Aberrant expression of miR‐451a contributes to 1,2‐dichloroethane‐induced hepatic glycerol gluconeogenesis disorder by inhibiting glycerol kinase expression in NIH Swiss mice

The identification of aberrant microRNA (miRNA) expression during chemical‐induced hepatic dysfunction will lead to a better understanding of the substantial role of miRNAs in liver diseases. 1,2‐Dichloroethane (1,2‐DCE), a chlorinated organic toxicant, can lead to hepatic abnormalities in occupationally exposed populations. To explore whether aberrant miRNA expression is involved in liver abnormalities mediated by 1,2‐DCE exposure, we examined alterations in miRNA expression patterns in the livers of NIH Swiss mice after dynamic inhalation exposure to 350 or 700 mg m^–3 1,2‐DCE for 28 days. Using a microarray chip, we discovered that only mmumiR‐451a was significantly upregulated in the liver tissue of mice exposed to 700 mg m^–3 1,2‐DCE; this finding was validated by quantitative real‐time polymerase chain reaction. In vitro study revealed that it was metabolite 2‐chloroacetic acid, not 1,2‐DCE that resulted in the upregulation of mmu‐miR‐451a in the mouse AML12 cell line. Furthermore, our data showed that the upregulation of mmu‐miR‐451a induced by 2‐chloroacetic acid could suppress the expression of glycerol kinase and lead to the inhibition of glycerol gluconeogenesis in mouse liver tissue and AML12 cells. These observations provide evidence that hepatic mmu‐miR‐451a responds to 1,2‐DCE exposure and might induce glucose metabolism disorders by suppressing the glycerol gluconeogenesis process.     

Di(2‐ethylhexyl) phthalate (DEHP) influences follicular development in mice between the weaning period and maturity by interfering with ovarian development factors and microRNAs

Although studies have shown that di(2‐ethylhexyl) phthalate (DEHP) can disrupt ovarian function, few reports have focused on follicular development in mice between the weaning period and maturity, especially with respect to microRNA (miRNA) expression. In this study, 21‐day‐old ICR mice were administered DEHP at doses of 0, 100, 400, and 1600 mg/(kg d) for 6 weeks by gavage. After DEHP administration, a significant decrease in the expression of follicle development‐related factors (including c‐kit, kitl, gdf9, and atm) was observed by quantitative real‐time PCR (RT‐PCR), but no significant difference in the proteins encoded by these genes was observed by Western blot. Bisulfite sequencing suggested that the total methylation percentages of promoter regions of these genes were not notably altered after DEHP exposure. However, RT‐PCR revealed a significantly increased expression of ovarian miRNAs (let‐7b, miR‐17‐5p miR‐181a, and miR‐151), which inhibit follicular granulosa cell proliferation. Overall, this study showed that DEHP administration from weaning to maturity could suppress the mRNA expression of follicular development factors, and this effect was not achieved through changes in the methylation of DNA in CpG islands of development factors. In addition, DEHP was shown to induce miRNAs to inhibit the proliferation of follicular granulosa cells and the anti‐apoptosis function of KITL and GDF9 while increasing bax/bcl₂ expression to further promote the apoptosis of granulosa cells.     

Differential expression of miRNAs in the visceral adipose tissue of patients with non‐alcoholic fatty liver disease

Aliment Pharmacol Ther 2010; 32: 487–497

Summary

Background Progression of non‐alcoholic fatty liver disease (NAFLD) can be facilitated by soluble molecules secreted by visceral adipose tissue (VAT). MicroRNAs (miRNAs) are likely to regulate some of these molecular pathways involved in pathogenesis of NAFLD. Aim To profile miRNA expression in the visceral adipose tissue of patients with NAFLD. Methods Visceral adipose tissue samples were collected from NAFLD patients and frozen. Patients with biopsy‐proven NAFLD were divided into non‐alcoholic steatohepatitis (NASH) (n = 12) and non‐NASH (n = 12) cohorts controlled for clinical and demographic characteristics. Extracted total RNA was profiled using TaqMan Human MicroRNA arrays. Univariate Mann–Whitney comparisons and multivariate regression analysis were performed to compare miRNA profiles. Results A total of 113 miRNA differentially expressed between NASH patients and non‐NASH patients (P < 0.05). Of these, seven remained significant after multiple test correction (hsa‐miR‐132, hsa‐miR‐150, hsa‐miR‐433, hsa‐miR‐28‐3p, hsa‐miR‐511, hsa‐miR‐517a, hsa‐miR‐671). Predicted target genes for these miRNAs include insulin receptor pathway components (IGF1, IGFR13), cytokines (CCL3, IL6), ghrelin/obestatin gene, and inflammation‐related genes (NFKB1, RELB, FAS). In addition, two miRNA species, hsa‐miR‐197 and hsa‐miR‐99, were significantly associated with pericellular fibrosis in NASH patients (P < 0.05). Levels of IL‐6 in the serum negatively correlated with the expression levels of all seven miRNAs capable of down regulating IL‐6 encoding gene. Conclusions miRNA expression from VAT may contribute to the pathogenesis of NAFLD – a finding which may distinguish relatively simple steatosis from NASH. This could help identify potential targets for pharmacological treatment regimens and candidate biomarkers for NASH.     

MicroRNAs – Novel Therapeutic Targets of Eicosanoid Signalling

MicroRNAs (miRNAs) have emerged as important regulators in human physiological and pathological processes. We summarize the current knowledge about the role of miRNA involved in the control of inflammatory responses with a special focus on eicosanoid signalling. Cyclooxygenase 2 – the key enzyme of the prostanoid pathway – is regulated by different miRNAs such as miRNA‐101, miR199a, miR26b and miR‐146a. In contrast to this, the understanding of miRNA regulation on enzymes of the leukotriene biosynthesis is just at the beginning. The knowledge of miRNAs regulating enzymes of the eicosanoid pathway offers a new way for the development of new therapeutic concepts for the treatment of inflammatory diseases.     

玉麦microRNAs类活性物质的筛选及功能分析

目的 鉴定玉麦中可吸收入血的microRNAs(miRNAs)类活性物质,并分析其靶基因功能。方法 采用二代高通量测序法,以正常小鼠血浆miRNAs为空白对照,测定喂食玉麦匀浆液小鼠血浆中miRNAs的总量,并与玉麦miRNAs比对,鉴定吸收入体内的玉麦miRNAs。采用TargetScan与MiRanda软件,预测玉麦miRNAs可能作用于人类mRNA靶基因,并通过Gene Ontology(GO)与Kyoto Encyclopedia of Genes and Genomes(KEGG)分析靶基因的相关功能和信号途径。结果 共鉴定出12个可吸收入体内的玉麦miRNAs。GO与KEGG分析显示这些miRNAs可能调控的靶基因及其参与的生物学功能包括胰岛素信号通路、癌症通路、MAPK通路等相关信号通路,其中miR4995、miR156a、miR396e为玉麦可能的主要核酸活性物质。结论 miRNAs可能为玉麦中的一种新的活性物质。     

CHANGES IN microRNA (miR) PROFILE AND EFFECTS OF miR-320 IN INSULIN-RESISTANT 3T3-L1 ADIPOCYTES

• 1 MicroRNAs (miRNAs) play essential roles in many biological processes. It is known that aberrant miRNA expression contributes to some pathological conditions. However, it is not known whether miRNAs play any role in the development of insulin resistance in adipocytes, a key pathophysiological link between obesity and diabetes.
• 2 To investigate the function of miRNAs in the development of insulin resistance, using miRNA microarray analysis we compared miRNA expression profiles between normal insulin‐sensitive 3T3‐L1 adipocytes and 3T3‐L1 adipocytes rendered insulin resistant following treatment with high glucose (25 mmol/L) and high insulin (1 µmol/L). Furthermore, adipocytes were transfected with specific antisense oligonucleotides against miRNA‐320 (anti‐miR‐320 oligo) and the effects on the development of insulin resistance were evaluated.
• 3 We identified 50 upregulated and 29 downregulated miRNAs in insulin‐resistant (IR) adipocytes, including a 50‐fold increase in miRNA‐320 (miR‐320) expression. Using bioinformatic techniques, the p85 subunit of phosphatidylinositol 3‐kinase (PI3‐K) was found to be a potential target of miR‐320. In experiments with anti‐miR‐320 oligo, insulin sensitivity was increased in IR adipocytes, as evidenced by increases in p85 expression, phosphorylation of Akt and the protein expression of the glucose transporter GLUT‐4, as well as insulin‐stimulated glucose uptake. These beneficial effects of anti‐miR‐320 oligo were observed only in IR adipocytes and not in normal adipocytes.
• 4 In conclusion, the miRNA profile changes in IR adipocytes compared with normal 3T3‐L1 adipocytes. Anti‐miR‐320 oligo was found to regulate insulin resistance in adipocytes by improving insulin–PI3‐K signalling pathways. The findings provide information regarding a potentially new therapeutic strategy to control insulin resistance.

     

Different regulation of miR‐29a‐3p in glomeruli and tubules in an experimental model of angiotensin II‐dependent hypertension: potential role in renal fibrosis

The aim of this study was to evaluate the role of the angiotensin II (Ang II) induced‐differential miRNA expression in renal glomerular and tubulo‐interstitial fibrosis in an experimental model of Ang II‐dependent hypertension. To clarify this issue, Sprague Dawley rats were treated with Ang II (200 ng/kg per minute, n = 15) or physiological saline (n = 14) for 4 weeks. Systolic blood pressure and albuminuria were measured every 2 weeks. At the end of the experimental period, renal glomerular and tubulo‐interstitial fibrosis was evaluated by histomorphometric analysis, after Sirius‐Red and Masson's trichrome staining. Ang II increased systolic blood pressure (P < 0.0001), albuminuria (P < 0.01) and both glomerular and tubulo‐interstitial fibrosis (P < 0.01). Using laser capture microdissection and miRNA microarray analysis this study showed that miR‐29a‐3p was down‐regulated in renal tubules and up‐regulated in glomeruli. Real‐time polymerase chain reaction (PCR) experiments confirmed in Ang II‐treated rats a down‐regulation of miR‐29a‐3p in tubules (P < 0.01), while no significant changes were observed in glomeruli. Matrix metalloproteinase‐2 (MMP‐2) was identified as putative miR‐29a‐3p target (by TargetScan, miRanda, Tarbase software) and functionally confirmed by luciferase activity assay. These data demonstrate that the effects of Ang II on miR‐29a‐3p expression in renal tubules is different from the one exerted in the glomeruli and that miR‐29a‐3p targets MMP‐2. These results suggest that the development of renal fibrosis at glomerular and tubulo‐interstitial level depends on different molecular mechanisms.     

A systematic review of microRNA expression studies with exposure to bisphenol A

Bisphenol A (BPA), as a common industrial component, is generally consumed in the synthesis of polymeric materials. To gain a deeper understanding of the detrimental effects of BPA, BPA‐induced microRNA (miRNA) alterations were investigated. A systematic search was performed in the PubMed, SCOPUS and Web of Science databases to evoke relevant published data up to August 10, 2019. We identified altered miRNAs that have been repeated in at least three studies. Moreover, miRNA homology analysis between human and nonhuman species was performed to determine the toxicity signatures of BPA in human exposure. In addition, to reflect the effects of environmental exposure levels of BPA, the study designs were categorized into two groups, including low and high doses according to the previous definitions. In total, 28 studies encountered our criteria and 17 miRNAs were identified that were differentially expressed in at least three independent studies. Upregulating miR‐146a and downregulating miR‐192, miR‐134, miR‐27b and miR‐324 were found in three studies. MiR‐122 and miR‐29a were upregulated in four studies after BPA exposure, and miR‐21 was upregulated in six studies. The results indicate that BPA at low‐level exposures can also alter miRNA expression in response to toxicity. Finally, the miRNA‐related pathways showed that BPA seriously can affect human health through various cell signaling pathways, which were predictable and consistent with existing studies. Overall, our findings suggest that further studies should be conducted to examine the role of miRNA level changes in human BPA exposure .     

CYP2E1 and miRNA‐378a‐3p contribute to acetaminophen‐ or tripterygium glycosides‐induced hepatotoxicity

Increased expression of CYP2E1 may represent the main factor contributing to oxidative stress‐mediated liver damage in drug‐induced liver injury (DILI). However, the regulation mechanism of CYP2E1 expression is poorly described. The present study was aimed to investigate the role of CYP2E1 in acetaminophen (APAP)‐ or tripterygium glycosides (TG)‐induced hepatotoxicity as well as the regulation of CYP2E1 and miR‐378a‐3p expression by APAP or TG. Rats were randomly divided and treated with APAP, TG, chlormethiazole (CMZ), APAP + CMZ and TG + CMZ, respectively, for 4 weeks. Then, blood and liver samples were collected. Serum and hepatic biochemical parameters were measured using commercial kits. Liver histopathology was tested by H&E staining. Expression levels of CYP2E1 mRNA and miR‐378a‐3p were detected by qRT‐PCR. CYP2E1 protein expression was determined by Western blot. Our results showed that CMZ effectively restored the hepatic histopathological changes, oxidative stress biomarkers and TNF‐α levels induced by APAP or TG. CYP2E1 mRNA and/or protein expression levels were dramatically increased after chronic APAP or TG treatment, while this induction was significantly reversed by CMZ co‐treatment. Of note, miR‐378a‐3p expression levels were significantly suppressed after APAP, TG and/or CMZ treatment. These results suggested that CYP2E1 were highly induced after chronic APAP or TG treatment, which in turn play an important role in APAP‐ or TG‐induced hepatotoxicity. These inductions of CYP2E1 expression were probably carried out by inhibition of miR‐378a‐3p. Our findings might provide a new molecular basis for DILI.     

神经母细胞瘤血浆外泌体差异表达miRNA靶基因预测及生物信息学分析

目的通过生物信息学分析筛选出神经母细胞瘤外周血血浆外泌体中差异表达miRNA,并对其靶基因功能进行分析预测。方法从高通量基因表达数据库下载数据集GSE128004,分析神经母细胞瘤血浆外泌体中miRNA的差异表达;通过miRTarBase数据库筛选差异表达miRNA的靶基因;进一步通过运用clusterProfiler进行靶基因的基因本体(GO)功能富集与京都基因与基因组百科全书数据库(KEGG)通路富集。结果经筛选发现,数据集GSE128004包含41个表达差异在2倍以上的血浆外泌体miRNA。靶基因预测显示,hsa-miR-199a-3p, hsa-miR-196b-5p, hsa-miR-127-3p, hsa-miR-410-3p和hsa-miR-487b-3p为靶基因最多的前5个差异表达miRNA。GO功能分析发现,这些靶基因大都在细胞运动正调节、细胞迁移正调节、血管生成等生物过程富集;在膜侧、细胞-基底连接、细胞-基底黏附连接、焦点黏连等细胞组成富集;在蛋白丝氨酸/苏氨酸激酶活性、蛋白异二聚体化活性、转录因子活性和RNA聚合酶Ⅱ核心启动子近端区序列特异性结合等分子功能富集。KEGG分析显示:这些差异表达miRNA的靶基因主要参与磷脂酰肌醇3激酶-蛋白激酶B信号通路、癌症相关miRNA、丝裂原活化蛋白激酶信号通路等通路富集。结论 hsa-miR-199a-3p, hsa-miR-127-3p和hsa-miR-410-3p可能作为神经母细胞瘤的潜在生物标志物或治疗靶标,进一步为该病的发病机制提供研究思路。     

MicroRNA‐652‐3p promotes the proliferation and invasion of the trophoblast HTR‐8/SVneo cell line by targeting homeobox A9 to modulate the expression of ephrin receptor B4

MicroRNAs (miRNAs) are emerging as novel modulators in the pathogenesis of preeclampsia (PE). Multiple miRNAs have been shown to regulate the proliferation and invasion of trophoblast cells, which play a critical role in successful pregnancies. miR‐652‐3p has been identified as a novel disease‐associated miRNA that is dysregulated in various pathological processes. However, whether miR‐652‐3p is dysregulated in PE and regulates the cellular function of trophoblast cells remains unknown. In the present study, we aimed to investigate the expression pattern of miR‐652‐3p in PE and explore its potential function in trophoblast cells. Herein, we found that miR‐652‐3p expression was significantly decreased in the placental tissues of pregnant women with PE. Cellular function experiments showed that overexpression of miR‐652‐3p promoted the viability, proliferation, and invasion of trophoblast cells in vitro. By contrast, inhibition of miR‐652‐3p had the opposite effect. Bioinformatics analysis predicted that homeobox A9 (HOXA9), a crucial regulator of trophoblast cell function, was a potential target gene of miR‐652‐3p. A luciferase reporter assay confirmed that miR‐652‐3p directly interacted with the 3′‐untranslated region of HOXA9. Moreover, miR‐652‐3p was shown to negatively regulate the expression of HOXA9 and ephrin receptor B4 (EphB4) in trophoblast cells. Notably, overexpression of HOXA9 or EphB4 significantly reversed the regulatory effect of miR‐652‐3p on proliferation and invasion of trophoblast cells. Taken together, our findings demonstrate that miR‐652‐3p regulates the proliferation and invasion of trophoblast cells, possibly through targeting HOXA9 and modulating EphB4 expression.     

mRNAs and miRNAs in whole blood associated with lung hyperplasia,fibrosis, and bronchiolo‐alveolar adenoma and adenocarcinoma after multi‐walled carbon nanotube inhalation exposure in mice

Inhalation exposure to multi‐walled carbon nanotubes (MWCNT) in mice results in inflammation, fibrosis and the promotion of lung adenocarcinoma; however, the molecular basis behind these pathologies is unknown. This study determined global mRNA and miRNA profiles in whole blood from mice exposed by inhalation to MWCNT that correlated with the presence of lung hyperplasia, fibrosis, and bronchiolo‐alveolar adenoma and adenocarcinoma. Six‐week‐old, male, B6C3F1 mice received a single intraperitoneal injection of either the DNA‐damaging agent methylcholanthrene (MCA, 10 µg g^?1 body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg m^?3, 5 hours per day, 5 days per week) or filtered air (control) for a total of 15 days. At 17 months post‐exposure, mice were euthanized and examined for the development of pathological changes in the lung, and whole blood was collected and analyzed using microarray analysis for global mRNA and miRNA expression. Numerous mRNAs and miRNAs in the blood were significantly up‐ or down‐regulated in animals developing pathological changes in the lung after MCA/corn oil administration followed by MWCNT/air inhalation, including fcrl5 and miR‐122‐5p in the presence of hyperplasia, mthfd2 and miR‐206‐3p in the presence of fibrosis, fam178a and miR‐130a‐3p in the presence of bronchiolo‐alveolar adenoma, and il7r and miR‐210‐3p in the presence of bronchiolo‐alveolar adenocarcinoma, among others. The changes in miRNA and mRNA expression, and their respective regulatory networks, identified in this study may potentially serve as blood biomarkers for MWCNT‐induced lung pathological changes. Copyright © 2015 John Wiley & Sons, Ltd.     

MiR‐21‐3p modulates lipopolysaccharide‐induced inflammation and apoptosis via targeting TGS4 in retinal pigment epithelial cells

Age‐related macular degeneration (AMD) is a major reason of blindness in the elderly. MicroRNAs are implicated in various pathological processes, including inflammation and apoptosis. In this study, we aim to investigate the biological functions of miR‐21‐3p in inflammation and apoptosis caused by lipopolysaccharide (LPS) in human retinal pigment epithelial (ARPE‐19) cells. The miR‐21‐3p inhibitor and mimic were transfected into ARPE‐19 cells for 48 hours, followed by exposed to LPS (10 μg/mL) for 24 hours. The mRNA and protein expression of IL‐6 and MCP‐1 were measured using real‐time PCR (RT‐PCR) and enzyme‐linked immunosorbent assays. Cell viability, apoptosis, caspase 3 activity, cleaved caspase‐3 and cleaved‐PARP protein levels were detected to evaluate the effects of miR‐21‐3p on apoptosis. Additionally, the target relationship between miR‐21‐3p and regulator of G‐protein signalling 4 (RGS4) was verified by dual luciferase reporter assay. RT‐PCR analysis demonstrated that LPS induced miR‐21‐3p expression. Inhibition of miR‐21‐3p reduced the mRNA and protein levels of IL‐6 and MCP‐1. Apoptosis, caspase‐3 activity, and cleaved‐caspase 3 and cleaved PARP protein levels were repressed by the miR‐21‐3p inhibitor. However, overexpression of miR‐21‐3p showed the opposite results. Furthermore, we identified that miR‐21‐3p directly targeted the 3′ untranslated region of RGS4. MiR‐21‐3p negatively regulated the expression of RGS4 both in mRNA and protein levels. Silencing RGS4 reduced the anti‐inflammatory and anti‐apoptotic effects of miR‐21‐3p inhibitor. Our results revealed that miR‐21‐3p inhibition targeted RGS4 to attenuate inflammatory responses and apoptosis caused by LPS in ARPE‐19 cells.