The Cystic Fibrosis Transmembrane Conductance Regulator Gene (CFTR) Is under Post-Transcriptional Control of microRNAs: Analysis of the Effects of agomiRNAs Mimicking miR-145-5p, miR-101-3p, and miR-335-5p.

(1) Background: MicroRNAs are involved in the expression of the gene encoding the chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator); the objective of this short report is to study the effects of the treatment of bronchial epithelial Calu-3 cells with molecules mimicking the activity of pre-miR-145-5p, pre-miR-335-5p, and pre-miR-101-3p, and to discuss possible translational applications of these molecules in pre-clinical studies focusing on the development of protocols of possible interest in therapy; (2) Methods: CFTR mRNA was quantified by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR). The production of the CFTR protein was assessed by Western blotting; (3) Results: The treatment of Calu-3 cells with agomiR-145-5p caused the highest inhibition of CFTR mRNA accumulation and CFTR production; (4) Conclusions: The treatment of target cells with the agomiR pre-miR-145-5p should be considered when CFTR gene expression should be inhibited in pathological conditions, such as polycystic kidney disease (PKD), some types of cancer, cholera, and SARS-CoV-2 infection.

SARS-CoV-2 viral entry and replication is impaired in Cystic Fibrosis airways due to ACE2 downregulation.

As an inherited disorder characterized by severe pulmonary disease, cystic fibrosis could be considered a comorbidity for coronavirus disease 2019. Instead, current clinical evidence seems to be heading in the opposite direction. To clarify whether host factors expressed by the Cystic Fibrosis epithelia may influence coronavirus disease 2019 progression, here we describe the expression of SARS-CoV-2 receptors in primary airway epithelial cells. We show that angiotensin converting enzyme 2 (ACE2) expression and localization are regulated by Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Consistently, our results indicate that dysfunctional CFTR channels alter susceptibility to SARS-CoV-2 infection, resulting in reduced viral entry and replication in Cystic Fibrosis cells. Depending on the pattern of ACE2 expression, the SARS-CoV-2 spike (S) protein induced high levels of Interleukin 6 in healthy donor-derived primary airway epithelial cells, but a very weak response in primary Cystic Fibrosis cells. Collectively, these data support that Cystic Fibrosis condition may be at least partially protecting from SARS-CoV-2 infection.

Teaching during COVID-19 pandemic in practical laboratory classes of applied biochemistry and pharmacology: A validated fast and simple protocol for detection of SARS-CoV-2 Spike sequences.

The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). We suggest that this laboratory practical exercises should be considered for face-to-face teaching especially if the emergency related to the COVID-19 pandemic is maintained. The teaching protocol here described might be considered in order to perform fast but meaningful in-presence teaching, making feasible the division of crowded classes in low-number cohorts of students, allowing the maintenance of the required social distance.

Possible effects of sirolimus treatment on the long­term efficacy of COVID­19 vaccination in patients with ß­thalassemia: A theoretical perspective.

The pandemic caused by the severe acute respiratory syndrome coronavirus (SARS­CoV­2), responsible for coronavirus disease 2019 (COVID­19) has posed a major challenge for global health. In order to successfully combat SARS­CoV­2, the development of effective COVID­19 vaccines is crucial. In this context, recent studies have highlighted a high COVID­19 mortality rate in patients affected by ß­thalassemia, probably due to their co­existent immune deficiencies. In addition to a role in the severity of SARS­CoV­2 infection and in the mortality rate of COVID­19­infected patients with thalassemia, immunosuppression is expected to deeply affect the effectivity of anti­COVID­19 vaccines. In the context of the interplay between thalassemia­associated immunosuppression and the effectiveness of COVID­19 vaccines, the employment of immunomodulatory molecules is hypothesized. For instance, short­term treatment with mammalian target of rapamycin inhibitors (such as everolimus and sirolimus) has been found to improve responses to influenza vaccination in adults, with benefits possibly persisting for a year following treatment. Recently, sirolimus has been considered for the therapy of hemoglobinopathies (including ß­thalassemia). Sirolimus induces the expression of fetal hemoglobin (and this may contribute to the amelioration of the clinical parameters of patients with ß­thalassemia) and induces autophagy (thereby reducing the excessive levels of α­globin). It may also finally contribute to the mobilization of erythroid cells from the bone marrow (thereby reducing anemia). In the present study, the authors present the hypothesis that sirolimus treatment, in addition to its beneficial effects on erythroid­related parameters, may play a crucial role in sustaining the effects of COVID­19 vaccination in patients with ß­thalassemia. This hypothesis is based on several publications demonstrating the effects of sirolimus treatment on the immune system.

In vitro induction of interleukin-8 by SARS-CoV-2 Spike protein is inhibited in bronchial epithelial IB3-1 cells by a miR-93-5p agomiR.

One of the major clinical features of COVID-19 is a hyperinflammatory state, which is characterized by high expression of cytokines (such as IL-6 and TNF-α), chemokines (such as IL-8) and growth factors and is associated with severe forms of COVID-19. For this reason, the control of the "cytokine storm" represents a key issue in the management of COVID-19 patients. In this study we report evidence that the release of key proteins of the COVID-19 "cytokine storm" can be inhibited by mimicking the biological activity of microRNAs. The major focus of this report is on IL-8, whose expression can be modified by the employment of a molecule mimicking miR-93-5p, which is able to target the IL-8 RNA transcript and modulate its activity. The results obtained demonstrate that the production of IL-8 protein is enhanced in bronchial epithelial IB3-1 cells by treatment with the SARS-CoV-2 Spike protein and that IL-8 synthesis and extracellular release can be strongly reduced using an agomiR molecule mimicking miR-93-5p.

Sulforaphane inhibits the expression of interleukin-6 and interleukin-8 induced in bronchial epithelial IB3-1 cells by exposure to the SARS-CoV-2 Spike protein.

BACKGROUND: A key clinical feature of COVID-19 is a deep inflammatory state known as "cytokine storm" and characterized by high expression of several cytokines, chemokines and growth factors, including IL-6 and IL-8. A direct consequence of this inflammatory state in the lungs is the Acute Respiratory Distress Syndrome (ARDS), frequently observed in severe COVID-19 patients. The "cytokine storm" is associated with severe forms of COVID-19 and poor prognosis for COVID-19 patients. Sulforaphane (SFN), one of the main components of Brassica oleraceae L. (Brassicaceae or Cruciferae), is known to possess anti-inflammatory effects in tissues from several organs, among which joints, kidneys and lungs. PURPOSE: The objective of the present study was to determine whether SFN is able to inhibit IL-6 and IL-8, two key molecules involved in the COVID-19 "cytokine storm". METHODS: The effects of SFN were studied in vitro on bronchial epithelial IB3-1 cells exposed to the SARS-CoV-2 Spike protein (S-protein). The anti-inflammatory activity of SFN on IL-6 and IL-8 expression has been evaluated by RT-qPCR and Bio-Plex analysis. RESULTS: In our study SFN inhibits, in cultured IB3-1 bronchial cells, the gene expression of IL-6 and IL-8 induced by the S-protein of SARS-CoV-2. This represents the proof-of-principle that SFN may modulate the release of some key proteins of the COVID-19 "cytokine storm". CONCLUSION: The control of the cytokine storm is one of the major issues in the management of COVID-19 patients. Our study suggests that SFN can be employed in protocols useful to control hyperinflammatory state associated with SARS-CoV-2 infection.

Tackling the COVID-19 "cytokine storm" with microRNA mimics directly targeting the 3'UTR of pro-inflammatory mRNAs.

COVID-19 is characterized by two major clinical phases, the SARS-CoV-2 infection of target cells and tissues, and a deep inflammatory state, known as "cytokine storm", caused by activation of pro-inflammatory genes, such as NF-kB, STAT-3, IL-6, IL-8, IL-1ß. Among possible anti-inflammatory agents, the "microRNA targeting" should be carefully considered, since it is well known that microRNAs are deeply involved in the expression of cytokines, chemokines and growth factors. The working general hypothesis is that targeting the microRNA network might be important for the development of therapeutic approaches to counteract the COVID-19 induction of inflammatory response. This hypothesis is based on several publications demonstrating the use of miRNA mimics for inhibitory effects on the production of proteins characterizing the COVID-19 "cytokine storm".