microRNA在小鼠乳腺不同发育时期差异表达谱及作用

microRNA是一类大小约22个核苷酸的非编码RNA分子,是一种广泛存在的对基因表达进行微调的分子。microRNA可以通过与靶基因mRNA的特定位点结合,抑制该蛋白的合成或诱导该mRNA的降解,从而参与基因的表达调控。一般来源于染色体的非编码区域,由大约70个核苷酸大小的可形成发夹结构的前体经Dicer酶加工而来。这类小RNA在表达上具有组织和时间的特异性,是调节其他功能基因表达的重要调控分子,在生物的生长发育过程中发挥着重要作用。因此,虽然microRNA的研究仅有很短的历史,但已成为基因表达调控研究的热点领域。以中国昆明小鼠不同发育时期的乳腺组织为实验材料,应用芯片技术及荧光定量PCR技术,分析发育不同时期的乳腺组织microRNA差异表达图谱。本文研究发现microRNA在乳腺不同的发育时期表达图谱不同;与青春期、退化期比较,妊娠期、哺乳期有十余种microRNAs表达上调,20余种microRNAs表达下调;microRNAs在乳腺发育和泌乳周期中发挥重要的作用。     

MicroRNA Profiles in Spontaneous Decidualized Menstrual Endometrium and Early Pregnancy Decidua with Successfully Implanted Embryos

To comparatively analyze the human microRNA (miRNA) profiles between spontaneous decidualized menstrual endometrium and early pregnancy decidua by an in-depth sequencing of miRNAs. The specific miRNAs expressed at conception might be involved in pregnancy establishment and expression of let-7f-5p and let-7g-5p was experimentally up-regulated or inhibited to assess the effect on the expression of IGF2BP-1 and IGF2R in vitro, respectively. Samples of endometria and deciduas were obtained from 25 women who suffered from tubal or male factor subfertility and from 35 early pregnant women who underwent pregnancy termination at 6–8 weeks gestation were irrespectively collected and comparatively analyzed by miRNA sequencing and differential expression of known and novel miRNAs was analyzed using bioinformatics. The 2042 miRNA expression was analyzed in the study and the differential expression of six miRNAs was validated by qRT-PCR. The expression of four miRNAs in decidua samples was down-regulated (miR-34c, miR-92a, miR-181a-5p, and miR-191), whereas the expression of miR-10a-5p and let-7f-5p was significantly up-regulated. The expression of IGF2BP-1 and IGF2R declined and increased with overexpression and inhibition of let-7f-5p and let-7g-5p, respectively. Changes in the expression of particular miRNAs might play a role in the physiology of decidualization following successful embryo implantation, ultimately resulting in continuous decidualization.     

High dietary intake of medium-chain fatty acids during pregnancy in rats prevents later-life obesity in their offspring

We investigated the effects of dietary fatty acids of different chain lengths during pregnancy in the rat on the susceptibility of offspring to later-life obesity and the underlying mechanisms. Pregnant rats were fed three different diets: standard (STD), high medium-chain fatty acids (MCFA); and high long-chain fatty acids (LCFA). The male offspring were assigned to three groups: STD control, MCFA and LCFA according to the maternal diets and suckled by dams fed with STD during pregnancy and lactation. After weaning, the offspring were fed with STD from 3 to 8 weeks of age. At the age of 8 weeks, rats in three groups: high-fat diet (HFD) control, MCFA and LCFA were fed with HFD until 14 weeks of age in an attempt to induce obesity, and rats in the HFD control group were selected randomly from the STD control group. Body weight and body fat content were decreased in the MCFA group accompanied by down-regulated mRNA expression of fatty acid synthase and acetyl-coA carboxylase 1, and increased mRNA and protein expression of adenosine monophosphate (AMP)-activated protein kinase (AMPK), carnitine palmitoyltransferase 1 and uncoupling protein 3 compared with the corresponding controls at 3, 8 and 14 weeks of age. The results suggested that the MCFA diet during pregnancy prevented later-life obesity in the offspring when they were exposed to HFD in later life, which might be related to programming of the expression of genes involved in fatty acid metabolism.     

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.     

Sox9-Regulated miRNA-574-3p Inhibits Chondrogenic Differentiation of Mesenchymal Stem Cells

The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.     

Triglyceridemia and peroxisome proliferator- activated receptor-α expression are not connected in fenofibrate-treated pregnant rats

To investigate the response to fenofibrate in pregnant rats, 0 mg, 100 mg or 200 mg of fenofibrate per kilogram body weight oral doses were given twice a day from day 16 of gestation and studied at day 20. Virgin rats were studied in parallel. Whereas in pregnant rats plasma triglycerides significantly increased, in virgin rats, fenofibrate decreased plasma triglycerides which accumulated in liver. Fenofibrate faithfully modulated the hepatic expression of PPAR responsive genes. Fenofibrate increased mRNA contents corresponding to both acyl-CoA oxidase, carnitine palmitoyltransferase (CPT), and peroxisome proliferator-activated receptor alpha (PPAR), and lowered mRNA amounts of apolipoproteins B and C-III, both in virgin and pregnant rats. However, genes related to hepatic lipogenesis, such as PPAR and stearoyl-CoA desaturase (SCD), showed an augmented expression by fenofibrate in virgin rats, but not in pregnant animals. We propose that the opposite effects of fenofibrate treatment in virgin and pregnant rats are a consequence of the enhanced capability for VLDL-triglyceride production in the latter, further promoted by the elevated amount of free fatty acids (FFA), which reach the liver in treated pregnant rats and were not sufficiently oxidized and/or stored, and therefore would have to be canalized as triglycerides to the plasma. Thus, the present study shows how fenofibrate, in spite of efficiently exerting its expected molecular effects in the liver (i.e., to induce fatty acid and lipoprotein catabolism, and to reduce TG-rich lipoprotein secretion), was unable to reverse the typical hypertriglyceridaemia of gestation.     

High fat diet induced downregulation of microRNA-467b increased lipoprotein lipase in hepatic steatosis

Non-alcoholic fatty liver disease (NAFLD) is characterized by hepatic fat accumulation and is presently the most common chronic liver disease. However, the mechanisms underlying the development of steatosis remain unclear. MicroRNAs (miRNAs) are small non-coding RNAs that modulate a variety of biological functions. We have investigated the role of miRNA in the development of steatosis. We found that miR-467b expression is significantly downregulated in liver tissues of high-fat diet fed mice and in steatosis-induced hepatocytes. The downregulation of miR-467b resulted in the upregulation of hepatic lipoprotein lipase (LPL), the direct target of miR-467b. Moreover, the interaction between miR-467b and LPL was associated with insulin resistance, a major cause of NAFLD. These results suggest that downregulation of miR-467b is involved in the development of hepatic steatosis by modulating the expression of its target, LPL.     

Temperature during early development has long-term effects on microRNA expression in Atlantic cod

Background

Environmental temperature has serious implications in life cycle of aquatic ectotherms. Understanding the molecular mechanisms of temperature acclimation and adaptation of marine organisms is of the uttermost importance for ecology, fisheries, and aquaculture, as it allows modeling the effects of global warming on population dynamics. Regulatory molecules are major modulators of acclimation and adaptation; among them, microRNAs (miRNAs) are versatile and substantial contributors to regulatory networks of development and adaptive plasticity. However, their role in thermal plasticity is poorly known. We have asked whether the temperature and its shift during the early ontogeny (embryonic and larval development) affect the miRNA repertoire of Atlantic cod (Gadus morhua), and if thermal experience has long-term consequences in the miRNA profile.

Results

We characterized miRNA during different developmental stages and in juvenile tissues using next generation sequencing. We identified 389 putative miRNA precursor loci, 120 novel precursor miRNAs, and 281 mature miRNAs. Some miRNAs showed stage- or tissue-enriched expression and miRNAs, such as the miR-17 ~ 92 cluster, myomiRs (miR-206), neuromiRs (miR-9, miR-124), miR-130b, and miR-430 showed differential expression in different temperature regimes. Long-term effect of embryonic incubation temperature was revealed on expression of some miRNAs in juvenile pituitary (miR-449), gonad (miR-27c, miR-30c, and miR-200a), and liver (let-7 h, miR-7a, miR-22, miR-34c, miR-132a, miR-192, miR-221, miR-451, miR-2188, and miR-7550), but not in brain. Some of differentially expressed miRNAs in the liver were confirmed using LNA-based rt-qPCR. The effect of temperature on methylation status of selected miRNA promoter regions was mostly inconclusive.

Conclusions

Temperature elevation by several degrees during embryonic and larval developmental stages significantly alters the miRNA profile, both short-term and long-term. Our results suggest that a further rise in seas temperature might affect life history of Atlantic cod.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1503-7) contains supplementary material, which is available to authorized users.     

Effect of maternal diet rich in omega‐6 and omega‐9 fatty acids on the liver of LDL receptor‐deficient mouse offspring

BACKGROUND: Omega‐6 fatty acids are important to fetal development. However, during gestation/lactation, these fatty acids may contribute toward the development of fat tissue. Omega‐9 fatty acids are associated with a reduction in serum lipids and protection from liver disease. OBJECTIVES: The present study investigated the effect of the maternal intake of omega‐6 and omega‐9 in hypercholesterolemic mothers on the liver of the offspring. METHODS: LDL receptor–deficient mice were fed a diet rich in either omega‐6 (E6D) or omega‐9 (E9D) for 45 days prior to mating and until the birth of the offspring, evaluating the effect on the offspring liver in comparison to a standard diet (STD). RESULTS: Mothers fed with the E6D experienced an increase in total cholesterol (TC) and the offspring exhibited an increase in TC, hepatic triglycerides (TG), and CC‐chemokine ligand (CCL)2/monocyte chemoattractant protein (MCP)‐1 as well as a reduction in HDL. Histological analysis on this group revealed steatosis, leukocyte infiltrate, and increased CCL2/MCP‐1 expression. The ultrastructural analysis revealed hepatocytes with lipid droplets and myofibroblasts. The offspring of mothers fed the standard diet exhibited low serum TC, but microvesicular steatosis was observed. The offspring of mothers fed the E9D exhibited lower serum and hepatic TG as well as higher LDL in comparison to the other diets. The histological analyses revealed lower steatosis and leukocyte infiltrate. CONCLUSIONS: The findings suggest that hypercholesterolemic mothers with a diet rich in omega‐6 fatty acids predispose their offspring to steatohepatitis, whereas a diet rich in omega‐9 has a protective effect. Birth Defects Res (Part B) 89:164–170, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of dietary n-3 PUFA levels in early life on susceptibility to high-fat-diet-induced metabolic syndrome in adult mice

The maternal nutritional status during pregnancy and lactation was closely related to the growth and development of the fetus and infants, which had a profound impact on the health of the offspring. N-3 polyunsaturated fatty acid (PUFA) had been proved to have beneficial effects on glucolipid metabolism. However, the effects of dietary different n-3 PUFA levels for mother during pregnancy and lactation on susceptibility to high-fat-diet-induced metabolic syndrome for offspring in adulthood are still unclear. The maternal mice were fed with control, n-3 PUFA-deficient or fish oil-contained n-3 PUFA-rich diets during pregnancy and lactation, and the weaned offspring were fed with high-fat or low-fat diet for 13 weeks, then were subjected to oral glucose tolerance tests. The results showed that dietary n-3 PUFA-deficiency in early life could aggravate the high-fat-diet-induced glucolipid metabolism disorders, including glucose intolerance, insulin resistance, obesity, and dyslipidemia, thus increased the susceptibility to metabolic syndrome of adult mice. Notably, nutritional supplementation with n-3 PUFA in early life could significantly alleviate the glucose metabolism disorders by increasing insulin sensitivity, inhibiting gluconeogenesis and promoting glycogenesis. In addition, administration with n-3 PUFA in early life remarkably reduced serum and hepatic lipid profiles by mediating the expression of genes related to lipogenesis and β-oxidation of fatty acids. Dietary n-3 PUFA-deficiency in early life increases the susceptibility to metabolic syndrome of adult offspring, and nutritional supplementation with n-3 PUFA enhances the tolerance to a high-fat diet of adult offspring.     

Effect of starvation on nutrition and insulin secretion in pregnant rats and their fetuses

Pregnancy is thought to create a metabolic condition of accelerated starvation. To clarify this idea, the effect of fasting on pregnant rats (day 21 of gestation) and their fetuses was examined. Although pregnancy significantly increased plasma insulin, plasma ketone body concentrations in fed pregnant rats were higher than those of age-matched fed virgin rats. After 48 hr fasting (i.e., fasting during days 19-21 of gestation), plasma insulin was markedly decreased in virgin rats compared with term pregnant rats, while ketone bodies were significantly higher in pregnant rats than in virgin rats. Body weight was lower in fetuses from fasted mothers than those from fed mothers. Starvation also markedly diminished the insulin response to glucose in isolated, perfused pancreases in both virgin and pregnant rats. The amount of insulin released during glucose stimulation was greater in pregnancy, and the inhibitory effect of 48 hr fasting on insulin release was greater in virgin rats than in pregnant rats. It is possible, therefore, that in term pregnant rats a decrease in insulin release caused by fasting may cause more profound catabolism than in nongravid rats.     

Growth and development of offspring following supplementation of sow diets with oil during early to mid gestation

The role of dietary fat during early pregnancy in sows has not yet been fully established. The aim of the study was to determine the consequences of altering the fatty acid profile of sow diets during the first half of gestation; oils of different fatty acid composition were chosen as energy supplements to provide diets with different fatty acid profiles. A group of 48 multiparous sows were used to evaluate the effects of supplemental feeding during the first 60 days of gestation (term ≈ 115 days). Sows were allocated (eight per treatment) to either 3 kg/day of commercial sow pellets (control; C) or an experimental diet consisting of 3 kg/day of commercial sow pellets supplemented with 10% extra energy in the form of excess pellets (E), palm oil (P), olive oil (O), sunflower oil (S) or fish oil (F). Differential effects were observed with respect to the fatty acid profile of the diet during the first half of gestation. P sows gave birth to the largest litters. Both P and O supplementation of the maternal diet resulted in heavier piglets at birth, after correction for differences in litter size. P piglets possessed the most fat at birth and remained fatter throughout the pre-weaning period; in contrast, the offspring of O sows contained the least fat throughout life (0 to 140 days of age). The offspring of F sows exhibited improved growth performance during the neonatal period. In conclusion, altering the fatty acid profile of sow diets during the first half of gestation has long-term consequences for the growth and development of their offspring.     

Differentially Expressed MicroRNAs in Postpartum Breast Cancer in Hispanic Women

The risk of breast cancer transiently increases immediately following pregnancy; peaking between 3-7 years. The biology that underlies this risk window and the effect on the natural history of the disease is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that have been shown to be dysregulated in breast cancer. We conducted miRNA profiling of 56 tumors from a case series of multiparous Hispanic women and assessed the pattern of expression by time since last full-term pregnancy. A data-driven splitting analysis on the pattern of 355 miRNAs separated the case series into two groups: a) an early group representing women diagnosed with breast cancer ≤ 5.2 years postpartum (n = 12), and b) a late group representing women diagnosed with breast cancer ≥ 5.3 years postpartum (n = 44). We identified 15 miRNAs with significant differential expression between the early and late postpartum groups; 60% of these miRNAs are encoded on the X chromosome. Ten miRNAs had a two-fold or higher difference in expression with miR-138, miR-660, miR-31, miR-135b, miR-17, miR-454, and miR-934 overexpressed in the early versus the late group; while miR-892a, miR-199a-5p, and miR-542-5p were underexpressed in the early versus the late postpartum group. The DNA methylation of three out of five tested miRNAs (miR-31, miR-135b, and miR-138) was lower in the early versus late postpartum group, and negatively correlated with miRNA expression. Here we show that miRNAs are differentially expressed and differentially methylated between tumors of the early versus late postpartum, suggesting that potential differences in epigenetic dysfunction may be operative in postpartum breast cancers.     

Gene pathways associated with mitochondrial function,oxidative stress and telomere length are differentially expressed in the liver of rats fed lifelong on virgin olive,sunflower or fish oils

This study investigates the effect of lifelong intake of different fat sources rich in monounsaturated (virgin olive oil), n6 polyunsaturated (sunflower oil) or n3 polyunsaturated (fish oil) fatty acids in the aged liver. Male Wistar rats fed lifelong on diets differing in the fat source were killed at 6 and at 24 months of age. Liver histopathology, mitochondrial ultrastructure, biogenesis, oxidative stress, mitochondrial electron transport chain, relative telomere length and gene expression profiles were studied. Aging led to lipid accumulation in the liver. Virgin olive oil led to the lowest oxidation and ultrastructural alterations. Sunflower oil induced fibrosis, ultrastructural alterations and high oxidation. Fish oil intensified oxidation associated with age, lowered electron transport chain activity and enhanced the relative telomere length. Gene expression changes associated with age in animals fed virgin olive oil and fish oil were related mostly to mitochondrial function and oxidative stress pathways, followed by cell cycle and telomere length control. Sunflower oil avoided gene expression changes related to age. According to the results, virgin olive oil might be considered the dietary fat source that best preserves the liver during the aging process.     

MicroRNA signature of air pollution exposure-induced congenital defects

Air pollution exposure has been increasing extensively and there are evidence suggesting that exposure to air pollution during pregnancy can lead to congenital defects in the offspring. Recent findings suggested that microRNAs (miRNAs) might play important roles in the pathogenesis of developmental defects. However, the miRNAs profile pattern in the air pollution-exposed embryos remains unknown. RNA sequencing was performed to determine the differentially expressed miRNAs in the rat embryos (gestation day 9) with or without air pollution exposure. The potential functions and the associated mechanisms of these differentially expressed miRNAs were determined using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analyses. The regulatory networks of mRNA–miRNA interactions were also reconstructed. As compared with the control group, a total of 291 miRNAs were differentially expressed in the rat embryos from the air pollution-exposed group, in which 204 and 87 miRNAs were significantly downregulated and upregulated, respectively. These miRNAs were predicted to deregulate mitotic spindle organization, cellular respiration, glycolate metabolism, and proteasome. Extensive regulation of target genes by miR-346, miR-504, miR-214-3p and miR-1224 was also predicted. Our results suggested that miRNAs may play crucial roles in the pathogenesis of air pollution-induced congenital spinal defects through deregulating multiple biological processes.