Direct Targets Of miRNAs Research Articles

Extensive transcriptional responses are co-ordinated by microRNAs as revealed by Exon-Intron Split Analysis (EISA).

Epithelial–mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate ‘indirect’ miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon–Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-β or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.

MicroRNAs related to cholesterol metabolism affected by vegetable diet in rainbow trout (Oncorhynchus mykiss) from control and selected lines

For the sustainable development of aquaculture, vegetable ingredients have been used to replace the traditional ingredients fishmeal and fish oil in aquafeeds. On the other hand, selective breeding has also been implemented at research level to obtain strains that are adapted to feeding on the plant-based diet. To better understand the underlying mechanisms prompting the adaptation to plant-based diets in fish, we investigated the hepatic expression of several microRNAs (miRNAs) that are involved in the post-transcriptional regulation of cholesterol and lipid metabolism at 8 h and 16 h after the last meal in two lines of rainbow trout: one selected for better adaptation to plant-based diets and the corresponding control line. Both groups were fed either a fishmeal and fish oil based diet or a 100% plant-based diet. Results showed that the expression of miR-33a in liver was greatly elevated in trout fed the plant-based diet, while the expression of miR-122 and miR-128 was much higher in the selected line at 8 h after the last meal regardless of the diet. Furthermore, our results indicated that some genes involved in immune processes (caspase 6 apoptosis-related cysteine peptidase like 2, casp6l2) and cAMP signal transduction (phosphodiesterase 4B cAMP-specific a, pde4ba) were also potentially regulated by miRNAs. They were newly identified as putative direct targets of miRNAs and affected in trout fed the plant-based diet. Though further investigations are still needed to establish a valid relationship between miRNAs and their target genes, our study found miR-33a, miR-122 and miR-128 as potential candidates for further study and provided new perspectives to understand the role of miRNAs in the selective breeding for adaptation to the plant-based diets.

MiFAST: A novel and rapid microRNA target capture method.

MicroRNAs (miRNAs), small 22-25 nucleotide non-coding RNAs, play important roles in cellular and tumor biology. However, characterizing miRNA function remains challenging due to an abundance of predicted targets and an experimental bottleneck in identifying biologically relevant direct targets. Here, we developed a novel technique (miFAST) to identify direct miRNA target genes. Using miFAST, we confirmed several previously reported miR-340 target genes and identified five additional novel direct miR-340 targets in melanoma cells. This methodology can also be efficiently applied for the global characterization of miRNA targets. Utilizing miFAST to characterize direct miRNA targetomes will further our understanding of miRNA biology and function.

MiR-33a inhibits cell proliferation and invasion by targeting CAND1 in lung cancer.

Lung cancer continues to be one of the top five causes of cancer-related mortality. This study aims to identify down- and upregulated miRNAs and mRNA which can be used as potential biomarkers and/or therapeutic targets for lung cancer. Integrated analysis of differential expression profiles of miRNA and mRNA in lung cancer was performed by searching Gene Expression Omnibus datasets. Based on miRNA expression profiles, direct mRNA targets of miRNAs with experimental support were identified through miRTarBase. The levels of representative miRNAs and mRNAs were confirmed through qualitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). The miR-33a was decreased in non-small cell lung cancer (NSCLC) tissues compared with the para-carcinoma tissues, whereas its target mRNA of cullin-associated NEDD8-dissociated protein 1 (CAND1) was increased in NSCLC tissues. Further research has shown that miR-33a can inhibit lung cancer cell proliferation, cell cycle progression, and migration by targeting CAND1. Moreover, the CAND1 knockout lung cancer cells showed similar results as cells transfected with miR-33a mimic. These results suggested that the data mining based on online databases was an effective method in finding novel target in cancer research, and the miR-33a and CAND1 played an important role in lung cancer proliferation and cell migration.

1235P - Post-transcriptional regulation of immune checkpoint genes by mir-16 in melanoma

Background: The complex interface between T lymphocytes and cancer ('the immunological synapse') comprises of both co-stimulatory and co-inhibitory proteins that modulate lymphocytes towards activation or anergy. 'Checkpoint inhibitors' have impressive activity in melanoma, but not all patients respond and drug resistance often develops. MiRNAs are master regulators of gene expression. Our aim is to study the regulation of the immunological synapse by miRNAs in melanoma. Methods: Bioinformatic analyses of mRNAs and miRNA expression in 451 samples from the melanoma TCGA database was performed. Spearman rho correlation coefficients were calculated and survival analysis was performed using the Kaplan-Meier method. Direct mRNA targets of miRNAs were found using luciferase reporter assays, and mRNA/miRNA expression was assessed by qRT-PCR following either ectopic expression or depletion of specific miRNAs. Results: Of 15 checkpoint mRNAs and 8 miRNAs examined, nine checkpoint mRNAs showed a highly statistically significant positive correlation to each other and, to a lesser extent, to mir-16. These results were fully corroborated in vitro. Mir-16 may potentially target the 3'UTR of 3 of these mRNAs. CD80 (B7.1) was found to a direct target of mir16 in vitro. Overexpression of mir-16 in melanoma cell lines led to downregulation of CD80, CD274 (PD-L1) and CD40, while downregulation of mir-16 increased the expression of these genes. Survival data from 163 stage III melanoma patients show that high levels of mir-16 and low levels of any of six checkpoint mRNAs (among them CD80) is significantly associated with poor prognosis. Conclusions: Our results suggest that mir-16 and many checkpoint mRNAs are generally under a strict joint transcriptional regulation. The ability of mir-16 to decrease CD80 expression suggests that it serves as a key regulator of the immunological sample. We hypothesize that in vivo, an aberrantly high expression of mir-16 decreases the expression of the co-stimulatory checkpoint CD80 in melanoma and other checkpoint mRNAs, leading to immune evasion and compromised outcome. Further elucidation of both the transcriptional and post-transcriptional regulation of the immunological synapse may help point to novel targets and means for immune modulation. Legal entity responsible for the study: Raya Leibowitz-Amit Funding: Israeli Scientific Foundation (ISF) Disclosure: All authors have declared no conflicts of interest.

MTDH and MAP3K1 are direct targets of apoptosis-regulating miRNAs in colorectal carcinoma

Artificially designed miRNAs mimics and inhibitors that specifically target known oncogenes have attracted significant research attention. Herein, we aimed to explore whether MIR-375, MIR-145, and MIR-224 are involved in induction of apoptosis of CRC cells by regulating apoptosis-mediating genes MTDH, MAP3K1, PDK1, BAX, and BCL-XL. MTT assay was used to assess cell growth. Apoptosis was determined in terms of caspase activity measurement and phosphatidylserine detection using annexin V staining by flow cytometry. Quantitative real time PCR, Western blotting, and luciferase reporter assay were carried out to validate genes regulation and targeting by miRNAs. We found that ectopic expression of MIR-375 and MIR-145, and inhibition of MIR-224 can decrease cell growth and induce cell ability to undergo early apoptosis. At mRNA level, transfected cells displayed down-regulation of MTDH, PDK1 and BCL-XL, while BAX and MAP3K1 were up-regulated. Protein expression of MTDH was decreased in cells transfected with MIR-145 mimic and MIR-224 inhibitor but remained unchanged in MIR-375 mimic-transfected cells. Furthermore, MAP3K1 protein expression exibited a decreased level after MIR-375 transient expression with no significant change after MIR-145 mimic or MIR-224 inhibitor transfection. Luciferase reporter assay revealed that MIR-375 and MIR-145 can bind to 3′UTR of MTDH, supporting that MTDH is directly targeted by both miRNAs. Similarly, MAP3K1 was found to be directly regulated by MIR-375. The study concluded that the expression modulation of tumor suppressors MIR-375 and MIR-145, and oncomiR MIR-224 have the ability to induce apoptosis of CRC cells through regulation of apoptosis mediating genes MTDH, MAP3K1, PDK1, BCL-XL and BAX.

MicroRNA Regulation of Skeletal Development.

Osteogenesis is a complex process involving the specification of multiple progenitor cells and their maturation and differentiation into matrix-secreting osteoblasts. Osteogenesis occurs not only during embryogenesis but also during growth, after an injury, and in normal homeostatic maintenance. While much is known about osteogenesis-associated regulatory genes, the role of microRNAs (miRNAs), which are epigenetic regulators of protein expression, is just beginning to be explored. While miRNAs do not abrogate all protein expression, their purpose is to finely tune it, allowing for a timely and temporary protein down-regulation. The last decade has unveiled a multitude of miRNAs that regulate key proteins within the osteogenic lineage, thus qualifying them as "ostemiRs." These miRNAs may endogenously target an activator or inhibitor of differentiation, and depending on the target, may either lead to the prolongation of a progenitor maintenance state or to early differentiation. Interestingly, cellular identity seems intimately coupled to the expression of miRNAs, which participate in the suppression of previous and subsequent differentiation steps. In such cases where key osteogenic proteins were identified as direct targets of miRNAs in non-bone cell types, or through bioinformatic prediction, future research illuminating the activity of these miRNAs during osteogenesis will be extremely valuable. Many bone-related diseases involve the dysregulation of transcription factors or other proteins found within osteoblasts and their progenitors, and the dysregulation of miRNAs, which target such factors, may play a pivotal role in disease etiology, or even as a possible therapy.

MicroRNA-132 and microRNA-223 control positive feedback circuit by regulating FOXO3a in inflammatory bowel disease.

Although many progresses have been achieved for inflammatory bowel disease (IBD), it is still remained as idiopathic disease to be completely controlled. MicroRNAs (miRNAs) have been identified as key players in many human diseases through degradation or translational inhibition of target genes. Because role of miRNAs in IBD is not completely understood yet, we need to identify miRNAs as novel targets for treatment of IBD. Microarray analysis for miRNAs was performed using dextran sulfate sodium-induced colitis samples and selected differentially regulated miRNAs. Candidate genes were validated using in vitro system and IBD patient samples. Molecular mechanism for regulation of inflammatory signaling was identified using gene modulation system of miRNAs. We selected 14 upregulated and 15 downregulated miRNAs through microarray analysis. Among candidate miRNAs, significant upregulation of miR-132 and miR-223 was confirmed in inflamed mouse tissues as well as human IBD patient tissues. Through bioinformatics analysis, we identified FOXO3a as direct target of miRNAs and confirmed regulatory mechanism using luciferase assay. Expression of miRNAs clearly suppressed the level of IκBα through downregulation of FOXO3a, leading to enhanced NF-κB signaling to promote the production of pro-inflammatory cytokines. The downregulation of FOXO3a concurrent with upregulation of cytokines was significantly reversed by sequestration of miRNAs with miRNA sponges. Our findings provided the evidences that miR-132 and 223 are critical mediators in positive circuit for pathogenesis of IBD by negatively regulating FOXO3a to enhance the expression of inflammatory cytokines and can be a good therapeutic target for IBD treatment.

Epstein-Barr viral microRNAs target caspase 3.

The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that transforms B cells and causes several malignancies including Burkitt’s lymphoma. EBV differentially expresses at least 49 mature microRNAs (miRNAs) during latency in various infected epithelial and B cells. Recent high-throughput studies and functional assays have begun to reveal the function of the EBV miRNAs suggesting roles in latency, cell cycle control, and apoptosis. In particular, the central executioner of apoptosis, Caspase 3 (CASP3), was proposed as a target of select EBV miRNAs. However, whether CASP3 is truly a target of EBV miRNAs, and if so, which specific miRNAs target CASP3 is still under debate. Based on previously published high-throughput biochemical data and a bioinformatic analysis of the entire CASP3 3′-UTR, we identified 12 EBV miRNAs that have one or more seed binding sites in the CASP3 3′-UTR. We individually tested all 12 miRNAs for repression of CASP3 in luciferase reporter assays, and nine showed statistically significant (P < 0.001) repression of a full-length CASP3 reporter. Further, three EBV miRNAs, including BART22, exhibited repression of endogenous CASP3 protein. These data confirm that CASP3 is a direct target of specific EBV BART miRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0602-7) contains supplementary material, which is available to authorized users.

MiR-508-3p concordantly silences NFKB1 and RELA to inactivate canonical NF-κB signaling in gastric carcinogenesis.

BackgroundNF-κB signaling pathway plays an important role in gastric carcinogenesis. The basic expression and functional role of NFKB1 and RELA (components of canonical NF-κB pathway) in gastric cancer (GC) have not been well elucidated. In this study, the role of NFKB1 and RELA in gastric tumorigenesis will be investigated and their regulation by microRNAs (miRNAs) will be deeply explored.MethodsThe mRNA and protein expression of NFKB1 and RELA were investigated by qRT-PCR and Western blot in GC cell lines and primary tumors. The functional roles of NFKB1 and RELA in GC were demonstrated by MTT proliferation assay, monolayer colony formation, cell invasion and migration, cell cycle analysis and in vivo study through siRNA mediated knockdown. Identification of NFKB1 as a direct target of tumor suppressor miRNA miR-508-3p was achieved by expression regulation assays together with dual luciferase activity experiments.ResultsNFKB1 and RELA were up-regulated in GC cell lines and primary tumors compared with normal gastric epithelium cells and their upregulation correlation with poor survival in GC. siRNA mediated knockdown of NFKB1 or RELA exhibited anti-oncogenic effect both in vitro and in vivo. NFKB1 was further revealed to be a direct target of miR-508-3p in gastric tumorigenesis and their expression showed negative correlation in primary GC samples. miR-508-3p was down-regulated in GC cells compared with normal gastric epithelium samples and its ectopic expression in GC cell lines also exerts tumor suppressor function. NFKB1 re-expression was found to partly abolish the tumor-suppressive effect of miR-508-3p in GC.ConclusionAll these findings supports that canonical NF-κB signaling pathway is activated in GC at least by the inactivation of miR-508-3p and this might have therapeutic potential in GC treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0493-7) contains supplementary material, which is available to authorized users.

Abstract 3998: miRNA associated with distal metastasis and local recurrence after post-operative radiotherapy in high-risk head and neck cancer

Abstract Distal metastasis and local-regional recurrence after radiation therapy is a major cause of treatment failure for patients with head and neck squamous cell carcinoma. miRNA have been proposed as biomarkers in cancer to predict therapeutic outcomes and has been associated with the cellular response to radiation. The advantages of miRNA as a biomarker lies in its stability in tissues as well as body fluids, hence the potential for non-invasive diagnosis and prognosis. In this study, we performed miRNA expression profiling using tumor samples from 114 head and neck cancer patients treated by post-operative radiotherapy (PORT) at M.D. Anderson Cancer Center from 1992 to 1999. All patients were considered to be at high-risk for recurrence having histologically proven advanced squamous cell carcinoma. Amongst these samples, 41 had distal metastasis (DM), 24 recurred locally or local/regional (LR) and 49 responded to PORT (no evidence of disease (NED)). Total RNA was extracted using a modified protocol of the Qiagen RNeasy Plus kit which retains small RNA fractions. miRNA profiling was performed using miRCURY 7th gen LNA™ microRNA Array from Exiqon. Microarray data were background-subtracted using mis-match control probes and normalized with median scaling. An ANOVA model based on clinical outcome was used to compare of miRNA expression between DM vs. NED and LR vs NED. There are 94 miRNAs that differentially expressed in DM and 60 miRNAs differentially expressed in LR with statistical significance (p &amp;lt; 0.05, Fold change &amp;gt; 1.4). Among them, miR-551a and miR-551b-3p are significantly associated with the DM group and have been validated using qPCR. By transfection of either mimic or an inhibitor of these miRNAs using the HN5 head and neck cancer cell line, we have confirmed that both miR-551a and miR-551b-3p promote cell proliferation, enhance cell migration and invasion. We have further discovered GLIPR2, an autophagy inhibitor, as a direct binding target of both miRNAs using luciferase assay. By over-expressing miR-551a/miR-551b-3p or suppressing GLIPR2 expression, we detected higher level autophagy with increased LC3 foci and overexpression of lipidated LC3B. Base on the above data, we propose a model that miR-551a and miR-551b-3p enhance radio-resistance and metastasis by promoting autophagy via suppressing GLIPR2 expression. A panel of 49 head and neck cancer cell lines were used for aCGH study and miRNA profiling. Further analysis is underway to correlate the patterns of DNA copy number variation and miRNA expression with radio-sensitivity. The findings in these cell lines will also be validated in tumor tissue samples. In summary, our data demonstrated the potential of miRNAs as biomarkers that predict therapeutic outcomes and mechanistic studies of miRNA markers will lead to novel pathway discovery and further improvement of cancer treatment. Citation Format: Lianghao Ding, Narasimha Kumar Karanam, John S. Yordy, Uma Giri, Michael D. Story. miRNA associated with distal metastasis and local recurrence after post-operative radiotherapy in high-risk head and neck cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3998. doi:10.1158/1538-7445.AM2015-3998

Abstract 3129: miR-340 is a modulator of oncogenic signaling in melanoma

Abstract Melanoma is responsible for only 4% of skin cancer diagnoses; nonetheless 80% of skin cancer deaths are attributed to this aggressive skin cancer each year. Growing evidence points to an important role miRNAs may play in the aberrant cellular signaling of cancers, including melanoma. microRNAs (miRNAs) are highly conserved 22-25nt non-coding RNAs involved in regulating RNA stability and translation and are predicted to regulate ∼60% of the human transcriptome. We are investigating the tumor suppressor role miR-340 plays in the development and progression of melanoma. A limited number of miR-340 targets were previously characterized, however, it is unlikely the effects of miR-340 on the melanoma phenotype are the result of directly targeting a few mRNAs, as miRNAs are likely to target upwards of 100 mRNAs each. Using an in silico target identification and pathway mapping system, we identified a multitude of potential miR-340 mRNA targets that encode proteins distributed amongst a variety of cellular signaling pathways. In silico analysis revealed a high proportion of miR-340 predicted mRNA targets that encode for members of the MAPK signaling pathway. Our results show that miR-340 modulated most of the assayed mRNAs that encode MAPK components. We also demonstrate that miR-340 inhibits the tumorigenic phenotype of melanoma cells. We recently developed a novel method to identify direct targets of miRNAs. Using this method, we demonstrate miR-340 directly targets mRNAs that encode protein kinases of the MAPK pathway. Further direct target identification is crucial toward understanding the role miR-340-directed regulation plays in triggering pleiotropic effects during melanoma development and progression. Elucidating the roles aberrant gene regulatory networks play in melanoma is critical for understanding the disease and developing treatment options to improve the melanoma prognosis. Supported by NIH grants ES007015 (to A.M.P.S.), AR063361 (to V.S.S.) and CA191550 (to V.S.S.). Citation Format: Ashley M. Poenitzsch Strong, Emily G. Adochio, Vijayasaradhi Setaluri, Vladimir S. Spiegelman. miR-340 is a modulator of oncogenic signaling in melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3129. doi:10.1158/1538-7445.AM2015-3129

MicroRNA-mediated regulation of p21 and TASK1 cellular restriction factors enhances HIV-1 infection.

MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in the regulation of gene expression by binding to target mRNAs. Several studies have revealed alterations in cellular miRNA profiles following HIV-1 infection, mostly for miRNAs involved in inhibiting viral infection. These miRNA expression modifications might also serve to block the innate HIV-1 inhibition mechanism. As a result, it is expected that during HIV-1 infection miRNAs target genes that hinder or prevent the progression of the HIV-1 replication cycle. One of the major sets of genes known to inhibit the progression of HIV-1 infection are cellular restriction factors. In this study, we identified a direct miRNA target gene that modulates viral spread in T-lymphocytes and HeLa-CCR5 cell lines. Following infection, let-7c, miR-34a or miR-124a were upregulated, and they targeted and downregulated p21 and TASK1 (also known as CDKN1A and KCNK3, respectively) cellular proteins. This eventually led to increased virion release and higher copy number of viral genome transcripts in infected cells. Conversely, by downregulating these miRNAs, we could suppress viral replication and spread. Our data suggest that HIV-1 exploits the host miRNA cellular systems in order to block the innate inhibition mechanism, allowing a more efficient infection process.

Hunting the needle in the haystack: a guide to obtain biologically meaningful microRNA targets.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of ~23 nucleotides in length that form up a novel class of regulatory determinants, with a large set of target mRNAs postulated for every single miRNA. Thousands of miRNAs have been discovered so far, with hundreds of them shown to govern biological processes with impact on disease. However, very little is known about how they specifically interfere with biological pathways and disease mechanisms. To investigate this interaction, the hunt for direct miRNA targets that mediate the miRNA effects—the “needle in the haystack”—is an essential step. In this review we provide a comprehensive workflow of successfully applied methods starting from the identification of putative miRNA-target pairs, followed by validation of direct miRNA–mRNA interactions, and finally presenting methods that dissect the impact of particular miRNA-target pairs on a biological process or disease. This guide allows the way to be paved for obtaining biologically meaningful miRNA targets.

Embryonic stem cell-specific microRNAs contribute to pluripotency by inhibiting regulators of multiple differentiation pathways.

The findings that microRNAs (miRNAs) are essential for early development in many species and that embryonic miRNAs can reprogram somatic cells into induced pluripotent stem cells suggest that these miRNAs act directly on transcriptional and chromatin regulators of pluripotency. To elucidate the transcription regulatory networks immediately downstream of embryonic miRNAs, we extended the motif activity response analysis approach that infers the regulatory impact of both transcription factors (TFs) and miRNAs from genome-wide expression states. Applying this approach to multiple experimental data sets generated from mouse embryonic stem cells (ESCs) that did or did not express miRNAs of the ESC-specific miR-290-295 cluster, we identified multiple TFs that are direct miRNA targets, some of which are known to be active during cell differentiation. Our results provide new insights into the transcription regulatory network downstream of ESC-specific miRNAs, indicating that these miRNAs act on cell cycle and chromatin regulators at several levels and downregulate TFs that are involved in the innate immune response.

Identification of hepatocyte nuclear factor‐1β gene regulatory networks in mouse kidney cells (690.9)

Hepatocyte nuclear factor‐1β (HNF‐1β) is a transcription factor that is expressed in renal tubules in the mammalian kidney. Mutations of HNF‐1β produce kidney cysts and electrolyte disorders. HNF‐1β has been shown to regulate the expression of cystic disease genes and solute transporters. The aim of this study was to identify the complete spectrum of genes that are regulated by HNF‐1β in the kidney. We performed genome‐wide chromatin immunoprecipitation and massively parallel sequencing (ChIP‐seq) in combination with microarray analysis to identify direct mRNA and miRNA targets in mouse mIMCD3 cells. ChIP‐seq analysis identified 4,725 mRNAs and 85 miRNAs as potential targets of HNF‐1β. Microarray analysis showed altered expression of 4,878 mRNAs and 51 miRNAs in cells expressing dominant‐negative mutant HNF‐1β. Combining the ChIP‐seq and microarray data identified 1,545 mRNAs and 22 miRNAs that were directly regulated by HNF‐1β. Pathway analysis revealed that HNF‐1β directly regulated the expression of multiple genes involved in cholesterol and sphingolipid metabolism. Liquid chromatography‐mass spectrometry showed corresponding alterations in the levels of sterols and sphingolipids in cells expressing mutant HNF‐1β. These findings provide new insights into the HNF‐1β transcriptional network in kidney cells and identify a novel role for HNF‐1β in regulating lipid metabolism in the kidney.

MiR-18b and miR-518b TargetFOXN1During Epithelial Lineage Differentiation in Pluripotent Cells

MicroRNAs (miRNAs) regulate myriad biological processes; however, their role in cell fate choice is relatively unexplored. Pluripotent NT2/D1 embryonal carcinoma cells differentiate into an epithelial/smooth muscle phenotype when treated with bone morphogenetic protein-2 (BMP-2). To identify miRNAs involved in epithelial cell development, we performed miRNA profiling of NT2/D1 cells treated with BMP-2 at 6, 12, and 24 h, and on days 6 and 10. Integration of the miRNA profiling data with previously obtained gene expression profiling (GEP) data of NT2/D1 cells treated with BMP-2 at the same time points identified miR-18b and miR-518b as the top two miRNAs with the highest number of up-regulated predicted targets with known functions in epithelial lineage development. Silencing of miR-18b and miR-518b in NT2/D1 cells revealed several up-regulated TFs with functions in epithelial lineage development; among these, target prediction programs identified FOXN1 as the only direct target of both miRNAs. FOXN1 has previously been shown to play an important role in keratinocyte differentiation and epithelial cell proliferation. NT2/D1 and H9 human embryonic stem cells with silenced miR-18b and miR-518b showed up-regulation of FOXN1 and the epithelial markers CDH1, EPCAM, KRT19, and KRT7. A 3'UTR luciferase assay confirmed FOXN1 to be a target of the two miRNAs, and up-regulation of FOXN1 in NT2/D1 cells led to the expression of epithelial markers. Overexpression of the two miRNAs in BMP-2-treated NT2/D1 cells led to down-regulation of FOXN1 and epithelial lineage markers. These results show that miR-18b and miR-518b are upstream controllers of FOXN1-directed epithelial lineage development.

Faster experimental validation of microRNA targets using cold fusion cloning and a dual firefly-Renilla luciferase reporter assay.

Different target prediction algorithms have been developed to provide a list of candidate target genes for a given animal microRNAs (miRNAs). However, these computational approaches provide both false-positive and false-negative predictions. Therefore, the target genes of a specific miRNA identified in silico should be experimentally validated. In this chapter, we describe a step-by-step protocol for the experimental validation of a direct miRNA target using a faster Dual Firefly-Renilla Luciferase Reporter Assay. We describe how to construct reporter plasmids using the simple, fast, and highly efficient cold fusion cloning technology, which does not require ligase, phosphatase, or restriction enzymes. In addition, we provide a protocol for co-transfection of reporter plasmids with either miRNA mimics or miRNA inhibitors in human embryonic kidney 293 (HEK293) cells, as well as a description on how to measure Firefly and Renilla luciferase activity using the Dual-Glo Luciferase Assay kit. As an example of the use of this technology, we will validate glucose-6-phosphate dehydrogenase (G6PD) as a direct target of miR-1207-5p.

Regulation of Cystic Fibrosis Transmembrane Conductance Regulator by MicroRNA-145, -223, and -494 Is Altered in ΔF508 Cystic Fibrosis Airway Epithelium

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is altered in individuals with the ΔF508 CFTR mutation. We previously reported differential expression of microRNA (miRNA) in CF airway epithelium; however, the role of miRNA in regulation of CFTR expression here remains unexplored. In this study, we investigated the role of upregulated miRNAs in CFTR regulation in vivo in bronchial brushings from individuals homozygous or heterozygous for ΔF508 CFTR, validated our observations in vitro, and assessed the impact of defective chloride ion conductance, genotype, and colonization status on miRNA expression. miRNA target prediction was performed in silico, and expression of miRNA and target genes were measured by quantitative real-time PCR and/or Western blotting. Overexpression and inhibition studies were performed with pre-miRs or antimiRs, respectively, and a luciferase reporter gene was used to elucidate direct miRNA-target interactions. miR-145, miR-223, and miR-494 were upregulated in CF versus non-CF bronchial brushings and cell lines; in ΔF508 CFTR homozygotes versus heterozygotes; in subjects positive for P. aeruginosa; and in cells treated with a CFTR inhibitor or IL-1β. Reciprocal downregulation or upregulation of CFTR gene and/or protein expression was observed after miRNA manipulation and direct miRNA-target relationships demonstrated via a reporter system containing a wild type or mutated full-length CFTR 3' untranslated region. Increased expression of miR-145, miR-223, and miR-494 in vivo in bronchial epithelium of individuals carrying the ΔF508 CFTR mutation correlates with decreased CFTR expression. Defective CFTR function, Pseudomonas colonization, and inflammation may affect miRNA expression and contribute to the regulation of ΔF508 CFTR.

Novel direct targets of miR-19a identified in breast cancer cells by a quantitative proteomic approach.

The miR-17–92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.