MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

PDGF enhances podosome formation and cell migration by regulating expression of the microRNAs miR-143 and -145, which target PDGF-R, PKC-ε, and fascin

High expression of HOXA13 correlates with poorly differentiated hepatocellular carcinomas and modulates sorafenib response in in vitro models

Hepatocellular carcinoma (HCC) represents the fifth and ninth cause of mortality among male and female cancer patients, respectively and typically arises on a background of a cirrhotic liver. HCC develops in a multi-step process, often encompassing chronic liver injury, steatosis and cirrhosis eventually leading to the malignant transformation of hepatocytes. Aberrant expression of the class I homeobox gene family (HOX), a group of genes crucial in embryogenesis, has been reported in a variety of malignancies including solid tumors. Among HOX genes, HOXA13 is most overexpressed in HCC and is known to be directly regulated by the long non-coding RNA HOTTIP. In this study, taking advantage of a tissue microarray containing 305 tissue specimens, we found that HOXA13 protein expression increased monotonically from normal liver to cirrhotic liver to HCC and that HOXA13-positive HCCs were preferentially poorly differentiated and had fewer E-cadherin-positive cells. In two independent cohorts, patients with HOXA13-positive HCC had worse overall survival than those with HOXA13-negative HCC. Using HOXA13 immunohistochemistry and HOTTIP RNA in situ hybridization on consecutive sections of 16 resected HCCs, we demonstrated that HOXA13 and HOTTIP were expressed in the same neoplastic hepatocyte populations. Stable overexpression of HOXA13 in liver cancer cell lines resulted in increased colony formation on soft agar and migration potential as well as reduced sensitivity to sorafenib in vitro. Our results provide compelling evidence of a role for HOXA13 in HCC development and highlight for the first time its ability to modulate response to sorafenib

Identification of cellular kinases responsible for Hepatitis C Virus NS5A hyperphosphorylation

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspect of the virus life cycle are still a mystery. The HCV Nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Efficient replication of HCV subgenomic RNA in cell culture requires the introduction of adaptive mutations. Some of the most effective adaptive mutations occur at the serine residues that have been shown to be implicated in NS5A hyperphosphorylation and adaptive mutations at these sites result in a significant reduction of NS5A hyperphosphorylation (p58). After screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro, as well as the formation of NS5A p58 in cell culture. Cells transfected with the HCV wild type replicon sequence supported HCV RNA replication upon addition of any of the three compounds. Thus, reduction of the formation of p58 below a certain threshold either by adaptive mutations or by inhibition of the NS5A–specific kinase(s) would enable HCV replication in cell culture. Although large amounts of NS5A-p58 appear to inhibit HCV RNA replication, the complete inhibition of NS5A hyperphosphotylation by the kinase inhibitors we identified abolishes HCV replication of already adapted replicons indicating that a small quantity of p58 is required for replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified CK1 kinase family as a target. NS5A-p58 increased upon overexpression of CK1alpha, CK1δ and CK1ε, whereas the RNA interference of only CK1alpha reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CK1 alpha overexpression, and we demonstrated that the CK1 alpha isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells and that the down-regulation of NS5A attenuates HCV RNA replication. Finally, we demonstrate here that NS5A is a direct substrate of CKI- alpha and phosphorylation of NS5A in vitro by CKI- alpha resulted in the production of two phosphorylated forms that resemble those products produced in cells. In vitro kinase reactions performed with NS5A peptides show that S2204 is a preferred substrate residue for CKI- alpha after pre-phosphorylation of S220

Epigenetics and Vascular Diseases: Influence of Non-coding RNAs and Their Clinical Implications

Epigenetics refers to heritable mechanisms able to modulate gene expression that do not involve alteration of the genomic DNA sequence. Classically, mechanisms such as DNA methylation and histone modifications were part of this classification. Today, this field of study has been expanded and includes also the large class of non-coding RNAs (ncRNAs). Indeed, with the extraordinary possibilities introduced by the next-generation sequencing approaches, our knowledge of the mammalian transcriptome has greatly improved. Today, we have identifying thousands of ncRNAs, and unsurprisingly, a direct association between ncRNA dysregulation and development of cardiovascular pathologies has been identified. This class of gene modulators is further divided into short-ncRNAs and long-non-coding RNAs (lncRNAs). Among the short-ncRNA sub-group, the best-characterized players are represented by highly conserved RNAs named microRNAs (miRNAs). miRNAs principally inhibit gene expression, and their involvement in cardiovascular diseases has been largely studied. On the other hand, due to the different roles played by lncRNAs, their involvement in cardiovascular pathology development is still limited, and further studies are needed. For instance, in order to define their roles in the cellular processes associated with the development of diseases, we need to better characterize the details of their mechanisms of action; only then might we be able to develop innovative therapeutic strategies. In this review, we would like to give an overview of the current knowledge on the function of ncRNAs and their involvement in the development of vascular diseases

Epigenetics and Vascular Diseases: Influence of Non-coding RNAs and Their Clinical Implications

Epigenetics refers to heritable mechanisms able to modulate gene expression that do not involve alteration of the genomic DNA sequence. Classically, mechanisms such as DNA methylation and histone modifications were part of this classification. Today, this field of study has been expanded and includes also the large class of non-coding RNAs (ncRNAs). Indeed, with the extraordinary possibilities introduced by the next-generation sequencing approaches, our knowledge of the mammalian transcriptome has greatly improved. Today, we have identifying thousands of ncRNAs, and unsurprisingly, a direct association between ncRNA dysregulation and development of cardiovascular pathologies has been identified. This class of gene modulators is further divided into short-ncRNAs and long-non-coding RNAs (lncRNAs). Among the short-ncRNA sub-group, the best-characterized players are represented by highly conserved RNAs named microRNAs (miRNAs). miRNAs principally inhibit gene expression, and their involvement in cardiovascular diseases has been largely studied. On the other hand, due to the different roles played by lncRNAs, their involvement in cardiovascular pathology development is still limited, and further studies are needed. For instance, in order to define their roles in the cellular processes associated with the development of diseases, we need to better characterize the details of their mechanisms of action; only then might we be able to develop innovative therapeutic strategies. In this review, we would like to give an overview of the current knowledge on the function of ncRNAs and their involvement in the development of vascular diseases

Arterial remodeling and atherosclerosis: MiRNAs involvement

Cardiometabolic diseases (CMD) (such as atherosclerosis, diabetes, and hypertension) are the primary cause of death and disability in the Western world. Although lifestyle programs and therapeutic approaches have significantly reduced the socio-economic burden of CMD, a large number of events still cannot be avoided (the so called residual risk). Recent developments in genetics and genomics provide a platform for investigating further this area with the aim of deepening our understanding of the atherosclerotic phenomena underlying CMD, for instance by providing better information on the type of subjects who would benefit the most from therapeutic interventions, or by discovering new genetic and metabolic derangements that may be targeted for the development of new interventions. MicroRNAs (miRNA) are short, non-coding RNAs that negatively regulate the expression of proteins by binding to specific sequences on the 3' region of target mRNAs. Bioinformatics analysis predicts that each miRNA may regulate hundreds of targets, suggesting that miRNAs may play roles in almost every biological pathway and process, including those of the cardiovascular system. Studies are beginning to unravel their fundamental importance in vessel biology. Here, we review recent advance regarding the involvement of miRNAs in arterial remodeling and atherosclerosis

The α Isoform of Protein Kinase CKI Is Responsible for Hepatitis C Virus NS5A Hyperphosphorylation

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified the CKI kinase family as a target. NS5A-p58 increased upon overexpression of CKI-α, CKI-δ, and CKI-ɛ, whereas the RNA interference of only CKI-α reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CKI-α overexpression, and we demonstrated that the CKI-α isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells. Finally, we showed that down-regulation of CKI-α attenuates HCV RNA replication

TGFβ triggers miR-143/145 transfer from smooth muscle cells to endothelial cells, thereby modulating vessel stabilization

Rationale: The miR-143/145 cluster is highly expressed in smooth muscle cells (SMCs), where it regulates phenotypic switch and vascular homeostasis. Whether it plays a role in neighboring endothelial cells (ECs) is still unknown. Objective: To determine whether SMCs control EC functions through passage of miR-143 and miR-145. Methods and Results: We used cocultures of SMCs and ECs under different conditions, as well as intact vessels to assess The transfer of miR-143 and miR-145 from one cell type to another. Imaging of cocultured cells transduced with fluorescent miRNAs suggested that miRNA transfer involves membrane protrusions known as tunneling nanotubes. Furthermore, we show that miRNA passage is modulated by The transforming growth factor (TGF) \u3b2 pathway because both a specific transforming growth factor-\u3b2 (TGF\u3b2) inhibitor (SB431542) and an shRNA against TGF\u3b2RII suppressed The passage of miR-143/145 from SMCs to ECs. Moreover, miR-143 and miR-145 modulated angiogenesis by reducing The proliferation index of ECs and their capacity to form vessel-like structures when cultured on matrigel. We also identified hexokinase II (HKII) and integrin \u3b2 8 (ITG\u3b28) -2 genes essential for The angiogenic potential of ECs -as targets of miR-143 and miR-145, respectively. The inhibition of these genes modulated EC phenotype, similarly to miR-143 and miR-145 overexpression in ECs. These findings were confirmed by ex vivo and in vivo approaches, in which it was shown that TGF\u3b2 and vessel stress, respectively, triggered miR-143/145 transfer from SMCs to ECs. Conclusions: Our results demonstrate that miR-143 and miR-145 act as communication molecules between SMCs and ECs to modulate The angiogenic and vessel stabilization properties of ECs