Downregulation Of miR-155 Research Articles

Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus-affected gestations: A focus on myogenic and neural differentiations.

Amniotic fluid-derived mesenchymal stem cells (AF-MSCs) are autologous to the fetus and represent a potential alternative source for the regenerative medicine and treatment of perinatal disorders. To date, AF-MSCs differentiation capacity to non-mesodermal lineages and epigenetic regulation are still poorly characterized. The present study investigated the differentiation potential of AF-MSCs toward neural-like cells in comparison to the mesodermal myogenic lineage and assessed epigenetic factors involved in tissue-specific differentiation. Myogenic and neural differentiation assays were performed by the incubation with specific induction media. Typical MSCs markers were determined by flow cytometry, the expression of lineage-specific genes, microRNAs and chromatin modifying proteins were examined by RT-qPCR and Western blot, respectively. AF-MSCs of normal and fetus-affected gestations had similar stem cells characteristics and two-lineage potential, as characterized by cell morphology and the expression of myogenic and neural markers. Two-lineage differentiation process was associated with the down-regulation of miR-17 and miR-21, the up-regulation of miR-34a, miR-146a and DNMT3a/DNMT3b along with the gradual decrease in the levels of DNMT1, HDAC1, active marks of chromatin (H4hyperAc, H3K9ac, H3K4me3) and the repressive H3K9me3 mark. Differentiation was accompanied by the down-regulation of PRC1/2 proteins (BMI1/SUZ12, EZH2) and the retention of the repressive H3K27me3 mark. We report that both AF-MSCs of normal and fetus-affected gestations possess differentiation capacity toward myogenic and neural lineages through rather similar epigenetic mechanisms that may provide potential applications for further investigation of the molecular basis of prenatal diseases and for the future autologous therapy.

MicroRNA‑223 attenuates LPS‑induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF‑κB signaling pathway via RHOB.

Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common and complex inflammatory lung diseases. MicroRNAs (miRs) have emerged as novel gene regulatory molecules, serving a crucial role in a variety of complex diseases, including ALI. In the present study, the anti-inflammatory action of miR-223 on inflammation in ALI was demonstrated and the possible mechanism was further examined. In lipopoly-saccharide-induced ALI, the expression of miR-223 was reduced compared with that in the control normal group. An in vitro model was used to analyze the effect of miR-223 downregulation on an ALI model, which increased inflammation, and induced the activation of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome and Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB signaling pathway via rho-related GTP-binding protein RhoB (RHOB). In addition, the overexpression of miR-223 reduced inflammation and suppressed the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB in the in vitro model. Furthermore, TLR4 inhibitor or NLRP3 inhibitor reduced the pro-inflammatory effect of miR-223 downregulation in ALI. In conclusion, the results of the present study indicated that miR-223 functioned as a biological indicator by regulating inflammation in ALI, and may represent a novel potential therapeutic target and prognostic marker of ALI.

Targeting astrocyte and motor neuron specific miRNAs to prevent neuro-immune dysregulation in ALS

Event Abstract Back to Event Targeting astrocyte and motor neuron specific miRNAs to prevent neuro-immune dysregulation in ALS Ana R. Vaz1, 2, Catarina Sequeira1, Daniela Vizinha1, Marta Barbosa1, Cátia Gomes1 and Dora Brites1, 2, 3* 1 Research Institute for Medicines (iMed.ULisboa), Portugal 2 Faculdade de Farmácia, Universidade de Lisboa, Department of Biochemistry and Human Biology, Portugal 3 Faculdade de Farmácia, Universidade de Lisboa, Portugal Amyotrophic lateral sclerosis (ALS) is characterized by motor neurons (MNs) loss and glial reactivity in brain and spinal cord (SC). A dysregulated expression of microRNAs (miRNAs) have been implicated in neurodegeneration and neuroinflammation in ALS. MiRNA(miR)-124 is elevated in the cerebrospinal fluid of ALS patients and in the SC of the ALS mice with the G93A mutation in human superoxide dismutase-1 (mSOD1). We demonstrated that this miRNA is upregulated in MN-NSC-34 overexpressing mSOD1 and in small extracellular vesicles isolated from cell secretome [1]. We also observed miR-155 upregulation in the SC of pe-symptomatic mSOD1 mice [2], often associated with mSOD1 microglia [3], but with unknown effect when expressed by astrocytes. In contrast, miR-146a was downregulated either in the cortical brain (CB) of symptomatic mSOD1 mice and in isolated astrocytes [4], suggesting distinct miRNA deregulation depending on the CNS region. Here, we evaluated the effects of miR-155/146a/124 modulation in astrocytic and MN function. For that, we used primary astrocytes from mSOD1 mice pups (CB or SC) and mSOD1 MN-NSC-34. Cells were transfected with anti-miR-155 (SC astrocytes), premiR-146a (CB astrocytes) or pre/anti-miR-124 (MN). We also explored whether changes in MN-miR-124 expression impact on secretome-mediated astrocytic function. Downregulation of miR-155 in mSOD1 SC astrocytes counteracted the inflammatory milieu, by decreasing TNF-α/IL-1β/HMGB1/miR-124, and increasing IL-6/IL-10/SOCS1/miR-146a expression, without changing the astrocytic aberrant phenotype (GFAPlow/Cx43high/S100Bhigh). However, miR-146a upregulation in mSOD1 CB astrocytes neutralized both the phenotypic aberrancy and the inflammatory profile. The upregulation of miR-124 in WT-MNs mimicked the pathological features of mSOD1-MNs, namely inflammatory-associated-miRNAs (increased miR-125b/decreased miR-146a/-21), synaptic dysfunction (increased synaptophysin/decreased PDS-95), neurite loss, reduced mitochondria viability and axonal transport deficits (increased dynein/decreased kinesin), while miR-124 downregulation was able to prevent those dysfunctionalities in mSOD1-MNs. The astrocytic aberrant phenotype of mSOD1 SC astrocytes was observed in WT cells upon incubation with secretome from mSOD1-MNs or pre-miR-124-WT-MN, but prevented in the presence of the secretome of anti-miR-124-mSOD1-MN. More importantly, secretome derived from anti-miR-124-mSOD1-MN rescued the aberrant phenotype of mSOD1 SC astrocytes by increasing GFAP/GLT-1 expression. Overall, our results highlight that combined modulation of specific miRNAs in different target cells may represent a promising therapeutic potential in ALS disease. Acknowledgements Funded by SCML (ALS Research Grant ELA-2015-002) and FCT (PTDC/MED-NEU/31395/2017 and LISBOA-01-0145-FEDER-031395 to DB, UID/DTP/04138/2013 to iMed.ULisboa, and fellowship ARV-SFRH/BPD/76590/2011).

Down-regulation of miR-9 promotes epithelial mesenchymal transition via regulating anoctamin-1 (ANO1) in CRC cells

MicroRNA-9 (miR-9) has been reported to play a suppressive or promoting role according to cancer type. In this study, we investigated the effects of anoctamin-1 (ANO1) and miR-9 on colorectal cancer (CRC) cell proliferation, migration, and invasion and determined the underlying molecular mechanisms.Thirty-two paired CRC tissues and adjacent normal tissues were analyzed for ANO1 expression using quantitative real-time PCR (qRT-PCR). HCT116 cells were transiently transfected with miR-9 mimic, miR-9 inhibitor, or si-ANO1. Cell proliferation was determined by MTT, and flow cytometric analysis, while cell migration and invasion were assayed by trans-well migration and invasion assay in HCT116 cells. ANO1 was validated as a target of miR-9 using luciferase reporter assay and bioinformatics algorithms.We found that ANO1 expression was up-regulated in CRC tissues compared with adjacent normal tissues. ANO1 expression was associated with advanced tumor stage and lymph node metastasis, and there was an inverse relationship between miR-9 and ANO1 mRNA expression in CRC specimens, but no significant difference was found between miR-9 and ANO1 expression. ANO1 is a direct target of miR-9, and overexpression of miR-9 suppressed both mRNA and protein expression of ANO1 and inhibited cell proliferation, migration, and invasion of HCT116 cells. We also showed that overexpression of miR-9 suppressed expression of p-AKT, cyclin D1, and p-ERK in HCT116 cells.We conclude that miR-9 inhibits CRC cell proliferation, migration, and invasion by directly targeting ANO1, and miR-9/ANO1 could be a potential therapeutic target for CRC.

Resveratrol Inhibits the TGF-β1-Induced Proliferation of Cardiac Fibroblasts and Collagen Secretion by Downregulating miR-17 in Rat.

Myocardial fibrosis (MF) can cause heart remodeling and it is an independent risk factor for malignant arrhythmias, sudden cardiac death, and other malignant cardiovascular events. It is often characterized by myocardial interstitial collagen deposition and hyperproliferation of cardiac fibroblasts (CFs). The transforming growth factor-β1 (TGF-β1) is the most influential profibrogenic factor. Resveratrol (RSV) is an active polyphenol substance that inhibits myocardial fibrosis. The mechanism of RSV-mediated inhibition of the proliferation of CFs at the microRNA level is not fully understood. We used TGF-β1 to induce CFs proliferation to simulate the pathogenesis of myocardial fibrosis. Neonatal rat CFs were treated with TGF-β1 in the presence or absence of resveratrol. Cell proliferation was measured using the CCK-8 and EdU assay. Collagen secretion was measured using hydroxyproline kit. Further, qPCR analysis was performed to determine microRNA levels after TGF-β1 or resveratrol treatment. To identify the target gene for miR-17, miR-17 was overexpressed or silenced, and the mRNA and protein levels of Smad7 were assessed. The effects of miR-17 silencing or Smad7 overexpression on cell proliferation and collagen secretion were also examined. Resveratrol treatment significantly decreased the TGF-β1-induced CF proliferation and collagen secretion. Resveratrol also decreased the levels of miR-17, miR-34a, and miR-181a in TGF-β1-treated CFs. Overexpression of miR-17 decreased the Smad7 mRNA and protein levels while silencing miR-17 increased them. Additionally, silencing miR-17 or overexpressing Smad7 decreased the TGF-β1-induced CFs proliferation and collagen secretion. In conclusion, resveratrol inhibits TGF-β1-induced CFs proliferation and collagen secretion. This inhibitory effect of resveratrol is orchestrated by the downregulation of miR-17 and the regulation of Smad7.

Antileukemic Activity of 8-Chloro-Adenosine (8-Cl-Ado) Is Mediated By Mir-155 Degradation and ErbB3 Binding Protein (Ebp1)-Dependent p53 Activation: A Novel Therapeutic Approach for FLT3-ITD Acute Myeloid Leukemia (AML)

AML patients (pts) carrying the fms-related tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) have a poor prognosis. Combination of chemotherapy with tyrosine kinase inhibitors (TKIs) has improved survival of these pts, but a large proportion of them still die of their disease. Nucleoside analogs (NAs) are the backbone of several upfront and salvage chemotherapy regimens for AML, including FLT3-ITD. Although these agents have significant antileukemic activity, they are not effective in eradicating leukemia stem cells (LSCs), the likely reason for treatment failures in AML. Consequently, new strategies are needed to improve the outcome for this and other molecular subsets of AML pts. 8-Cl-Ado is a novel, ribose-containing, RNA-directed nucleoside analog which, different from other NAs, is incorporated into newly transcribed RNA rather than in DNA, causing inhibition of RNA transcription. Among the AML molecular subsets, we identified FLT3-ITD blasts as one of the most sensitive to 8-Cl-Ado; however the mechanism of this differential effect remains unknown. Ex-vivo treatment with 8-Cl-Ado induced dose-dependent growth inhibition and apoptosis in FLT3-ITD AML cell lines and primary blasts including the LSC-enriched CD34+CD38- immunophenotypic subpopulation, with IC50s ranging from 200 nM to 1400 nM and a negligible effect on normal hematopoietic stem cells. In an orthotopic murine model, mice xenografted with FLT3-ITD-positive MV4-11 cells and treated (upon engraftment) with 75 mg 8-Cl-Ado/kg/day through an implanted osmotic pump survived significantly longer than vehicle-treated controls (median survival 45 days vs. 27.3 days, p=0.002). MicroRNA-155 (miR-155) is the most over-expressed miRNA in FLT3-ITD and reportedly plays a key role in FLT3-ITD blast hyper-proliferation [PMID 20656931, PMID 25971362]. Thus, silencing of miR-155 has been proposed as a novel therapeutic approach for FLT3-ITD AML [PMID 25971362]. As 8-Cl-Ado is incorporated mainly into RNA, we reasoned that it could also be incorporated into miR-155 (and other miRNAs). Consistent with our hypothesis, we detected co-localization of 8-Cl-Ado and miR-155 in FLT3-ITD primary blast cells and MV4-11, using fluorescence-labeled 8-Cl-Ado (8-Cl-Ado-FAM) and miR-155 staining (SmartFlare probes), suggesting that 8-Cl-Ado interacts directly with miR-155. Using quantitative RT-PCR we demonstrated ~50% miR-155 down-regulation in 8-Cl-Ado-FAM or 8-Cl-Ado-treated MV4-11 cells and FLT3-ITD primary blast cells, compared to vehicle-treated controls. On a molecular level, 8-Cl-Ado-dependent miR-155 down-regulation was accompanied by up-regulation of SHIP1, a bona fide miR-155 target phosphatase that decreased p-AKT levels thereby negatively regulating FLT3-ITD-dependent AKT signaling required for leukemia cell growth and survival. This effect also disrupted the interaction of AKT and ErbB3 binding protein (Ebp1), a highly expressed protein that regulates p53 expression and prevents DNA fragmentation and apoptosis in normal and leukemic cells. Thus, we hypothesized by disrupting the AKT/Ebp1 interplay via miR-155 down-regulation, 8-Cl-Ado induces pro-apoptotic Ebp1-dependent p53 expression and activation and leukemia cell death. Indeed, we showed that 8-Cl-Ado treatment caused AKT/Ebp1 dissociation and p53 activation in primary FLT3-ITD AML blasts. Conversely, overexpression of miR-155 reversed 8-Cl-Ado-induced apoptosis. By combining 8-Cl-Ado with the TKI quizartinib we elicited a synergistic anti-leukemic effect in primary AML blasts [combination index (CI) values <1 at doses required to inhibit 50, 75, and 90% of cell growth (ED50, ED75, ED90)]. In an orthotopic mouse model where FLT3-ITD-positive MV4-11 cells were xenografted, mice treated with 75 mg 8-Cl-Ado/kg/day plus quizartinib (0.5 mg/kg/day) survived significantly longer than mice treated with the individual agents or vehicle alone (median survival 54.2 days vs. 27.3 days for controls, p=0.016).Taken together, these results support 8-Cl-Ado as a promising novel agent via a unique mechanism of action for FLT3-ITD AML, mediated by miR-155 degradation. 8-Cl-Ado also synergizes with TKIs both in vitro and in vivo thereby representing a potentially novel clinical approach for FLT3-ITD AML. A single-agent phase I/II clinical trial in relapsed/refractory AML is already ongoing at our institution. [Display omitted] DisclosuresWennerberg:Novartis: Research Funding. Gandhi:Pharmacyclics: Research Funding.

Effects of miR-155 on hypertensive rats via regulating vascular mesangial hyperplasia.

Vascular smooth muscle cell (VSMC) excessive proliferation is related to hypertension. The cell cycle inhibitory factor (p27) can arrest cell cycle, while its down-regulation is associated with hypertension. It is found that microRNA-155 (miR-155) plays a regulatory role in VSMC proliferation, while its relationship with hypertension is still unclear. Bioinformatics analysis reveals the targeted relationship between miR-155 and the 3'-UTR of p27 mRNA. This study aims to explore the role of miR-155 in regulating p27 expression, VSMC proliferation and apoptosis, and the pathogenesis of hypertension. Dual luciferase reporter gene assay confirmed the relationship between miR-155 and p27. MiR-155, p27, α-smooth muscle actin (α-SMA), and Ki-67 expressions in the thoracic aorta media of rat hypertension model were detected. VSMCs were cultured in vitro and divided into five groups, including anti-miR-NC, anti-miR-155, pIRES2-blank, pIRES2-p27, and anti-miR-155 + pIRES2-p27 groups. Cell cycle was evaluated by using flow cytometry. Cell proliferation was detected with EdU staining. Hypertension rats were randomly divided into antagomir-155 and antagomir-control. Caudal artery systolic and diastolic pressures were measured. MiR-155 targeted suppressed p27 expression. MiR-155 and Ki-67 expressions significantly enhanced, while p27 and α-SMA levels reduced in the tunica media from hypertension rats compared with control. Down-regulation of miR-155 and/or up-regulation of p27significantly declined cell proliferation and arrested cell cycle in G1 phase. Antagomir-155 injection markedly decreased systolic and diastolic pressures, elevated p27 and α-SMA expressions in media, and reduced the thickness of tunica media. MiR-155 promoted VSMC proliferation by targeting p27. MiR-155 enhancement was related to hypertension. MiR-155 played a therapeutic effect on hypertension.

A negative feedback loop between nuclear factor 90 (NF90) and an anti-oncogenic microRNA, miR-7

Alterations in microRNAs (miRNAs) levels deeply correlate with tumorigenesis. However, the molecular mechanism for the regulation of the miRNA production in tumors is not fully understood. We previously reported that downregulation of miR-7, which is an anti-oncogenic miRNA, was caused by overexpression of the nuclear factor 90 (NF90)-nuclear factor 45 (NF45) complex through the binding of double-stranded (ds) RNA-binding proteins to primary miR-7, resulting in promotion of tumorigenesis (Higuchi et al 2016). During this study, we found that the level of NF90 protein was dramatically decreased by overexpression of miR-7. Interestingly, the miR-7-mediated reduction in NF90 family proteins was only observed in NF90 protein, but not in NF110 protein, which is a longer form of the NF90 gene. Luciferase reporter analysis indicated that the overexpression of miR-7 significantly repressed the luciferase activity in the coding region of NF90 mRNA harboring a predicted target sequence of miR-7. The luciferase activity of the reporter vector, which has a mutated miR-7 target site in the coding region, was the same in the control and miR-7 overexpressed cells. Furthermore, the translation of TARGET-tagged NF90 mRNA without the 3′UTR of the NF90 mRNA was inhibited by the overexpression of miR-7. These results imply that miR-7 suppresses NF90 at the protein level through the binding of miR-7 to the complementary site of the seed sequence in the coding region of the NF90 mRNA. We further confirmed increased endogenous NF90 protein levels in SK-N-SH cells transfected with antisense oligonucleotides targeting miR-7, indicating that miR-7-mediated translational repression of NF90 is a physiological event. Taken together with our previous findings (Higuchi et al 2016), it suggests that the level of NF90 is increased by a negative feedback loop between NF90 and miR-7 in tumor tissues under physiological conditions.

Role of circulating miR-182 and miR-150 as biomarkers for cirrhosis and hepatocellular carcinoma post HCV infection in Egyptian patients

In Egypt, liver diseases are exceptionally high, maintaining the highest prevalence of hepatitis C virus (HCV) worldwide, and increasing rates of hepatocellular carcinoma (HCC). Available diagnostic methods show poor performance in early diagnosis of HCC. Definite pathogenic factors contributing in the development of HCV are still lacking. MicroRNAs have been reported as promising biomarkers for cancers diagnosis and in virus-host interaction.This study was conducted to detect the role of miR-182 and miR-150 as biomarkers for development of cirrhosis and malignant transformation in HCV infected patients.The expression of miR-182 and miR-150 was evaluated using real-time quantitative PCR (qRT-PCR) in 120 subjects: 40 HCC patients, 40 hepatitis C patients (20 cirrhotic and 20 non-cirrhotic HCV genotype 4) and 40 healthy controls.In HCC, statistically significant decrease of miR-182 and miR-150 compared to non-cirrhotic HCV patients (p = 0.015, p = 0.006 respectively) and of miR-150 compared to controls (p = 0.039). In cirrhotic HCV patients, significant down regulation of miR-182 and miR-150 compared to non-cirrhotic HCV (p = 0.003, p = 0.024 respectively). On the other hand, significant upregulation of miR-182 was observed in non-cirrhotic HCV compared to controls (p = 0.036). Alpha-fetoprotein (AFP) showed sensitivity 15% for HCC diagnosis at the cut-off value of 400 ng/ml, while combining AFP with miR-182 and miR-150, resulted in improving sensitivity to (90%) and diagnostic accuracy to (80%).miR-182 and miR-150 can be used as non invasive biomarkers for HCC and combination of these miRNAs and AFP markedly improve the diagnosis of HCC. Both miR-182 and miR-150 can also be used as predictive markers for detection of cirrhosis progression in HCV infected patients.

Abstract 5389: Circulating miRNAs as potential targets for the protection of radiation-induced immunologic injury

Abstract MicroRNAs (miRNAs) are non-coding RNAs of 19-22 nucleotides which regulate a diverse set of physiological processes including immune cell development by post-transcriptional gene regulation. Circulating miRNAs in blood have been assessed as biomarkers of disease diagnosis and are capable of modulating a wide-range of biological functions through targeting the molecules of recipient cells. In our previous work, some radiosensitive circulating miRNAs have been identified, and most of them were closely associated with immune and hematopoietic system. Here, we further investigated the effects of radioprotector on the changes of miRNA expression levels after exposure of mice to X-rays or carbon ion beam, aiming to explore novel potential therapeutic targets. Kunming mice were whole-body exposed to 2 and 4 Gy of X-rays generated by Faxitron RX650 or 0.1 and 0.5 Gy of carbon ion beam generated by the HIRFL (Institute of Modern Physics, Lanzhou, China). Angelica sinensis polysaccharide (ASP) which is able to promote the hematopoietic function in vivo and GANRA agent which can scavenge the ROS were administrated to mice by gavage for 7 days before exposure. The expression of circulating miRNAs were detected by qRT-PCR analysis. Our results confirmed that ASP significantly protected the immune and hematopoietic systems of irradiated mice by recovering the numbers of bone marrow hematopoietic cells, peripheric leucocytes, lymphocytes and atrophy of the spleen. GANRA increased the number of lymphocytes, leucocytes and the ratio of CD4+/CD8+ cells in the spleen of irradiated mice. Moreover, GANRA could eliminate ROS which was produced by X-rays and carbon ion beam, and increased the cell viability. Meanwhile, the expression levels of circulating miRNAs such as let-7a, miR-34a, miR-223, miR-150 and miR-574 were distinctively different between ASP or GANRA treated and the control group. ASP or GANRA treatment could rescue the up-regulation of let-7a, miR-34a and miR-223, and the down-regulation of miR-150 and miR-574 that were induced by radiation, indicating that circulating miRNAs could be possible targets of radioprotector responding to radiation exposure. In conclusion, ASP and GANRA treatments have significant protective effects on the immune and hematopoietic system. The differential expressions of circulating miRNAs in radioprotector treated and non-treated groups indicate that miRNAs are potential targets for the protection of radiation-induced immunologic injury. By further exploring the function of these miRNA and their influence on the immune and hematopoietic systems, it will accelerate the development of new biomarkers and drug targets. Citation Format: Heng Zhou, Wenjun Wei, Jufang Wang. Circulating miRNAs as potential targets for the protection of radiation-induced immunologic injury [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5389.

Long Noncoding RNA Zfas1 in Diabetic Cardiomyopathy

Fibrosis is a significant problem in diabetic cardiomyopathy (DCM). Both long noncoding RNAs (LncRNAs) and miRNAs (miR) play important roles in cellular processes. miR-9 targets important extracellular matrix(ECM) protein, such as fibronectin(FN) and collagen(Col), which are upregulated in DCM. The role of ZNFX1-AS1 (Zfas1), a LncRNA targeting miR-9 in DCM is not clear. We investigated Zfas1 in vitro and in vivo and its regulatory mechanism on ECM protein through miR-9. Microarrays were used to examine LncRNAs and miRNAs expression in ECs exposed to 25 mM glucose (HG) compare to 5 mM glucose and were validated by qPCR. Expression of ECM proteins targeted by miR-9 were examined. Luciferase assay was used to determine the interaction between miR-9 and its target genes. Cardiac tissues from streptozotocin-induced diabetic mice were examined similarly. The array and qPCR analysis showed glucose-induced upregulation of LncRNA Zfas1 and downregulation of miR-9 in ECs. FN mRNA, Col1&amp;4 mRNA and protein levels were increased in ECs treated with HG. Luciferase assay showed the binding of miR-9 to FN 3’-UTR and miR-9. Zfas1 silencing expression of its target genes expression in HG. Furthermore miR-9 mimics co-transfection with Zfas1 siRNA partially recovered these ECM protein expression. Interestingly, methylation blockade also reduced ECM protein expression. In the hearts of the diabetic animals, Zfas1 expression was increased and miR-9 was decreased and expression of transcripts regulated by miR-9 were increased. These studies indicate a novel glucose-induced mechanism of increased ECM protein production, which is regulated by Zfas1 through miR-9 in diabetes. Identifying such mechanisms may lead to potential RNA based treatment for diabetic complications. Disclosure B. Feng: None. S. Chen: None. S. Chakrabarti: None.

MiR-182 regulates trastuzumab resistance by targeting MET in breast cancer cells.

It has been found that microRNAs (miRNAs) play a key role in drug resistance. The purpose of the current study was to investigate the function of miR-182 in trastuzumab resistance in breast cancer cells. The results showed that both breast cancer SKBR3 trastuzumab-resistant cells (SKBR3/TR) and BT474 trastuzumab-resistant cells (BT474/TR) were associated with miR-182 downregulation compared with their parental cells. Ectopic expression of the miR-182 mimic inhibited trastuzumab resistance, decreasing the invasion and migration of these trastuzumab-resistant cells. However, the miR-182 inhibitor increased trastuzumab resistance, cell invasion, and migration in the parental cells. In addition, MET is a directly targeted gene of miR-182 in breast cancer cells. MET knockdown showed an inhibitory effect of trastuzumab resistance on trastuzumab-resistant cells. In contrast, MET overexpression in SKBR3 cells produced an effect that promotes resistance to trastuzumab. Moreover, we revealed that overexpression of miR-182 reduced trastuzumab resistance in trastuzumab-resistant cells due in part to MET/PI3K/AKT/mTOR signaling pathway inactivation. Furthermore, miR-182 could also sensitize SKBR3/TR cells to trastuzumab in vivo. In conclusion, our results suggest that the activation of miR-182 or inactivation of its target gene pathway could be used as a new method to reverse trastuzumab resistance in breast cancer.

Methylation-mediated miR-155-FAM133A axis contributes to the attenuated invasion and migration of IDH mutant gliomas

Gliomas with isocitrate dehydrogenases gene mutations (IDHMT) were found to be less aggressive than their wildtype (IDHWT) counterparts. However, the mechanism remains unclear. The current study aims to investigate the role of silenced oncogenic microRNAs in IDHMT gliomas, which were largely ignored and may contribute to the less aggressive behavior of IDHMT gliomas. Microarrays, bioinformatics analysis of the data from TCGA and qPCR analysis of samples from our experimental cohort (LGG: IDHWT = 10, IDHMT = 31; GBM: IDHWT = 34, IDHMT = 9) were performed. The results show that miR-155 was consistently down-regulated in IDHMT gliomas. Establishment of IDH1R132H overexpressing glioma cell line and bisulfite sequencing PCR suggested that miR-155 down-regulation was associated with IDH1R132H mutation induced promoter CpG islands methylation. The cancer testis antigen FAM133A is a direct downstream target of miR-155 and is a negative regulator of glioma invasion and migration possibly by regulating matrix metallopeptidase 14 (MMP14). Together, we found that methylation-regulated miR-155-FAM133A axis may contribute to the attenuated invasion and migration of IDHMT gliomas by targeting MMP14.

Eriocheir sinensis microRNA-7 targets crab Myd88 to enhance white spot syndrome virus replication

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at the posttranscriptional level. In this study, the function of microRNA-7 (miR-7) in host-virus interaction was investigated. Replication of White spot syndrome virus (WSSV) was enhanced with the overexpression of miR-7 and inhibited with the downregulation of miR-7 by using anti-miRNA oligonucleotide AMO-miR-7. The target gene of miR-7 was predicted using bioinformatics methods. Results showed that crab myeloid differentiation factor 88 (Myd88) could be targeted by miR-7. When the expression of Myd88 was knocked down by sequence-specific siRNA, WSSV copies in crabs were significantly increased. Further findings revealed that knockdown of Myd88, Tube, or Pelle inhibited the expressions of interleukin enhancer-binding factor 2 homolog (ILF2) and interleukin-16-like gene (IL-16L). While ILF2 was silenced, IL-16L expression was inhibited. The overexpression of miR-7 inhibited the expressions of ILF2 and IL-16L. Moreover, when ILF2 or IL-16L was silenced, WSSV copies in crabs were increased. Thus, the upregulated expression of miR-7 during WSSV challenge suppressed the host Myd88-ILF2-(IL-16L) signaling pathway in crabs and enhanced WSSV replication. Our study indicated that WSSV utilized crab miR-7 to enhance virus replication during infection.

Long non-coding RNA UCA1/miR-182/PFKFB2 axis modulates glioblastoma-associated stromal cells-mediated glycolysis and invasion of glioma cells

Glioblastoma (GB) is the most common and deadliest malignant primary brain tumor with a high recurrence. In this study, lncRNA UCA1/miR-182 axis has been regarded as a nodal driver of glioma invasion mediated by GB-associated stromal cells (GASCs) and GASC-secreted chemokine CXCL14. In clinical specimens, CXCL14 upregulation in GASCs also correlated with poor prognosis. Notably, CXCL14-high GASCs mediated lncRNA UCA1 upregulation and miR-182 downregulation in glioma cells. Moreover, miR-182 directly bound to the fructose-2,6-biphosphatase PFKFB2; UCA1/miR-182 axis thereby modulated GASC-induced glycolysis in glioma cells. Overall, UCA1/miR-182/PFKFB2 axis modulates chemokine CXCL14 secretion, glycolysis and invasion of glioma cells in GASCs.

Analysis of microRNA expression in the thymus of Myasthenia Gravis patients opens new research avenues.

In early-onset Myasthenia Gravis (MG) with anti-acetylcholine receptor antibodies, thymic abnormalities associated with ectopic germinal centers are frequent. miRNAs by acting as post-transcriptional regulators are involved in autoimmunity. To investigate the implication of miRNAs in thymic changes associated with early-onset MG, we performed a miRnome study and data were analyzed with different approaches. miRNAs of interest were further investigated by RT-PCR and transfection experiments for functional tests. First, analyzing specific dysregulated miRNAs, we focused our attention on miR-7-5p and miR-125a-5p, and confirmed by RT-PCR their respective down- and up-regulation in MG thymuses. miR-7 was the most down-regulated thymic miRNA in MG and we observed an inverse correlation between its expression and CCL21 mRNA expression. We next showed that miR-7 down-regulation was due to thymic epithelial cells and by transfecting these cells with miR-7, we demonstrated that it controlled CCL21 release. As CCL21 is essential for germinal center development, we suggested that miR-7 could be involved in thymic changes associated with MG. miR-125a was up-regulated in MG thymuses and is of great interest as it is known to regulate FoxP3 expression, and to modulate the different inflammatory signaling pathways. Thanks to this thymic miRnome study, we also showed the specific dysregulation of miRNA clusters. In particular, we observed that miRNAs localized at the extremity of the X chromosome were down-regulated. This effect seemed linked to their close localization to the fragile X mental retardation 1 gene (FMR1) and the DNA methylation status. Altogether, this miRnome analysis demonstrated that specific thymic miRNAs can be associated with MG and provides novel insights into the pathogenesis of MG.

Peripheral Chemoreceptor Ablation Modulates Renal MiR‐155/KLF4 Expression in Chronic Heart Failure

Background and SignificanceRecent evidence suggests aberrant carotid body chemoreceptor (CBC) function contributes to tonic elevation of renal nerve activity and reductions in renal blood flow in chronic heart failure (CHF). These changes combined with anemia and capillary rarefaction likely contribute to tissue hypoxia, and may drive renal fibrosis and attendant dysfunction. Previous work indicates that stable expression of hypoxia‐inducible factor 1‐α (HIF1α) and down regulation of MiR‐155 drive gene programs that mediate epithelial‐mesenchymal transition (EMT) in renal tubular epithelial cells, including increases in Krüppel‐like factor 4 (KLF4) and matrix metalloproteinase 9 (MMP9). To date, no studies have examined this signaling pathway in CHF, thus our objective was to determine the influence of CBC on renal cortical expression of HIF1α, KLF4, MiR‐155, MMP9, and α‐smooth muscle actin (SMA) in CHF.HypothesisWe hypothesize that CBC‐mediated reductions in renal blood flow activate EMT pathways in CHF by exacerbating tissue hypoxia, leading to sustained accumulation of HIF1α, downregulation of MiR‐155, and KLF4/MMP9/SMA induction.MethodsTo address this hypothesis, we measured expression of HIF1α, MiR‐155, KLF4, MMP9, and SMA in renal cortical tissue from sham, and CHF animals (n=4–8 per group) with and without ablation of the CBC (CBA). CHF was induced in rats by coronary artery ligation (CAL) and CBA was performed (4 weeks post‐CAL) by cryogenic ablation. CHF was confirmed via echocardiography under isoflurane anesthesia (1–1.5%). At 8‐weeks post‐CAL, rats were humanely euthanized and renal cortical tissue was collected and analyzed for HIF1α, KLF4, MMP9, and SMA expression via western blot and MiR‐155 expression by RT‐qPCR.ResultsEjection fraction was 72±2% in sham animals, 34±2% in CHF animals, and 32±2% in CHF‐CBA animals (p&lt;0.05 sham vs. CHF and CHF‐CBA). Expression of HIF1α was 1.37±0.33 in CHF rats relative to sham, and 1.25±0.15 in CHF‐CBA relative to sham. KLF4 was 1.64±0.25 in CHF rats relative to sham, and this effect was attenuated in CHF‐CBA (1.06±0.16 relative to sham, p&lt;0.05 vs. CHF). MMP9 expression in CHF rats was 1.79±0.43 relative to sham and 1.092±0.23 in CHF‐CBA relative to sham. SMA expression in CHF rats was 0.58±0.17 relative to sham and 0.83±0.15 relative to sham in CHF‐CBA. MiR‐155 expression in CHF rats was 0.48±0.11 relative to sham, and this effect was attenuated by CBA (0.73±0.12 relative to sham, p&lt;0.05 vs. CHF).ConclusionThese results suggest a novel relationship between CBC function and expression of renal cortical MiR‐155 and KLF4 in CHF, and that CBA has the potential to influence EMT pathways in the kidney in this pathophysiological setting.Support or Funding InformationThis work was supported by a grant from NHLBI (1 R15 HL138600‐01)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MiR-155 targets PTCH1 to mediate endothelial progenitor cell dysfunction caused by high glucose

Endothelial progenitor cells (EPCs) are involved in diabetes-associated complications, including diabetic foot ulcer (DFU). Recent reports showed that miR-155 downregulation promotes wound healing in diabetic rats and ameliorates endothelial injury induced by high glucose, but its role in DFU is unknown. We found that miR-155 was overexpressed in EPCs from patients with DFU and in high glucose–induced EPCs from healthy people. Reductions in cell viability, migration, tube formation and nitric oxide production, as well as increases in lactated hydrogenase, cell apoptosis, and reactive oxygen species induced by high glucose, were enhanced by miR-155 overexpression and restrained by miR-155 inhibition. Additionally, dual-luciferase reporter assay demonstrated that miR-155 directly targeted the 3′ untranslated region of patched-1 (PTCH1), a receptor of the sonic hedgehog signaling pathway, and downregulated the mRNA and protein expression of PTCH1. qRT-PCR and Western blot results revealed that the PTCH1 was downregulated in EPCs treated with high glucose. Silencing PTCH1 by PTCH1 siRNA alleviated the protective effect of anti-miR-155 on high glucose–induced EPC dysfunction. Our results indicate that miR-155 worsened high glucose–induced EPC function by downregulating PTCH1. These findings suggest that miR-155 may be a potential therapeutic target for DFU.

RTP801 is a critical factor in the neurodegeneration process of A53T α-synuclein in a mouse model of Parkinson's disease under chronic restraint stress.

Recently, the incidence of Parkinson's disease has shown a tendency to move to a younger population, linked to the constantly increasing stressors of modern society. However, this relationship remains obscure. Here, we have investigated the contribution of stress and the mechanisms underlying this change. Ten-month-old α-synuclein A53T mice, a model of Parkinson's disease (PD), were treated with chronic restraint stress (CRS) to simulate a PD-sensitive person with constant stress stimulation. PD-like behavioural tests and pathological changes were evaluated. Differentiated PC12-A53T cells were treated with corticosterone in vitro. We used Western blot, microRNA expression analysis, immunofluorescence staining, dual luciferase reporter assay and HPLC electrochemical detection to assess cellular and molecular networks after stress treatment. In vivo, stereotaxic injection of shRNA lentivirus was used to confirm our in vitro results. The protein RTP801 is encoded by DNA-damage-inducible transcript 4, and it was specifically increased in dopaminergic neurons of the substantia nigra after CRS treatment. RTP801 was post-transcriptionally inhibited by the down-regulation of miR-7. Delayed turnover of RTP801, through the inhibition of proteasome degradation also contributed to its high content. Elevated RTP801 blocked autophagy, thus increasing accumulation of oligomeric α-synuclein and aggravating endoplasmic reticulum stress. RTP801 inhibition alleviated the symptoms of neurodegeneration during this process. RTP801 is a promising target for the treatment of PD, especially for PD-sensitive patients who live under increased social pressure. Down-regulation of RTP801 could inhibit the current tendency to an earlier onset of PD.

Acute downregulation of miR-155 leads to a reduced collagen synthesis through attenuating macrophages inflammatory factor secretion by targeting SHIP1.

Fibrosis, tightly associated with fibroblasts collagen synthesis, is related closely with inflammatory response. Our previously study found that acute downregulation of miR-155 at wound sites leads to a reduced fibrosis, however its particular mechanism is unclear. Herein, we aimed to explore the mechanism of miR-155 in reducing fibrosis. We first found that down-regulation of miR-155 inhibited macrophages transforming growth factor-β1 (TGF-β1) and IL-1β secretion. Next, we found that co-cultured with macrophages increased the proliferation and collagen synthesis of fibroblasts, and downregulation of miR-155 in macrophages could effectively attenuate the accelerative effects. We further identified SH2 domain containing inositol-5-phosphatase 1 (SHIP1) as a direct target of miR-155 in macrophages, and the expression of SHIP1 was negatively correlated with the level of miR-155. We further confirmed that PI3K/Akt pathway was involved in this process. Last, we found that downregulation of miR-155 leads to a reduced fibrosis in sever burn rat. Taken together, these results indicate that down-regulation of miR-155 leads to a reduced fibroblasts proliferation and collagen synthesis through attenuating macrophages TGF-β1 and IL-1β secretion by targeting SHIP1 via PI3K/Akt pathway, suggesting its potential therapeutic effects on the treatment of skin fibrosis.