Downregulation Of miR-145 Research Articles

MicroRNA-126 Targeting PIK3R2 Inhibits NSCLC A549 Cell Proliferation, Migration, and Invasion by Regulation of PTEN/PI3K/AKT Pathway

BackgroundOur study explored whether the microRNA-126 (miR-126)-mediated PTEN/PI3K/AKT (phosphatase and tensin homology deleted on chromosome 10/phosphatidylinositol 3-kinase regulatory subunit-β/AKT) signaling pathway by targeting PIK3R2 affects the proliferation, migration, and invasion of non–small-cell lung cancer (NSCLC) A549 cells. Materials and MethodsQuantitative real-time polymerase chain reaction was used to measure the expression of miR-126 in A549 cells. The MTT (methyl thiazolyl tetrazolium) assay, cell scratch test, Transwell assay, and Western blot were used to detect the proliferation, migration, and invasion of A549 cells and protein expression in A549 cells, respectively. ResultsThe expression of miR-126 decreased and the expression of PIK3R2 increased in A549 cells (P < .05, for both). Upregulation of miR-126 resulted in the decrease of the proliferation, migration, and invasive abilities of A549 cells, the downregulation of the expression of PIK3R2, PI3K, and phosphorylated Akt (p-Akt) protein, and the upregulation of PTEN expression (P < .05 for all). Also, these abilities of A549 cells increased, and the expression of these 3 proteins was upregulated with downregulation of miR-126 (P < .05 for all). The results of the dual luciferase reporter gene assay showed that PIK3R2 was the target gene of miR-126. PIK3R2, PI3K, and p-Akt proteins were downregulated, but PTEN protein was upregulated as PIK3R2 was silenced or the inhibitor of the PTEN/PI3K/AKT signaling pathway increased. Also, downregulation of miR-126 with silencing of PIK3R2 or increasing the inhibitor of the pathway caused increased PI3K and p-Akt protein expression and increased active proliferation, migration, and invasive abilities of A549 cells (P < .05 for all). ConclusionThe upregulation of miR-126 in NSCLC A549 cells can reduce the expression of the target gene PIK3R2 and influence the PTEN/PI3K/AKT signaling pathway, suppressing the proliferation, migration, and invasive abilities of A549 cells.

655 Grainyhead-Like 2, a Novel Hepatic Transcription Factor, Promotes Alcohol-induced Liver Injury, Steatosis and Inflammation via miR-122 Down-regulation

655 Grainyhead-Like 2, a Novel Hepatic Transcription Factor, Promotes Alcohol-induced Liver Injury, Steatosis and Inflammation via miR-122 Down-regulation

MiR-21 and miR-150 downregulation correlates with oxaliplatin-related sinusoidal obstruction syndrome and impaired survival

miR-21 and miR-150 downregulation correlates with oxaliplatin-related sinusoidal obstruction syndrome and impaired survival

Mast cells promote proliferation and migration and inhibit differentiation of mesenchymal stem cells through PDGF

BackgroundMast cells (MCs) dynamically participate in wound healing after myocardial infarction (MI) by releasing cytokines. Indeed, MC-deficient mice undergo rapid left ventricular dilation post-MI. Mesenchymal stem cells (MSCs) are recruited to the injured region following an MI and have potential for cardiac repair. In the current study, we evaluate the effect of MCs on MSC proliferation and myogenic differentiation. Methods and resultsMCs were cultured from mouse bone marrow and MC granulate (MCG) was extracted from MCs via freeze-thaw cycles followed by filtration. α-SMA (smooth muscle actin) expression was examined as an indicator of myogenic differentiation. MSC/MC co-culture resulted in decreased MSC differentiation indicated by α-SMA suppression in MSCs. MCG also suppressed α-SMA expression and increased MSC migration and proliferation in a dose-dependent manner. Removal of MCG rescued α-SMA expression and MSC differentiation. Platelet derived growth factor (PDGF) receptor blockade using AG1296 also rescued MSC differentiation even after MCG treatment. Real-time PCR and Western blot showed that MCG exerted its effects on MSCs via downregulation of miR-145 and miR-143, downregulation of myocardin, upregulation of Klf4, and increased Erk and Elk1 phosphorylation. ConclusionsMCs promote MSC proliferation and migration by suppressing their myogenic differentiation. MCs accomplish this via activation of the PDGF pathway, downregulation of miR-145/143, and modulation of the myocardin-Klf4 axis. These data suggest a potential role for MSC/MC interaction in the infarcted heart where MCs may inhibit MSCs from differentiation and promote their proliferation whereby increased cardiac MSC accumulation promotes eventual cardiac regeneration after MCs cease activity.

Longitudinal Examination of the Intestinal Lamina Propria Cellular Compartment of Simian Immunodeficiency Virus-Infected Rhesus Macaques Provides Broader and Deeper Insights into the Link between Aberrant MicroRNA Expression and Persistent Immune Activation.

Chronic immune activation/inflammation driven by factors like microbial translocation is a key determinant of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) disease progression. Although extensive research on inflammation has focused on studying protein regulators, increasing evidence suggests a critical role for microRNAs (miRNAs) in regulating several aspects of the immune/inflammatory response and immune cell proliferation, differentiation, and activation. To understand their immunoregulatory role, we profiled miRNA expression sequentially in intestinal lamina propria leukocytes (LPLs) of eight macaques before and at 21, 90, and 180 days postinfection (dpi). At 21 dpi, ∼20 and 9 miRNAs were up- and downregulated, respectively. However, at 90 dpi (n = 60) and 180 dpi (n = 44), ≥75% of miRNAs showed decreased expression. Notably, the T-cell activation-associated miR-15b, miR-142-3p, miR-142-5p, and miR-150 expression was significantly downregulated at 90 and 180 dpi. Out of ∼10 downregulated miRNAs predicted to regulate CD69, we confirmed miR-92a to directly target CD69. Interestingly, the SIV-induced miR-190b expression was elevated at all time points. Additionally, elevated lipopolysaccharide (LPS)-responsive miR-146b-5p expression at 180 dpi was confirmed in primary intestinal macrophages following LPS treatment in vitro Further, reporter and overexpression assays validated IRAK1 (interleukin-1 receptor 1 kinase) as a direct miR-150 target. Furthermore, IRAK1 protein levels were markedly elevated in intestinal LPLs and epithelium. Finally, blockade of CD8(+) T-cell activation/proliferation with delta-9 tetrahydrocannabinol (Δ(9)-THC) significantly prevented miR-150 downregulation and IRAK1 upregulation. Our findings suggest that miR-150 downregulation during T-cell activation disrupts the translational control of IRAK1, facilitating persistent gastrointestinal (GI) inflammation. Finally, the ability of Δ(9)-THC to block the miR-150-IRAK1 regulatory cascade highlights the potential of cannabinoids to inhibit persistent inflammation/immune activation in HIV/SIV infection. Persistent GI tract disease/inflammation is a cardinal feature of HIV/SIV infection. Increasing evidence points to a critical role for miRNAs in controlling several aspects of the immune/inflammatory response. Here, we show significant dysregulation of miRNA expression exclusively in the intestinal lamina propria cellular compartment through the course of SIV infection. Specifically, the study identified miRNA signatures associated with key pathogenic events, such as viral replication, T-cell activation, and microbial translocation. The T-cell-enriched miR-150 showed significant downregulation throughout SIV infection and was confirmed to target IRAK1, a critical signal-transducing component of the IL-1 receptor and TLR signaling pathways. Reduced miR-150 expression was associated with markedly elevated IRAK1 expression in the intestines of chronically SIV-infected macaques. Finally, Δ(9)-THC-mediated blockade of CD8(+) T-cell activation in vitro significantly inhibited miR-150 downregulation and IRAK1 upregulation, suggesting its potential for targeted immune modulation in HIV infection.

MicroRNA-145 Regulates Neural Stem Cell Differentiation Through the Sox2–Lin28/let-7 Signaling Pathway

MicroRNAs (miRNAs or miRs) regulate several biological functions, including cell fate determination and differentiation. Although miR-145 has already been described to regulate glioma development, its precise role in neurogenesis has never been addressed. miR-145 represses sex-determining region Y-box 2 (Sox2), a core transcription factor of embryonic stem cells (ESCs), to inhibit pluripotency and self-renewal in human ESCs. In addition, the Sox2-Lin28/let-7 signaling pathway regulates proliferation and neurogenesis of neural precursors. In this study, we aimed to investigate the precise role of miR-145 in neural stem cell (NSC) fate decision, and the possible involvement of the Sox2-Lin28/let-7 signaling pathway in miR-145 regulatory network. Our results show for the first time that miR-145 expression significantly increased after induction of mouse NSC differentiation, remaining elevated throughout this process. Forced miR-145 downregulation decreased neuronal markers, namely βIII-tubulin, NeuN, and MAP2. Interestingly, throughout NSC differentiation, protein levels of Sox2 and Lin28, a well-known suppressor of let-7 biogenesis, decreased. Of note, neuronal differentiation also resulted in let-7a and let-7b upregulation. Transfection of NSCs with anti-miR-145, in turn, increased both Sox2 and Lin28 protein levels, while decreasing both let-7a and let-7b. More importantly, Sox2 and Lin28 silencing partially rescued the impairment of neuronal differentiation induced by miR-145 downregulation. In conclusion, our results demonstrate a novel role for miR-145 during NSC differentiation, where miR-145 modulation of Sox2-Lin28/let-7 network is crucial for neurogenesis progression. Stem Cells 2016;34:1386-1395.

Chronic inflammation contributes to the development of hepatocellular carcinoma by decreasing miR-122 levels.

Persistent inflammation in chronic hepatitis plays a major role in the development of hepatocellular carcinoma (HCC). In this study, the major inflammatory cytokines expressed in chronic hepatitis, IL-6 and TNF-α, induced a marked decrease in microRNA-122 (miR-122) levels, and miR-122 expression was downregulated in the livers of chronic hepatitis B (CHB) patients. The decrease of miR-122 caused upregulation of the proinflammatory chemokine CCL2. IL-6 and TNF-α suppressed miR-122 both by directly downregulating the transcription factor C/EBPα and indirectly upregulating c-myc, which blocks C/EBPα-mediated miR-122 transcription. In addition, IL-6 and TNF-α levels were elevated and miR-122 levels were decreased in mouse and rat models of diethylnitrosamine (DEN)-induced HCC. Restoration of miR-122 levels through delivery of agomir-122 suppressed DEN-induced hepatocarcinogenesis in mice. Our results show that inflammation-induced miR-122 downregulation in hepatitis contributes to carcinogenesis and suggest that increasing miR-122 may be an effective strategy for preventing HCC development in CHB patients.

Downregulation of Microrna-126 Contributes to Tumorigenesis of Squamous Tongue Cell Carcinoma via Targeting KRAS.

BackgroundmiR-126 has been reported to be differentially expressed in various malignancies, whereas its role in the pathogenesis of tongue squamous cell carcinoma (TSCC) remains largely unknown.Material/MethodsIn this study, we collected 21 pairs of TSCC cancerous and adjacent non-cancerous tissue samples, with which we performed real-time PCR to determine and compare the expression of 6 candidate miRNAs that are reportedly associated with tumorigenesis of TSCC, including miR-100, miR-451, miR-221, let-7a, miR-21, and miR-126. We further performed luciferase assay to validate KRAS as a target of miR-126, and conducted transfection to study the effect of miR-126 on proliferation and apoptosis of the cells.ResultsWe identified that miR-126 was significantly downregulated in the cancerous tissue samples compared with the non-cancerous control tissue samples. By using computational analysis, we identified that KRAS is a virtual target of miR-126, and such association was verified by using luciferase assay. In addition, we found that mRNA and protein expression level of KRAS was significantly higher in the tumor tissue than the control tissue samples.ConclusionsThe following in vitro experiment showed that both mRNA and protein KRAS expression were significantly decreased in SCC-15 cells in which miR-126 was overexpressed, in comparison with similar cells transfected with a negative control, while downregulation of miR-126 by transfecting the cells with miR-126 inhibitors significantly upregulated the mRNA and protein expression of KRAS. Conclusions: miR-126 might be a promising diagnostic and therapeutic target in the prevention and management of TSCC patients.

Age-related changes in miR-143-3p:Igfbp5 interactions affect muscle regeneration.

A common characteristic of aging is defective regeneration of skeletal muscle. The molecular pathways underlying age-related decline in muscle regenerative potential remain elusive. microRNAs are novel gene regulators controlling development and homeostasis and the regeneration of most tissues, including skeletal muscle. Here, we use satellite cells and primary myoblasts from mice and humans and an in vitro regeneration model, to show that disrupted expression of microRNA-143-3p and its target gene, Igfbp5, plays an important role in muscle regeneration in vitro. We identified miR-143 as a regulator of the insulin growth factor-binding protein 5 (Igfbp5) in primary myoblasts and show that the expression of miR-143 and its target gene is disrupted in satellite cells from old mice. Moreover, we show that downregulation of miR-143 during aging may act as a compensatory mechanism aiming at improving myogenesis efficiency; however, concomitant upregulation of miR-143 target gene, Igfbp5, is associated with increased cell senescence, thus affecting myogenesis. Our data demonstrate that dysregulation of miR-143-3p:Igfbp5 interactions in satellite cells with age may be responsible for age-related changes in satellite cell function.

MiR-145 promotes TNF-α-induced apoptosis by facilitating the formation of RIP1-FADDcaspase-8 complex in triple-negative breast cancer.

Researches indicate that the dysregulation of microRNA (miRNA) is involved in tumorigenesis. Among such dysregulated miRNAs in cancer, miR-145 is reported to be downregulated in multiple cancers. In this study, we demonstrated the downregulation of miR-145 in triple-negative breast cancer (TNBC) tissues and TNBC cell lines by quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Furthermore, we found that the tumor necrosis factor-alpha (TNF-α)-induced apoptosis was expanded by the transfection of miR-145 in MDA-MB-231 which belongs to the TNBC cell lines. We then indicated that the mechanism by which miR-145 promotes the TNF-α-induced apoptosis is dependent on the formation of RIP1-FADD-caspase-8 complex. The cellular inhibitor of apoptosis (cIAP1), which is the inhibitor of apoptosis protein, was found to be a target of miR-145 in MDA-MB-231 cells. As a result of cIAP1 overexpression, the promotion of miR-145 on TNF-α-induced apoptosis was inhibited in MDA-MB-231 cells. Therefore, our results indicate that miR-145 acts as a tumor suppressor in TNBC, suggesting that the miR-145-cIAP1 axis might be a potential therapeutic target for TNBC.

FRI-058 - MIR-21 and MIR-150 Downregulation is Associated with Oxaliplatin-Related Sinusoidal Obstruction Syndrome and Impaired Survival

FRI-058 - MIR-21 and MIR-150 Downregulation is Associated with Oxaliplatin-Related Sinusoidal Obstruction Syndrome and Impaired Survival

MicroRNA-126 targeting PIK3R2 promotes rheumatoid arthritis synovial fibro-blasts proliferation and resistance to apoptosis by regulating PI3K/AKT pathway

ObjectiveThe purpose of our study was to elucidate the impact of microRNA-126 (miR-126) targeting PIK3R2 gene on cell proliferation and apoptosis of rheumatoid arthritis synovial fibro-blasts (RASFs) by regulating PI3K/AKT signal pathway. MethodsThe synovial tissue samples of this study were from 55 RA patients undergoing joint replacement and 27 healthy people undergoing joint repair due to trauma. The target genes of miR-126 were collected by the TargetScan and PIK3R2 as the direct target gene of miR-126 was confirmed by dual-luciferase reporter assay system. Our experiment had five groups including the blank control, miR-126 mimic, miR-126 mimic control, miR-126 inhibitor and miR-126 inhibitor control groups. Additionally, real-time quantitative polymerase chain reaction (RT-qPCR), Western-Blot, cell counting kit (CCK-8) and flow cytometry were carried out in this study. ResultsCompared with healthy individuals, the RA patients had increased miR-126, but decreased PIK3R2 mRNA expressions in the synovial tissues. Pearson correlation analysis indicated that miR-126 expression was negatively correlated with PIK3R2 mRNA expression (all P<0.05). When compared with the blank group respectively, the miR-126 mimic group had raising cell proportions in S and G2/M phases with reduced rate of cell apoptosis, while the miR-126 inhibitor group had raising cell proportions in G0/G1 and G2/M phases with increased rate of cell apoptosis (all P<0.05). Besides, compared with the blank control group, the miR-126 mimic group had declined expression of PIK3R2 protein with ascended expression of PI3K and p-AKT (all P<0.05), while the miR-126 inhibitor group had increased expression of PIK3R2 protein with decreased expression of PI3K and p-AKT (all P<0.05). ConclusionOur study demonstrated that down-regulation of miR-126 may indirectly inhibit PI3K/AKT signaling pathway to disrupt the imbalance between growth and death of RASFs by targeting PIK3R2, which may be clinically helpful to find therapeutic strategies directed toward miR-126 function for RA patients.

Upregulation of microRNA-375 increases the cisplatin-sensitivity of human gastric cancer cells by regulating ERBB2.

Resistance to chemotherapy is a major challenge in the effective treatment of patients with gastric cancer; however, the mechanisms underlying chemoresistance in gastric cancer cells are yet to be elucidated. MicroRNAs (miRNAs) have previously been associated with cancer progression and sensitivity to chemotherapy; therefore, the present study aimed to identify miRNAs that may influence the sensitivity of human gastric cancer cells to cisplatin (DDP) treatment. Initially, miRNAs that were differentially expressed between the DDP-sensitive SGC7901 human gastric cancer cell line and DDP-resistant SGC7901/DDP cell line were identified using high-throughput sequencing technology. miRNA-375 (miR-375), which was shown to be downregulated in the SGC7901/DDP cells, has previously been associated with numerous types of cancer; however, to the best of our knowledge, a role for miR-375 in the DDP-sensitivity of gastric cancer cells has yet to be explored. In the present study, the expression levels of miR-375 were significantly downregulated in the SGC7901/DDP cells, as compared with the SGC7901 cells, as demonstrated by reverse transcription-quantitative polymerase chain reaction. In addition, upregulation of miR-375 significantly enhanced the anti-proliferative and apoptosis-inducing effects of DDP, whereas downregulation of miR-375 decreased these effects. Subsequently, western blot analysis demonstrated that upregulation of miR-375 in the SGC7901/DDP cells increased their sensitivity to DDP treatment via regulating the protein expression levels of erb-b2 receptor tyrosine kinase 2 and phosphorylated-Akt. The results of the present study indicate that the expression levels of miR-375 may influence the sensitivity of human gastric cancer cells to the effects of DDP; thus suggesting that a combination of miR-375 regulation and DDP may be considered a novel strategy in the treatment of patients with chemoresistant gastric cancer.

Knockdown (KD) of Mir-126 Expression Enhances Tyrosine Kinase Inhibitor (TKI)-Mediated Targeting of Chronic Myelogenous Leukemia (CML) Stem Cells

BCR-ABL tyrosine kinase inhibitors (TKI), although highly effective in inducing remission and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse. The long-term goal of our research is to improve the understanding of molecular mechanisms regulating LSC growth and develop effective mechanism-based therapeutic strategies to target LSC in CML. MicroRNAs (miRNAs) are short non-coding RNAs that regulate translation of target protein-coding messenger RNAs (mRNAs) and levels of the corresponding encoded proteins. Among distinct miRNAs, miR-126-3p (miR-126) is expressed in normal hematopoietic stem cells (HSCs) and early hematopoietic progenitor cells (HPCs) and plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo (Cell Stem Cell, 2012). We previously reported that miR-126 expression varies among AML patients; higher level of miR-126 associated with poor outcome, a LSC-associated gene expression signature and increased LSC quiescence; and miR-126 down-regulation decreased LSC self-renewal activity in serial transplant experiments (Leukemia, 2015). Here, we report that the more primitive LSK (Lin-Sca-1+Kit+) from SCL-tTA-BCR/ABL mouse model of CML had higher miR-126 expression compared to more differentiated subpopulations, i.e. common myeloid progenitors (CMP) (3.2 fold) and granulocyte-macrophage progenitors (GMP) (6.4 fold). Among LSK subpopulations, LSC (Flt3-CD150+CD48- LSK) demonstrated the highest miR-126 levels (P<0.01). Furthermore, quiescent (Hoechst-Pyronin-) LSC demonstrated higher miR-126 expression (2.6 fold, p<0.01), generated significantly higher long-term engraftment of donor CML cells in recipient mice (58.6±4.4% vs 33±3.4% in BM at 16 wks after transplanted with 100 LSC/mouse, p=0.003) and enhanced leukemogenic capacity (5 out of 9 mice receiving quiescent LSC developed CML within 14 wks after transplantation vs. none of 10 mice receiving proliferating LSC) compared with proliferating (Hoechst+/-Pyronin+) LSC. Human primary untreated chronic phase (CP) CML CD34+CD38- primitive progenitors also showed higher miR-126 expression compared with CD34+CD38+ committed progenitors. KD of miR-126 in LSK cells of CML mouse model and in primary CML CD34+CD38- cells by using either miRZip-126-3p anti-miR lentivirus (System Biosciences) or a novel, myeloid cell-specific CpG-miR-126 oligonucleotide (ODN) inhibitor increased LSC cell-cycle entry as demonstrated by increased Ki67 staining (p<0.05) and EdU labeling (p<0.05), but affected apoptosis only slightly compared to scramble-treated controls. More importantly, miR-126 KD either by lentivirus or CpG-miR-126 ODN inhibitor combined with Nilotinib (NIL) significantly increased apoptosis of LSC compared with NIL alone (34% vs 19%; p=0.01 for lentivirus KD; and 40% vs 35%; p=0.03 for CpG-miR-126 inhibitor KD), resulting in significant reduction of cell growth and colony forming cell (CFC) frequency in methylcellulose progenitor assays. Conversely, enforced miR-126 expression by miR-126 precursor lentivirus (System Biosciences) impaired cell-cycle entry (28% vs 33%; p=0.05) and led to significantly reduced apoptosis (27% vs 19%; P=0.01) in NIL (5µM) treated CML CD34+CD38- cells compared to cells treated with control lentivirus and NIL. We further investigated the effect of miR-126 KD on growth of primary CML LSC and therapeutic response in vivo. CP CML CD34+ cells were cultured with CpG-scrambled RNA or CpG-miR-126 ODN inhibitor (500nM), in the presence or absence of NIL (5µM) for 4 days, and then transplanted into irradiated NSG mice. While the experiment is still ongoing, we have already observed a significantly reduced engraftment in blood at 4 wks in recipient mice receiving cells treated with CpG-miR-126 inhibitor and NIL compared with those receiving cells treated with CpG-scrambled RNA and NIL (0.16±0.03% vs 0.4±0.04%; P=0.01). Final results of this in vivo experiment and in vivo treatment of SCLtTA/BCR-ABL mice with CpG-miR-126 inhibitor and NIL will be reported at the meeting. Altogether, these observations suggest that miR-126 play a role in CML LSC homeostasis and down-regulation of miR-126 may decrease CML LSC quiescence, increase LSC proliferation and in turn enhance their sensitivity to TKI. miR-126 therefore may represent a novel therapeutic target in CML. DisclosuresStein:Amgen: Speakers Bureau. Snyder:BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Forman:Mustang: Research Funding; Amgen: Consultancy.

MicroRNA-122 confers sorafenib resistance to hepatocellular carcinoma cells by targeting IGF-1R to regulate RAS/RAF/ERK signaling pathways

Sorafenib is the first-line treatment for advanced hepatocellular carcinoma (HCC), but the clinical response to sorafenib is seriously limited by drug resistance. In this study, we investigated the molecular mechanisms of sorafenib resistance in HCC cells. Our miRNA microarray data indicate that liver-specific miR-122 expression was significantly reduced in sorafenib-resistant cells. Overexpression of miR-122 made drug-tolerant cells sensitive to sorafenib and induced apoptosis. Insulin-like growth factor 1 receptor (IGF-1R) was validated as a target of miR-122 and was repressed by this miRNA. miR-122-induced apoptosis was repressed by the IGF-1R activator IGFI or IGFII. Conversely, the IGF-1R inhibitor PPP or NVP-AEW541 in combination with sorafenib significantly induced cell apoptosis and disrupted tolerance to drugs in vitro. These results indicated that activation of IGF-1R by ectopic down-regulation of miR-122 counteracted the effects of sorafenib-induced apoptosis, thus conferring sorafenib resistance. Further study revealed that activation of IGF-1R by miR-122 down-regulation contributed to activation of RAS/RAF/ERK signaling, which was associated with drug resistance. Our data imply that an intimate correlation between miR-122 and IGF-1R abnormal expression is a critical determinant of sorafenib tolerance.

Downregulation of MIR-145 predicts a worse outcome in upper tract urothelial carcinomas

Downregulation of MIR-145 predicts a worse outcome in upper tract urothelial carcinomas

Abstract 16139: Mast Cells Promote Proliferation and Suppress Myogenic Differentiation of Mesenchymal Stem Cells

Introduction: Efficient scar formation post-myocardial infarction (MI) depends on fibroblast/mesenchymal stem cell (MSC) accumulation and myofibroblast transdifferentiation. Mast cell (MC)-deficient mice exhibit reduced cardiac myofibroblast accumulation and undergo rapid left ventricular dilation post-MI. Thus, we sought to investigate how MCs modulate MSC proliferation and their myogenic differentiation into myofibroblasts. Methods: MC/MSC co-culture was used to examine MC effects on MSC proliferation and differentiation. MC granulate (MCG) was then used to further examine the mechanisms by which MCs exerted their effects on MSC proliferation, migration, and myogenic differentiation. The effects of MCs on MSC activity in vivo was evaluated by immunohistological analysis on MC- and saline-treated infarcted mouse hearts at 3 and 7 days post-MI Results: MC co-culture suppressed expression of the myogenic differentiation marker α-smooth muscle actin in MSCs. Similarly, MCG dose-dependently decreased myogenic differentiation by up to ~90% (P&lt;0.01, n=4) and increased MSC proliferation ~two-fold (P&lt;0.01, n=5) and migration by ~75% (P&lt;0.01, n=4). Pharmacological antagonism of platelet-derived growth factor receptor (PDGFR) rescued MSC differentiation despite MCG treatment, suggesting MC suppression of MSC differentiation occurs through PDGFR. MCG treatment resulted in up to ~70% decreased miR-145 and -143 expression (P&lt;0.05, n=4), as well as increased Klf4 (~25%) and decreased myocardin (~50%) protein expression in MSCs, indicating that the myocardin-Klf4 axis mediates MSC proliferation/differentiation. Finally, infarcted hearts from mice treated with MCs showed ~five-fold increased CD29+ MSC proliferation at day 3 vs saline-treated animals (P&lt;0.05, n=3). This effect was absent by day 7 post-MI. Conclusions: MSCs can oscillate between proliferative and differentiated states. MCs promote proliferation at the expense of differentiation early after MI through PDGFR, downregulation of miR-145 and -143, and the Klf4-myocardin signalling axis to promote MSC accumulation. This in turn results in a larger cardiac MSC pool for later myofibroblast differentiation, facilitating improved wound healing.

Abstract 13844: Microrna-126 and Microrna-132 Abrogates Deleterious Effects of Diabetes-induced Impaired Angiogenesis

Background: Diabetes-induced microvascular abnormalities are implicated in altering cardiac function and structure, a condition referred as diabetic microangiopathy in heart (DMAH). Endothelial-enriched microRNAs (angiomiRs) have been recently demonstrated to play a major role in regulating angiogenesis. However, their pathophysiological role in development of DMAH is elusive. Objective: To investigate the modulation of angiomiRs (miR-126, miR-132) with evolution of DMAH and demonstrate if restoration of angiomiRs can improve functional properties of high glucose treated endothelial cells. Methods &amp; Results: Cardiac tissues were collected from type-2 diabetic mice (BKS.Cg-m+/+Leprdb/J) every 4Wks, from 8 to 32Wks of age. qPCR revealed significant downregulation of miR-126 and -132 starting at 12Wks and continued till 32Wks (miR-126: 0.30±0.03; miR-132: 0.22±0.05,p&lt;0.001 vs. age matched non-diabetic). This was associated with concomitant dysfunction of cardiac functions (assessed through echocardiography). Western blotting analysis confirmed marked dysregulation of VEGF and p120RasGap (P&lt;0.05 vs. non-diabetic), the target proteins for miR-126 and -132, respectively. Importantly, immunohistochemical analysis did not show any noticeable changes in capillary density and arterioles until 20Wks in diabetes. To determine if restoration of miR-126 and -132 could reduce the development of DMAH, high glucose (HG)-treated Human Umbilical Vein Endothelial Cells (HUVECs) were transfected with either miR-126 or -132 mimics. Results showed that restoration of both the miRs markedly improved angiogenesis, assessed by tube formation assay (NG-Control- 8824±212; HG-Control: 3570±304; HG-126: 8970±184; HG-132: 7227±228), proliferation (CyQUANT cell proliferation assay) and reduced cell death (caspase-3/7 assay), p&lt;0.05 for all the parameters. Of note, overexpression of miR-126 alone tends to restore the expression of miR-132 and vice versa in HG-treated HUVECs, suggesting an orchestrated role of miRs in regulating angiogenesis. Conclusion: Modulation of miRs occur much earlier before the development of DMAH and restoration of DMAH from early stages in HUVECs exposed to high glucose markedly improved their angiogenic potential.

MicroRNA-100 suppresses the migration and invasion of breast cancer cells by targeting FZD-8 and inhibiting Wnt/β-catenin signaling pathway.

Wnt/β-catenin signaling pathway plays a major role in the cancer metastasis. Several microRNAs (miRNAs) are contributed to the inhibition of breast cancer metastasis. Here, we attempted to find novel targets and mechanisms of microRNA-100 (miR-100) in regulating the migration and invasion of breast cancer cells. In this study, we found that miR-100 expression was downregulated in human breast cancer tissues and cell lines. The overexpression of miR-100 inhibited the migration and invasion of MDA-MB-231 breast cancer cells. Inversely, the downregulation of miR-100 increased the migration and invasion of MCF-7 breast cancer cells. Furthermore, FZD-8, a receptor of Wnt/β-catenin signaling pathway, was demonstrated a direct target of miR-100. The overexpression of miR-100 decreased the expression levels not only FZD-8 but also the key components of Wnt/β-catenin pathway, including β-catenin, metalloproteniase-7 (MMP-7), T-cell factor-4 (TCF-4), and lymphoid enhancing factor-1 (LEF-1), and increased the protein expression levels of GSK-3β and p-GSK-3β in MDA-MB-231 cells, and the transfection of miR-100 inhibitor in MCF-7 cells showed the opposite effects. In addition, the expression of miR-100 was negatively correlated with the FZD-8 expression in human breast cancer tissues. Overall, these findings suggest that miR-100 suppresses the migration and invasion of breast cancer cells by targeting FZD-8 and inhibiting Wnt/β-catenin signaling pathway and manipulation of miR-100 may provide a promoting therapeutic strategy for cancer breast treatment.

Overexpression of miR-100 inhibits cell proliferation, migration, and chemosensitivity in human glioblastoma through FGFR3.

BackgroundGlioblastoma multiforme is one of the most deadly forms of brain cancer. We investigated the regulatory effects of microRNA-100 (miR-100) on cell proliferation, migration, and chemosensitivity in human glioblastoma.MethodsmiR-100 expression was assessed by quantitative real-time polymerase chain reaction in both glioblastoma cells and human tumors. Lentiviruses of miR-100 mimics and inhibitors were transfected into U251 and T98G cells. The regulatory effects of either overexpressing or downregulating miR-100 on glioblastoma were evaluated by a viability assay, growth assay, migration assay, chemosensitivity assay, and an in vivo tumor transplantation assay. Expression of fibroblast growth factor receptor 3 (FGFR3), the bioinformatically predicted target of miR-100, was examined by Western blot in glioblastoma. FGFR3 was then ectopically overexpressed in U251 and T98G cells, and its effects on miR-100-mediated cancer regulation were evaluated by growth, migration, and chemosensitivity assays.ResultsMiR-100 was markedly downregulated in both glioblastoma cell lines and human tumors. Overexpressing miR-100 through lentiviral transfection in U251 and T98G cells significantly inhibited cancer growth (both in vitro and in vivo) and migration and increased chemosensitivity to cisplatin and 1, 3-bis (2-chloroethyl)-l-nitrosourea, whereas downregulation of miR-100 had no effects on development of cancer. FGFR3 was directly regulated by miR-100 in glioblastoma. Ectopically overexpressing FGFR3 was able to ameliorate the anticancer effects of upregulation of miR-100 on glioblastoma growth, migration, and chemosensitivity.ConclusionMiR-100 was generally downregulated in glioblastoma. Overexpressing miR-100 had anticancer effects on glioblastoma, likely through regulation of FGFR3. The MiR-100/FGFR3 signaling pathway might be a biochemical target for treatment in patients with glioblastoma.