MicroRNA expression profiling of Chinese follicular lymphoma by microarray: A preliminary study

BackgroundMicroRNAs (miRNAs) have been widely regarded as crucial regulators in various biological processes involved in carcinogenesis. However, the comprehensive miRNA profiles of Chinese follicular lymphoma (FL) remains completely unknown.MethodsThe Exiqon miRCURY LNA™ microRNA Array (v.18.0) was used to detect the miRNA expression profiles of three Chinese FL samples, and compared to three reactive lymphatic nodes (RLN). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to confirm the selected miRNAs in different series. Three databases (miRAnda, miRBase and TargetScan) were used to predict the putative target genes. Bioinformatic analysis (gene ontology analysis and pathway analysis) was performed for further evaluation.ResultsThe microarray assay demonstrated that 1643 miRNAs were expressed; in which 103 miRNAs were upregulated and 68 miRNAs were downregulated, according to P-value (< 0.05) and fold change (FC > 2-fold). Furthermore, qRT-PCR was used to confirm that miR-17-5p, miR-20a-5p and miR-19a-3p were upregulated, and miR-3615 was downregulated (P < 0.05). Bioinformatic analysis (gene ontology analysis and pathway analysis) was used for further evaluation. Pathway analysis indicated that 25 pathways corresponded to differentially expressed miRNAs (P-value cut-off is 0.05). Furthermore, miR-17-5p, miR-20a-5p and miR-19a-3p were validated by qRT-PCR in an independent series including five FL3a and five RLN cases. Data analysis revealed that the changing trend of miR-19a-3p and miR-17-5p expression in the independent series was basically identical with that of the microarray data.ConclusionsOur results are the first to reveal the miRNA expression profiling of Chinese FL and three upregulated miRNAs. Furthermore, the expression of miR-19a-3p and miR-17-5p were found to be significantly upregulated in FL3a. Further study needs to be urgently performed to reveal its potential role in the pathogenesis of FL in the near future.     

Effect of c-fos gene silence on PM2.5-induced miRNA alteration in human bronchial epithelial cells

Human bronchial epithelial (HBE) cells and c-fos-silenced HBE cells were first exposed to fine particulate matter (PM_2.5) and the resulting miRNA sequenced. Thereafter, a weighted gene co-expression network analysis was performed using Cytoscape software to visualize the interactions between identified hub miRNAs and their target genes. Nine differentially expressed miRNAs in hub miRNAs were identified in the different treatment groups, of which miR-25−3p, miR-215−5p, and miR-145−5p were selected for further study. Following qPCR validation, both miR-25−3p and miR-215−5p were found to be significantly up-regulated whilst, miR-145−5p was significantly down-regulated (p < 0.05) in the PM_2.5 group. Furthermore, miR-25−3p and miR-145−5p were also significantly down-regulated in the untreated group of c-fos silenced HBE cells. However, miR-215−5p was significantly down-regulated in both the untreated and PM_2.5-treated groups of c-fos silenced HBE cells. Subsequent analysis of their target genes also illustrated differential gene expression when comparing the treatment groups of the two cell types. The present data indicated that the c-fos gene has an important effect on the miRNA expression profiles and the related signaling pathways in PM_2.5-treated HBE cells. Therefore, each of miR-25−3p, miR-145−5p, and miR-215−5p may potentially provide future research information for additional exploration of a PM_2.5-induced carcinogenesis mechanism.     

Involvement of miR-326 in chemotherapy resistance of breast cancer through modulating expression of multidrug resistance-associated protein 1

Multidrug resistance-associated protein (MRP-1/ABCC1) transports a wide range of therapeutic agents and may play a critical role in the development of multidrug resistance (MDR) in tumor cells. However, the regulation of MRP-1 remains controversial. To explore whether miRNAs are involved in the regulation of MRP-1 expression and modulate the sensitivity of tumor cells to chemotherapeutic agents, we analyzed miRNA expression levels in VP-16-resistant MDR cell line, MCF-7/VP, in comparison with its parent cell line, MCF-7, using a miRNA microarray. MCF-7/VP overexpressed MRP-1 mRNA and protein not MDR-1 and BCRP. miR-326 was downregulated in MCF-7/VP compared to MCF-7. Additionally, miR-326 was downregulated in a panel of advanced breast cancer tissues and consistent reversely with expression levels of MRP-1. Furthermore, the elevated levels of miR-326 in the mimics-transfected VP-16-resistant cell line, MCF-7/VP, downregulated MRP-1 expression and sensitized these cells to VP-16 and doxorubicin. These findings demonstrate for the first time the involvement of miRNAs in multidrug resistance mediated by MRP-1 and suggest that miR-326 may be an efficient agent for preventing and reversing MDR in tumor cells.     

Paraquat inhibited differentiation in human neural progenitor cells (hNPCs) and down regulated miR-200a expression by targeting CTNNB1

Paraquat (PQ) exposure influences central nervous system and results in serious neurotoxicity in vitro and in vivo. However, the role of PQ exposure in the development of CNS remains unclear. In present study, we investigated microRNAs (miRNAs) expression profiling and cell differential status following PQ treatment in human neural progenitor cells (hNPCs) as well as involved mechanism. Microarray profiling of miRNAs expression of PQ treated cell line and their corresponding control was determined. Differentially expression miRNAs were confirmed by quantitative real time PCR. Neural cell differentiation was performed with immunocytochemical analysis. Predicated target of miRNA was identified with luciferase reports and quantitatively analyzed using western blotting. Our results found PQ dramatically suppressed neural cell differentiation ability. 43 differentially expressed miRNAs were identified in PQ treated cells. The expression levels were over expressed in 25 miRNAs, whereas 18 miRNAs were suppressed. More importantly, we observed that miR-200a expression level to be lower in PQ treated cells. Luciferase assay and protein expression results confirmed the direct binding effect between CTNNB1 and miR-200a following PQ exposure. Collectively, our data suggested that down regulation of miR-200a in the PQ treated neural stem cell significantly participated in the differentiation processes and subsequently resulting in decreased cell viability, increased epithelial-mesenchymal transition process and the inhibited differential through CTNNB1 pathway.     

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.     

基于miRNAs的知母水提物体外抑制胃癌细胞SGC7901增殖作用机制

目的 从微小核苷酸（miRNAs）的角度研究知母水提物（aqueous extract from Anemarrhenae Rhizoma,ARAE）体外抑制SGC7901细胞增殖的作用机制。方法 采用MTT法检测ARAE对SGC7901细胞增殖的影响;采用流式细胞术检测ARAE对SGC7901细胞凋亡的影响;采用高通量测序法检测细胞中miRNAs的表达丰度差异;采用miranda、mirbase和targetscan 3个数据库信息比对,预测差异miRNAs的靶基因,并通过KEGG分析靶基因的相关功能;采用实时荧光定量PCR法验证主要靶基因的表达变化。结果 ARAE能明显抑制SGC7901细胞的增殖并诱导其凋亡,调节细胞miR-16-5p、miR-20a-5p、miR-26b-5p和miR-15b-5p的表达水平;并提示这些miRNAs所调控的靶基因主要富集于PI3K-Akt、JAK-STAT和MAPK等信号通路。结论 ARAE可能通过调节SGC7901细胞内miRNAs的表达从而抑制SGC7901细胞增殖,并诱导其凋亡。     

Chir99021联合PD0325901对小鼠胚胎干细胞中miRNAs差异表达的影响

目的：通过研究Chir99021联合PD0325901对小鼠胚胎干细胞中miRNAs差异表达的影响,为揭示胚胎干细胞的自我更新和分化的机制提供更多线索。方法采用miRNA基因芯片技术检测Chir99021联合PD0325901处理组和PD0325901处理组miRNAs的表达谱差异。选取3倍以上及通过查文献与胚胎干细胞自我更新相关的1.5倍以上3倍以下的miRNAs,采用实时荧光定量PCR法验证,利用miRDB、Miranda两个数据库交叉预测差异表达的靶基因,并应用KEGG Pathway进行靶基因功能富集分析。结果与PD0325901单独处理相比,Chir99021联合PD0325901处理组有47种miRNAs上调1.5倍以上,75种miRNAs下调1.5倍以上;用实时荧光定量PCR验证差异表达的miRNAs,结果显示13个miRNAs与芯片结果相符。靶基因预测分析显示,miR-466a-5p、miR-466d-5p处于重要位置,Plcb1、Prkcb处于关键基因位置。结论 Chir99021可引起小鼠胚胎干细胞中的miRNAs差异表达,差异表达的miRNAs可能通过调控Plcb1、Prkcb基因而影响胚胎干细胞的自我更新。     

Breast cancer resistance protein BCRP/ABCG2 regulatory microRNAs (hsa-miR-328, -519c and -520h) and their differential expression in stem-like ABCG2+ cancer cells

Recent studies have shown that a number of microRNAs (miRNA or miR) may regulate human breast cancer resistance protein (BCRP/ABCG2), an important efflux transporter responsible for cellular drug disposition, whereas their effects on ABCG2 protein expression are not compared. In this study, we first identified a new proximal miRNA response element (MRE) for hsa-miR-519c within ABCG2 3′-untranslated region (3′UTR) through computational analyses. This miR-519c MRE site was confirmed using dual luciferase reporter assay and site-directed mutagenesis. Immunoblot analyses indicated that ABCG2 protein expression was significantly down-regulated in MCF-7/MX100 cells after transfection with hsa-miR-328- or -519c expression plasmids, and was markedly up-regulated in MCF-7 cells after transfection with miR-328 or -519c antagomir. However, ABCG2 protein expression was unchanged in MCF-7/MX100 cells after transfection with hsa-miR-520h expression plasmids, which was associated with undetectable miR-520h expression. Furthermore, ABCG2 mRNA degradation was accelerated dramatically in cells transfected with miR-519c expression plasmid, suggesting the involvement of mRNA degradation mechanism. Intervention of miR-328 or -519c signaling led to significant change in intracellular mitoxantrone accumulation, as determined by flow cytometry analyses. In addition, we separated RB143 human retinoblastoma cells into stem-like (ABCG2+) and non-stem-like (ABCG2−) populations through immunomagnetic selection, and found that miR-328, -519c and -520h levels were 9-, 15- and 3-fold lower in the ABCG2+ cells, respectively. Our data suggest that miR-519c and -328 have greater impact on ABCG2 expression than miR-520h in MCF-7 human breast cancer cells, and the presence of proximal miR-519c MRE explains the action of miR-519c on shortened ABCG2 3′UTR.     

Angiotensin II type 1 receptor signalling regulates microRNA differentially in cardiac fibroblasts and myocytes

BACKGROUND AND PURPOSE

The angiotensin II type 1 receptor (AT₁R) is a key regulator of blood pressure and cardiac contractility and is profoundly involved in development of cardiac disease. Since several microRNAs (miRNAs) have been implicated in cardiac disease, we determined whether miRNAs might be regulated by AT₁R signals in a Gαq/11-dependent or -independent manner.

EXPERIMENTAL APPROACH

We performed a global miRNA array analysis of angiotensin II (Ang II)-mediated miRNA regulation in HEK293N cells overexpressing the AT₁R and focused on separating the role of Gαq/11-dependent and -independent pathways. MiRNA regulation was verified with quantitative PCR in both HEK293N cells and primary cardiac myocytes and fibroblasts.

KEY RESULTS

Our studies revealed five miRNAs (miR-29b, -129-3p, -132, -132* and -212) that were up-regulated by Ang II in HEK293N cells. In contrast, the biased Ang II analogue, [Sar1, Ile4, Ile8] Ang II (SII Ang II), which selectively activates Gαq/11-independent signalling, failed to regulate miRNAs in HEK293N cells. Furthermore, Ang II-induced miRNA regulation was blocked following Gαq/11 and Mek1 inhibition. The observed Ang II regulation of miRNA was confirmed in primary cultures of adult cardiac fibroblasts. Interestingly, Ang II did not regulate miRNA expression in cardiac myocytes, but SII Ang II significantly down-regulated miR-129-3p.

CONCLUSIONS AND IMPLICATIONS

Five miRNAs were regulated by Ang II through mechanisms depending on Gαq/11 and Erk1/2 activation. These miRNAs may be involved in Ang II-mediated cardiac biology and disease, as several of these miRNAs have previously been associated with cardiovascular disease and were found to be regulated in cardiac cells.     

Integrated mRNA and micro RNA profiling reveals epigenetic mechanism of differential sensitivity of Jurkat T cells to AgNPs and Ag ions

In our previous in vitro study of the toxicity on silver nanoparticles (AgNPs), we observed a dramatically higher sensitivity of Jurkat T cells to AgNPs than to Ag ions, and DNA damage and apoptosis were found to be involved in that toxicity. In this study, to understand underlying mechanism of different sensitivity of Jurket T cells to AgNPs and Ag ions, mRNA microarray and micro RNA microarray were concomitantly conducted on AgNPs and Ag ions exposed Jurkat T cells. Surprisingly only a small number of genes were differentially expressed by exposure to each of the silver (15 altered mRNA by AgNPs exposure, whereas 4 altered mRNA by Ag ions exposure, as determined 1.5-fold change as the cut-off value). miRNA microarray revealed that the expression of 63 miRNAs was altered by AgNPs exposure, whereas that of 32 miRNAs was altered by Ag ions exposure. An integrated analysis of mRNA and miRNA expression revealed that the expression of hsa-miR-219-5p, was negatively correlated with the expression of metallothionein 1F (MT1F) and tribbles homolog 3 (TRIB3), in cells exposed to AgNPs; whereas, the expression of hsa-miR-654-3p was negatively correlated with the expression of mRNA, endonuclease G-like 1 (EDGL1) in cells exposed to Ag ions. Network analysis were further conducted on mRNA-miRNA pairs, which revealed that miR-219-5p–MT1F and –TRIB3 pairs by AgNPs are being involved in various cellular processes, such as, oxidative stress, cell cycle and apoptosis, whereas, miR-654-3p and ENDOGL1 pair by Ag ions generated a much simpler network. The putative target genes of AgNPs-induced miR-504, miR-33 and miR-302 identified by Tarbase 6.0 are also found to be involved in DNA damage and apoptosis. These results collectively suggest that distinct epigenetic regulation may be an underlying mechanism of different sensitivity of Jurkat T cells to AgNPs and Ag ion. Further identification of putative target genes of DE miRNA by AgNPs and Ag ions may provide additional clues for the mechanism of differential toxicity. Overall results suggest that epigenetic mechanism is involved in toxicity of AgNPs and Ag ions in Jurkat T cells.     

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。     

Underexpression of miR-224 in methotrexate resistant human colon cancer cells

MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatments. The aim of this work was to detect differentially expressed miRNAs in MTX-resistant cells. Thus, miRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed miRNAs in resistant cells were identified and miR-224, which was one of the most differentially expressed miRNAs and with high raw signal values, was selected for further studies. The underexpression of miR-224 was also observed in CaCo-2 and K562 cells resistant to MTX. Putative targets were predicted using TargetScan 5.1 software and integrated with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells towards MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells.