Identification of new RAD51D-regulating microRNAs that also emerge as potent inhibitors of the Fanconi anemia/homologous recombination pathways.

The Fanconi anemia (FA) and homologous recombination (HR) pathways, which partially overlap and include RAD51 and its paralogs, are key for the repair of different types of DNA damage, such as DNA interstrand crosslinks. First, to broadly assess the impact of microRNA-mediated regulation, we examined microRNA expression profiles in five isogenic fibroblast cell pairs, either deficient in DNA repair due to germline mutations in FANCA, FANCB, FANCC, FANCI or BRIP1/FANCJ or proficient due to correction with retroviral vectors. In each pair, we observed lower abundance of specific microRNAs in the FA-deficient cells. From the list of microRNAs, we experimentally confirmed the effects of miR-141-3p and miR-369-3p targeting RAD51B and miR-15a-5p, miR-494-3p as well as miR-544a targeting RAD51D. However, by western blotting, only RAD51D protein was reduced by a mixture of its regulating microRNAs. Gene ontology analyses and identification of additional FA/HR factors as targets of miR-15a-5p, miR-494-3p and miR-544a strongly suggested the widespread influence of these microRNAs on HR. Interestingly, only miR-494-3p directly reduced RAD51 foci formation, while a mixture of miR-15a-5p, miR-494-3p and miR-544a strongly reduced HR activity in green fluorescent protein (GFP) repair assays. In summary, by successfully employing this novel loss- and gain-of-function strategy, we have identified new microRNAs strongly inhibiting HR in mammalian cells. Understanding and modulating such miRNA regulation of DNA repair genes/pathways might help to overcome the reduced repair capacity of FA patients with biallelic hypomorphic mutations or help to engineer synthetic lethality strategies for patients with mutations in cancer-associated FA/HR genes.

MicroRNAs miR-26a, miR-26b, and miR-29b accelerate osteogenic differentiation of unrestricted somatic stem cells from human cord blood.

BACKGROUND: MicroRNAs are a population of short non-coding RNAs with widespread negative regulatory impact on mRNA translation. Unrestricted somatic stem cells (USSC) are a rare population in human cord blood that can be induced into cells representative of all three germinal layers. Here we analyzed the functional impact of miRNAs on the osteogenic differentiation in USSC. RESULTS: Gene expression profiling identified 20 microRNAs that were consistently upregulated during osteogenic differentiation of two different USSC cell lines (SA5/73 and SA8/25). Bioinformatic target gene prediction indicated that among these microRNAs, miR-10a, -22, -26a, -26b, and -29b recognize transcripts that encode a set of proteins inhibiting osteogenesis. We subsequently verified osteo-inhibitory CDK6, CTNNBIP1, HDAC4, and TOB1 and osteo-promoting SMAD1 as targets of these microRNAs. In Western blot analyses demonstrated that endogenous levels of CDK6 and HDAC4 were downregulated during osteogenic differentiation of USSC and reduced following ectopic expression of miR-26a/b and miR-29b. In contrast, endogenous expression of SMAD1, targeted by miR-26a/b, was unaltered during osteogenic differentiation of USSC or following ectopic expression of miR-26a/b. Functional overexpression analyses using microRNA mimics revealed that miR-26a/b, as well as miR-29b strongly accelerated osteogenic differentiation of USSC as assessed by Alizarin-Red staining and calcium-release assays. CONCLUSIONS: miR-26a/b and miR-29b are upregulated during osteogenic differentiation of USSC and share target genes inhibiting osteogenesis. Furthermore, these microRNAs accelerate osteogenic differentiation, likely mediated by osteo-inhibitory proteins such as CDK6 and HDAC4.

MicroRNAs MiR-17, MiR-20a, and MiR-106b act in concert to modulate E2F activity on cell cycle arrest during neuronal lineage differentiation of USSC.

BACKGROUND: MicroRNAs are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Here the functional impact of microRNAs on cell cycle arrest during neuronal lineage differentiation of unrestricted somatic stem cells from human cord blood (USSC) was analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Expression profiling revealed downregulation of microRNAs miR-17, -20a, and -106b in USSC differentiated into neuronal lineage but not in USSC differentiated into osteogenic lineage. Transfection experiments followed by Ki67 immunostainings demonstrated that each of these microRNAs was able to promote proliferation of native USSC and to prevent in part cell cycle arrest during neuronal lineage differentiation of USSC. Bioinformatic target gene predictions followed by experimental target gene validations revealed that miR-17, -20a, and -106b act in a common manner by downregulating an overlapping set of target genes mostly involved in regulation and execution of G(1)/S transition. Pro-proliferative target genes cyclinD1 (CCND1) and E2F1 as well as anti-proliferative targets CDKN1A (p21), PTEN, RB1, RBL1 (p107), RBL2 (p130) were shown as common targets for miR-17, -20a, and -106b. Furthermore, these microRNAs also downregulate WEE1 which is involved in G(2)/M transition. Most strikingly, miR-17, -20a, and -106b were found to promote cell proliferation by increasing the intracellular activity of E2F transcription factors, despite the fact that miR-17, -20a, and -106b directly target the transcripts that encode for this protein family. CONCLUSIONS/SIGNIFICANCE: Mir-17, -20a, and -106b downregulate a common set of pro- and anti-proliferative target genes to impact cell cycle progression of USSC and increase intracellular activity of E2F transcription factors to govern G(1)/S transition.

Network-like impact of MicroRNAs on neuronal lineage differentiation of unrestricted somatic stem cells from human cord blood.

Unrestricted somatic stem cells (USSCs) represent an intrinsically multipotent CD45-negative fetal population from human cord blood. They show differentiation into neuronal cells of a dopaminergic phenotype, which express neuronal markers such as synaptophysin, neuronal-specific nuclear protein, and neurofilament and release the neurotransmitter dopamine accompanied by expression of dopaminergic key factors tyrosine hydroxylase and Nurr1 (NR4A2). MicroRNA expression analysis highlighted their importance in neural development but their specific functions remain poorly understood. Here, downregulation of a set of 18 microRNAs during neuronal lineage differentiation of unrestricted somatic stem cells, including members of the miR-17-92 family and additional microRNAs such as miR-130a, -138, -218, and -335 as well as their target genes, is described. In silico target gene predictions for this microRNA group uncovered a large set of proteins involved in neuronal differentiation and having a strong impact on differentiation-related pathways such as axon guidance and TGFß, WNT, and MAPK signaling. Experimental target validations confirmed approximately 35% of predictions tested and revealed a group of proteins with specific impact on neuronal differentiation and function including neurobeachin, neurogenic differentiation 1, cysteine-rich motor neuron protein 1, neuropentraxin 1, and others. These proteins are combined targets for several subgroups from the set of 18 downregulated microRNAs. This finding was further supported by the observed upregulation of a significant amount of predicted and validated target genes based on Illumina Beadstudio microarray data. Confirming the functional relationship of a limited panel of microRNAs and predicted target proteins reveals a clear network-like impact of the group of 18 downregulated microRNAs on proteins involved in neuronal development and function.

Role of DNA methylation in miR-200c/141 cluster silencing in invasive breast cancer cells.

BACKGROUND: The miR-200c/141 cluster has recently been implicated in the epithelial to mesenchymal transition (EMT) process. The expression of these two miRNAs is inversely correlated with tumorigenicity and invasiveness in several human cancers. The role of these miRNAs in cancer progression is based in part on their capacity to target the EMT activators ZEB1 and ZEB2, two transcription factors, which in turn repress expression of E-cadherin. Little is known about the regulation of the mir200c/141 cluster, whose targeting has been proposed as a promising new therapy for the most aggressive tumors. FINDINGS: We show that the miR-200c/141 cluster is repressed by DNA methylation of a CpG island located in the promoter region of these miRNAs. Whereas in vitro methylation of the miR-200c/141 promoter led to shutdown of promoter activity, treatment with a demethylating agent caused transcriptional reactivation in breast cancer cells formerly lacking expression of miR-200c and miR-141. More importantly, we observed that DNA methylation of the identified miR-200c/141 promoter was tightly correlated with phenotype and the invasive capacity in a panel of 8 human breast cancer cell lines. In line with this, in vitro induction of EMT by ectopic expression of the EMT transcription factor Twist in human immortalized mammary epithelial cells (HMLE) was accompanied by increased DNA methylation and concomitant repression of the miR-200c/141 locus. CONCLUSIONS: The present study demonstrates that expression of the miR-200c/141 cluster is regulated by DNA methylation, suggesting epigenetic regulation of this miRNA locus in aggressive breast cancer cell lines as well as untransformed mammary epithelial cells. This epigenetic silencing mechanism might represent a novel component of the regulatory circuit for the maintenance of EMT programs in cancer and normal cells.