Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.     

MiRNA-181b suppresses IGF-1R and functions as a tumor suppressor gene in gliomas

MicroRNAs (miRNAs) are single-stranded, 18- to 23-nt RNA molecules that function as regulators of gene expression. Previous studies have shown that microRNAs play important roles in human cancers, including gliomas. Here, we found that expression levels of miR-181b were decreased in gliomas, and we identified IGF-1R as a novel direct target of miR-181b. MiR-181b overexpression inhibited cell proliferation, migration, invasion, and tumorigenesis by targeting IGF-1R and its downstream signaling pathways, PI3K/AKT and MAPK/ERK1/2. Overexpression of IGF-1R rescued the inhibitory effects of miR-181b. In clinical specimens, IGF-1R was overexpressed, and its protein levels were inversely correlated with miR-181b expression. Taken together, our results indicate that miR-181b functions in gliomas to suppress growth by targeting the IGF-1R oncogene and that miR-181b may serve as a novel therapeutic target for gliomas.     

MicroRNA-1291-mediated silencing of IRE1α enhances Glypican-3 expression

MicroRNAs (miRNA) are generally described as negative regulators of gene expression. However, some evidence suggests that they may also play positive roles. As such, we reported that miR-1291 leads to a GPC3 mRNA expression increase in hepatoma cells through a 3′ untranslated region (UTR)-dependent mechanism. In the absence of any direct interaction between miR-1291 and GPC3 mRNA, we hypothesized that miR-1291 could act by silencing a negative regulator of GPC3 mRNA expression. Based on in silico predictions and experimental validation, we demonstrate herein that miR-1291 represses the expression of the mRNA encoding the endoplasmic reticulum (ER)-resident stress sensor IRE1α by interacting with a specific site located in the 5′ UTR. Moreover, we show, in vitro and in cultured cells, that IRE1α cleaves GPC3 mRNA at a 3′ UTR consensus site independently of ER stress, thereby prompting GPC3 mRNA degradation. Finally, we show that the expression of a miR-1291-resistant form of IRE1α abrogates the positive effects of miR-1291 on GPC3 mRNA expression. Collectively, our data demonstrate that miR-1291 is a biologically relevant regulator of GPC3 expression in hepatoma cells and acts through silencing of the ER stress sensor IRE1α.     

miR-223 contributes to the AGE-promoted apoptosis via down-regulating insulin-like growth factor 1 receptor in osteoblasts

Advanced glycation end products (AGEs) have been confirmed to induce bone quality deterioration in diabetes mellitus (DM), and to associate with abnormal expression of miRNAs in DM patients or in vitro. Recently, miRNAs have been recognized to mediate the onset or progression of DM. In the present study, we investigated the regulation on miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells, with real-time quantitative PCR assay. And then we examined the inhibition of insulin-like growth factor 1 receptor (IGF-1R) expression by miR-223, via targeting of the 3′ UTR of IGF-1R with real-time quantitative PCR, western blotting and luciferase reporter assay. Then we explored the regulation of miR-223 and IGF-1R levels, via the lentivirus-mediated miR-223 inhibition and IGF-1R overexpression in the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It was demonstrated that AGE-BSA treatment with more than 100 μg/ml significantly up-regulated miR-223 level, whereas down-regulated IGF-1R level in MC3T3-E1 cells. And the up-regulated miR-223 down-regulated IGF-1R expression in both mRNA and protein levels, via targeting the 3′ UTR of IGF-1R. Moreover, though the AGE-BSA treatment promoted apoptosis in MC3T3-E1 cells, the IGF-1R overexpression or the miR-223 inhibition significantly attenuated the AGE-BSA-promoted apoptosis in MC3T3-E1 cells. In summary, our study recognized the promotion of miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells. The promoted miR-223 targeted IGF-1R and mediated the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It implies that miR-223 might be an effective therapeutic target to antagonize the AGE-induced damage to osteoblasts in DM.     

ATM-NFκB axis-driven TIGAR regulates sensitivity of glioma cells to radiomimetics in the presence of TNFα

Gliomas are resistant to radiation therapy, as well as to TNFα induced killing. Radiation-induced TNFα triggers Nuclear factor κB (NFκB)-mediated radioresistance. As inhibition of NFκB activation sensitizes glioma cells to TNFα-induced apoptosis, we investigated whether TNFα modulates the responsiveness of glioma cells to ionizing radiation-mimetic Neocarzinostatin (NCS). TNFα enhanced the ability of NCS to induce glioma cell apoptosis. NCS-mediated death involved caspase-9 activation, reduction of mitochondrial copy number and lactate production. Death was concurrent with NFκB, Akt and Erk activation. Abrogation of Akt and NFκB activation further potentiated the death inducing ability of NCS in TNFα cotreated cells. NCS-induced p53 expression was accompanied by increase in TP53-induced glycolysis and apoptosis regulator (TIGAR) levels and ATM phosphorylation. siRNA-mediated knockdown of TIGAR abrogated NCS-induced apoptosis. While DN-IκB abrogated NCS-induced TIGAR both in the presence and absence of TNFα, TIGAR had no effect on NFκB activation. Transfection with TIGAR mutant (i) decreased apoptosis and γH2AX foci formation (ii) decreased p53 (iii) elevated ROS and (iv) increased Akt/Erk activation in cells cotreated with NCS and TNFα. Heightened TIGAR expression was observed in GBM tumors. While NCS induced ATM phosphorylation in a NFκB independent manner, ATM inhibition abrogated TIGAR and NFκB activation. Metabolic gene profiling indicated that TNFα affects NCS-mediated regulation of several genes associated with glycolysis. The existence of ATM-NFκB axis that regulate metabolic modeler TIGAR to overcome prosurvival response in NCS and TNFα cotreated cells, suggests mechanisms through which inflammation could affect resistance and adaptation to radiomimetics despite concurrent induction of death.     

RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

Aerobic glycolysis or the Warburg effect contributes to cancer cell proliferation; however, how this glucose metabolism pathway is precisely regulated remains elusive. Here we show that receptor-interacting protein 1 (RIP1), a cell death and survival signaling factor, regulates mitochondrial oxidative phosphorylation and aerobic glycolysis. Loss of RIP1 in lung cancer cells suppressed peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) expression, impairing mitochondrial oxidative phosphorylation and accelerating glycolysis, resulting in spontaneous DNA damage and p53-mediated cell proliferation inhibition. Thus, although aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis causes cytostasis. Our data suggest that maintenance of glycolysis by RIP1 is pivotal to cancer cell energy homeostasis and DNA integrity and may be exploited for use in anticancer therapy.     

Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach

Recent studies demonstrated that miR-152 overexpression down-regulates the nonclassical human leukocyte antigen (HLA) class I molecule HLA-G in human tumors thereby contributing to their immune surveillance. Using two-dimensional gel electrophoresis followed by MALDI-TOF mass spectrometry, the protein expression profile of HLA-G⁺, miR-152^low cells, and their miR-152-overexpressing (miR^high) counterparts was compared leading to the identification of 24 differentially expressed proteins. These were categorized according to their function and localization demonstrating for most of them an important role in the initiation and progression of tumors. The novel miR-152 target 14-3-3 protein β/α/YWHAB (14-3-3β) is down-regulated upon miR-152 overexpression, although its overexpression was often found in tumors of distinct origin. The miR-152-mediated reduction of the 14-3-3β expression was accompanied by an up-regulation of BAX protein expression resulting in a pro-apoptotic phenotype. In contrast, the reconstitution of 14-3-3β expression in miR-152^high cells increased the expression of the anti-apoptotic BCL2 gene, enhances the proliferative activity in the presence of the cytostatic drug paclitaxel, and causes resistance to apoptosis induced by this drug. By correlating clinical microarray data with the patients'' outcome, a link between 14-3-3β and HLA-G expression was found, which could be associated with poor prognosis and overall survival of patients with tumors. Because miR-152 controls both the expression of 14-3-3β and HLA-G, it exerts a dual role in tumor cells by both altering the immunogenicity and the tumorigenicity.     

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α

Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in context-dependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of pre-miRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5′ hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p; and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-β signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.     

MicroRNA-21 regulates T-cell apoptosis by directly targeting the tumor suppressor gene Tipe2

MicroRNAs (MiRs) are short noncoding RNAs that can regulate gene expression. It has been reported that miR-21 suppresses apoptosis in activated T cells, but the molecular mechanism remains undefined. Tumor suppressor Tipe2 (or tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TNFAIP8L2)) is a newly identified anti-inflammatory protein of the TNFAIP8 family that is essential for maintaining immune homeostasis. We report here that miR-21 is a direct target of nuclear factor-κB and could regulate Tipe2 expression in a Tipe2 coding region-dependent manner. In activated T cells and macrophages, Tipe2 expression was markedly downregulated, whereas miR-21 expression was upregulated. Importantly, Tipe2-deficient T cells were significantly less sensitive to apoptosis. Conversely, overexpression of Tipe2 in EL-4 T cells increased their susceptibility to activation-induced apoptosis. Therefore, Tipe2 provides a molecular bridge between miR-21 and cell apoptosis; miR-21 suppresses apoptosis in activated T cells at least in part through directly targeting tumor suppressor gene Tipe2.     

Reciprocal inhibition between miR-26a and NF-κB regulates obesity-related chronic inflammation in chondrocytes

Obesity is causally linked to osteoarthritis (OA), with the mechanism being not fully elucidated. miRNAs (miRs) are pivotal regulators of various diseases in multiple tissues, including inflammation in the chondrocytes. In the present study, we for the first time identified the expression of miR-26a in mouse chondrocytes. Decreased level of miR-26a was correlated to increased chronic inflammation in the chondrocytes and circulation in obese mouse model. Mechanistically, we demonstrated that miR-26a attenuated saturated free fatty acid-induced activation of NF-κB (p65) and production of proinflammatory cytokines in chondrocytes. Meanwhile, NF-κB (p65) also suppressed miR-26a production by directly binding to a predicted NF-κB binding element in the promoter region of miR-26a. Finally, we observed a negative correlation between NF-κB and miR-26a in human patients with osteoarthritis. Thus, we identified a reciprocal inhibition between miR-26a and NF-κB downstream of non-esterified fatty acid (NEFA) signalling in obesity-related chondrocytes. Our findings provide a potential mechanism linking obesity to cartilage inflammation.     

Gadd45α: A Novel Diabetes-Associated Gene Potentially Linking Diabetic Cardiomyopathy and Baroreflex Dysfunction

Both diabetic cardiomyopathy (DCM) and baroreflex dysfunction independently contribute to sudden cardiac death (SCD), however the inherent connections between them under diabetic state remains unclear. As microRNAs (miRNAs) have been reported to participate in various physiological and pathological processes, we presume they may also be involved in DCM and DM-induced impairment of baroreflex sensitivity. Two sets of gene expression profiles data from streptozotocin (STZ)-induced diabetic heart and diabetic dorsal root ganglia (DDRG) were retrieved from GEO and ArrayExpress. Co-differentially-expressed genes in diabetic heart and DDRG were identified by t test and intersection analysis. Human Protein Reference Database (HPRD) was applied to find direct interacting proteins of Gadd45α. Differentially-expressed miRNAs in left ventricle from 4-week STZ-induced diabetic rats were screened by miRNA microarray. Expression of miR-499 and its regulating effect on Gadd45α were then verified by quantitative real-time PCR (qRT-PCR), western blot, computational predication, and dual-luciferase reporter analysis. Four co-differentially-expressed genes in DCM and DDRG were identified. Among these genes, Gadd45α has 16 direct interacting proteins and 11 of them are documentedly associated with DM. Accompanied with significantly increased miR-499 expression, Gadd45α expression was increased at mRNA level but decreased at protein level in both diabetic heart and nucleus ambiguous. Furthermore, miR-499 was confirmed negatively regulating Gadd45α by targeting its 3′UTR. Collectively, reduced Gadd45α protein expression by forced miR-499 expression indicated it''s a diabetes-associated gene which might potentially be involved in both DCM and DM-induced baroreflex dysfunction.