Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin,promotes cell senescence and enhances senolysis

A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.     

Elevated miR-34c-5p Mediates Dermal Fibroblast Senescence by Ultraviolet Irradiation

Previous studies showed that several miRNAs can regulate pathways involved in UVB-induced premature senescence and response to ultraviolet irradiation. It has also been reported that miR-34c-5p may be involved in senescence-related mechanisms. We propose that miR-34c-5p may play a crucial role in senescence of normal human primary dermal fibroblasts. Here, we explored the roles of miR-34c-5p in UVB-induced premature senescence on dermal fibroblasts. MiR-34c-5p expression was increased in dermal fibroblasts after repeated subcytotoxic UVB treatments. Underexpression of miR-34c-5p in dermal fibroblasts led to a marked delay of many senescent phenotypes induced by repeated UVB treatments. Furthermore, underexpression of miR-34c-5p in dermal fibroblasts can antagonize the alteration of G1-arrested fibroblasts. Moreover, E2F3, which can inactivate p53 pathway and play a role in cell cycle progression, is a down-stream target of miR-34c-5p. Forced down-expression of miR-34c-5p decreased the expression of UVB-SIPS induced P21 and P53 at both mRNA and protein levels. Our data demonstrated that down-regulation of miR-34c-5p can protect human primary dermal fibroblasts from UVB-induced premature senescence via regulations of some senescence-related molecules.     

Integration of SNP and mRNA Arrays with MicroRNA Profiling Reveals That MiR-370 Is Upregulated and Targets NF1 in Acute Myeloid Leukemia

Background

Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied.

Results

Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1.

Conclusions

NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors.     

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.     

Transcriptional changes in response to X chromosome dosage in the mouse: implications for X inactivation and the molecular basis of Turner Syndrome

Background

In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.

Results

Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.

Conclusions

The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.     

Integrated microRNA–mRNA analysis of coronary artery disease

Although patients with coronary artery disease (CAD) have a high mortality rate, the pathogenesis of CAD is still poorly understood. The purpose of this study was to explore the underlying molecular mechanisms and potential target molecules for CAD. The platelet miRNA (GSE28858) and blood mRNA (GSE42148) expression profiles of patients with CAD and healthy controls were downloaded from Gene Expression Omnibus. Differentially expressed miRNAs and genes (DEGs) were identified by significant analysis of microarray algorithm after data preprocessing. Furthermore, the miRNA-target gene regulatory network was constructed based on miRecords database. The spearman correlation coefficients (ρ) between miRNAs and their target genes were calculated. Six up- (miR-340, miR-545, miR-451, miR454-5p, miR-624 and miR-585) and four down-regulated (miR-199a, miR-17-3p, miR-154 and miR-339) miRNAs were screened. Total 295 target genes of miR-545, miR-451, miR-585 and miR-154 were predicted. Among these 295 target genes, 7 genes were DEGs. Further analysis showed miR-545-TFEC and miR-585-SPOCK1 were highly positively correlated (ρ = 0.808091264; ρ = 0.874680776) in CAD samples. Therefore, differentially expressed miRNAs might participate in the pathogenesis of CAD by regulating their target genes.     

MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.     

MicroRNAs 130a/b are regulated by BCR-ABL and downregulate expression of CCN3 in CML

Chronic Myeloid Leukaemia (CML) is a myeloproliferative disorder characterized by the expression of the oncoprotein, Bcr-Abl kinase. CCN3 normally functions as a negative growth regulator, but it is downregulated in CML, the mechanism of which is not known. MicroRNAs (miRNAs) are small non-coding RNAs, which negatively regulate protein translation by binding to the complimentary sequences of the 3′ UTR of messenger RNAs. Deregulated miRNA expression has emerged as a hallmark of cancer. In CML, BCR-ABL upregulates oncogenic miRNAs and downregulates tumour suppressor miRNAs favouring leukaemic transformation. We report here that the downregulation of CCN3 in CML is mediated by BCR-ABL dependent miRNAs. Using the CML cell line K562, we profiled miRNAs, which are BCR-ABL dependent by transfecting K562 cells with anti-BCR-ABL siRNA. MiRNA expression levels were quantified using the Taqman Low Density miRNA array platform. From the miRNA target prediction databases we identified miRNAs that could potentially bind to CCN3 mRNA and reduce expression. Of these, miR-130a, miR-130b, miR-148a, miR-212 and miR-425-5p were significantly reduced on BCR-ABL knockdown, with both miR-130a and miR-130b decreasing the most within 24 h of siRNA treatment. Transfection of mature sequences of miR-130a and miR-130b individually into BCR-ABL negative HL60 cells resulted in a decrease of both CCN3 mRNA and protein. The reduction in CCN3 was greatest with overexpression of miR-130a whereas miR-130b overexpression resulted only in marginal repression of CCN3. This study shows that miRNAs modulate CCN3 expression. Deregulated miRNA expression initiated by BCR-ABL may be one mechanism of downregulating CCN3 whereby leukaemic cells evade negative growth regulation.     

Eimeria papillata: upregulation of specific miRNA-species in the mouse jejunum

Increasing evidence indicates miRNAs as critical regulators of gene expression, but little information is available for miRNAs in intestinal diseases. Here, we investigated intestinal infections of male Balb/c mice with the coccidian parasite Eimeria papillata. On day 4 after oral infection, mice were shedding 3150 ± 430 oocysts per gram feces. This was associated with a low inflammatory response of the jejunum of mice evidenced by histology, non-response of IL-1β mRNA, even slight downregulation of IL-6 mRNA, only slight increases in iNOS mRNA, nitrate/nitrate, malondialdehyde, and a small decrease in glutathione, respectively. Only IFNγ mRNA was strongly induced. Using miRNA microarray technology, there were significantly upregulated the four miRNA species miR-1959, MCMV-miR-M23-1-5P, miR-203, and miR-21 out of 634 miRNAs, which was also confirmed by quantitative RT-PCR. Our data provide evidence that E. papillata parasites are able to induce specific miRNA species in their host target organ.     

Anti-Argonaute RIP-Chip shows that miRNA transfections alter global patterns of mRNA recruitment to microribonucleoprotein complexes

MicroRNAs (miRNAs) play key roles in gene expression regulation by guiding Argonaute (AGO)-containing microribonucleoprotein (miRNP) effector complexes to target polynucleotides. There are still uncertainties about how miRNAs interact with mRNAs. Here we employed a biochemical approach to isolate AGO-containing miRNPs from human H4 tumor cells by co-immunoprecipitation (co-IP) with a previously described anti-AGO antibody. Co-immunoprecipitated (co-IPed) RNAs were subjected to downstream Affymetrix Human Gene 1.0 ST microarray analysis. During rigorous validation, the “RIP-Chip” assay identified target mRNAs specifically associated with AGO complexes. RIP-Chip was performed after transfecting brain-enriched miRNAs (miR-107, miR-124, miR-128, and miR-320) and nonphysiologic control miRNA to identify miRNA targets. As expected, the miRNA transfections altered the mRNA content of the miRNPs. Specific mRNA species recruited to miRNPs after miRNA transfections were moderately in agreement with computational target predictions. In addition to recruiting mRNA targets into miRNPs, miR-107 and to a lesser extent miR-128, but not miR-124 or miR-320, caused apparent exclusion of some mRNAs that are normally associated with miRNPs. MiR-107 and miR-128 transfections also result in decreased AGO mRNA and protein levels. However, AGO mRNAs were not recruited to miRNPs after either miR-107 or miR-128 transfection, confirming that miRNAs may alter gene expression without stable association between particular mRNAs and miRNPs. In summary, RIP-Chip assays constitute an optimized, validated, direct, and high-throughput biochemical assay that provides data about specific miRNA:mRNA interactions, as well as global patterns of regulation by miRNAs.     

Testosterone-induced upregulation of miRNAs in the female mouse liver

Testosterone (T) regulates expression of protein-encoding genes directly through androgen receptor (AR) targeting androgen response element (ARE) in gene promoters or indirectly through non-genotropic mechanisms, but only limited information is available about T effects on expression of gene-regulatory non-coding miRNAs. Here, we investigate the effect of T on miRNA expression profiles in the female mouse liver using miRXplore microarrays and quantitative RT-PCR. T treatment for 3 weeks induced upregulation of the 6 miRNAs miR-22, miR-690, miR-122, let-7A, miR-30D and let-7D, reaching maximal expression at different time-points during T treatment. This upregulation was transient, i.e. it disappeared after T withdrawal for 12 weeks, and it was rather robust since it was not essentially affected by blood-stage infections with Plasmodium chabaudi malaria. In silico analysis revealed an ARE in the miR-122 promoter, while the other 5 miRNAs did not contain any ARE in their 2000 bp promoters. The T-induced upregulation of the 6 miRNAs coincided with a downregulation of some of their target protein-encoding genes, the majority of which did incidentally not contain any ARE in their promoters. T treatment did not affect expression of AR and estrogen receptor β (ERβ), but significantly downregulated the miR-22 target genes ERα and aromatase. This downregulation is presumably not caused by T after its aromatase-mediated conversion to E₂ through ER, but rather by the T-induced upregulation of miR-22. Collectively, our data suggest that T can regulate expression of distinct miRNAs in vivo by both genotropic and non-genotropic mechanisms.     

MicroRNA-340 suppresses osteosarcoma tumor growth and metastasis by directly targeting ROCK1

MicroRNAs (miRNAs) play key roles in cancer development and progression. In the present study, we investigated the role of miR-340 in the progression and metastasis of osteosarcoma (OS). Our results showed that miR-340 was frequently downregulated in OS tumors and cell lines. Overexpression of miR-340 in OS cell lines significantly inhibited cell proliferation, migration, and invasion in vitro, and tumor growth and metastasis in a xenograft mouse model. ROCK1 was identified as a target of miR-340, and ectopic expression of miR-340 downregulated ROCK1 by direct binding to its 3′ untranslated region. siRNA-mediated silencing of ROCK1 phenocopied the effects of miR-340 overexpression, whereas restoration of ROCK1 in miR-340-overexpressing OS cells reversed the suppressive effects of miR-340. Together, these findings indicate that miR-340 acts as a tumor suppressor and its downregulation in tumor tissues may contribute to the progression and metastasis of OS through a mechanism involving ROCK1, suggesting miR-340 as a potential new diagnostic and therapeutic target for the treatment of OS.