MicroRNA-101 downregulates Alzheimer's amyloid-β precursor protein levels in human cell cultures and is differentially expressed

The full repertoire of regulatory interactions utilized by human cells to control expression of amyloid-β precursor protein (APP) is still undefined. We investigated here the contribution of microRNA (miRNA) to this regulatory network. Several bioinformatic algorithms predicted miR-101 target sites within the APP 3′-untranslated region (3′-UTR). Using reporter assays, we confirmed that, in human cell cultures, miR-101 significantly reduced the expression of a reporter under control of APP 3′-UTR. Mutation of predicted site 1, but not site 2, eliminated this reporter response. Delivery of miR-101 directly to human HeLa cells significantly reduced APP levels and this effect was eliminated by co-transfection with a miR-101 antisense inhibitor. Delivery of a specific target protector designed to blockade the interaction between miR-101 and its functional target site within APP 3′-UTR enhanced APP levels in HeLa. Therefore, endogenous miR-101 regulates expression of APP in human cells via a specific site located within its 3′-UTR. Finally, we demonstrate that, across a series of human cell lines, highest expression of miR-101 levels was observed in model NT2 neurons.     

miR-122 targets an anti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma cell lines

miR-122, a hepato-specific microRNA (miRNA), is frequently down-regulated in human hepatocellular carcinoma (HCC). In an effort to identify novel miR-122 targets, we performed an in silico analysis and detected a putative binding site in the 3′-untranslated region (3′-UTR) of Bcl-w, an anti-apoptotic Bcl-2 family member. In the HCC-derived cell lines, Hep3B and HepG2, we confirmed that miR-122 modulates Bcl-w expression by directly targeting binding site within the 3′-UTR. The cellular mRNA and protein levels of Bcl-w were repressed by elevated levels of miR-122, which subsequently led to reduction of cell viability and activation of caspase-3. Thus, Bcl-w is a direct target of miR-122 that functions as an endogenous apoptosis regulator in these HCC-derived cell lines.     

MicroRNAs function as cis- and trans-acting modulators of peripheral circadian clocks

Based on their extracellular expression and targeting of the clock gene Bmal1, miR-142-3p and miR-494 were analyzed for evidence of vesicle-mediated communication between cells and intracellular functional activity. Our studies demonstrate that: miR-142-3p + miR-494 overexpression decreases endogenous BMAL1 levels, increases the period of Per2 oscillations, and increases extracellular miR-142-3p/miR-494 abundance in conditioned medium; miRNA-enriched medium increases intracellular expression of miR-142-3p and represses Bmal1 3′-UTR activity in naïve cells; and inhibitors of vesicular trafficking modulate intercellular communication of these miRNAs and ensemble Per2 rhythms. Thus, miR-142-3p and miR-494 may function as cis- and trans-acting signals contributing to local temporal coordination of cell-autonomous circadian clocks.     

FOXO1 3′UTR functions as a ceRNA in repressing the metastases of breast cancer cells via regulating miRNA activity

The competitive endogenous RNAs (ceRNAs) are RNA molecules that affect each other’s expression through competition for their shared microRNAs (miRNAs). In this study we explored whether FOXO1 3′UTR can function as a ceRNA in repressing epithelial-to-mesenchymal transition (EMT) and metastasis of breast cancer cells via regulating miR-9 activity. We found that miR-9 binds to both the FOXO1- and E-cadherin-3′UTR, indicating that the FOXO1- and E-cadherin-3′UTR can be linked through miR-9. Follow-up analyses showed that there existed a competition of miR-9 between FOXO1 and E-cadherin-3′UTR. Thus FOXO1 3′UTR inhibits the metastases of breast cancer cells via induction of E-cadherin expression. Our results suggest that FOXO1 3′UTR may function as a miRNA-inhibitor in modulating metastasis of breast cancer cells.     

Preliminary validation of ERBB2 expression regulated by miR-548d-3p and miR-559

ERBB2 overexpression occurs in numerous types of primary human tumors and alterations in microRNA (miRNA) expression have been associated with tumor suppression or tumorigenesis in human cancer, nevertheless, little is known about natural miRNAs acting on ERBB2. In this study, bioinformatical analysis of the 3′-UTRs of ERBB2 revealed the target elements for miR-548d-3p and miR-559. Moreover, a predicted miRNA/mRNA interaction experimental validation showed that both miR-548d-3p and miR-559 can interact specifically with the 3′-UTR of the ERBB2 mRNA. And miR-548d-3p plus miR-559 transfection showed a cooperative regulation of translationally repressing ERBB2 mRNA rather than by either miR-548d-3p or miR-559 alone. These results not only support the idea that different miRNAs can simultaneously and cooperatively repress a given target mRNA but also preliminarily validate the role of miR-548d-3p and miR-559 in regulating the ERBB2 expression. These data provide molecular basis for the application of miRNAs in ERBB2-targeted therapy.     

Induction of growth arrest by miR-542-3p that targets survivin

Survivin is a protein which functions as a mitotic regulator as well as apoptosis inhibitor. In this study, we show that introduction of synthetic miR-542-3p mimetic reduced both mRNA and protein levels of survivin. In A549 cells, luciferase reporter assay revealed that miR-542-3p targeted predicted binding sites in the 3′-untranslated region (3′-UTR) of survivin. We also demonstrate that ectopic expression of miR-542-3p inhibited cell proliferation by inducing Gap 1 (G1) and Gap 2/Mitosis (G2/M) cell cycle arrest. Collectively, these results suggest that survivin is a direct target of miR-542-3p and growth inhibition by miR-542-3p may have a potential utility as an anti-cancer therapy.     

MicroRNA-383 inhibits anchorage-independent growth and induces cell cycle arrest of glioma cells by targeting CCND1

In recent years, microRNAs (miRNAs) have been proved to be closely related to the tumorigenesis and progression. An increasing number of researches have shown that microRNAs function as oncogenes or tumor suppressor genes in human malignant tumors. This study aims to explore the effects of microRNA-383 (miR-383) on malignant biological function of human gliomas. We detected the expression of miR-383 in glioma tissues and normal brain tissues by quantitative real-time PCR. Anchorage-independent growth assays, and flow cytometry were used to evaluate the functions of miR-383 that involves in cell growth and cell cycle. Western blotting assay was used to examine protein expression levels of Cyclin D1 (CCND1), a cell cycle-associated oncogene which has a predicted binding site of miR-383 within its 3′-untranslated region (3′-UTR), and luciferase activity assay was used to evaluate the 3′-UTR activity of CCND1. In this study, we found that miR-383 expression level was lower in gliomas than normal brain tissues. Overexpression of miR-383 in U251 and U87 cells showed a significant inhibitory effect on cell growth, which accompanied with cell cycle G0/G1 arrest as well as downregulation of CCND1 expression. Moreover, CCND1 was verified to be one of the direct targets of miR-383. In summary, this study suggested that miR-383 plays the role of tumor suppressor by targeting CCND1 in glioma cells, and may be useful for developing a new therapeutic strategy for gliomas.     

Single and Compound Knock-outs of MicroRNA (miRNA)-155 and Its Angiogenic Gene Target CCN1 in Mice Alter Vascular and Neovascular Growth in the Retina via Resident Microglia

The response of the retina to ischemic insult typically leads to aberrant retinal neovascularization, a major cause of blindness. The epigenetic regulation of angiogenic gene expression by miRNAs provides new prospects for their therapeutic utility in retinal neovascularization. Here, we focus on miR-155, a microRNA functionally important in inflammation, which is of paramount importance in the pathogenesis of retinal neovascularization. Whereas constitutive miR-155-deficiency in mice results in mild vascular defects, forced expression of miR-155 causes endothelial hyperplasia and increases microglia count and activation. The mouse model of oxygen-induced retinopathy, which recapitulates ischemia-induced aberrant neovessel growth, is characterized by increased expression of miR-155 and localized areas of microglia activation. Interestingly, miR-155 deficiency in mice reduces microglial activation, curtails abnormal vessel growth, and allows for rapid normalization of the retinal vasculature following ischemic insult. miR-155 binds to the 3′-UTR and represses the expression of the CCN1 gene, which encodes an extracellular matrix-associated integrin-binding protein that both promotes physiological angiogenesis and harnesses growth factor-induced abnormal angiogenic responses. Single CCN1 deficiency or double CCN1 and miR-155 knock-out in mice causes retinal vascular malformations typical of faulty maturation, mimicking the vascular alterations of miR-155 gain of function. During development, the miR-155/CCN1 regulatory axis balances the proangiogenic and proinflammatory activities of microglia to allow for their function as guideposts for sprout fusion and anastomosis. Under ischemic conditions, dysregulated miR-155 and CCN1 expression increases the inflammatory load and microglial activation, prompting aberrant angiogenic responses. Thus, miR-155 functions in tandem with CCN1 to modulate inflammation-induced vascular homeostasis and repair.     

MiR-29b inhibits collagen maturation in hepatic stellate cells through down-regulating the expression of HSP47 and lysyl oxidase

Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3′-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl₄-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3′-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3′-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.     

MicroRNA-302a inhibits adipogenesis by suppressing peroxisome proliferator-activated receptor γ expression

The present study explored the involvement of miR-302a in adipocyte differentiation via interaction with 3′-untranslated region of peroxisome proliferator-activated receptor gamma (PPARγ) mRNA. In differentiating 3T3-L1 adipocytes, expression of miR-302a was negatively correlated with that of the adipogenic gene aP2 and PPARγ. Overexpression of miR-302a inhibited adipogenic differentiation with lipid accumulation, and inversely anti-miR-302a increased the differentiation. In silico analysis revealed a complementary region of miR-302a seed sequence in 3′-UTR of PPARγ mRNA. Luciferase assay showed the direct interaction of miR-302a with PPARγ at the cellular level. The miR-302a inhibition of adipocyte differentiation was reversed by PPARγ overexpression. These findings suggest that miR-302a might be a negative regulator of adipocyte differentiation and that the dysregulation of miR-302a should lead to metabolic disorders.     

Downregulation of miR-638 promotes invasion and proliferation by regulating SOX2 and induces EMT in NSCLC

Aberrant expression of microRNAs has been shown to regulate the biological processes of lung cancer cells. However, the role of miR-638 in the development of NSCLC is still unclear. In this study, low miR-638 and high SOX2 were shown to be associated with tumor size and metastasis of NSCLC patients. Downregulated miR-638 could promote cell invasion and proliferation, while high miR-638 expression reversed the effect. Furthermore, miR-638 could regulate SOX2 by directly binding to its 3′-UTR. Silencing of SOX2 by siRNA partially abolished the enhancement of cell invasion and proliferation induced by downregulated miR-638. Aberrant miR-638 expression could modulate the expression levels of markers of epithelial-to-mesenchymal transition. Our results indicate that miR-638 may play a pivotal role in the development of NSCLC.