UTR Reporter Activity Research Articles

Post-transcriptional regulation of MRTF-A by miRNAs during myogenic differentiation of myoblasts.

The differentiation and regeneration of skeletal muscle from myoblasts to myotubes involves myogenic transcription factors, such as myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF). In addition, post-transcriptional regulation by miRNAs is required during myogenesis. Here, we provide evidence for novel mechanisms regulating MRTF-A during myogenic differentiation. Endogenous MRTF-A protein abundance and activity decreased during C2C12 differentiation, which was attributable to miRNA-directed inhibition. Conversely, overexpression of MRTF-A impaired differentiation and myosin expression. Applying miRNA trapping by RNA affinity purification (miTRAP), we identified miRNAs which directly regulate MRTF-A via its 3′UTR, including miR-1a-3p, miR-206-3p, miR-24-3p and miR-486-5p. These miRNAs were upregulated during differentiation and specifically recruited to the 3′UTR of MRTF-A. Concomitantly, Ago2 recruitment to the MRTF-A 3′UTR was considerably increased, whereas Dicer1 depletion or 3′UTR deletion elevated MRTF-A and inhibited differentiation. MRTF-A protein expression was inhibited by ectopic miRNA expression in murine C2C12 and primary human myoblasts. 3′UTR reporter activity diminished upon differentiation or miRNA expression, whereas deletion of the predicted binding sites reversed these effects. Furthermore, TGF-β abolished MRTF-A reduction and decreased miR-486-5p expression. Our findings implicate miR-24-3p and miR-486-5p in the repression of MRTF-A and suggest a complex network of transcriptional and post-transcriptional mechanisms regulating myogenesis.

Epstein-Barr virus-encoded miR-BART5-5p upregulates PD-L1 through PIAS3/pSTAT3 modulation, worsening clinical outcomes of PD-L1-positive gastric carcinomas.

Epstein-Barr virus (EBV) is etiologically associated with ~ 10% of all gastric carcinomas. However, the molecular mechanisms and roles of EBV miRNAs in gastric carcinoma oncogenesis are yet to be elucidated. MicroRNA microarray and TaqMan quantitative real-time RT-PCR were conducted. RT-PCR and luciferase reporter assay for PIAS3, western blotting for 20 proteins, immunofluorescence for STAT3, transfection with miRBART5-5p-plasmid, STAT3-plasmid, miRBART5-5p mimic, or PIAS3-siRNA, and in vitro assays for biological effects of PD-L1 were implemented. In situ hybridization for EBV-encoded small RNAs and immunohistochemistry were performed on gastric carcinoma tissues. Transfecting miR-BART5-5p into EBV(-) gastric carcinoma cell lines caused a decrease in PIAS3 3'-UTR reporter activity, PIAS3 downregulation, and subsequent STAT3 activation followed by PIAS3/pSTAT3-dependent PD-L1 upregulation. Interestingly, due to PD-L1 knockdown, apoptosis was increased, while the rate of cell proliferation, invasion capacity, and migration were decreased in miR-BART5-5p-transfected cells. In EBV(+) gastric carcinoma cells, anti-miR-BART5-5p reduced PD-L1 levels through PIAS3/pSTAT3 control. Among 103 patients with EBV-associated gastric carcinomas, overall survival was significantly shortened for those with PD-L1(+) tumors compared to those with PD-L1(-) tumors (P = 0.049). Our findings imply that miR-BART5-5p directly targets PIAS3 and augments PD-L1 through miR-BART5/PIAS3/pSTAT3/PD-L1 axis control. This contributes to antiapoptosis, tumor cell proliferation, invasion and migration, as well as immune escape, furthering gastric carcinoma progression and worsening the clinical outcome, especially in the PD-L1(+) group of patients with EBV-associated gastric carcinomas. miR-BART5-5p may, therefore, be amenable to PD-1/PD-L1 immune checkpoint inhibitor therapy.

Analysis of the relationship between microRNA-31 and interferon regulatory factor-1 in hepatocellular carcinoma cells.

MicroRNAs (miRNAs) play a role in the pathogenesis of hepatocellular carcinoma (HCC). This study was designed to elucidate the role of microRNA-31 (miR-31) in HCC. HuH7 cell lines were transfected with miR-31 mimic or miR-31 inhibitor to investigate the role of miR-31 in regulating interferon regulatory factor-1 (IRF-1). The mRNA and protein expression levels of IRF-1 were quantitatively detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Subsequently, Dual-Luciferase reporter assay was also performed. The expression level of miR-31 was significantly up-regulated in HuH7 cells when compared with that in primary human hepatocytes (hHC). Dual-Luciferase reporter assay indicated that IRF-1 was the direct target of miR-31. The expression levels of IRF-1 were decreased in HuH7 and HepG2 cell lines. IRF-1 was negatively correlated with miR-31 in HCC tissues and paired adjacent tissues. The expression level of miR-31 was inversely correlated with IRF-1. MiR-31 inhibitor up-regulated the expression levels of IRF-1 in HuH7 cells, whereas miR-31 mimic down-regulated the expression levels of IRF-1. Furthermore, the miR-31 mimic repressed IRF-1-3'UTR reporter activity, whereas the miR-31 inhibitor enhanced IRF-1-3'UTR reporter activity depending on the concentration of miR-31 mimic and miR-31 inhibitor. These results indicated that miR-31 can regulate the expression level of IRF-1 in HCC, which probably provided novel theoretical evidence for the application of target miR-31 treatment of HCC.

Full sequence of mutant huntingtin 3'-untranslated region and modulation of its gene regulatory activity by endogenous microRNA.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). Since the entire course of the disease starts from this dominant gain-of-function mutation, lowering total or mutant huntingtin mRNA/protein has emerged as an appealing therapeutic strategy. We reasoned that endogenous mechanisms underlying HTT gene regulation may inform strategies to target the source of the disease. As part of our investigation to understand how the expression of HTT is controlled, we performed (1) complete sequencing analysis for mutant HTT 3'-UTR and (2) unbiased screening assays to identify naturally-occurring miRNAs that could lower the HTT mRNA levels. By sequencing HD families inheriting the major European mutant haplotype, we determined the full sequence of HTT 3'-UTRs of the most frequent mutant (i.e., hap.01) and normal (i.e., hap.08) haplotypes, revealing 5 sites with alternative alleles. In subsequent miRNA activity assays using the full-length hap.01 and hap.08 3'-UTR reporter vectors and follow-up validation experiments, hsa-miR-4324 and hsa-miR-4756-5p significantly reduced HTT 3'-UTR reporter activity and endogenous HTT protein levels. However, those miRNAs did not show strong haplotype-specific effects. Nevertheless, our data highlighting full sequences of HTT 3'-UTR haplotypes, effects of miRNAs on HTT levels, and potential interaction sites provide rationale and promising targets for total and mutant-specific HTT lowering intervention strategies using endogenous and artificial miRNAs, respectively.

TGF‐β1‐induced miR‐424 Mediates Pulmonary Myofibroblast Differentiation via Targeting Slit2

Background and objectiveIdiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal interstitial lung disease. Transforming growth factor (TGF)‐β1, a major profibrogenic cytokine, induces differentiation of pulmonary fibroblasts to myofibroblasts that produce high levels of extracellular matrix, leading to loss of lung elasticity and function. Emerging evidence has implicated the importance of microRNAs (miRNAs) in the development of IPF but their regulation and individual pathological roles remain largely unknown. The objective of our study was to define the pro‐fibrotic roles and mechanisms of miRNAs in TGF‐β1‐induced pulmonary fibroblast differentiation.MethodsWe used both RNA sequencing and quantitative RT‐PCR strategies to systematically study TGF‐β1‐induced alternations of miRNAs in human lung fibroblasts (HLF). Significantly upregulated miRNAs in HLF were confirmed in lung tissue of IPF patients. Then, we overexpressed the identified miRNAs or their inhibitors to determine their importance in TGF‐β1‐induced HLF differentiation. Western blot and immunofluorescence staining were used to measure the changes in TGF‐β1 signaling and HLF differentiation. Finally, we searched for the putative targets of these miRNAs using online algorithms (Targetscan and mirSVR) and luciferase reporter assays were performed to determine whether the predicted binding site is directly targeted.ResultsWe identified miR‐424 as an important TGF‐β1‐induced miRNA in HLF. Quantitative RT‐PCR confirmed that miR‐424 expression was increased by 2.5‐fold in HLF in response to TGF‐β1 and was 2‐fold higher in IPF patients as compared to normal subjects. Overexpression of a miR‐424 inhibitor in HLF reduced TGF‐β1‐induced expression of classic myofibroblast differentiation markers including α‐smooth muscle actin (α‐SMA) and connective tissue growth factor (CTGF), whereas a miR‐424 mimic significantly enhanced TGF‐β1‐induced differentiation. Interestingly, upregulation of miR‐424 was blocked by the Smad3 inhibitor SIS3, suggesting the involvement of the canonical TGF‐β1 signaling pathway. In addition, TGF‐β1‐induced Smad3 phosphorylation in HLF was reduced by the miR‐424 inhibitor. Among the putative targets of miR‐424, Slit2 inhibits TGF‐β1‐induced Smad3 phosphorylation. Luciferase reporter assays showed that miR‐424 represses wild‐type Slit2‐3′UTR reporter activity without effects on a mutant Slit2‐3′UTR reporter, indicating the direct regulation by miR‐424 of the 3′UTR of Slit2 mRNA. Indeed, overexpression of the miR‐424 mimic down‐regulated Slit2 expression in HLF, consistent with the reduced Slit2 expression in IPF patients.ConclusionMiR‐424 plays a pro‐fibrotic role in TGF‐β1‐induced HLF differentiation. It functions as a positive feed‐back regulator of the TGF‐β1 signaling pathway via targeting Slit2. Thus, inhibition of miR‐424 may provide a new therapeutic strategy to prevent IPF progression.Support or Funding InformationSupported by grants from NIH R01HL116849 to YT and AG053553 to KB, Nebraska State LB595 to YT and PWAThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-135a regulates sodium–calcium exchanger gene expression and cardiac electrical activity

Complete atrioventricular block (CAVB) causes arrhythmogenic remodeling and increases the risk of torsades de pointes arrhythmias. MicroRNAs (miRNAs) are key regulators of gene expression that contribute to cardiac remodeling. The purpose of this study was to assess miRNA changes after CAVB and identify novel candidates potentially involved in arrhythmogenic cardiac remodeling. CAVB was induced in mice via His-bundle ablation. Expression of miRNAs was evaluated by pan-miRNA microarray with quantitative polymerase chain reaction (qPCR) confirmation, on samples obtained 24 hours and 4 weeks post-CAVB. MiRNA target prediction algorithms were used to identify potential target genes. Targets confirmed by luciferase assays in HEK293 cells were followed up with overexpression studies in neonatal rat ventricular myocytes to evaluate regulation using real time- quantitative polymerase chain reaction (RT-qPCR), western blots, cell shortening measurements, and fura-2 Ca2+ fluorescence imaging. Of >400 miRNAs assayed, only miRNA-135a (miR-135a) was altered at 24 hours, down-regulated 78% (P <.001). Algorithms predicted miR-135a regulation of the sodium-calcium exchanger type 1 (NCX1). miR-135a transfection suppressed NCX1 3'UTR reporter activity by 42% (P <.001), mRNA expression by 34% (P <.001), and protein levels by 45% (P <.001) vs noncoding miRNA control. miR-135a overexpression reduced spontaneous beating frequency of neonatal rat ventricular myocytes by 63% (P<.001) while slowing decay (by 56%, P <.05) of caffeine-induced Ca2+ transients. miR-135a also suppressed the Ca2+ loading effects of ouabain and ouabain-induced spontaneous Ca2+ release events. NCX1 is negatively regulated by miR-135a, a microRNA that is down-regulated in the heart after CAVB in mice. By controlling NCX1 expression, miR-135a modulates cardiomyocyte automaticity, Ca2+ extrusion, and arrhythmogenic Ca2+ loading/spontaneous Ca2+ release events. Therefore, miR-135a may contribute to proarrhythmic remodeling after CAVB.

Gga-miR-21 inhibits chicken pre-adipocyte proliferation in part by down-regulating Kruppel-like factor 5

Gga-miR-21 is abundantly expressed in chicken pre-adipocytes, but its role is unclear. The present study investigated the role of gga-miR-21 in chicken pre-adipocyte proliferation. Cell proliferation assay and gene expression analysis of proliferating cell nuclear antigen (PCNA) showed that the gga-miR-21 mimic inhibited pre-adipocyte proliferation. In contrast, the gga-miR-21 inhibitor enhanced pre-adipocyte proliferation. The subsequent investigation identified Kruppel-like factor 5 (KLF5) mRNA as a target of gga-miR-21. The gga-miR-21 mimic inhibited KLF5 3′ UTR reporter activity and decreased endogenous KLF5 expression in primary pre-adipocytes. KLF5 knockdown using RNAi had a similar effect to that of the gga-miR-21 mimic on cell proliferation. The promoting effect of the gga-miR-21 inhibitor on pre-adipocyte proliferation was partially attenuated by KLF5 knockdown. Taken together, our results demonstrated that miR-21 inhibits chicken pre-adipocyte proliferation, at least in part, by targeting KLF5.

The tumor suppressor miR-124 inhibits cell proliferation and invasion by targeting B7-H3 in osteosarcoma.

Our previous studies have shown that the expression level of B7 homolog 3 (B7-H3) was correlated with clinical staging and prognosis of osteosarcoma (OS) patients, and its silencing inhibited the proliferation and invasion of OS cells in vitro. However, its overexpression mechanism behind was far from elucidated. On the basis of bioinformatics and the preliminary screening data, we hypothesized that miR-124 might play an important role in OS development and as a lead candidate for modulating B7-H3 expression. In this study, we found that miR-124 was downregulated significantly in OS tumor tissue, compared to normal adjacent tissues (NATs). Lower miR-124 expression levels were associated with advanced Ennecking stage, lower tumor differentiation, and common pulmonary metastasis. The 5-year overall survival rate in the miR-124 upregulated group was 61.5%, while with low miR-124 expression, only 11.8% survived. Further studies in vitro showed that B7-H3 was a direct target of miR-124. Overexpression of miR-124 decreased B7-H3 mRNA and protein level and inhibited B7-H3 3'-UTR reporter activity. Treatment of OS cells with miR-124 mimics induced the inhibition of cell growth and invasion in vitro, which could be abrogated by transfected by B7-H3 expression vector. Our findings highlight the potential application of miR-124 as a novel onco-miRNA in OS, and its oncogenic effects are mediated chiefly through downregulation of B7-H3, thus suggesting a model for identifying miR-124 that can be exploited to improve the therapeutic potential efficacy of mAb targeting to B7-H3.

MicroRNA-23a downregulates the expression of interferon regulatory factor-1 in hepatocellular carcinoma cells.

Interferon regulatory factor-1 (IRF-1) is a tumor-suppressor gene induced by interferon-γ (IFNγ) and plays an important role in the cell death of hepatocellular carcinoma (HCC). HCC tumors evade death in part by downregulating IRF-1 expression, yet the molecular mechanisms accounting for IRF-1 suppression in HCC have not yet been characterized. Previous studies have shown that microRNA-23a (miR-23a) can suppress apoptosis by targeting IRF-1. Therefore, we hypothesized that miR-23a promotes HCC growth by down-regulating IRF-1. For the in vivo studies, 7 cases of resected HCC and adjacent liver samples were analyzed. For the in vitro studies, IRF-1 mRNA and protein were examined in HepG2 and Huh-7 HCC cells after IFNγ stimulation by real-time PCR and western blotting, respectively. To determine the role of miR-23a in regulating IRF-1, HepG2 cells were transfected with an miR-23a mimic or inhibitor, and IRF-1 expression was examined. Binding of miR-23a was assessed by cloning the 528-bp human IRF-1 3′-untranslated region (3′UTR) into luciferase reporter plasmid pMIR-IRF-1-3′UTR. The results showed that IRF-1 mRNA expression was down-regulated in the human HCC tumor tissues compared to that in the adjacent background liver tissues. IFNγ-induced IRF-1 protein was less in the HepG2 tumor cells compared to that in the primary human hepatocytes. miR-23a expression was inversely correlated with IRF-1, and addition of the miR-23a inhibitor increased basal IRF-1 mRNA and protein. Likewise, the miR-23a mimic downregulated IFNγ-induced IRF-1 protein expression, while the miR-23a inhibitor increased IRF-1. Furthermore, the miR-23a mimic repressed IRF-1-3′UTR reporter activity, while the miR-23a inhibitor increased the reporter activity. These results demonstrated that IRF-1 expression is downregulated in human HCC tumors compared to that noted in the background liver. miR-23a downregulates the expression of IRF-1 in HCC cells, and the IRF-1 3′UTR has an miR-23a binding site that binds miR-23a and decreases reporter activity. These findings suggest that the targeting of IRF-1 by miR-23a may be the molecular basis for IRF-1 downregulation in HCC and provide new insight into the regulation of HCC by miRNAs.

25-methoxyl-dammarane-3β, 12β, 20-triol and artemisinin synergistically inhibit MDA-MB-231 cell proliferation through downregulation of testes-specific protease 50 (TSP50) expression.

While the incidence of cancer continues to increase, the current therapeutic options remain imperfect. Therefore, there is an urgent need to discover new targeted anti-cancer therapies. Testes-specific protease 50 (TSP50) is abnormally expressed in most cancer tissues and downregulation of TSP50 expression can reduce cell proliferation and induce cell apoptosis, which makes it a potential target for cancer therapy. In this study, we constructed a firefly luciferase reporter pGL3-TSP50-3'-UTR as a drug screening model to screen potential candidate compounds that target TSP50 mRNA. We identified the compound 7P3A, which consists of 70% 25-methoxyl-dammarane-3β, 12β, 20-triol and 30% artemisinin, as being capable of inhibiting the TSP50-3'-UTR reporter activity, as well as the expression of TSP50. Further investigation revealed that 7P3A could inhibit MDA-MB-231 cell proliferation and induce cell cycle arrest, and over-expression of TSP50 partially reversed the effect of 7P3A. In vivo investigation showed that 7P3A could inhibit tumor growth in a xenograft model of breast cancer. These results suggest that 7P3A exhibits anti-cancer effects, in part, through downregulation of TSP50 expression.

MicroRNA-23b Promotes Avian Leukosis Virus Subgroup J (ALV-J) Replication by Targeting IRF1.

Avian leukosis virus subgroup J (ALV-J) can cause several different leukemia-like proliferative diseases in the hemopoietic system of chickens. Here, we investigated the transcriptome profiles and miRNA expression profiles of ALV-J-infected and uninfected chicken spleens to identify the genes and miRNAs related to ALV-J invasion. In total, 252 genes and 167 miRNAs were differentially expressed in ALV-J-infected spleens compared to control uninfected spleens. miR-23b expression was up-regulated in ALV-J-infected spleens compared with the control spleens, and transcriptome analysis revealed that the expression of interferon regulatory factor 1 (IRF1) was down-regulated in ALV-J-infected spleens compared to uninfected spleens. A dual-luciferase reporter assay showed that IRF1 was a direct target of miR-23b. miR-23b overexpression significantly (P = 0.0022) decreased IRF1 mRNA levels and repressed IRF1-3′-UTR reporter activity. In vitro experiments revealed that miR-23b overexpression strengthened ALV-J replication, whereas miR-23b loss of function inhibited ALV-J replication. IRF1 overexpression inhibited ALV-J replication, and IRF1 knockdown enhanced ALV-J replication. Moreover, IRF1 overexpression significantly (P = 0.0014) increased IFN-β expression. In conclusion, these results suggested that miR-23b may play an important role in ALV-J replication by targeting IRF1.

Abstract P4-04-15: Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression

Abstract Background: Increasing evidence has demonstrated that stromal cells play a pivotal role to promote breast cancer progression and metastasis. Breast cancer stroma is comprised mainly of Cancer Associated Fibroblasts (CAFs). Upon interaction with tumor cells, CAFs promote tumor progression by providing paracrine oncogenic signals mediated by activation of various pathways including developmental pathways, integrin signaling, and the MAPK pathway in tumor cells. CAFs have also been shown to promote the survival of CTCs and help them in metastasis at distant sites. Using breast cancer patient tumor datasets, we have previously identified a microRNA signature reflective of hyperactive MAPK signaling and that is significantly associated with reduced recurrence-free and overall survival. We have established 3 primary breast CAF lines, one from a Luminal A breast cancer, one from an ER-/Her2 amplified cancer, and one from a triple negative cancer, along with several primary tumor-derived dissociated tumor (DT) culture models that are tumorigenic in vivo and vary in metastatic ability. The CAFs express several hMAPK-microRNAs preferentially compared to the DTs. In addition to the paracrine interaction of stromal and tumor cells mediated by chemokines or hormones, miRNA cross talk between stromal and tumor cells can also occur. Results: To further investigate the connection between our miRNA signature and stroma, we analyzed the TCGA and METABRIC breast cancer datasets and found that the hMAPK-miRNA identifies tumors that with signficant stromal cell infiltrate. To investigate the role of specific expression of hMAPK-miRNAs in the CAFs, CM was isolated from CAFs from "aggressive" tumors, from the "indolent" tumor, and from normal human mammary fibroblasts (HMFs) and analyzed for exosome and microRNA secretion. CAFs from the aggressive tumors secrete more exosomes and more hMAPK-microRNAs into the CM than do HMFs or CAFs from the indolent tumor. Importantly, conditioned media (CM) from the "aggressive" CAFs activate MAPK and repress ER protein, mRNA and ER 3’UTR-reporter activity in ER+ MCF-7 breast cancer cells, while HMFs and "indolent" CAFs did not. Exosomes from the "aggressive" CAFs were responsible for the ER repression. To determine if the secreted miRNA differences exhibited by the CAFs could be seen in patients, serum from breast cancer patients with metastatic breast cancer and patients without metastases was analyzed for microRNA expression. Differentially expressed circulating hMAPK-miRNAs were identified in serum from metastatic breast cancer patients compared with patients without metastatses. Further analyses of the serum for CTCs and CAFs show that serum samples from metastatic patients had a significantly higher number of CTCs with CAFs compared to serum from patients without metastases. Conclusions: Collectively these data suggest that different CAF populations have distinct abilities to influence the phenotype and behavior of associated cancer cells and that CAF secreted hMAPK-miRNAs may play important roles in breast cancer progression. They further suggest that these CAF secreted miRNAs can be found in patient serum along with circulating CAFs. Citation Format: Sanket H Shah, Phil Miller, Zheng Ao, Emilio Issa, Katherine Drews Elger, Ram Datar, Marc Lippman, Dorraya El-Ashry. Hierarchy of breast cancer associated fibroblasts communicate with cancer cells via microRNAs to drive breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-04-15.

MicroRNA-31 controls G protein alpha-13 (GNA13) expression and cell invasion in breast cancer cells.

BackgroundGα13 (GNA13) is the α subunit of a heterotrimeric G protein that mediates signaling through specific G protein-coupled receptors (GPCRs). Our recent study showed that control of GNA13 expression by specific microRNAs (miRNAs or miRs) is important for prostate cancer cell invasion. However, little is known about the control of GNA13 expression in breast cancers. This project was carried out to determine (i) whether enhanced GNA13 expression is important for breast cancer cell invasion, and (ii) if so, the mechanism of deregulation of GNA13 expression in breast cancers.MethodsTo determine the probable miRNAs regulating GNA13, online miRNA target prediction tool Targetscan and Luciferase assays with GNA13-3′-UTR were used. Effect of miRNAs on GNA13 mRNA, protein and invasion was studied using RT-PCR, western blotting and in vitro Boyden chamber assay respectively. Cell proliferation was done using MTT assays.ResultsOverexpression of GNA13 in MCF-10a cells induced invasion, whereas knockdown of GNA13 expression in MDA-MB-231 cells inhibited invasion. Expression analysis of miRNAs predicted to bind the 3′-UTR of GNA13 revealed that miR-31 exhibited an inverse correlation to GNA13 protein expression in breast cancer cells. Ectopic expression of miR-31 in MDA-MB-231 cells significantly reduced GNA13 mRNA and protein levels, as well as GNA13-3′-UTR-reporter activity. Conversely, blocking miR-31 activity in MCF-10a cells induced GNA13 mRNA, protein and 3′-UTR reporter activity. Further, expression of miR-31 significantly inhibited MDA-MB-231 cell invasion, and this effect was partly rescued by ectopic expression of GNA13 in these cells. Examination of 48 human breast cancer tissues revealed that GNA13 mRNA levels were inversely correlated to miR-31 levels.ConclusionsThese data provide strong evidence that GNA13 expression in breast cancer cells is regulated by post-transcriptional mechanisms involving miR-31. Additionally our data shows that miR-31 regulates breast cancer cell invasion partially via targeting GNA13 expression in breast cancer cells. Loss of miR-31 expression and increased GNA13 expression could be used as biomarkers of breast cancer progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0337-x) contains supplementary material, which is available to authorized users.

Abstract 11940: Mineralocorticoid Receptor Dysregulation by MicroRNA-135a in Bradycardic Arrhythmogenic Remodeling

Introduction: Complete atrioventricular block (CAVB) causes arrhythmogenic remodeling and increases the risk of Torsades de Pointes (TdP) arrhythmias. MicroRNAs (miRs) are key regulators of gene expression that contribute to cardiac remodeling. Here, we assess microRNA changes after CAVB and their potential significance in arrhythmogenic cardiac remodeling. Methods: CAVB was induced in mice via His bundle ablation. Expression of miRs was evaluated by pan-microRNA microarray with qPCR confirmation on samples from controls and at 24 hrs and 4 wks post-CAVB. Arrhythmias were detected by continuous monitoring. MicroRNA target prediction algorithms were used to identify potential target genes. Biological confirmation of targets was by luciferase assay and overexpression (OE) studies in HEK and H9c2 cells respectively. Results: TdP occurred within 24 hrs in 6/17 CAVB mice, vs no controls. Ventricular tachyarrhythmia episodes were frequent at 4 wks CAVB (212±83 per 24 hrs) and were not seen in controls. CAVB was followed by increased left ventricular (LV) dimension (by 31%, ***p&lt;.001), LV mass (by 33%, *p&lt;.05) and LV fractional shortening (by 33%*) at 4 wks. Of &gt;400 miRs assayed, only miR-135a was altered at 24 hrs (downregulated by 96%***, Fig. A), with 70% decrease at 4 wks. TargetScan predicted miR-135a regulation of the mineralocorticoid receptor (MCR), encoded by the NR3C2 gene. miR-135a OE suppressed NR3C2 3’UTR reporter activity 3.2-fold* (n=5, Fig. B). miR-135a OE did not affect NR3C2 mRNA (Fig. C) but significantly reduced NR3C2 protein expression (n=4, Fig. D). Coexpression with anti-miR-135a eliminated the NR3C2 downregulating effect of miR-135a OE. Conclusions: The mineralocorticoid receptor, encoded by the NR3C2 gene, is translationally regulated by miR-135a, a microRNA that is downregulated in the heart following CAVB in mice. These results implicate miR-135a/mineralocorticoid modulation in arrhythmogenic ventricular remodeling caused by CAVB.

Negative auto-regulation of myostatin expression is mediated by Smad3 and microRNA-27.

Growth factors, such as myostatin (Mstn), play an important role in regulating post-natal myogenesis. In fact, loss of Mstn has been shown to result in increased post-natal muscle growth through enhanced satellite cell functionality; while elevated levels of Mstn result in dramatic skeletal muscle wasting through a mechanism involving reduced protein synthesis and increased ubiquitin-mediated protein degradation. Here we show that miR-27a/b plays an important role in feed back auto-regulation of Mstn and thus regulation of post-natal myogenesis. Sequence analysis of Mstn 3′ UTR showed a single highly conserved miR-27a/b binding site and increased expression of miR-27a/b was correlated with decreased expression of Mstn and vice versa both in vitro and in mice in vivo. Moreover, we also show that Mstn gene expression was regulated by miR-27a/b. Treatment with miR-27a/b-specific AntagomiRs resulted in increased Mstn expression, reduced myoblast proliferation, impaired satellite cell activation and induction of skeletal muscle atrophy that was rescued upon either blockade of, or complete absence of, Mstn. Consistent with this, miR-27a over expression resulted in reduced Mstn expression, skeletal muscle hypertrophy and an increase in the number of activated satellite cells, all features consistent with impaired Mstn function. Loss of Smad3 was associated with increased levels of Mstn, concomitant with decreased miR-27a/b expression, which is consistent with impaired satellite cell function and muscular atrophy previously reported in Smad3-null mice. Interestingly, treatment with Mstn resulted in increased miR-27a/b expression, which was shown to be dependent on the activity of Smad3. These data highlight a novel auto-regulatory mechanism in which Mstn, via Smad3 signaling, regulates miR-27a/b and in turn its own expression. In support, Mstn-mediated inhibition of Mstn 3′ UTR reporter activity was reversed upon miR-27a/b-specific AntagomiR transfection. Therefore, miR-27a/b, through negatively regulating Mstn, plays a role in promoting satellite cell activation, myoblast proliferation and preventing muscle wasting.

MicroRNA-433 Inhibits Liver Cancer Cell Migration by Repressing the Protein Expression and Function of cAMP Response Element-binding Protein

We show for the first time that potent microRNA-433 (miR-433) inhibition of expression of the cAMP response element-binding protein CREB1 represses hepatocellular carcinoma (HCC) cell migration. We identified a miR-433 seed match region in human and mouse CREB1 3'-UTRs. Overexpression of miR-433 markedly decreased human CREB1 3'-UTR reporter activity, and the inhibitory effect of miR-433 was alleviated upon mutation of its binding site. Ectopic expression of miR-433 reduced CREB1 protein levels in a variety of human and mouse cancer cells, including HeLa, Hepa1, Huh7, and HepG2. Human CREB1 protein levels in highly invasive MHCC97H cells were diminished by expression of miR-433 but were induced by miR-433 antagomir (anti-miR-433). The expression of mouse CREB1 protein negatively correlated with miR-433 levels in nuclear receptor Shp(-/-) liver tissues and liver tumors compared with wild-type mice. miR-433 exhibited a significant repression of MHCC97H cell migration, which was reversed by anti-miR-433. Overexpressing miR-433 inhibited focus formation dramatically, demonstrating that miR-433 may exert a tumor suppressor function. Knockdown of CREB1 by siRNAs impeded MHCC97H cell migration and invasion and antagonized the effect of anti-miR-433. Interestingly, CREB1 siRNA decreased MHCC97H cell proliferation, which was not influenced by anti-miR-433. Overexpressing CREB1 decreased the inhibitory activity of miR-433. The CpG islands surrounding miR-433 were hypermethylated, and the DNA methylation agent 5'-aza-2'-deoxycytidine, but not the histone deacetylase inhibitor trichostatin A, drastically stimulated the expression of miR-433 and miR-127 in HCC cells. The latter is clustered with miR-433. The results reveal a critical role of miR-433 in mediating HCC cell migration via CREB1.

Proteome Modulation in H9c2 Cardiac Cells by microRNAs miR-378 and miR-378

MicroRNAs are a novel class of powerful endogenous regulators of gene expression. MiR-378 and miR-378* are localized in the first intron of the Ppargc1b gene that codes the transcriptional co-activator PGC-1β. The latter regulates energy expenditure as well as mitochondrial biogenesis. The miR-378:miR-378* hairpin is highly expressed in cardiac cells. To better assess their role in cardiomyocytes, we identified miR-378 and miR-378* targets via a proteomic screen. We established H9c2 cellular models of overexpression of miR-378 and miR-378* and identified a total of 86 down-regulated proteins in the presence of either one of these miRs. Functional annotation clustering showed that miR-378 and miR-378* regulate related pathways in cardiomyocytes, including energy metabolism, notably glycolysis, cytoskeleton, notably actin filaments and muscle contraction. Using bioinformatics algorithms we found that 20 proteins were predicted as direct targets of the miRs. We validated eight of these targets by quantitative RT-PCR and luciferase reporter assay. We found that miR-378 targets lactate dehydrogenase A and impacts on cell proliferation and survival whereas miR-378* targets cytoskeleton proteins actin and vimentin. Proteins involved in endoplasmic reticulum stress response such as chaperone and/or calcium buffering proteins GRP78, PPIA (cyclophilin A), calumenin, and GMMPA involved in glycosylation are repressed by these miRs. Our results show that the miR-378/378* hairpin establishes a connection among energy metabolism, cytoskeleton remodeling, and endoplasmic reticulum function through post-transcriptional regulation of key proteins involved in theses pathways.

Tumor Suppressor Functions of miR-133a in Colorectal Cancer

Dysregulated microRNA (miRNA) expression was profiled through a miRNA array comparison between human colorectal cancer tumors and their adjacent normal tissues. Specifically, using laser capture micro-dissection, miR-133a was shown to be significantly downregulated in primary colorectal cancer specimens compared with matched adjacent normal tissue. Ectopic expression of miR-133a significantly suppressed colorectal cancer cell growth in vitro and in vivo. Cell-cycle analysis revealed that miR-133a induced a G0/G1-phase arrest, concomitant with the upregulation of the key G1-phase regulator p21(Cip1). We further revealed that miR-133a markedly increased p53 protein and induced p21(Cip1) transcription. Studies in silico revealed that the 3'UTR of the ring finger and FYVE-like domain containing E3-ubiquitin protein ligase (RFFL), which regulates p53 protein, contains an evolutionarily conserved miR-133a binding site. miR-133a repressed RFFL-3'UTR reporter activity and reduced RFFL protein levels, indicating that miR-133a directly bound to RFFL mRNA and inhibited RFFL translation. Moreover, miR-133a sensitized colon cancer cells to doxorubicin and oxaliplatin by enhancing apoptosis and inhibiting cell proliferation. These data add weight to the significance of miR-133a in the development of CRC. miR-133a serves as a potential tumor suppressor upstream of p53 in colorectal cancer and may sensitize cells to therapeutics.

MicroRNA-187, down-regulated in clear cell renal cell carcinoma and associated with lower survival, inhibits cell growth and migration though targeting B7-H3

Aberrantly expressed microRNAs (miRNAs) are frequently associated with the aggressive malignant behavior of human cancers, including clear cell renal cell carcinoma (ccRCC). Based on the preliminary deep sequencing data, we hypothesized that miR-187 may play an important role in ccRCC development. In this study, we found that miR-187 was down-regulated in both tumor tissue and plasma of ccRCC patients. Lower miR-187 expression levels were associated with higher tumor grade and stage. All patients with high miR-187 expression survived 5years, while with low miR-187 expression, only 42% survived. Suppressed in vitro proliferation, inhibited in vivo tumor growth, and decreased motility were observed in cells treated with the miR-187 expression vector. Further studies showed that B7 homolog 3 (B7-H3) is a direct target of miR-187. Over-expression of miR-187 decreased B7-H3 mRNA level and repressed B7-H3-3′-UTR reporter activity. Knockdown of B7-H3 using siRNA resulted in similar phenotype changes as that observed for overexpression of miR-187. Our data suggest that miR-187 is emerging as a novel player in the disease state of ccRCC. miR-187 plays a tumor suppressor role in ccRCC.

MicroRNA-15b contributes to ginsenoside-Rg1-induced angiogenesis through increased expression of VEGFR-2

Ginsenoside-Rg1 (Rg1) has been identified as potent proangiogenic agent, which plays an important role in wound healing promotion or treatment of ischemic injury. We previously reported that miR-214/eNOS pathway was involved in Rg1-induced angiogenesis. Following the same microRNA microarray profiling data, we proposed miR-15b would be another microRNA candidate involved in Rg1-induced angiogenesis. Using human umbilical vein endothelial cells (HUVECs), it was showed that Rg1 could reduce miR-15b expression rapidly and steadily, leading to a temporal induction of vascular endothelial growth factor receptor-2 (VEGFR-2). The in vitro motility and tubulogenesis via VEGFR-2 in Rg1-treated HUVECs were also demonstrated. Besides, the reduction of VEGFR-2 3′-UTR reporter activity by miR-15b in the luciferase reporter gene assay clearly indicated that miR-15b could affect the VEGFR-2 transcript through targeting its 3′-UTR region. Diminishing expression of endogenous miR-15b could increase VEGFR-2 expression and HUVECs migration and tubulogenesis; while over-expression of miR-15b was found to associate with the reduction of VEGFR-2 expression as well as cellular migration and tubulogenesis. In vivo, artificial increment of miR-15b by injecting Pre-miR-15b precursor into zebrafish embryos was also found to significantly suppress the subintestinal vessels formation. In conclusion, our results further demonstrated the involvement of microRNAs in Rg1-induced angiogenesis.