Extracellular vesicles (EVs) are membrane-released vesicles acting as transporters of proteins, lipids and short/long non-coding RNA (miRNAs and lncRNAs). They are released by normal and pathological ...cells, including hepatocellular carcinoma (HCC). To date, studies focused on miRNAs and lncRNAs contained in EVs derived from HCC are limited. Our aim was to analyze the transcriptional profile of potential regulating miRNAs and lncRNAs in EVs secreted by HCC tumor cell line (HepG2,
n
= 6), and from a non-tumorigenic hepatocyte cell line (WRL68,
n
= 6), to compare their differential expression profile and to identify novel molecular diagnostic markers of HCC. EVs were isolated from the conditioned medium, through differential centrifugations. The expression profile of miRNAs (miR-23a, miR-16–2, miR-181a, miR-373, miR-205, miR-27a, miR-1323, and miR-532) and lncRNAs (HULC, HOTAIR, XIST, MALAT-1, GAS-5, H19) was performed in Real-time PCR, and their transcript was found both in HepG2 and WRL68 EVs. Lower miR-181a, miR-205 and miR-1323 expression were detected in EVs secreted by HepG2 compared to WRL68, while an opposite trend was observed for miR-23a, miR-16–2, miR-373, miR-27a, and miR-532. Several significant correlations were found between miRNA and lncRNA. An in silico analysis was also performed. The results obtained could identified them as new potential diagnostic and prognostic biomarkers of HCC.
Long non-coding RNAs (lncRNAs) have been recently described as key mediators in the development of hematological malignancies. In the last years, circulating lncRNAs have been proposed as a new class ...of non-invasive biomarkers for cancer diagnosis and prognosis and to predict treatment response. The present study is aimed to investigate the potential of circulating lncRNAs as non-invasive prognostic biomarkers in myelofibrosis (MF), the most severe among Philadelphia-negative myeloproliferative neoplasms. We detected increased levels of seven circulating lncRNAs in plasma samples of MF patients (n = 143), compared to healthy controls (n = 65). Among these, high levels of LINC01268, MALAT1 or GAS5 correlate with detrimental clinical variables, such as high count of leukocytes and CD34+ cells, severe grade of bone marrow fibrosis and presence of splenomegaly. Strikingly, high plasma levels of LINC01268 (p = 0.0018), GAS5 (p = 0.0008) or MALAT1 (p = 0.0348) are also associated with a poor overall-survival while high levels of LINC01268 correlate with a shorter leukemia-free-survival. Finally, multivariate analysis demonstrated that the plasma level of LINC01268 is an independent prognostic variable, suggesting that, if confirmed in future in an independent patients’ cohort, it could be used for further studies to design an updated classification model for MF patients.
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•Circulating microRNAs are potential biomarkers of metabolic disease and obesity•CNP: important natural regulator of adipogenesis during pediatric obesity•In silico analysis ...identified miR-33a-3p, miR-223-5p, miR-142-5p as circulating CNP-linked miRNAs•A significant association was found between circulating miR-199-5p, miR-142-5p, miR-33a-3p, miR-223-5p and CNP•Circulating miRNAs may have a promising role for early identification of obese children at risk of cardiometabolic alterations
Circulating microRNAs (miRNAs) are potential biomarkers of metabolic disease implicated in the pathogenesis of obesity and at present, no data are available on a possible contribution of C-type natriuretic peptides (CNP)-linked miRNAs to childhood obesity. Our aims were to 1) perform an in silico-analysis to identify miRNAs targeting CNP gene; 2) recognize CNP-linked miRNAs associated with obesity; 3) characterize their circulating profiling in normal-weight (N) and obese adolescents (O). A clinical examination was performed in 25 N and 52 O adolescents. CNP plasma levels were detected by immunometric assay while miRNA expression was carried out on peripheral blood using Real-Time PCR. Plasma CNP resulted significantly lower in O than in N (5.58 ± 0.62 vs.14.78 ± 1.35 pg/mL, p < 0.0001). In silico-analysis disclosed several specific circulating CNP-linked miRNAs among which miR-33a-3p, miR-223−5p and miR-142−5p also associated with obesity. MiR-199−5p and miR-4454, known to be associated with obesity but not with CNP, were also studied. miR-223−5p and miR-33a-3p resulted significantly (p = 0.05) higher in O (0.97 ± 0.1; 0.85 ± 0.1, respectively) than in N (0.66 ± 0.11; 0.51 ± 0.08, respectively). Plasma CNP correlated inversely with miR-33a-3p (p = 0.036), miR-223−5p (p = 0.004), miR-199−5p (p = 0.003) and miR-4454 (p < 0.0001). Significantly positive correlations were observed between miR-33a-3p and miR-223−5p (p = 0.002) and between miR-199−5p and miR-4454 (p = 0.0001). Applying a multiple linear regression model, miR-142−5p, miR-199a-5p, miR-223−5p, miR33a-3p, diastolic blood pressure (DBP) and age were independent determinants of CNP. Our results underline the concept that expanding our knowledge on the behaviour of circulating miRNA profile may have a promising role for early identification of obese children at increased risk of cardiometabolic alterations.
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•Aging is caused by the dysfunction of physiological and metabolic processes.•Adrenomedullin and ANP as suitable markers for a successful aging.•Decrease in ADM expression levels as a ...function of age observed by us for the first time.•Non-coding RNAs have been shown to be excellent therapeutic targets.•Importance of a multi-label biomolecular approach to study aging.
Chronological age is considered one of the major risk factors for cardiovascular disease and mortality. The study aimed to evaluate the transcriptional levels of the natriuretic peptides (NP), endothelin (ET)-1, adrenomedullin (ADM), their receptors and long non-coding (Lnc) RNA MIAT, MALAT-1, CARMEN and XIST in rat cardiac tissue as cardiovascular biomarkers of aging. Three groups of male Wistar rats were studied: A (n = 6; young), B (n = 13; adult), C (n = 10; old). Total RNA was extracted from left ventricle and analyzed by Real-Time PCR. Echocardiographic and histological analyses were performed. A significant increase of Atrial NP (ANP) and Brain NP (BNP) mRNA was observed in C while C-type NP (CNP) remained in a steady-state in B and C; ET-1 mRNA increased significantly as a function of age. Any difference was observed for NP receptors. ETA expression was statistically lower in B than A while ETB were similar in all the three groups. The ADM showed an opposite trend to that of the other peptides decreasing significantly as a function of age and presenting a counter-regulation of calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP)-2. LncRNA transcripts decreased significantly as a function of age except for XIST. ADM and LncRNA trend suggest that the animals are subjected to “successful aging” as also confirmed by histological analysis. Applying a multivariate logistic regression analysis, only LnANP (p = 0.003) and LnADM (p = 0.023) resulted significantly associated with aging identifying them, for the first time, as independent markers of aging. The study underlining the importance of a multi-label biomolecular approach in the evaluation of aging.
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Introduction: Myelofibrosis (MF), whether primary (PMF) or secondary (SMF) to polycythemia vera or essential thrombocytemia, is characterized by a complex and partially undeciphered molecular ...architecture. Besides mutations in driver genes (JAK2, CALR, MPL), somatic mutations in selected myeloid-associated genes have been shown to impact prognosis of MF patients (pts). Among these, ASXL1 mutations (ASXL1MTs) are associated with poor outcomes in myeloid malignancies including PMF, where they are included in the category of “high molecular risk” (HMR) mutations along with EZH2MTs, IDH1/2MTs, and SRSF2MTs (Vannucchi AM, Leukemia 2013). However, a recent study (Luque Paz D, Blood Adv 2021) questioned the value of ASXL1MTs in MF. The current study aimed at further characterizing the prognostic role of ASXL1MTs in MF.
Methods: After IRB approval, pts with WHO-defined MF were included in the study. Mutational analysis by targeted NGS was performed as previously described (Guglielmelli P, JCO 2017). All deposited variants were manually curated to assess pathogenicity. In this study, we also used the molecular model proposed by Luque Paz et al. that identifies 4 genetic groups: TP53MT; High-risk (≥1 mutation in EZH2, CBL, U2AF1, SRSF2, IDH1/2); ASXL1MT-only; and “Others”.
Results: A total of 525 pts were included in the study, including 331 (63%) PMF and 194 (37%) SMF. Median age at diagnosis was 89 (18-90) years, 314 (60%) were male. The median follow-up time was 80 (98% CI, 68-90) months. Overall, 324 (62%) pts were JAK2MT, 126 (24%) CALRMT, 24 (5%) MPLMT, 40 (8%) triple negative (TN), and 11 (2%) double mutated. Among non-driver genes, ASXL1MTs were found in 158 (30%) pts, EZH2MTs in 45 (9%), SRSF2MTs in 37 (7%), NRASMTs in 30 (6%) U2AF1MTs in 27 (5%), TP53MTs and CBLMTs in 25 (5%) each, IDH1/2MTs in 18 (3%), and KRASMTs in 15 (3%). Pts in the HMR category were 125 (38%) in PMF and 63 (32%) in SMF.
According to the above model, distribution of pts was as follows: TP53MT n=25 (5%), High-risk n=137 (26%), ASXL1MT-only n=64 (12%), and Others n=299 (57%). Pts in the TP53MT and ASXL1MT-only groups were more likely to be diagnosed with SMF compared to pts in the High-risk and Others groups (44% and 48% vs 28% and 38%, respectively). In addition, the High-risk group was enriched in TN pts (16%), while CALRMTs were more common in the ASXL1MT-only and Others compared to the TP53MT and High-risk groups (25% and 27% vs 12% and 18%, respectively).
In univariate analysis, the TP53MT and High-risk groups were associated with the worst overall survival (OS), with median values of 38 (14-110) and 55 (45-85) months (P=.0039), respectively (Fig 1A). Albeit remarkably better, the OS of pts in the ASXL1MT-only group was inferior compared to pts in the Others group (median 124 91-156 vs 193 142-NR months; P=.0118) (Fig 1A).
We then analyzed separately PMF and SMF cohorts. In the former, the TP53MT and High-risk groups remained associated with the worst OS (median 58 20-126 vs 55 36-85 months), although with no significant difference, likely due to the low frequency (4%) of TP53MTs mutations in PMF (Fig 1B). Concurrently, the negative prognostic impact of the ASXL1MT-only group was confirmed in comparison to the Others group (median 103 78-NR vs 320 178-NR months; P=.0170). In pts with SMF, while the TP53MT group (6%) had by far the worst OS (median 13 6-NR months), the OS of the ASXL1MT-only group (median 141 56-171 months) was comparable to that of the Others group (median 131 106-NR months; P=.5188) and not different from the High-risk group (median 58 45-174 months; P=.3606) (Fig 1C). In a further analysis including only pts in the High-risk group, ASXL1MTs were found in 62% and 63% of patients with PMF and SMF, respectively. In survival analysis, the presence of ASXL1MTs was associated with an increased risk of death only in PMF (median OS 47 31-73 vs 102 34-317 months; P=.0240), unlike in SMF (median OS 90 47-174 vs 25 16-338 months; P=.3296) (Fig 1D-E).
Conclusion: In the current study, we critically re-addressed the prognostic impact of ASXL1MTs by applying a genetic model recently developed by Luque Paz et al. to our cohort of molecularly annotated, WHO-defined MF pts. Overall, our results confirm that ASXL1MTs -even in the absence of other co-occurring high-risk mutations- harbor a negative prognostic impact mainly in PMF. These findings also reinforce the idea that PMF and SMF represent two different biological entities.
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Vannucchi: Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.
The transcriptional profile of adrenomedullin (AM), a new metastasis-related factor involved in hepatocellular carcinoma (HCC), and its specific receptors (CLR, RAMP1, RAMP3) were evaluated in liver ...tissues of HCV-positive HCC subjects undergoing liver transplantation (LR) and in donors (LD). AM and its specific receptor expression were also assessed in extracellular vesicles (EVs) secreted by tumorigenic (HepG2) and non-tumorigenic (WRL68) cells by Real-Time PCR. AM expression resulted significantly elevated in LR concerning LD (p = 0.0038) and, for the first time, significantly higher levels in HCC patients as a function of clinical severity (MELD score), were observed. RAMP3 and CLR expression increased in LR as a function of clinical severity while RAMP1 decreased. Positive correlations were found among AM, its receptors, and apoptotic markers. No AM mRNA expression difference was observed between HepG2 and WRL68 EVs. RAMP1 and RAMP3 resulted lower in HepG2 concerning WRL68 while significantly higher levels were observed for CLR. While results at tissue level characterize AM as a regulator of carcinogenesis-tumor progression, those obtained in EVs do not indicate AM as a target candidate, neither as a pathological biomarker nor as a marker involved in cancer therapy.
Recent evidence suggested the role of secreted extracellular vesicles (EVs) in the intracellular signalling within the liver becoming a promising candidate as biomarker in hepatocellular carcinoma ...(HCC). Osteopontin (OPN) seems to play a relevant role both for early diagnosis of HCC than on the mechanisms that drive oncogenesis but, to date, information on the expression levels of OPN in EVs secreted by HCC tumor cell line are missing. The study aimed to verify, by transcriptional and proteomic study, the presence of OPN in EVs secreted by tumorigenic (HepG2) and non-tumorigenic hepatocyte cell line (WRL68), and to analyse the expression variations of OPN, its isoforms and miRNA-181a in both these EVs. “In silico analysis” was also performed via the Gene expression Profiling Interactive analysis (GEPIA) and Hepatocellular Carcinoma Database (HCCDB). An up-regulation of OPN in EVs secreted by HepG2 with respect to WRL68 was found in line with the results obtained by the “in silico analysis”. The study demonstrates, for the first time, the OPN isoforms and its modulator miRNA-181a expression in EVs secreted by both cell lines, highlighting high levels of OPN isoforms in EVs secreted by HepG2 and identifying OPN as a promising biomarker for HCC diagnosis.
Osteopontin (OPN), a novel hepatic damage marker, as well as several non-coding RNA seem to be associated with liver aging, a little studied and not yet defined process. The aim of the study was to ...evaluate the expression variations of OPN and miRNA-181a, the transcriptional profiling of long non coding (lnc) RNA GAS-5/miRNA-222 axis and lncRNA NEAT-1 in liver tissue of rats. In addition, to monitor the senescence process, the telomere shortening and TERT/TERC gene expression were also measured.
Three groups of male Wistar rats were studied: A (n = 6, young); B (n = 13, adult); C (n = 10, old). Total RNA, including miRNAs, was extracted from liver and analysed by Real-Time PCR. Ultrasound and biochemical evaluation were performed in all rats as well as the histological analysis.
OPN mRNA resulted lower in C with respect to A and B while miRNA-181a expression was significantly increased as a function of age. An increasing of both NEAT-1 and miRNA-222 expression as a function of age in parallel with a decreasing of GAS-5 expression in young and old rats, but not in the adults, was observed. A positive correlation was detected between miRNA-181a and miRNA-222. The hepatic ultrasound analysis revealed areas of hyperechogenicity distributed as a function of age. A significant telomere shortening was measured as a function of age while the two subunits TERT and TERC expressions showed an opposite trend.
This work could provide a valid starting point to better understand the physiopathological changes during aging, pinpointing in the OPN/miRNA-181a axis significant predictors of successful aging.
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•Aging is attended by a low-grade systemic inflammation.•OPN counter-regulation mediated by miR-181a: hepatic markers of inflamm-aging•GAS-5 is a target of miRNA-222: reciprocal repression regulatory loop.•Low-grade inflammation associated with development of age-related conditions•Multivariate analysis: OPN/miRNA-181a significant predictors of successful aging
Obesity has been implicated in the development of many cancers. This can lead to genome damage, especially in the form of double-strand break, the presence of which is now easily detected through ...nuclear phosphorylation of histone H2AX (γ-H2AX) focus assay. Recently, the endothelin (ET) axis has also been shown to have a role in the growth and progression of several tumors, including lung cancer. The aim of this study was to evaluate the ET-1 system transcriptional alterations and γ-H2AX in lung tissue of Zucker rats subdivided into obese (O, n=22) and controls (CO, n=18) rats: under either fasting conditions (CO(fc)-O(fc)) or acute hyperglycemia (CO(AH)-O(AH)). Significantly higher prepro-ET-1 (p=0.05) and ET-converting enzyme (ECE)-2 mRNA expression was observed in O with respect to CO. A significant positive association was observed between prepro-ET-1 and ET-A in the whole rat population (p=0.009) or in the obese group alone (p=0.007). The levels of γ-H2AX in O and in O(AH) rats were significantly higher (p=0.019) than in the corresponding CO and CO(AH) rats (p=0.038). The study shows an inappropriate secretion of ET-1 in O animals with a parallel DNA damage in their lungs, providing novel mechanisms by which ET receptor antagonist may exert organ protection.
: Many prognostic factors for CDH patients are described and validated in the current literature: the size of diaphragmatic defects, need for patch repair, pulmonary hypertension and left ventricular ...dysfunction are recognized as the most influencing outcomes. The aim of this study is to analyze the influence of these parameters in the outcome of CDH patients in our department and identify any further prognostic factors.
: An observational retrospective single-center study was conducted including all patients treated at our centre with posterolateral CDH between 01.01.1997 and 12.31.2019. The main outcomes evaluated were mortality and length of hospital stay. A univariate and multivariate analysis was performed.
: We identified 140 patients with posterolateral CDH; 34.8% died before discharge. The overall median length of stay was 24 days. A univariate analysis confirmed that both outcomes are associated with the size of diaphragmatic defects, need for patch repair and presence of spleen-up (
< 0.05). A multivariate analysis identified that the need for patch repair and maximum dopamine dose used for cardiac dysfunction are independent parameters associated with the length of stay only (
< 0.001).
: In our series, the duration of hospitalization is longer for newborns with CDH treated with higher doses of dopamine for left ventricular dysfunction or needing patch repair in large diaphragmatic defects.
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