Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines

<div><p>Hepatocellular carcinoma (HCC) is a multi-factorial cancer with a very poor prognosis; therefore, there are several investigations aimed at the comprehension of the molecular mechanisms leading to development and progression of HCC and at the definition of new therapeutic strategies. We have recently evaluated the expression of selenoproteins in HCC cell lines in comparison with normal hepatocytes. Recent results have shown that some of them are down- and others up-regulated, including the selenoprotein K (SELK), whose expression was also induced by sodium selenite treatment on cells. However, so far very few studies have been dedicated to a possible effect of microRNAs on the expression of selenoproteins and their implication in HCC. In this study, the analysis of SELK 3’UTR by bioinformatics tools led to the identification of eight sites potentially targeted by human microRNAs. They were then subjected to a validation test based on luciferase reporter constructs transfected in HCC cell lines. In this functional screening, miR-544a was able to interact with SELK 3’UTR suppressing the reporter activity. Transfection of a miR-544a mimic or inhibitor was then shown to decrease or increase, respectively, the translation of the endogenous SELK mRNA. Intriguingly, miR-544a expression was found to be modulated by selenium treatment, suggesting a possible role in SELK induction by selenium.</p></div

miR-544a expression in three common HCC cell lines.

<p>miR-544a expression levels were evaluated by RT-qPCR and reported as fold change relative to that of HuH-7.</p

Human MiR-544a Modulates SELK Expression in Hepatocarcinoma Cell Lines - Fig 5

<p><b>Effect of sodium selenite on miR-544a expression and SELK</b> (A) Cells were cultured for 24 h and 48 h in standard condition or in presence of increasing sodium selenite doses; miR-544a expression was determined by RT-qPCR and reported as fold change of expression in treated samples vs untreated ones. (B) Upper panel, representative Western blot analysis of protein extracts from cells cultured in absence (-) or presence (+) of 1 μM sodium selenite for 24h and 48 h; lower panel, quantification of protein bands; values represent the mean ± s.d. of three independent experiment.</p

The silencing effect of miR-544a on SELK in HuH-7 cells.

<p>Representative Western blot analysis of protein extracts at 48 h after transfection with miR-544a mimic (A) or miR-544a inhibitor (B) and their relative control molecules at 50 nM. (C) Quantification of SELK protein band normalized to that of tubulin; signal intensity values determined for the control experiments (Ctrl-miR and Ctrl-anti-miR) were set at 1; values represent the mean ± s.d. of three independent experiment. P-values are indicated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156908#pone.0156908.g003" target="_blank">Fig 3</a>.</p

Interfering activity of miR-544a in HCC cell lines.

<p>HuH-7 and HepG2 cells were transfected with the luciferase-based reporter plasmid psiCheck-2 containing the SELK target sequence for miR-544a (WT) or a control DNA with inverted sequence (I), along with 50 nM miR-544a mimic (miR-544a, A and B) or 50 nM miR-544a inhibitor (anti-miR-544a, C and D). Ctrl-miRNA and Ctrl-anti-miRNA are their relative unrelated molecules used as negative controls. Significant p-values evaluated by Student’s t-test were indicated with *, ** and *** when P < 0.05, or < 0.01 or < 0.001.</p

Additional file 4: Figure S2. of Targeting the cross-talk between Urokinase receptor and Formyl peptide receptor type 1 to prevent invasion and trans-endothelial migration of melanoma cells

Proliferation rate of melanoma cells. Cell proliferation of the indicated melanoma cell lines assessed by monitoring impedance by RTCA xCELLigence system. The reported doubling times were calculated from the cell growth curves, during exponential growth. Data represent mean ± SD from a quadruplicate experiment representative of 3 replicates. (PDF 120 kb

Additional file 1: Figure S1. of Targeting the cross-talk between Urokinase receptor and Formyl peptide receptor type 1 to prevent invasion and trans-endothelial migration of melanoma cells

Uncropped images of immunoblots. Full blots from Fig. 1b, Inset of the Fig. 2a, c, Fig. 4b, and Fig. 7a. (PDF 164 kb

DataSheet_1_The impact of the COVID-19 pandemic on diagnosis and treatment of patients with soft tissue and bone sarcomas or aggressive benign musculoskeletal diseases: A single-center retrospective study (SarCorD study).pdf

BackgroundThe COVID-19 pandemic led to a rapid reorganization of healthcare activities, leading to reduced access to clinics, interruption of screenings, and treatment schedule modifications in several cancer types. Few data are available on sarcomas. We analyzed COVID-19-related diagnostic delay in a sarcoma referral center in Italy.MethodsWe retrospectively enrolled in this study patients with histological diagnosis of soft tissue or bone sarcoma and aggressive benign musculoskeletal diseases obtained during the first year of the pandemic (Covid group) or the year before (Control group) and followed at the Regina Elena National Cancer Institute in Rome. The primary endpoint was the time from the first symptom to histological diagnosis.ResultsWe evaluated 372 patients, 185 of whom were eligible for primary endpoint analysis (92 patients in the Control group and 93 patients in the Covid group). The patients were affected by soft tissue sarcoma in most cases (63.0% and 66.7% in Covid and Control groups, respectively). We observed a diagnostic delay in the Covid group with a median time from the first symptom to the definitive histological diagnosis of 103.00 days (95% CI 92.77–113.23) vs. 90.00 days (95% CI 69.49–110.51) in the Control group (p = 0.024), but not a delay in treatment beginning (151 days, 95% CI 132.9–169.1 vs. 144 days, 95% CI 120.3–167.7, respectively, p = 0.208). No differences in stage at diagnosis were observed (12% vs. 16.5% of patients with metastatic disease at diagnosis in the Covid and Control groups, respectively, p = 0.380). Progression-free survival (p = 0.897) and overall survival (p = 0.725) were comparable in the subgroup of patients affected by soft tissue sarcoma.ConclusionsA delay in sarcoma diagnosis but not in starting treatment has been observed during the first year of the COVID-19 pandemic. Nevertheless, no difference in stage at diagnosis or in terms of survival has been observed.</p

Additional file 1: of Serum miR-22 as potential non-invasive predictor of poor clinical outcome in newly diagnosed, uniformly treated patients with diffuse large B-cell lymphoma: an explorative pilot study

Fig. S1. Kaplan-Meier Overall Survival curves. Correlation between the indicated miRNAs and the Overall Survival of 47 DLBCL patients from TCGA data analysis. (PDF 204 kb

MOESM2 of Human lung adenocarcinoma cell cultures derived from malignant pleural effusions as model system to predict patients chemosensitivity

Additional file 2. Distribution in the chromosomes of the common non-synonymous variants