Aberrant expression of miRNA-192-5p contributes to N,N-dimethylformamide-induced hepatic apoptosis

Excessive exposure to N,N-dimethylformamide (DMF) can lead to occupational liver poisoning in workers; however, the underlying mechanism is not fully clarified. The importance of microRNAs (miRNAs) in chemical-induced hepatotoxicity has been demonstrated. To determine whether miRNAs are also involved in DMF-induced hepatotoxicity, we systematically analyzed the miRNA expression profiles in DMF-treated (75 and 150 mm ) HL-7702 liver cells and controls by high-throughput sequencing. Among the altered miRNAs, miR-192-5p was the most significantly upregulated in HL-7702 cells after DMF exposure and was involved in DMF-mediated cell apoptosis. By contrast, suppression of miR-192-5p in HL-7702 cells attenuated the apoptosis induced by DMF. Furthermore, the anti-apoptotic gene (NIN1/RPN12 binding protein 1 homolog [NOB1]) was predicted to be a potential miR-192-5p target according to bioinformatics analysis. The direct interaction between miR-192-5p and NOB1 was confirmed by the dual-luciferase activity assay in HEK293FT cells. Overexpression of miR-192-5p efficiently reduced NOB1 mRNA and protein expression in HL-7702 cells. Alteration in NOB1 expression influenced DMF-induced hepatotoxicity by affecting hepatic apoptosis. In addition, the inverse correlation between miR-192-5p expression levels and NOB1 expression was further confirmed in DMF-exposed mouse liver tissue samples. These observations demonstrated that promotion of apoptosis from the suppression of NOB1 by miR-192-5p overexpression was responsible for the DMF-induced hepatotoxicity. This work provides the molecular mechanism at the miRNA level for hepatic apoptosis induced by DMF.     

The role of ROS in microcystin-LR-induced hepatocyte apoptosis and liver injury in mice

Microcystin-LR (MC-LR) produced by cyanobacteria in diverse water systems is a potent specific hepatotoxin and has been documented to induce hepatocyte apoptosis and liver injury; however, the mechanisms have not been fully elucidated. In the present study, we investigated whether MC-LR stimulated ROS generation in the liver of mice and the role of ROS in the pathogenesis of MC-LR-induced liver injury in vivo. MC-LR treatment (60 microg/kg of body weight) for 12h prompted large amount of ROS generation in mice liver, upregulated the expression of Bax and Bid, caused the mitochondrial membrane potential (MMP) loss and hepatocyte apoptosis as well as liver injury. While pretreatment with antioxidants, oral administration of vitamin C (250mg/kg of body weight, dissolved in double distill water) and vitamin E (200mg/kg of body weight, dissolved in corn oil) per day for 3 days continually, significantly reduced the generation of ROS and effectively inhibited the MC-LR-induced hepatocyte apoptosis and liver injury, suggesting that ROS played a critical role in MC-LR-induced hepatocyte apoptosis and liver injury. The protective effect of vitamin C and E also suggested the potential interest in the clinical treatment of MC-LR-induced liver injury and hepatotoxicity.     

鼠异种心脏移植miRNA表达谱分析*

【摘要】 目的 研究小鼠-大鼠异种异位心脏移植后供心miRNA表达谱的变化,为有效控制异种移植排斥反应奠定实验基础。方法 采用改良Cuff袖套法建立小鼠-大鼠异位心脏移植模型,分为同系对照组、异种24h组和异种停跳组。通过miRNA芯片杂交筛选出异种心脏移植排斥反应中显著差异表达的miRNA,选取芯片结果中差异表达显著的miR-146a和miR-451进行相对定量研究,通过TaqMan miRNA Assays技术验证芯片结果。结果 异种24h组与同系对照组比较有24个miRNA差异表达显著,其中11个下调,13个上调。异种停跳组与同系对照组比较有25个miRNA差异表达显著,其中12个下调,13个上调。异种组的miR-146a表达水平高于同系对照组,miR-451表达水平低于同系对照组(F分别为15.530、13431.6,均P<0.05)。结论 在小鼠-大鼠异种心脏移植排斥反应中出现多个miRNA显著差异表达,其中miR-146a高表达,miR-451低表达,提示miRNA在异种心脏移植排斥反应中发挥着重要的调控作用。     

Circulating microRNA profiles altered in mice after 28 d exposure to perfluorooctanoic acid

Perfluorooctanoic acid (PFOA) is a stable man-made compound with many industrial and commercial uses. Recently, however, concern has been raised that it may induce various toxicological effects such as hepatotoxicity, immunotoxicity, and developmental toxicity. Because levels of circulating microRNAs (miRNAs) can be altered in several clinical diseases, they may serve as potential novel biomarkers. Here, we explored differences in the profiles of circulating miRNAs in mice after PFOA exposure. Using TaqMan miRNA arrays, we determined that the levels of 24 circulating miRNAs were altered in mice dosed with PFOA at 1.25 mg/kg/d and 73 were altered in mice dosed with 5 mg/kg/d. Eight miRNAs were further validated using TaqMan Real-Time PCR assays. Results were consistent with those obtained from the TaqMan miRNA arrays, except for miR-199a-3p. The most remarkable of the circulating miRNAs (miR-26b-5p and miR-199a-3p) were also up-regulated in the serum of occupational workers in our previous epidemiological study. We also found similar patterns in mice exposed to PFOS. These results demonstrated that circulating miRNA profiles were altered after exposure to high concentrations of PFOA and miR-28-5p, miR-32-5p, miR-122-5p, miR-192-5p, and miR-26b-5p in serum may be linked to effects of PFOA, especially in occupationally exposed people.     

Microcystin-LR and embryo-larval development of medaka fish, Oryzias latipes. I. Effects on the digestive tract and associated systems.

The cyanobacterial hepatotoxin microcystin-LR (MC-LR) is a specific potent PP1 and PP2A protein phosphatase inhibitor. In view to obtain an integrated whole-body, understanding of the key target organs of MC-LR subsequent to embryonic exposure on the anatomy of medaka fish hatchlings, embryos at stage 19 were microinjected with a sublethal dose of MC-LR corresponding to 0.2 pg/vitellus. MC-LR-induced histo-pathological modifications of the alimentary system (i.e. digestive tract, pancreas, liver) were analysed in newly hatched embryos. Our data are indicative of an MC-LR-induced inhibition of both yolk sac resorption and gas concentrating swimbladder expansion. In contrast to control hatchlings, (i) no mucus-secreting cells in the oesophagus, (ii) a decreased folding of the stomach and intestine, (iii) a clear reduction in size of the exocrine pancreas associated with a destructuration of acinar units, and (iv) a strong decrease in the mass and size of the liver were observed in MC-LR treated embryos. Furthermore, as an indication of MC-LR-induced hepatic glycogen store depletion, unstained cytoplasmic areas present in control hatchling hepatocytes, were fully absent in all liver examined in treated embryos. Finally, as a general observation in MC-LR-treated embryos, our data clearly indicated terminal differentiation disorders in all organs associated with the digestive tract.     

Evaluation of microRNA responses in ARPE-19 cells against the oxidative stress

Purpose: This study aimed to determine microRNA (miRNA) expression profile of human retinal pigment epithelium cell (ARPE-19) against the oxidative stress induced by hydrogen peroxide (H₂O₂).

Methods: ARPE-19 cells were incubated with different concentrations of H₂O₂ (200, 600 and 800?μM) for 18?h, and then cell viability, vascular endothelial growth factor levels and total oxidant status were evaluated. Expressions of 1152 miRNA were determined by quantitative real-time PCR in each group.

Results: Expressions of 90 miRNA were significantly changed in the ARPE-19 cells incubated with H₂O₂ compared to control group. However, miR-143-3p was only found to be expressed in groups incubated with H₂O₂. While 24 miRNA (hsa-miR-200c-3p, miR-192-5p, miR-194-5p, miR-141-3p, miR-658, miR-18?b-5p, miR-486-5p, miR-525-3p, miR-493-3p, miR-518d-3p, miR-29?b-1-5p, miR-675-3p, miR-1238-3p, miR-195-3p, miR-1539, miR-490-5p, miR-3200-5p, miR-1273d, miR-130a-5p, miR-30?b-5p, miR-1247-5p, miR-1910-5p, miR27a-5p and miR-200?b-3p) upregulated due to the increased dose of H₂O₂, nine miRNA (hsa-miR-96-5p, miR-33a-5p, miR-345-5p, miR-106?b-3p, miR-1285-3p, miR-23?b-5p, miR-27?b-5p, miR-103a-3p and miR-4289) were also found to be downregulated.

Conclusion: This study suggests that oxidative stress may be an important factor on expression of miRNAs in ARPE-19 cells. These miRNAs may have a role in the pathogenesis of age-related macular degeneration related to oxidative stress. However, this relationship needs to be examined in new studies by evaluation of pathways and target genes.     

Differential and unique patterns of synaptic miRNA expression in dorsolateral prefrontal cortex of depressed subjects

Altered synaptic plasticity is often associated with major depressive disorder (MDD). Disease-associated changes in synaptic functions are tightly correlated with altered microRNA (miRNA) expression. Here, we examined the role of miRNAs and their functioning at the synapse in MDD by examining miRNA processing machinery at synapse and sequencing miRNAs and analyzing their functions in synaptic and total tissue fractions obtained from dorsolateral prefrontal cortex (dlPFC) of 15 MDD and 15 matched non-psychiatric control subjects. A total of 333 miRNAs were reliably detected in the total tissue fraction. Multiple testing following the Benjamini–Hochberg false discovery rate [FDR] showed that 18 miRNAs were significantly altered (1 downregulated 4 up and 13 downregulated; p < 0.05) in MDD subjects. Out of 351 miRNAs reliably expressed in the synaptic fraction, 24 were uniquely expressed at synapse. In addition, 8 miRNAs (miR-215-5p, miR-192-5p, miR-202-5p, miR-19b-3p, miR-423-5p, miR-219a-2-3p; miR-511-5p, miR-483-5p showed significant (FDR corrected; p < 0.05) differential regulation in the synaptic fraction from dlPFC of MDD subjects. In vitro transfection studies and gene ontology revealed involvement of these altered miRNAs in synaptic plasticity, nervous system development, and neurogenesis. A shift in expression ratios (synaptic vs. total fraction) of miR-19b-3p, miR-376c-3p, miR-455-3p, and miR-337-3p were also noted in the MDD group. Moreover, an inverse relationship between the expression of precursor (pre-miR-19b-1, pre-miR-199a-1 and pre-miR-199a-2) and mature (miR-19b-3p, miR-199a-3p) miRNAs was found. Although not significantly, several miRNA processing enzymes (DROSHA [95%], DICER [17%], TARBP2 [38%]) showed increased expression patterns in MDD subjects. Our findings provide new insights into the understanding of the regulation of miRNAs at the synapse and their possible roles in MDD pathogenesis.Subject terms: Depression, Depression     

Regulation of heat shock protein 27 phosphorylation during microcystin-LR-induced cytoskeletal reorganization in a human liver cell line

Acute exposure to microcystin-LR (MC-LR) can induce the reorganization or disruption of the cytoskeleton, but proteins or enzymes correlated with this stress response have not been fully identified. Here, we report alterations to HSP27 during MC-LR-induced cytoskeletal reorganization in the human liver cell line HL7702. The cells incubated with MC-LR exhibited the rearrangement of filamentous actins and microtubules. The activity of protein phosphatase 2A was greatly decreased by MC-LR exposure. Furthermore, MC-LR markedly increased the level of HSP27 phosphorylation with the enhanced distribution of phosphorylated HSP27 to the cytoskeleton. To further determine the regulation of MC-LR-induced HSP27 phosphorylation, the activation of the MAPK superfamily was assessed. The result showed phospho-activation of p38 MAPK, JNK and ERK1/2 by MC-LR. Increases in HSP27 phosphorylation were suppressed by pretreating cells with SB203580 or SP600125, which are inhibitors of p38 MAPK or JNK, respectively. These data suggest that phosphorylated HSP27 is involved in cytoskeletal reorganization and is regulated by MAPKs, possibly as a consequence of PP2A inhibition. Moreover, the regulation of HSP27 phosphorylation may be important in MC-LR-induced cytoskeleton reassembly, which may provide helpful insights into the mechanism of MC-LR toxicity.     

Endoplasmic reticulum stress in murine liver and kidney exposed to microcystin-LR

To investigate the effect of microcystin-LR (MC-LR) on apoptosis based on the endoplasmic reticulum stress (ERS) pathway in mouse liver and kidney, male ICR mice were intraperitoneally injected with 20 μg kg⁻¹ body weight MC-LR for 21 days, and mRNA and protein levels of ERS special molecules in liver and kidney were analyzed using quantitative real-time PCR and western blotting. MC-LR significantly improved mRNA and protein expression of C/EBP homologous protein (CHOP) and cleaved caspase-12 in liver, whereas it inhibited expression of CHOP and caspase-12 in kidney. MC-LR also induced significant down-regulation of B-cell lymphoma/leukemia-2 (Bcl-2) mRNA expression in liver and weak up-regulation in kidney. These results indicated the involvement of the ERS pathway in MC-LR-induced apoptosis of hepatic cells but not in renal cells of mice. The weight changes and histological damage of liver and kidney were in accordance with the appearance of ERS. Our results indicate that ERS plays an important role in hepatic cell apoptosis induced by MC-LR, and is considered as a new pathway of liver toxicity. Its relative special genes might be considered as potentially new biomarkers used for risk assessment of MC-LR in the environment.     

MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e.g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e.g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity.     

Differential microRNAs expression in seminal plasma of normospermic patients with different sperm DNA fragmentation indexes

Sperm DNA fragmentation index (SDF), as an important supplement to routine semen parameters, has been proposed to discriminate between fertile and infertile men, and predicts the outcomes of natural conception and in vitro fertilization. Unfortunately there are uncertainty and contradictory evidences regarding the importance of SDF. An important reason is the fact that significant and fundamental research about SDF is rare. This study was designed to characterize the microRNA (miRNA) expression profile in seminal plasma of normospermic patients with different SDF and their implications in human fertility. Using next-generation sequencing (NGS), a total of 897 human miRNAs were detected from 10 seminal plasma samples, out of which 431 differentially expressed miRNAs in 5 pairs of seminal plasma samples (each pair of seminal plasma samples obtained from the same male), with 14 miRNAs were identified in all the pairs. According to the fold change and expression level, 7 miRNAs including miR-374b-5p, miR-429, hsa-miR-26b-5p, miR-21−5p, miR-4257, miR-135b-5p and miR-134−5p were selected for further excavation. MiR-374b-5p and miR-26b-5p were significantly different in 3 sets of individual seminal plasma samples with different SDF from total 90 infertile patients (30 patients each set). Our results demonstrate that the profile of miR-374b and miR-26b with significantly decreased expression could be used as a first indication of increased SDF. And miR-374b and miR-26b could serve as adjunct biomarkers for the diagnosis of idiopathic infertile males.     

Aberrant expression of miR-638 contributes to benzo(a)pyrene-induced human cell transformation

Identification of aberrant microRNA (miRNA) expression during chemical carcinogen-induced cell transformation will lead to a better understanding of the substantial role of miRNAs in cancer development. To explore whether aberrant miRNAs expression can be used as biomarkers of chemical exposure in risk assessment of chemical carcinogenesis, we analyzed miRNA expression profiles of human bronchial epithelial cells expressing an oncogenic allele of H-Ras (HBER) at different stages of transformation induced by benzo(a)pyrene (BaP) by miRNA array. It revealed 12 miRNAs differentially expressed in HBER cells at both pretransformed and transformed stages. Differentially expressed miRNAs were confirmed in transformed cells and examined in 50 pairs of primary human non-small-cell lung cancer (NSCLC) tissues using real-time PCR. Among these miRNAs, downregulation of miR-638 was found in 68% (34/50) of NSCLC tissues. However, the expression of miR-638 in HBER cells increased upon treatment of BaP in a dose-dependent manner. The expression of miR-638 was also examined in peripheral lymphocytes from 86 polycyclic aromatic hydrocarbons (PAHs)-exposed (PE) workers. We found that the average expression level of miR-638 in peripheral lymphocytes from 86 PE workers increased by 72% compared with control group. The levels of miR-638 were correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) and external levels of PAHs. Overexpression of miR-638 aggravated cell DNA damage induced by BaP, which might be mediated by suppression of breast cancer 1 (BRCA1), one of the target genes of miR-638. In summary, we suggest that miR-638 is involved in the BaP-induced carcinogenesis by targeting BRCA1.     

Protective effects of green tea polyphenols against subacute hepatotoxicity induced by microcystin-LR in mice

Green tea polyphenols (GTP) have been shown to possess anti-oxidative, anti-mutagenic and anti-carcinogenic activities. The present study aimed to evaluate the chemopreventive efficacy of GTP against subacute hepatotoxicity induced by microcystin-LR (MC-LR) in mice and also elucidates the underlying mechanisms. In this study, healthy Kunming male mice (24-26gbw) were randomly assigned to five groups. Group I was fed on normal diet and water ad libitum as control. Group II was maintained on normal diet and received MC-LR intraperitoneal injection (10μg/kg/day) from day 6 till sacrifice. Mice in groups III, IV and V were daily pre-treated with GTP through intragastric administration at doses of 50, 100 and 200mg/kg/day from day 0 prior to MC-LR intoxication, consecutively 18 days. The results showed MC-LR alone led to oxidative stress and to damage antioxidant defense system, as evidenced by elevation of serum and liver lipid peroxidation. Additionally, hepatocellular apoptosis and injury were significantly observed. GTP pre-treatment caused a significant elevation in serum antioxidant enzymes GSH and SOD activities as well as a decrease in hepatic lipid peroxidation MDA level and serum ALT, AST, ALP activities. GTP pre-treatment obviously inhibited hepatocellular apoptosis and up-regulated Bcl-2 protein expression. The damages in liver were less severe in GTP pre-treated mice in correlation with the biochemical parameters. In summary, this study confirmed that repeated exposure to MC-LR could induce hepatotoxicity. Our study demonstrated that GTP can reduce MC-LR-induced oxidant stress and prevent biochemical parameters and pathological changes caused by MC-LR in a dose-dependent manner. The results indicated that tea polyphenols have a potential to be developed as a preventive agent against MC-LR-induced toxicity and the mechanism involved in the protection could be due to their antioxidant activities.     

MiR-33b-5p sensitizes gastric cancer cells to chemotherapy drugs via inhibiting HMGA2 expression

MicroRNAs (miRNAs) are internal, non-coding, and ～22?nt small RNAs that display cell- and tissue-specific expression. They play important regulatory roles in cell proliferation and chemo-sensitivity. This study focused on tumor-suppressive miR-33b-5p expression as well as its role in gastric cancer. MiR-33b-5p was found low expression in gastric cancer cell lines. Functionally, western blots and the luciferase reporter assay were used to confirm that HMGA2 was the potential target of miR-33b-5p. Next, we used CCK-8 kits to analyze the effect of miR-33b-5p combined chemotherapy drugs on cell inhibition rate, and flow cytometry to analyze cells apoptosis. Colony formation ability was determined by plating at 500 cells per well into six-well plates and culturing for 15?d. The results showed that upregulation of miR-33b-5p decreased expression of HMGA2 and inhibited gastric cancer cell growth as well as sensitized gastric cancer cells to chemotherapy drugs. MiR-33b-5p overexpression hindered luciferase activity of HMGA2,3′-untranslated region-based reporter construct in 293?T cells. These data demonstrate that miR-33b-5p may be a potential therapeutic target for gastric cancer and function as tumor-suppressive miRNA through targeting HMGA2 in gastric cancer.