MicroRNA expression profiling involved in MC-LR-induced hepatotoxicity using high-throughput sequencing analysis

Microcystin-LR (MC-LR), the most common microcystin (MC) present in water is known to pose a significant threat to human health especially hepatotoxicity. However, the specific molecular mechanisms underlying MC-LR-induced hepatic cellular damage still remain to be determined. MicroRNAs (miRNAs) are known to play key roles in cellular processes including development, cell proliferation and responsiveness to stress. Thus, this study aimed to examine, whether miRNAs were involved in the observed MC-LR-mediated liver damage using miRNA profiling of a human normal liver cell line HL7702 using high-throughput sequencing techniques. Protein phosphatase 2A (PP2A) activity, an established biomarker of microcystin toxicity, was determined 24 hr following treatment with the algal toxin to confirm responsiveness. Data demonstrated that MC-LR significantly inhibited PP2A activity in a concentration-dependent manner with inhibitory concentration (IC₅₀) value of 4.6 μM. Compared with control cells, treatment with MC-LR at concentrations of 1, 2.5, 5 or 10 μM significantly modified expression of levels of 3, 10, 9, and 99 miRNAs, respectively. Expression levels of miR-15b-3p were significantly increased in all 4 treatment groups, while miR-4521 expression levels were markedly reduced. In the case of miR-451a, 1, 5 or 10 μM also significantly lowered expression levels. However, a significant rise in miR-451a was noted in cells exposed to 2.5 μM toxin. The results obtained from miRNA differential expression levels were confirmed by real-time fluorescent quantitative PCR (qPCR). Gene Ontology (GO) enrichment analysis of hepatic cells demonstrated that miRNAs significantly altered were involved in systems development, metabolism, and protein binding. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis data showed that target genes of differentially expressed miRNAs in liver cells predominantly participated in mechanistic target of rapamycin kinase (mTOR), Ras, Ras-related protein 1 (Rap1), hypoxia inducible factor 1 (HIF-1), and cancer development. In summary, evidence indicates that MC-LR-induced hepatotoxicity may be associated with alterations in miRNAs. Evidence indicates that alterations in miR-451a, miR-4521 and miR-15b-3p may be involved in the observed MC-LR- induced hepatotoxicity     

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.     

MiR-195 regulates cell apoptosis of human hepatocellular carcinoma cells by targeting LATS2

Recently, we have reported tissue- and stage-specific expression of miR-195 in human hepatocellular carcinoma cells and so far, not many reports discuss the function of this microRNA (miRNA). Expression profiling of miRNAs revealed a limited set of miRNAs with altered expression in drug resistant hepatocellular carcinoma cell line BEL-7402/5-FU compared to its parental BEL-7402 cell line. Real-time PCR confirmed down-regulation of miR-195 in BEL-7402/5-FU cells. Western blots were performed to determine protein levels of LATS2, P53 and CDK2. MTT analysed the cell proliferation activity. Flow cytometry were performed to determine apoptosis rate. Up-regulation of miR-195 increased expression of LATS2 and increased apoptosis of HCC cells, while Anti-miR-195 treatment inhibited expression of LATS2. miR-195 over-expression inhibited the luciferase activity of a LATS2 3' untranslated region-based reporter construct in BEL-7402/5-FU cells. These results indicate that miR-195 could increase cell apoptosis by targeting LATS2 in hepatocellular carcinoma cells.     

The changes of miRNA expression in rat hippocampus following chronic lead exposure

miRNAs have been found to contribute to normal brain functions, nervous system diseases, as well as neurotoxicities induced by external agents. However, whether they are involved in lead-induced neurotoxicities is still not clear. To identify that, a lead-induced chronic neurotoxicity model of rats was built. Both miRNA microarray analysis and qRT-PCR were performed to determine the change of miRNA expression in hippocampus. Then 3 bioinformatics databases were used to analyze the relative target genes of these miRNA, which were further confirmed by qRT-PCR and Western blot. In the present study, lead exposure resulted in the changed expression of 7 miRNAs: miR-204, miR-211, miR-448, miR-449a, miR-34b, and miR-34c were greatly up-regulated while miR-494 was greatly down-regulated. Bioinformatics analysis results showed that the target genes of 6 up-regulated miRNAs were related to neural injury and neurodegeration, axon and synapse function, neural development and regeneration. Correspondingly, the expression levels of mature mRNAs and proteins of three target genes (Bcl-2, Itpr1, and Map2k1) were greatly repressed, verifying the results of bioinformatics analysis. Taken together, our results showed that the expression of several miRNAs reported to be associated with neurophysiological pathways and neurodegenerative diseases changed in rat hippocampus following chronic lead exposure. These miRNAs may play important roles in lead-induced neurotoxicity.     

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。     

Aberrant expression of microRNAs in gastric cancer and biological significance of miR-574-3p

The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanisms, diagnosis and treatments of cancer. In this study, we employed AFFX miRNA expression chips to search for miRNAs that may be aberrantly expressed in gastric cancer tissues and to investigate the potential roles that miRNAs may play in the development and progression of gastric cancer. 14 miRNAs were found to be down-regulated and 2 miRNAs up-regulated in gastric cancer tissues compared to the normal gastric tissues. Among the aberrantly expressed miRNAs, miR-574-3p was selected to further study its expression features and functional roles. Interestingly, the reduced expression of miR-574-3p occurred mainly in the early stages of gastric cancer or in cancers with high level of differentiation, suggesting that it can be used as a marker for a mild case of gastric cancer. Functional study revealed that cell proliferation, migration and invasion were significantly inhibited in miR-574-3p-transfected gastric cancer SGC7901 cells. Computational prediction and experimental validation suggest that Cullin2 may be one of the targets of miR-574-3p. Overall our study suggests that the aberrantly expressed miRNAs may play regulatory and functional roles in the development and progression of gastric cancer.     

Epirubicin-mediated expression of miR-302b is involved in osteosarcoma apoptosis and cell cycle regulation

Epirubicin is widely used in osteosarcoma chemotherapy. Growing evidence indicates that the microRNA (miRNA) expression levels which are induced by chemotherapeutic agents play an important role in osteosarcoma development and progression. In this study we investigate the alterations of miRNA expression in the osteosarcoma cells after epirubicin treatment and whether miRNAs can enhance its anti-osteosarcoma effect. After epirubicin exposure, microarray shows 40 miRNAs are differentially expressed in osteosarcoma cells including 24 down-regulated miRNAs. Notably, miR-302b, which is stably low-expressed in osteosarcoma, could be induced by the epirubicin. Furthermore, we find that miR-302b can inhibit the osteosarcoma cell proliferation, promote cell apoptosis and cell cycle arrest MiR-302b can activate caspase-3 and regulate the Akt/pAkt, Bcl-2, Bim expression to increase the cell apoptosis. Meanwhile, miR-302b also attenuates cyclin D1 and CDKs expression to induce cell cycle arrest. Therefore, our results suggest miR-302b can play an essential role in osteosarcoma treatment as a potential tumor suppressor.     

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.     

MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells

MicroRNAs (miRNAs) can regulate cell survival and death by targeting apoptosis-related gene expression. miR-210 is one of the most hypoxia-sensitive miRNAs. In this study, we evaluated the roles of miR-210 in hypoxia-induced insults to neural cells. Treatment of neuro-2a cells with oxygen/glucose deprivation (OGD) induced cell apoptosis in a time-dependent manner. In parallel, OGD time-dependently increased cellular miR-210 levels. Knocking down miR-210 expression using specific antisenses significantly attenuated OGD-induced neural apoptosis. Concurrently, OGD increased hypoxia-inducible factor (HIF)-1α mRNA and protein syntheses. Pretreatment with YC-1, an inhibitor of HIF-1α, reduced OGD-caused cell death. Sequentially, OGD specifically decreased antiapoptotic Bcl-2 mRNA and protein levels in neuro-2a cells. A search by a bioinformatic approach revealed that miR-210-specific binding elements exist in the 3′-untranslated region of Bcl-2 mRNA. Application of miR-210 antisenses simultaneously alleviated OGD-involved inhibition of Bcl-2 mRNA expression. In comparison, overexpression of miR-210 synergistically diminished OGD-caused inhibition of Bcl-2 mRNA expression and consequently induced greater cellular insults. Taken together, this study shows that OGD can induce miR-210 expression through activating HIF-1α. And miR-210 can mediate hypoxia-induced neural apoptosis by targeting Bcl-2.     

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.     

Inhibitory effects of anti-miRNA oligonucleotides (AMOs) on A549 cell growth

MicroRNAs (miRNAs) are a new class of non-protein-coding, endogenous small RNA molecules of 18–24 nt in size. miRNAs can specifically down-regulate gene expression involved in proliferation, apoptosis, and differentiation in cancer cells. Our purpose was to identify several functional miRNAs as potential drug targets by using specific antisense-microRNA oligonucleotides (AMOs), and to study the inhibitory effects of these AMOs on A549 cell growth. miR-16, miR-21, miR-214, and miR-181a were selected as target candidates, based on which specific AMOs were designed, synthesized, and transfected into A549 cells. The viable cells were counted by using trypan blue dye exclusion assay. Apoptosis of A549 cells were determined flowcytometrically, and miR-21 expression levels in A549 cells were determined by real-time PCR. The results showed that AMO-miR-21, AMO-miR-16, and AMO-miR-181a inhibited A549 cell growth by inducing apoptosis and S-phase arrest. These inhibitory effects increased with dose and time. It was found that AMO-miR-21 down-regulated miR-21 expression in A549 cells. We conclude that miR-21, miR-16, and miR-181a are potential targets for lung cancer therapy, and specific AMOs can be a powerful technique for miRNA inhibition.     

Inhibition of connective tissue growth factor attenuates paraquat‐induced lung fibrosis in a human MRC‐5 cell line

Chronic exposure to Paraquat (PQ) may result in progressive pulmonary fibrosis and subsequent chronic obstructive pulmonary malfunction. Connective tissue growth factor (CTGF) has been proposed as a key determinant in the development of lung fibrosis. We investigated thus whether knock down of CTGF can prevent human lung fibroblasts (MRC‐5) activation and proliferation with the subsequent inhibition of PQ‐induced fibrosis. MRC‐5 was transfected with CTGF‐siRNAs and exposed to different concentrations of PQ. The siRNA‐silencing efficacy was evaluated using western blotting analyses, qRT‐PCR and flow cytometry. Next, the viability and migration of MRC‐5 was determined. MMP‐2, MMP‐9, and TIMP‐1 accumulation were quantified to evaluate the lung fibrosis exposure to PQ. Over expression of CTGF mRNA was observed in human MRC‐5 cell as early as 6 h following PQ stimulation. CTGF gene expression in MRC‐5 cells was substantially reduced by RNAi, which significantly suppressed the expression of the lung fibrosis markers such as tissue inhibitor of metalloproteinase‐2 (TIMP‐2), Matrix metalloproteinase‐2 (MMP‐2) and Matrix metalloproteinase‐9 (MMP‐9) that were stimulated by PQ. Inhibition of CTGF expression suppressed impeded the proliferation and migration ability of MRC‐5 cells and resulted in cell‐extracellular matrix (ECM) protein accumulation in cells. Our results suggest that CTGF promoted the development of PQ‐induced lung fibrosis in collaboration with transforming growth factor β1 (TGFβ1). Furthermore, the observed arresting effects of CTGF knock down during this process suggested that CTGF is the potential target site for preventing PQ‐induced pulmonary fibrosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1620–1626, 2016.     

MiR-320 and miR-494 affect cell cycles of primary murine bronchial epithelial cells exposed to benzo[a]pyrene

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules with profound impact on various biological processes. Some miRNAs are involved in tumorigenesis by regulation of cell cycle progression. Here, we cultured primary murine bronchial epithelial cells and then examined the expression of miR-320 and miR-494 in cells exposed to benzo[a]pyrene (B[a]P). To better characterize roles of miR-320 and miR-494 in cell cycle progression, we used miRNA inhibitors to downregulate expression of miRNAs and determined cell cycle distribution and expression of cyclin-dependent kinases 6 (CDK6) by flow cytometric analysis. Treating cells with 1 μM B[a]P for 24 h resulted in time-dependent increases in miR-320 and miR-494 expression. Moreover, G1 arrest and downregulated expression of CDK6 were shown in the treated cells. Flow cytometric analysis indicated a relief of G1 arrest and an elevated expression of CDK6 after inhibition of the expressions of miR-320 and miR-494 in cells exposed to B[a]P. These results suggest that expression levels of miRNA-320 and miR-494, which regulate B[a]P-exposed cell cycle progression, may impact G1/S transition through CDK6, and provide further insights into functions of miRNAs in cell cycle of primary murine bronchial epithelial cells exposed to B[a]P.     

莱菔硫烷经由miR-124抑制胶质瘤干细胞的增殖

摘要：目的 研究莱菔硫烷对SWO-38胶质瘤干细胞增殖的影响及其机制。方法 细胞增殖实验检测莱菔硫烷对SWO-38细胞增殖的影响;克隆形成实验、肿瘤球形成实验、蛋白印迹法等检测并比较莱菔硫烷处理前后SWO-38细胞克隆形成能力、肿瘤球形成能力及干性相关基因（如β-catenin、Oct4、Sox-2、c-Myc）表达水平等改变,比较莱菔硫烷和（或）miR-124抑制物处理对干性相关基因表达水平的影响。实时荧光定量PCR检测莱菔硫烷对miRNA-9、21、221、124、128、181等转录水平的影响。结果 莱菔硫烷有效抑制SWO-38细胞增殖,其半数抑制浓度为（26.41±2.13）μmol/L。莱菔硫烷呈现剂量依赖性地削弱SWO-38细胞克隆和肿瘤球形成的能力。莱菔硫烷下调β-catenin、Oct4、Sox-2和c-Myc等干性相关基因的表达。同时,莱菔硫烷还影响miR-9、21、221、124、128、181等miRNA的转录水平, 其中miR-124转录水平增高约5.9倍,miR-128增高约2.6倍。miR-124抑制物组β-catenin、Oct4、Sox-2等基因表达较空白对照组显著增高,而miR-124抑制物与莱菔硫烷联合组上述基因表达水平高于空白对照组,但低于miR-124抑制物组。结论 莱菔硫烷有效抑制SWO-38胶质瘤干细胞的增殖,其机制可能与miR-124/ (β-catenin/ Sox-2/Oct4)通路有关。     

miR-21 regulates N-methyl-N-nitro-N′-nitrosoguanidine-induced gastric tumorigenesis by targeting FASLG and BTG2

MicroRNAs (miRNAs) are recently discovered regulators of gene expression and are important in the regulation of many cellular events. Evidence collected to date shows that miRNAs are altered after exposure to environmental toxicants. However, the role that miR-21 plays in the gastric tumorigenesis induced by environmental carcinogens remains largely unknown. The aim of this study was to characterize the regulatory role of miR-21 in the carcinogenic processes following exposure to the N-nitroso carcinogen N-methyl-N-nitro-N′-nitrosoguanidine (MNNG). We found a progressive dose- and time-dependent increase in miR-21 expression following treatment with MNNG. Dysregulated miR-21 affected both cell growth in GES-1 cells and the gastric tumorigenesis induced with MNNG. These data demonstrate the involvement of miR-21 in the malignant transformation and tumorigenesis activated by MNNG. We also established that the Fas ligand (FASLG) and B-cell translocation gene 2 (BTG2), regulated by miR-21, contribute to the transformation induced by MNNG in GES-1 cells. This is the first study to show that miR-21 is involved in chemical carcinogenesis in vivo and in vitro. The regulation by miR-21 of the gastric carcinogenesis induced by MNNG highlights the functional roles of miRNAs in chemical carcinogenesis, and offers a new explanation of the mechanisms underlying chemical carcinogenesis.     

Genome-wide miRNA expression profiling of human lymphoblastoid cell lines identifies tentative SSRI antidepressant response biomarkers

Aim: Over 30% of patients with major depression do not respond well to first-line treatment with selective serotonin reuptake inhibitors (SSRIs). Using genome-wide expression profiling of human lymphoblastoid cell lines (LCLs) CHL1 was identified as a tentative SSRI sensitivity biomarker. This study reports on miRNAs implicated in SSRI sensitivity of LCLs. Methods: Eighty LCLs were screened from healthy adult female individuals for growth inhibition by paroxetine. Eight LCLs exhibiting high or low sensitivities to paroxetine were chosen for genome-wide expression profiling with miRNA microarrays. Results: The miRNA miR-151-3p had 6.7-fold higher basal expression in paroxetine-sensitive LCLs. This corresponds with lower expression of CHL1, a target of miR-151-3p. The additional miRNAs miR-212, miR-132, miR-30b*, let-7b and let-7c also differed by >1.5-fold (p < 0.05) between the two LCL groups. Conclusion: The potential value of these miRNAs as tentative SSRI response biomarkers awaits validation with lymphocyte samples of major depression patients. Original submitted 28 March 2012; Revision submitted 21 May 2012.