Efficient inhibition of miR-155 function in vivo by peptide nucleic acids

MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use.     

Induction of MiR-17-3p and MiR-106a [corrected] by TNFα and LPS

MicroRNAs (miRNAs), small non-coding molecules, regulate gene expression in response to stimuli. Lipopolysaccharide (LPS) was reported to induce the expression of miR-146 and miR-155 in HL-60. The effects of LPS and the related stimulus, tumour necrosis factor alpha (TNFα), on miRNA expression required to be further studied. Using T7-oligo ligation assay (OLA)-based miRNA array, we profiled the expression of 132 miRNAs and identified a number of TNFα-regulated miRNAs in HeLa cells, including miR-17-3p and miR-106a. TNFα induction of miR-17-3p and miR-106a was verified by Northern blot analysis with RNU48 normalization. Northern blot analysis also showed that LPS was able to induce the expression of both miR-17-3p and miR-106a in HeLa cells. Furthermore, both array assay and Northern blot analysis showed that the expression levels of miR-146 and miR-155 were either low or undetectable in HeLa cells and TNFα- and LPS-mediated induction of these two miRNAs was not found. Luciferase reporter analysis confirmed the induction of miR-17-3p and miR-106a in response to TNFα and LPS treatment in HeLa cells. These results suggested that the expression of miR-17-3p and miR-106a is regulated by TNFα and LPS in HeLa cells.     

MicroRNA-21 promotes wound healing via the Smad7-Smad2/3-Elastin pathway

Wound healing is regulated by a complex network of cells, molecules, and cytokines, as well as microRNAs (miRNAs). miRNAs were confirmed to influence the wound healing process, and miR-21, an important member of the miRNA family, was also shown to regulate wound healing. The aim of the present study was to investigate the role of miR-21 in the wound healing process and the possible underlying cell signaling pathways. We isolated GMSCs from WT and miR-21-KO mouse gingiva. Flow cytometric analysis and immunocytofluorescense staining were used to identify the GMSCs acquired from WT and miR-21-KO mice. RT-PCR, western blot analysis and immunohistofluorescence staining were performed to examine the expression of extracellular matrix components and key proteins of cell signaling pathways. TargetScan and pmiR-RB-REPORT vectors were used to verify that Smad7 was a direct target of miR-21. Compared to WT mice, miR-21-KO mice showed slower wound healing. RT-PCR and western blot analysis indicated that Elastin expression was downregulated in miR-21-deficient samples. We confirmed that Smad7 was a direct target of miR-21. miR-21 knockout resulted in increased expression of Smad7 and impaired phosphorylation of the Smad2/3 complex. The expression of the Smad7-Smad2/3-Elastin axis in palate tissues sections acquired from WT and miR-21-KO mice showed the same trend. Based on all these results, we demonstrated that miR-21 promoted the wound healing process via the Smad7-Smad2/3-Elastin pathway.     

MicroRNA-148/152 impair innate response and antigen presentation of TLR-triggered dendritic cells by targeting CaMKIIα

MicroRNAs (miRNAs) are involved in the regulation of immunity, including the lymphocyte development and differentiation, and inflammatory cytokine production. Dendritic cells (DCs) play important roles in linking innate and adaptive immune responses. However, few miRNAs have been found to regulate the innate response and APC function of DCs to date. Calcium/calmodulin-dependent protein kinase II (CaMKII), a major downstream effector of calcium (Ca(2+)), has been shown to be an important regulator of the maturation and function of DCs. Our previous study showed that CaMKIIα could promote TLR-triggered production of proinflammatory cytokines and type I IFN. Inspired by the observations that dicer mutant Drosophila display defect in endogenous miRNA generation and higher CaMKII expression, we wondered whether miRNAs can regulate the innate response and APC function of DCs by targeting CaMKIIα. By predicting with software and confirming with functional experiments, we demonstrate that three members of the miRNA (miR)-148 family, miR-148a, miR-148b, and miR-152, are negative regulators of the innate response and Ag-presenting capacity of DCs. miR-148/152 expression was upregulated, whereas CaMKIIα expression was downregulated in DCs on maturation and activation induced by TLR3, TLR4, and TLR9 agonists. We showed that miR-148/152 in turn inhibited the production of cytokines including IL-12, IL-6, TNF-α, and IFN-β upregulation of MHC class II expression and DC-initiated Ag-specific T cell proliferation by targeting CaMKIIα. Therefore, miRNA-148/152 can act as fine-tuner in regulating the innate response and Ag-presenting capacity of DCs, which may contribute to the immune homeostasis and immune regulation.     

Real-Time Monitoring of miRNA Function in Pancreatic Cell Lines Using Recombinant AAV-Based miRNA Asensors

Background

MicroRNAs (miRNAs) are reportedly involved in pancreatic ductal adenocarcinoma (PDAC) development. Current methods do not allow us to reliably monitor miRNA function. Asensors are adeno-associated virus (AAV) vector miRNA sensors for real-time consecutive functional monitoring of miRNA profiling in living cells.

Methods

miR-200a, -200b, -21, -96, -146a, -10a, -155, and -221 in three PDAC cell lines (BxPC-3, CFPAC-1, SW1990), pancreatic epithelioid carcinoma cells (PANC-1), and human pancreatic nestin-expressing cells (hTERT-HPNE) were monitored by Asensors. Subsequently, the real-time consecutive functional profile of all miRNAs was evaluated.

Results

Selected miRNAs were detectable in all cell lines with high sensitivity and reproducibility. In the three PDAC cell lines, BxPC-3, CFPAC-1, and SW1990, the calibrated signal unit of the eight miRNAs Asensors was significantly lower than that of the Asensor control. However, in PANC-1 cells, miR-200a and -155 showed upregulation of target gene expression at 24 hours after infection with the sensors; at 48 hours, miR-200b and -155 displayed upregulation of reporter expression; and at 72 hours, reporter gene expression was upregulated by miR-200a and -200b. The result that miRNA could upregulate gene expression was further confirmed in miR-155 of hTERT-HPNE cells. Furthermore, miRNA activity varied among cell/tissue types and time.

Conclusion

It is possible that miRNA participates in the pathophysiology of pancreatic cancer, but the current popular methods do not accurately reveal the real-time miRNA function. Thus, this report provided a convenient, accurate, and sensitive approach to miRNA research.     

Pathophysiology of translational regulation by microRNAs in multiple sclerosis

MicroRNAs (miRNAs) comprise a group of several hundred, small non-coding RNA molecules with a fundamental influence on the regulation of gene expression. Certain miRNAs are altered in blood cells of multiple sclerosis (MS), and active and inactive MS brain lesions have distinct miRNA expression profiles. Several miRNAs such as miR-155 or miR-326 are considerably overexpressed in active MS lesions versus controls, and mice lacking these miRNAs either through knock-out (miR-155) or by in vivo silencing (miR-326) show a reduction of symptoms in experimental autoimmune encephalomyelitis (EAE), a model system for multiple sclerosis. This review describes miRNAs regulated in the blood or in brain lesions of MS patients in the context of their previously described functions in physiology and pathophysiology.     

U6 is not a suitable endogenous control for the quantification of circulating microRNAs

Recently, microRNAs have been detected in serum and plasma, and circulating microRNA (miRNA) profiles have now been associated with many diseases such as cancers and heart disease, as well as altered physiological states. Because of their stability and disease resistance, circulation miRNAs appear to be an ideal material for biomarkers of diseases and physiological states in blood. However, the lack of a suitable internal reference gene (internal reference miRNA) has hampered research and application of circulating miRNAs. Currently, U6 and miR-16 are the most common endogenous controls in the research of miRNAs in tissues and cells. We performed microarray-based serum miRNA profiling on the serum of 20 nasopharyngeal carcinoma patients and 20 controls to detect the expressions of U6 and miRNAs. Profiling was followed by real-time quantitative Polymerase Chain Reaction (qPCR) in 80 patients (20 each with gastric cancer, nasopharyngeal carcinoma, colorectal cancer, and breast cancer) and 30 non-cancerous controls. qPCR was also performed to detect miRNAs in serum with repeated freezing and thawing. The results of microarray showed that with the exception of U6, Ct values of miR-16, miR-24, miR-142-3p, miR-19b and miR-192 in serum samples of nasopharyngeal carcinoma were greater than control samples. The results of 110 cases showed large fluctuations in U6 expression. The difference between the greatest and the least levels of expression was 3.29 for delta Ct values, and 1.23 for miR-16. The expressions of U6, miR-16 and miR-24 in serum subjected to different freeze–thaw cycles showed that U6 expression gradually decreased after 1, 2, and 4 cycles of freezing and thawing, while the expression of miR-16 and miR-24 remained relatively stable. Collectively, our results suggested that U6 is unsuitable as an internal reference gene in the research of circulating miRNAs.     

Identification of postoperative prognostic microRNA predictors in hepatocellular carcinoma

Comparison of microRNA (miRNA) expression profiles in the noncancerous liver tissues adjacent to hepatocelluar carcinomas (HCCs) was a strategy to identify postoperative prognostic predictors in this study. Expression profiles of 270 miRNAs were determined in the paraneoplastic liver tissues of 12 HCC patients with known postoperative prognosis. A panel of candidate miRNA predictors was identified. The prognostic predictive value of these candidate miRNAs was then verified in 216 postoperative HCC patients. Univariate analysis identified 8 and 3 miRNA predictors for recurrence-free (RFS) and overall (OS) survivals, respectively. Multivariate analysis revealed high expression levels of miR-155 (HR, 2.002 [1.324-3.027]; P?=?.001), miR-15a (HR, 0.478 [0.248-0.920]; P?=?.027), miR-432 (HR, 1.816 [1.203-2.740]; P?=?.015), miR-486-3p (HR, 0.543 [0.330-0.893]; P?=?.016), miR-15b (HR, 1.074 [1.002-1.152]; P?=?.043) and miR-30b (HR, 1.102 [1.025-1.185]; P?=?.009) were significantly associated with RFS. When clinicopathological predictors were included, multivariate analysis revealed that tumor number and miR-432, miR-486-3p, and miR-30b expression levels remained significant as independent predictors for RFS. Additionally, expression knockdown of miR-155 in J7 and Mahlavu hepatoma cells resulted in decreased cell growth and enhanced cell death in xenograft tumors, suggesting an oncogenic effect of miR-155. In conclusion, significant prognostic miRNA predictors were identified through examination of miRNA expression levels in paraneoplastic liver tissues. Functional analysis of a miRNA predictor, miR-155, suggested that the prognostic miRNA predictors identified under this strategy could serve as potential molecular targets for anticancer therapy.