Alessia Finotti

Mapping the Transcriptional Machinery of the IL-8 Gene in Human Bronchial Epithelial Cells

IL-8 released from bronchial epithelial cells infected with Pseudomonas aeruginosa plays a crucial role in the chronic lung pathology of patients affected by cystic fibrosis. Novel anti-inflammatory approaches will benefit from a thorough understanding of the regulatory mechanisms involved in the transcription of this chemokine to identify potential pharmacological targets. We addressed this issue by investigating the role of phosphoproteins and transcription factors (TFs) on transcription of IL-8 gene in the human bronchial epithelial IB3-1, CuFi-1, and Calu-3 cells. P. aeruginosa increased the basal phosphorylation of the ERK1/2 pathway components 90-kDa ribosomal S6 kinase (RSK)1/2 and mitogen- and stress-activated kinase-2 and of the p38 MAPK pathway components p38α/δ/γ and heat shock protein 27 (HSP27). The involvement of these kinases in the expression of IL-8 gene was confirmed with pharmacological inhibitors of ERK1/2, RSK, p38, and HSP27 both at transcription and secretion levels. Transfection of TF decoy oligodeoxynucleotides, designed to interfere with the interaction of the TFs NF-κB, NF-IL6, AP-1, CREB, and CHOP with the corresponding consensus sequences identified in the IL-8 promoter, reduced the P. aeruginosa-dependent transcription of IL-8, suggesting their participation in the transcriptional machinery. Stimulation of IB3-1 cells with IL-1β led to a similar pattern of activation, whereas the pattern of phosphoproteins and of TFs modulated by TNF-α differentiated sharply. In conclusion, the results highlight a novel role for RSK1/2 and HSP27 phosphoproteins and of the cooperative role of the TFs NF-κB, NF-IL6, AP-1, CHOP, and CREB in P. aeruginosa-dependent induction of transcription of the IL-8 gene in human bronchial epithelial cells.

Targeting microRNAs involved in human diseases: a novel approach for modification of gene expression and drug development.

The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson-Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo.

Effects of rapamycin on accumulation of α-, β- and γ-globin mRNAs in erythroid precursor cells from β-thalassaemia patients

Abstract:  We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 β‐thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two‐phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the β‐thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to γ‐globin mRNA accumulation, being only minor for β‐globin and none for α‐globin mRNAs. The ability of rapamycin to preferentially increase γ‐globin mRNA content and production of HbF in erythroid precursor cells from β‐thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti‐rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in β‐thalassaemia and sickle cell anaemia.

Modulation of the biological activity of microRNA-210 with peptide nucleic acids (PNAs).

Herein we describe the activity of a peptide nucleic acid (PNA) that targets microRNA‐210 (miR‐210), which is associated with hypoxia and is modulated during erythroid differentiation. PNAs directed against miR‐210 were designed to bind with high affinity to the target RNA strand and to undergo efficient uptake in target cells. A polyarginine–PNA conjugate directed against miR‐210 (Rpep‐PNA‐a210) showed both very high affinity for RNA and efficient uptake into target cells without the need for transfection reagents. An unmodified PNA of the same sequence displayed the ability to bind RNA, but cellular uptake was very poor. Consistent with this, only Rpep‐PNA‐a210 strongly inhibited miR‐210 activity, as evaluated by assays on undifferentiated K562 cells and on cells treated with mithramycin, which was found to induce erythroid differentiation and miR‐210 overexpression. Targeting miR‐210 by Rpep‐PNA‐a210 resulted in: 1) a decrease in miR‐210 levels as measured by RT‐PCR, 2) up‐regulation of raptor mRNA, 3) a decrease in γ‐globin mRNA, and 4) decreased expression of differentiated functions (i.e., proportion of benzidine‐positive cells, content of embryo‐fetal hemoglobins). The efficient delivery of anti‐miR PNAs through a suitable peptide carrier (Rpep‐PNA‐a210) leads to the inhibition of miR‐210 activity, altering the expression of miR‐210‐regulated erythroid functions.

Peptide nucleic acids targeting miR-221 modulate p27Kip1 expression in breast cancer MDA-MB-231 cells.

The activity of a peptide nucleic acid (PNA) targeting cancer-associated microRNA-221 is described. PNAs against miR-221 were designed in order to bind very efficiently to the target RNA strand and to undergo efficient uptake in the cells. A polyarginine-PNA conjugate targeted against miR-221 (Rpep-PNA-a221) showed both very high affinity for RNA and efficient cellular uptake without the addition of transfection reagents. Unmodified PNA with the same sequence displayed RNA binding, but cellular uptake was very poor. Consistently, only Rpep-PNA-a221 strongly inhibited miR-221. Targeting miR-221 by PNA resulted in i) lowering of the hybridization levels of miR-221 measured by RT-qPCR, ii) upregulation of p27Kip1 gene expression, measured by RT-qPCR and western blot analysis. The major conclusion of this study is that efficient delivery of anti‑miR PNA through a suitable peptide carrier (Rpep‑PNA-a221) leads to inhibition of miR-221 activity, altering the expression of miR-221-regulated functions in breast cancer cells.

Involvement of miRNA in erythroid differentiation

miRNAs are a family of small ncRNAs that regulate gene expression by targeting mRNAs in a sequence-specific manner, inducing translational repression or mRNA degradation. In this review, we present and discuss the available literature on the expression of miRNAs in erythroid cells. There are several experimental systems that can be employed for studies focusing on the relationship between miRNAs and erythroid differentiation, including human embryonic stem cells forced to erythroid differentiation, K562 and UT-7 cells induced to hemoglobin production by chemical compounds, erythropoietin-treated erythroid precursor cells from normal subjects or patients affected by hematological disease and in vivo systems, such as zebrafish embryos. Several miRNAs were identified as deeply involved in the erythroid phenotype, including miR-15a, miR-16-1, miR-126, miR-144, miR-451 and miR-210. Several functions related with erythroid cells were demonstrated to be regulated by these miRNAs, including maturation and proliferation of early erythroid cells, expression of fetal γ-globin genes and enucleation. These identified erythroid specific miRNAs represent the starting point to develop new protocols for miRNA therapeutics, based on both anti-miR molecules or miRNA replacement.

Resveratrol: Antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and β-thalassemia patients

Thalassemia and sickle-cell anemia (SCA) present a major public health problem in countries where the number of carriers and affected individuals is high. As a result of the abnormalities in hemoglobin production, cells of thalassemia and SCA patients exhibit oxidative stress, which ultimately is responsible for the chronic anemia observed. Therefore, identification of compounds exhibiting both antioxidant and hemoglobin-inducing activities is highly needed. Our results demonstrate resveratrol to be such a compound. This was shown both in the human K562 cell line, as well as in erythroid precursors derived from normal donors and β-thalassemia patients. Resveratrol was shown to exhibit antioxidant activity and to stimulate the expression of the γ-globin genes and the accumulation of fetal hemoglobin (HbF). To the best of our knowledge, this is the first report pointing to such a double effect of resveratrol. Since this natural product is already marketed as an antioxidant, future investigations should concentrate on demonstrating its potential to augment HbF production in experimental animal models (e.g., thalassemia and SCA mice) as well as in patients. We believe that the potential of clinical use of resveratrol as an antioxidant and HbF stimulator may offer a simple and inexpensive treatment to patients.

miRNA therapeutics: delivery and biological activity of peptide nucleic acids targeting miRNAs.

Peptide nucleic acids (PNAs) are DNA/RNA mimics extensively used for pharmacological regulation of gene expression in a variety of cellular and molecular systems, and they have been described as excellent candidates for antisense and antigene therapies. At present, very few data are available on the use of PNAs as molecules targeting miRNAs. miRNAs are a family of small nc RNAs that regulate gene expression by sequence-selective targeting of mRNAs, leading to a translational repression or mRNA degradation to the control of highly regulated biological functions, such as differentiation, cell cycle and apoptosis. The aim of this article is to present the state-of-the-art concerning the possible use of PNAs to target miRNAs and modify their biological metabolism within the cells. The results present in the literature allow to propose PNA-based molecules as very promising reagents to modulate the biological activity of miRNAs. In consideration of the involvement of miRNAs in human pathologies, PNA-mediated targeting of miRNAs has been proposed as a potential novel therapeutic approach.

Effects of decoy molecules targeting NF-kappaB transcription factors in Cystic fibrosis IB3-1 cells: recruitment of NF-kappaB to the IL-8 gene promoter and transcription of the IL-8 gene.

One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3–1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.

Recent trends in the gene therapy of β-thalassemia.

The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases.