Urokinase‐type plasminogen activator (uPA) and c‐met play a major role in cancer invasion and metastasis. Evidence has suggested that uPA and c‐met overexpression may be coordinated in human hepatocellular carcinoma (HCC). In the present study, to understand whether the expression of these genes might be coregulated by specific microRNAs (miRs) in human cells, we predicted that Homo sapiens microRNA‐23b could recognize two sites in the 3′‐UTR of uPA and four sites in the c‐met 3′‐UTR by the algorithm pictar. The miR‐23b expression analysis in human tumor and normal cells revealed an inverse trend with uPA and c‐met expression, indicating that uPA and c‐met negative regulation might depend on miR‐23b expression. Transfection of miR‐23b molecules in HCC cells (SKHep1C3) led to inhibition of protein expression of the target genes and caused a decrease in cell migration and proliferation capabilities. Furthermore, anti‐miR‐23b transfection in human normal AB2 dermal fibroblasts upregulated the expression of endogenous uPA and c‐met. Cotransfection experiments in HCC cells of the miR‐23b with pGL4.71 Renilla luciferase reporter gene constructs, containing the putative uPA and c‐met 3′‐UTR target sites, and with the pGL3 firefly luciferase‐expressing vector showed a decrease in the relative luciferase activity. This would indicate that miR‐23b can recognize target sites in the 3′‐UTR of uPA and of c‐met mRNAs and translationally repress the expression of uPA and c‐met in HCC cells. The evidence obtained shows that overexpression of miR‐23b leads to uPA and c‐met downregulation and to decreased migration and proliferation abilities of HCC cells.

CDK5R1 encodes p35, a specific activator of the serine/threonine kinase CDK5, which plays crucial roles in CNS development and maintenance. CDK5 activity strongly depends on p35 levels and p35/CDK5 misregulation is deleterious for correct CNS function, suggesting that a tightly controlled regulation of CDK5R1 expression is needed for proper CDK5 activity. Accordingly, CDK5R1 expression was demonstrated to be controlled at both transcriptional and post-transcriptional levels, but a possible regulation through microRNAs (miRNAs) has never been investigated. We predicted, within the large CDK5R1 3′UTR several miRNA target sites. Among them, we selected for functional studies miR-103 and miR-107, whose expression has shown a strong inverse correlation with p35 levels in different cell lines. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection of SK-N-BE neuroblastoma cells with the miR-103 or miR-107 precursor (pre-miR-103 or pre-miR-107). Conversely, p35 levels significantly increased following transfection of the corresponding antagonists (anti-miR-103 or anti-miR-107). Moreover, the level of CDK5R1 transcript shifts from the polysomal to the subpolysomal mRNA fraction after transfection with pre-miR-107 and, conversely, from the subpolysomal to the polysolmal mRNA fraction after transfection with anti-miR-107, suggesting a direct action on translation efficiency. We demonstrate, by means of luciferase assays, that miR-103 and miR-107 are able to directly interact with the CDK5R1 3′-UTR, in correspondence of a specific target site. Finally, miR-103 and miR-107 overexpression, as well as CDK5R1 silencing, caused a reduction in SK-N-BE migration ability, indicating that these miRNAs affect neuronal migration by modulating CDK5R1 expression. These findings indicate that miR-103 and miR-107 regulate CDK5R1 expression, allowing us to hypothesize that a miRNA-mediated mechanism may influence CDK5 activity and the associated molecular pathways.

Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, the main activatory subunit of cyclin-dependent kinase 5 (CDK5). The p35/CDK5 active complex plays a fundamental role in brain development and functioning, but its deregulated activity has also been implicated in various neurodegenerative disorders, including Alzheimer's disease (AD). CDK5R1 displays a large and highly evolutionarily conserved 3′-untranslated region (3′-UTR), a fact that has suggested a role for this region in the post-transcriptional control of CDK5R1 expression. Our group has recently demonstrated that two miRNAs, miR-103 and miR-107, regulate CDK5R1 expression and affect the levels of p35. MiR-103 and miR-107 belong to the miR-15/ 107 family, a group of evolutionarily conserved miRNAs highly expressed in human cerebral cortex. In this work, we tested the hypothesis that other members of this group of miRNAs, in addition to miR-103 and miR-107, were able to modulate CDK5R1 expression. We provide evidence that several miRNAs belonging to the miR-15/107 family regulate p35 levels. BACE1 expression levels were also found to be modulated by different members of this family. Furthermore, overexpression of these miRNAs led to reduced APP phosphorylation levels at the CDK5-specific Thr668 residue. We also show that miR-15/107 miRNAs display reduced expression levels in hippocampus and temporal cortex, but not in cerebellum, of AD brains. Moreover, increased CDK5R1 mRNA levels were observed in AD hippocampus tissues. Our results suggest that the downregulation of the miR-15/ 107 family might have a role in the pathogenesis of AD by increasing the levels of CDK5R1/p35 and consequently enhancing CDK5 activity.

Mental retardation (MR) is displayed by 57% of NF1 patients with microdeletion syndrome as a result of 17q11.2 region haploinsufficiency. We considered the cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) and oligodendrocyte-myelin glycoprotein (OMG) genes, mapping in the NF1 microdeleted region, as candidate genes for MR susceptibility. CDK5R1 encodes for a neurone-specific activator of cyclin-dependent kinase 5 (CDK5) involved in neuronal migration during central nervous system development. OMG encodes for an inhibitor of neurite outgrowth by the binding to the Nogo-66 receptor (RTN4R). CDK5R1 and OMG genes are characterized by large 3′ and 5′ untranslated regions (UTRs), where we predict the presence of several transcription/translation regulatory elements. We screened 100 unrelated Italian patients affected by unspecific MR for mutations in CDK5R1 and OMG coding regions and in their 3′ or 5′ UTRs. Four novel mutations and two novel polymorphisms for CDK5R1 and three novel mutations for OMG were detected, including two missense changes (c.323C>T; A108V in CDK5R1 and c.1222A>G; T408A in OMG), one synonymous codon variant (c.532C>T; L178L in CDK5R1), four variants in CDK5R1 3′UTR and two changes in OMG 5′UTR. All the mutations were absent in 370 chromosomes from normal subjects. The allelic frequencies of the two novel polymorphisms in CDK5R1 3′UTR were established in both 185 normal and 100 mentally retarded subjects. Prediction of mRNA and protein secondary structures revealed that two changes lead to putative structural alterations in the mutated c.2254C>G CDK5R1 3′UTR and in OMG T408A gene product.

Noonan syndrome is a genetic condition characterized by congenital heart defects, short stature, and characteristic facial features. Familial or de novo mutations in PTPN11, RAF1, SOS1, KRAS, and NRAS are responsible for 60-75% of the cases, thus, additional genes are expected to be involved in the pathogenesis. In addition, the genotype-phenotype correlation has been hindered by the highly variable expressivity of the disease. For all these reasons, expanding the genotyped and clinically evaluated case numbers will benefit the clinical community. A mutation analysis has been performed on RAF1, SOS1, and GRB2, in 24 patients previously found to be negative for PTPN11 and KRAS mutations. We identified four mutations in SOS1 and one in RAF1, while no GRB2 variants have been found. Interestingly, the RAF1 mutation was present in a patient also carrying a newly identified p.R497Q familial SOS1 mutation, segregating with a typical Noonan Syndrome SOS1 cutaneous phenotype. Functional analysis demonstrated that the R497Q SOS1 mutation leads to Jnk activation, but has no effect on the Ras effector Erk1. We propose that this variant might contribute to the onset of the peculiar ectodermal traits displayed by the propositus amidst the more classical Noonan syndrome presentation. To our knowledge, this is the first reported case of a patient harboring mutations in two genes, with an involvement of both Ras and Rac1 pathways, indicating that SOS1 may have a role of modifier gene that might contribute the variable expressivity of the disease, evidencing a genotype-phenotype correlation in the family.