to induce a strong humoral immune response in monkeys. This work suggests that microRNA targeting to control flavivirus tissue tropism and pathogenesis might represent a rational approach for virus attenuation and vaccine development.     

抑制小鼠乳腺肿瘤转移 microRNA 的筛选

目的：鉴定25种microRNA的功能及其在乳腺癌中发挥的作用,以筛选新的抑制乳腺癌转移的microRNA分子。方法利用脂质体2000将25种鼠源microRNA表达载体转染至4TO7细胞,经G418筛选结合流式细胞仪分选获绿色荧光细胞得稳定表达鼠源microRNA 的细胞株。将细胞2＆#215;105个/只尾静脉注射接种于BALB/C小鼠,14 d后解剖分离肺组织,统计小鼠肺组织结节数目。结果和接种阴性对照细胞小鼠相比,接种mir-449a稳转细胞的小鼠肿瘤肺转移减少。而接种 mir-1935稳转细胞的小鼠肿瘤肺转移增多。其它23种microRNA稳转细胞接种小鼠肿瘤肺转移既不增加也不减少。结论从25种鼠源microRNA中筛选到2种与乳腺癌肿瘤转移相关的microRNA：mir-449a抑制乳腺癌细胞肺转移,mir-1935则促进癌细胞肺转移。     

阿尔茨海默病中mir-222作用机制的初步研究

目的探讨mir-222在阿尔茨海默病发病机制中的作用。方法取6月龄APPswe／PSAE9小鼠脑组织,进行microRNA芯片的检测;利用real—timePCR对芯片检测结果进行验证;构建mir-222表达载体,将其转染至SH-SY5Y细胞中,转染后48小时提取蛋白检测p27kipl的表达情况。结果芯片结果显示,与野生型C57BL／6J小鼠相比,6月龄APPswe／PSAE9小鼠脑组织中mir-222表达明显下降,经real-timePCR验证,差别具有统计学意义（P=O．012）;将mir-222表达载体转染至SH—SY5Y细胞后,p27kipl表达减少。结论mir-222可能通过调节细胞周期抑制因子p27kipl的表达参与阿尔茨海默病的发生。     

Interplay between HIV-1 infection and host microRNAs

Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.