Expression of Micro‐RNA‐145 is regulated by a highly conserved genomic sequence 3′ to the pre‐miR

Micro‐RNA‐145 (miR145), a tumor suppressor miR, dramatically inhibits growth of cancer cells in culture and plays a significant role in human stem cells differentiation. We have isolated a human genomic sequence of 864 bp comprising the pre‐miR and its flanking sequences. The cloned miR145 genomic sequence expresses a mature miR145 in transfected cells. We show here that flanking sequences on either side of the pre‐miR sequence can modulate its expression levels. Surprisingly, a highly conserved sequence 3′ to the pre‐miR plays a crucial role in miR145 expression. J. Cell. Physiol. 226: 602–607, 2011. © 2010 Wiley‐Liss, Inc.     

Mechanism of growth inhibition by MicroRNA 145: The role of the IGF‐I receptor signaling pathway

MicroRNA 145 (miR145) has been proposed as a tumor suppressor. It was previously shown that miR145 targets the 3′ UTR of the insulin receptor substrate‐1 (IRS‐1) and dramatically inhibits the growth of colon cancer cells. miR145 also targets the type 1 insulin‐like growth factor receptor (IGF‐IR). We show here that an IRS‐1 lacking its 3′ UTR is no longer down‐regulated by miR145 and rescues colon cancer cells from miR145‐induced inhibition of growth. An IGF‐IR resistant to miR145 (again by elimination of its 3′ UTR) is not down‐regulated by miR145 but fails to rescue colon cancer cells from growth inhibition. These and other results, taken together, indicate that down‐regulation of IRS‐1 plays a significant role in the tumor suppressor activity of miR145. J. Cell. Physiol. 220: 485–491, 2009. © 2009 Wiley‐Liss, Inc.     

MiRNA‐145‐5p prevents differentiation of oligodendrocyte progenitor cells by regulating expression of myelin gene regulatory factor

The roles of specific microRNAs (miRNA) in oligodendrocyte (OL) differentiation have been studied in depth. However, miRNAs in OL precursors and oligodendrocyte progenitor cells (OPCs) have been less extensively investigated. MiR‐145‐5p is highly expressed in OPCs relative to differentiating OLs, suggesting this miRNA may serve a function specifically in OPCs. Knockdown of miR‐145‐5p in primary OPCs led to spontaneous differentiation, as evidenced by an increased proportion of MAG⁺ cells, increased cell ramification, and upregulation of multiple myelin genes including MYRF, TPPP, and MAG, and OL cell cycle exit marker Cdkn1c. Supporting this transition to a differentiating state, proliferation was reduced in miR‐145‐5p knockdown OPCs. Further, knockdown of miR‐145‐5p in differentiating OLs showed enhanced differentiation, with increased branching, myelin membrane production, and myelin gene expression. We identified several OL‐specific genes targeted by miR‐145‐5p that exhibited upregulation with miR‐145‐5p knockdown, including myelin gene regulatory factor (MYRF), that could be regulating the prodifferentiation phenotype in both miR‐145 knockdown OPCs and OLs. Indeed, spontaneous differentiation with knockdown of miR‐145‐5p was fully rescued by concurrent knockdown of MYRF. However, proliferation rate was only partially rescued with MYRF knockdown, and overexpression of miR‐145‐5p in OPCs increased proliferation rate without affecting expression of already lowly expressed differentiation genes. Taken together, these data suggest that in OPCs miR‐145‐5p both prevents differentiation at least in part by preventing expression of MYRF and promotes proliferation via as‐yet‐unidentified mechanisms. These findings clarify the need for differential regulation of miR‐145‐5p between OPCs and OLs and may have further implications in demyelinating diseases such as multiple sclerosis where miR‐145‐5p is dysregulated.     

Micro RNA 145 targets the insulin receptor substrate-1 and inhibits the growth of colon cancer cells

The insulin receptor substrate-1 (IRS-1), a docking protein for both the type 1 insulin-like growth factor receptor (IGF-IR) and the insulin receptor, is known to send a mitogenic, anti-apoptotic, and anti-differentiation signal. Several micro RNAs (miRs) are suggested by the data base as possible candidates for targeting IRS-1. We show here that one of the miRs predicted by the data base, miR145, whether transfected as a synthetic oligonucleotide or expressed from a plasmid, causes down-regulation of IRS-1 in human colon cancer cells. IRS-1 mRNA is not decreased by miR145, while it is down-regulated by an siRNA targeting IRS-1. Targeting of the IRS-1 3'-untranslated region (UTR) by miR145 was confirmed using a reporter gene (luciferase) expressing the miR145 binding sites of the IRS-1 3'-UTR. In agreement with the role of IRS-1 in cell proliferation, we show that treatment of human colon cancer cells with miR145 causes growth arrest comparable to the use of an siRNA against IRS-1. Taken together, these results identify miR145 as a micro RNA that down-regulates the IRS-1 protein, and inhibits the growth of human cancer cells.     

Knock‐out of MicroRNA 145 impairs cardiac fibroblast function and wound healing post‐myocardial infarction

Prevention of infarct scar thinning and dilatation and stimulation of scar contracture can prevent progressive heart failure. Since microRNA 145 (miR‐145) plays an important role in cardiac fibroblast response to wound healing and cardiac repair after an myocardial infarction (MI), using a miR‐145 knock‐out (KO) mouse model, we evaluated contribution of down‐regulation of miR‐145 to cardiac fibroblast and myofibroblast function during adverse cardiac remodelling. Cardiac function decreased more and the infarct size was larger in miR‐145 KO than that in WT mice after MI and this phenomenon was accompanied by a decrease in cardiac fibroblast‐to‐myofibroblast differentiation. Quantification of collagen I and α‐SMA protein levels as well as wound contraction revealed that transdifferentiation of cardiac fibroblasts into myofibroblasts was lower in KO than WT mice. In vitro restoration of miR‐145 induced more differentiation of fibroblasts to myofibroblasts and this effect involved the target genes Klf4 and myocardin. MiR‐145 contributes to infarct scar contraction in the heart and the absence of miR‐145 contributes to dysfunction of cardiac fibroblast, resulting in greater infarct thinning and dilatation. Augmentation of miR‐145 could be an attractive target to prevent adverse cardiac remodelling after MI by enhancing the phenotypic switch of cardiac fibroblasts to myofibroblasts.     

microRNA‐145‐3p inhibits non‐small cell lung cancer cell migration and invasion by targeting PDK1 via the mTOR signaling pathway

The mammalian target of rapamycin (mTOR) pathway is dysregulated in more than 50% of all human malignancies and is a major target in cancer treatment. In this study, we explored the underlying mechanism involving microRNA‐145‐3p (miR‐145‐3p) in the development and progression of non‐small cell lung cancer (NSCLC) by targeting PDK1 via the mTOR signaling pathway. NSCLC tissues and adjacent normal tissues were obtained from 83 NSCLC patients. miR‐145‐3p, PDK1, and mTOR levels were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and immunohistochemistry. Human NSCLC cell lines A549 and H1299 were transfected with miR‐145‐3p and siPDK1 to confirm the effect of miR‐145‐3p and PDK1 on NSCLC cells in vitro. Cell growth was evaluated by a CCK8 assay. Cell motility and chemotaxis analysis were determined by the scratch test and chemotaxis assay, respectively. The protein levels of PDK1 and mTOR were measured using the western blotting. Results showed lower level of miR‐145‐3p and higher levels of PDK1 and mTOR in NSCLC tissues compared to the adjacent normal tissues. In vitro results showed that cell growth, cell motility, and chemotaxis were all inhibited in cells transfected with miR‐145‐3p and those transfected with siPDK. Additionally, dual luciferase reporter gene assay helped confirmed that PDK1 is a target of miR‐145. Finally, levels of PDK1, mTOR, and phosphorylated‐mTOR were lower in cells transfected with miR‐145‐3p as well as those with siPDK1. These findings indicate that miR‐145‐3p may inhibit cell growth, motility, and chemotaxis in NSCLC by targeting PDK1 through suppressing the mTOR pathway.     

Generation of transgenic mice that conditionally express microRNA miR‐145

MicroRNAs are modulators of cellular phenotypes and their functions contribute to development, homeostasis, and disease. miR‐145 is a conserved microRNA that has been implicated in regulating an array of phenotypes. These include supporting smooth muscle differentiation, repression of stem cell pluripotency, and inhibition of tumor growth and metastasis. Previously, our lab demonstrated that miR‐145 acts to suppress cardiac fibrosis through inhibition of the TGF‐β signaling pathway. The range of effects that miR‐145 has on different cell types makes it an attractive microRNA for further study. Here we describe the generation of transgenic mice that conditionally express miR‐145 through Cre recombinase‐mediated activation. Characterization of individual founder lines indicates that overexpression of miR‐145 in the developing cardiovascular system has detrimental effects, with three independent miR‐145 transgenic lines exhibiting Cre‐dependent lethality. Expression analysis demonstrates that the transgene is robustly expressed and our analysis reveals a novel downstream target of miR‐145, Tnnt2. The miR‐145 transgenic mice represent a valuable tool to understand the role of miR‐145 in diverse cell types and to address its potential as a therapeutic mediator for the treatment of disease.     

miR‐145 inhibits the proliferation and migration of vascular smooth muscle cells by regulating autophagy

miR‐145, the most abundant miRNA in the vascular smooth muscle cells (VSMCs), regulates VSMC function in intimal hyperplasia. It has been reported that autophagy participates in the regulation of proliferation and migration of VSMCs. However, the effect of miR‐145 on autophagy and related mechanism in the proliferation and migration of VSMCs remains unclear. Therefore, we aimed to determine the effect of miR‐145 on autophagy and the mechanism in VSMCs. Cell autophagy was determined by transmission electron microscope, mRFP‐GFP‐LC3 assay and Western blotting. A recombinant lentivirus containing miR‐145 was used to construct VSMCs with miR‐145 overexpression. We found that miR‐145 expression was decreased, and autophagy was increased in the carotid arteries of C57BL/6J mice with intimal hyperplasia and TGF‐β1‐stimulated VSMCs. Furthermore, miR‐145 overexpression inhibited cell autophagy, whereas miR‐145 inhibition promoted autophagy in TGF‐β1‐stimulated VSMCs. Meanwhile, miR‐145 inhibited the proliferation and migration of VSMCs. More importantly, our study showed that autophagy inhibition augmented the inhibitory effect of miR‐145 on the proliferation and migration of VSMCs. In addition, we found that the sirtuins are not direct targets of miR‐145 in the proliferation and migration of VSMCs. These results suggest that miR‐145 inhibits the proliferation and migration of VSMCs by suppressing the activation of autophagy.     

HEF1 promotes epithelial mesenchymal transition and bone invasion in prostate cancer under the regulation of microRNA‐145

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bones, and it's crucial to understand the mechanism of tumor progression to metastasis in order to develop therapies that may reduce the morbidity and mortality of PCa patients. Although we had identified that microRNA(miR)‐145 could repress bone metastasis of PCa via regulating epithelial–mesenchymal transition (EMT) in previous study, it is still unknown how miR‐145 regulated EMT. In the present study, we constructed a luciferase reporter system and identified HEF1 as a direct target of miR‐145. More importantly, HEF1 was shown to promote migration, invasion and EMT of PC‐3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. And HEF1 was also shown to partially mediate miR‐145 suppression of EMT and invasion. Furthermore, inhibition of HEF1 repressed bone invasion of PC‐3 cells in vivo. Expression of HEF1 was negatively correlated with miR‐145 in primary PCa and bone metastatic specimens, but HEF1 was higher in samples which were more likely to commit to bone metastasis or those with higher free prostate‐specific antigen (fPSA) levels and Gleason scores. Taken together, these findings indicate that HEF1 promotes EMT and bone invasion in prostate cancer by directly targeted by miR‐145, and miR‐145 suppresses EMT and invasion, at least in part, through repressing HEF1. J. Cell. Biochem. 114: 1606–1615, 2013. © 2013 Wiley Periodicals, Inc.     

Expression profiles of micro RNA in proliferating and differentiating 32D murine myeloid cells

32D cells are murine myeloid cells that grow indefinitely in Interleukin-3 (IL-3). In these cells, the type 1 insulin-like growth factor (IGF-I) and granulocytic-colony stimulating factor (G-CSF) induce differentiation to granulocytes. 32D cells do not express insulin receptor substrate-1 (IRS-1) or IRS-2, docking proteins of the IGF-I receptor. Ectopic expression of IRS-1 in these cells inhibits differentiation, the cells become IL-3 independent and IGF-1 dependent and can form tumors in mice. 32D and 32D-derived cells offer a good model in which to study the expression profiles of Micro Rna (miR) related to sustained proliferation or differentiation. We present here the data obtained with miR micro-arrays and identify the miR that are regulated by IGF-1 or G-CSF and are associated with either differentiation or indefinite cell proliferation of 32D murine myeloid cells.     

MiR‐199b‐5p inhibits osteogenic differentiation in ligamentum flavum cells by targeting JAG1 and modulating the Notch signalling pathway

Ossification of the ligamentum flavum (OLF) is a pathology almost only reported in East Asian countries. The leading cause of OLF is thoracic spinal canal stenosis and myelopathy. In this study, the role of miR‐199b‐5p and jagged 1 (JAG1) in primary ligamentum flavum cell osteogenesis was examined. MiR‐199b‐5p was found to be down‐regulated during osteogenic differentiation in ligamentum flavum cells, while miR‐199b‐5p overexpression inhibited osteogenic differentiation. In addition, JAG1 was found to be up‐regulated during osteogenic differentiation in ligamentum flavum cells, while JAG1 knockdown via RNA interference caused an inhibition of Notch signalling and osteogenic differentiation. Moreover, target prediction analysis and dual luciferase reporter assays supported the notion that JAG1 was a direct target of miR‐199b‐5p, with miR‐199b‐5p found to down‐regulate both JAG1 and Notch. Further, JAG1 knockdown was demonstrated to block the effect of miR‐199b‐5p inhibition. These findings imply that miR‐199b‐5p performs an inhibitory role in osteogenic differentiation in ligamentum flavum cells by potentially targeting JAG1 and influencing the Notch signalling pathway.     

miR‐145 is differentially regulated by TGF‐β1 and ischaemia and targets Disabled‐2 expression and wnt/β‐catenin activity

The effect of wnt/β‐catenin signalling in the response to acute myocardial infarction (AMI) remains controversial. The membrane receptor adaptor protein Disabled‐2 (Dab2) is a tumour suppressor protein and has a critical role in stem cell specification. We recently demonstrated that down‐regulation of Dab2 regulates cardiac protein expression and wnt/β‐catenin activity in mesenchymal stem cells (MSC) in response to transforming growth factor‐β₁ (TGF‐β₁). Although Dab2 expression has been shown to have effects in stem cells and tumour suppression, the molecular mechanisms regulating this expression are still undefined. We identified putative binding sites for miR‐145 in the 3′‐UTR of Dab2. In MSC in culture, we observed that TGF‐β₁ treatment led to rapid and sustained up‐regulation of pri–miR‐145. Through gain and loss of function studies we demonstrate that miR‐145 up‐regulation was required for the down‐regulation of Dab2 and increased β‐catenin activity in response to TGF‐β₁. To begin to define how Dab2 might regulate wnt/β‐catenin in the heart following AMI, we quantified myocardial Dab2 as a function of time after left anterior descending ligation. There was no significant Dab2 expression in sham‐operated myocardium. Following AMI, Dab2 levels were rapidly up‐regulated in cardiac myocytes in the infarct border zone. The increase in cardiac myocyte Dab2 expression correlated with the rapid and sustained down‐regulation of myocardial pri–miR‐145 expression following AMI. Our data demonstrate a novel and critical role for miR‐145 expression as a regulator of Dab2 expression and β‐catenin activity in response to TGF‐β₁ and hypoxia.     

Long noncoding RNA MALAT1 enhances the docetaxel resistance of prostate cancer cells via miR‐145‐5p‐mediated regulation of AKAP12

Our present work was aimed to study on the regulatory role of MALAT1/miR‐145‐5p/AKAP12 axis on docetaxel (DTX) sensitivity of prostate cancer (PCa) cells. The microarray data (GSE33455) to identify differentially expressed lncRNAs and mRNAs in DTX‐resistant PCa cell lines (DU‐145‐DTX and PC‐3‐DTX) was retrieved from the Gene Expression Omnibus (GEO) database. QRT‐PCR analysis was performed to measure MALAT1 expression in DTX‐sensitive and DTX‐resistant tissues/cells. The human DTX‐resistant cell lines DU145‐PTX and PC3‐DTX were established as in vitro cell models, and the expression of MALAT1, miR‐145‐5p and AKAP12 was manipulated in DTX‐sensitive and DTX‐resistant cells. Cell viability was examined using MTT assay and colony formation methods. Cell apoptosis was assessed by TUNEL staining. Cell migration and invasion was determined by scratch test (wound healing) and Transwell assay, respectively. Dual‐luciferase assay was applied to analyse the target relationship between lncRNA MALAT1 and miR‐145‐5p, as well as between miR‐145‐5p and AKAP12. Tumour xenograft study was undertaken to confirm the correlation of MALAT1/miR‐145‐5p/AKAP12 axis and DTX sensitivity of PCa cells in vivo. In this study, we firstly notified that the MALAT1 expression levels were up‐regulated in clinical DTX‐resistant PCa samples. Overexpressed MALAT1 promoted cell proliferation, migration and invasion but decreased cell apoptosis rate of PCa cells in spite of DTX treatment. We identified miR‐145‐5p as a target of MALAT1. MiR‐145‐5p overexpression in PC3‐DTX led to inhibited cell proliferation, migration and invasion as well as reduced chemoresistance to DTX, which was attenuated by MALAT1. Moreover, we determined that AKAP12 was a target of miR‐145‐5p, which significantly induced chemoresistance of PCa cells to DTX. Besides, it was proved that MALAT1 promoted tumour cell proliferation and enhanced DTX‐chemoresistance in vivo. There was an lncRNA MALAT1/miR‐145‐5p/AKAP12 axis involved in DTX resistance of PCa cells and provided a new thought for PCa therapy.     

Bioimaging of the unbalanced expression of microRNA9 and microRNA9* during the neuronal differentiation of P19 cells

Generally, the 3'-end of the duplex microRNA (miR) precursor (pre-miR) is known to be stable in vivo and serve as a mature form of miR. However, both the 3'-end (miR9) and 5'-end (miR9*) of a brain-specific miR9 have been shown to function biologically in brain development. In this study, real-time PCR analysis and in vitro/in vivo bioluminescent imaging demonstrated that the upstream region of a primary miR9-1 (pri-miR9-1) can be used to monitor the highly expressed pattern of endogenous pri-miR9-1 during neurogenesis, and that the Luciferase reporter gene can image the unequal expression patterns of miR9 and miR9* seen during the neuronal differentiation of P19 cells. This demonstrates that our bioimaging system can be used to study the participation of miRs in the regulation of neuronal differentiation.