Molecular profile of the NF-κB signalling pathway in human colorectal cancer.

The development and progression of colorectal cancer (CRC) have been associated with inflammation processes that involve the overactivation of the NF-κB signalling pathway. The characterization of the NF-κB expression profile in CRC is an important topic since the suppression of NF-κB represents a potential therapeutic approach. In this study, we assessed the expression levels of 84 NF-κB-related genes in paired tumoral (T) and peritumoral (PT) tissues from 18 CRC patients and 18 normal colonic mucosae, and the expression levels of three miRNAs targeting the most dysregulated genes revealed by the case-control analysis. Comparing the gene expression profile of T and controls, 60 genes were dysregulated. The comparison of T and PT revealed 17 dysregulated genes in the tumoral tissues, with IL1B, CXCL8, IL1A, and CSF2 being the most upregulated. Notably, through a bioinformatics analysis, the differential gene expression of 11 out of the 17 genes was validated on a larger cohort of 308 CRC patients compared with 41 controls. Moreover, a decrease in the levels of RELA, NOD1, CASP8, BCL2L1, ELK1, and IKBKB was identified in poorly differentiated tumours compared to moderately differentiated tumours. The analysis of the three miRNAs targeting IL1B, CXCL8, IL1A, and CSF2 showed that miR-182-5p was upregulated in T compared with PT, whereas miR-10b-5p was downregulated in T compared with PT and control tissues. Our results may contribute to the design of new experimental therapeutic strategies based on endogenous molecules, such as miRNAs, to target the genetic key players of the NF- κB pathway.

Expression of Cellular and Extracellular TERRA, TERC and TERT in Hepatocellular Carcinoma.

Non-coding RNAs are transcribed from telomeres and the telomeric repeat-containing RNAs (TERRA) are implicated in telomere homeostasis and in cancer. In this study, we aimed to assess in hepatocellular carcinoma (HCC) the cellular and extracellular expression of TERRA, the telomerase RNA subunit (TERC) and the telomerase catalytic subunit (TERT). We determined by qPCR the expression level of TERRA 1_2_10_13q, TERRA 15q, TERRA XpYp, TERC and of TERT mRNA in HCC tissues and in the plasma of HCC patients. Further, we profiled the same transcripts in the HCC cell lines, HA22T/VGH and SKHep1C3, and in the extracellular vesicles (EVs) derived from their secretomes. We found that the expression of TERRA and TERT mRNA was significantly deregulated in HCC, being TERRA downregulated and TERT mRNA upregulated in HCC tissues vs. the peritumoral (PT) ones, and the receiver operating characteristic (ROC) curve analyses revealed a significant ability in discriminating HCC from PT tissue. Further, the determinations of circulating TERRA and TERC showed higher amounts of these transcripts in the plasma of HCC patients vs. controls and ROC analyses gave significant results. The expression characterization of the cultured HCC cells showed their ability to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that was not detectable in the EVs; and the ability to respond to sorafenib treatment increasing TERRA expression. Our results highlight that: (i) both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC as well as the cellular expression of TERT mRNA and (ii) the combined detection of TERRA and TERC in plasma may represent a promising approach for non-invasive diagnostic molecular indicators of HCC.

Study of the in vitro modulation exerted by the antidepressant drug escitalopram on the expression of candidate microRNAs and their target genes.

Recent studies indicated a role of microRNAs (miRNAs, small non-coding RNAs which regulate the expression of target genes by acting on mRNAs) in several neural processes, in the pathogenetic mechanisms of neuropsychiatric diseases and in the action of psychotropic drugs. A modulation induced by the antidepressant drug escitalopram on the expression levels of 30 miRNAs was highlighted in the blood of patients suffering from major depressive disorder. With the aim to investigate the effects of escitalopram in an in vitro model, we performed an analysis of the effects produced by escitalopram on the profiles of the 6 miRNAs found to be more significantly modulated in the above-mentioned study (miR-130b, miR-26a and -26b, let-7f, miR-770-5p, miR-34c-5p) in human U87 glioblastoma cells. Cells were treated with the drug for 24, 48 and 72h. The obtained results confirmed a significant increase of let-7f, both after 48 (p=0.031) and 72h (p=0.022), and of miR-26a after 48h (p=0.032). On the same experimental model, a transcriptome analysis was conducted after 72h, highlighting a drug-induced modulation of 1184 protein-coding genes, 207 of which represent let-7f targets. Particularly interesting was the downregulation of BCOR, CCND1 and ATR, validated let-7f targets, which play a key role in the mechanisms of neurogenesis, neuroplasticity and protection from oxidative stress in the brain, indicating that escitalopram could exert downstream effects on gene expression through the regulation of specific miRNAs.

Cultured human amniocytes express hTERT, which is distributed between nucleus and cytoplasm and is secreted in extracellular vesicles.

BACKGROUND: An increasing number of studies on stem cells suggests that the therapeutic effect they exert is primarily mediated by a paracrine regulation through extracellular vesicles (EVs) giving solid grounds for stem cell EVs to be exploited as agents for treating diseases or for restoring damaged tissues and organs. Due to their capacity to differentiate in all embryonic germ layers, amniotic fluid stem cells (AFCs), represent a highly promising cell type for tissue regeneration, which however is still poorly studied and in turn underutilized. In view of this, we conducted a first investigation on the expression of human hTERT gene - known to be among the key triggers of organ regeneration - in AFCs and in the EVs they secrete. METHODS: Isolated AFCs were evaluated by RT-qPCR for hTERT expression. The clones expressing the highest levels of transcript, were analyzed by Immunofluorescence imaging and Nuclear/cytoplasmic fractionation in order to evaluate hTERT subcellular localization. We then separated EVs from FBS depleted culture medium by serial (ultra) centrifugations steps and characterized them using Western blotting, Atomic force Microscopy and Nanoplasmonic assay. RESULTS: We first demonstrated that primary cultures of AFCs express the gene hTERT at different levels. Then we evidenced that in AFCs with the higher transcript levels, the hTERT protein is present in the nuclear and cytoplasmic compartment. Finally, we found that cytosolic hTERT is embodied in the EVs that AFCs secrete in the extracellular milieu. CONCLUSIONS: Our study demonstrates for the first time the expression of the full protein hTERT by AFCs and its release outside the cell mediated by EVs, indicating a new extra telomeric role for this protein. This finding represents an initial but crucial evidence for considering AFCs derived EVs as new potential sources for tissue regeneration.

Long-term evolution and prognostic stratification of biopsy-proven active myocarditis.

BACKGROUND: Active myocarditis is characterized by large heterogeneity of clinical presentation and evolution. This study describes the characteristics and the long-term evolution of a large sample of patients with biopsy-proven active myocarditis, looking for accessible and valid early predictors of long-term prognosis. METHODS AND RESULTS: From 1981 to 2009, 82 patients with biopsy-proven active myocarditis were consecutively enrolled and followed-up for 147±107 months. All patients underwent clinical and echocardiographic evaluation at baseline and at 6 months. At this time, improvement/normality of left ventricular ejection fraction (LVEF), defined as a LVEF increase > 20 percentage points or presence of LVEF≥50%, was assessed. At baseline, left ventricular dysfunction (LVEF<50%) and left atrium enlargement were independently associated with long-term heart transplantation-free survival, regardless of the clinical pattern of disease onset. At 6 months, improvement/normality of LVEF was observed in 53% of patients. Persistence of New York Heart Association III to IV classes, left atrium enlargement, and improvement/normality of LVEF at 6 months emerged as independent predictors of long-term outcome. Notably, the short-term reevaluation showed a significant incremental prognostic value in comparison with the baseline evaluation (baseline model versus 6 months model: area under the curve 0.79 versus 0.90, P=0.03). CONCLUSIONS: Baseline left ventricular function is a marker for prognosis regardless of the clinical pattern of disease onset, and its reassessment at 6 months appears useful for assessing longer-term outcome.

Evaluation of DNA methylation levels of SEPT9 and SHOX2 in plasma of patients with head and neck squamous cell carcinoma using droplet digital PCR.

Head and neck squamous cell carcinoma (HNSCC) is the seventh most commonly diagnosed cancer globally. HNSCC develops from the mucosa of the oral cavity, pharynx and larynx. Methylation levels of septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) genes in circulating cell­free DNA (ccfDNA) are considered epigenetic biomarkers and have shown predictive value in preliminary reports in HNSCC. Liquid biopsy is a non­invasive procedure that collects tumor­derived molecules, including ccfDNA. In the present study, a droplet digital PCR (ddPCR)­based assay was developed to detect DNA methylation levels of circulating SEPT9 and SHOX2 in the plasma of patients with HNSCC. The assay was first set up using commercial methylated and unmethylated DNA. The dynamic changes in the methylation levels of SEPT9 and SHOX2 were then quantified in 20 patients with HNSCC during follow­up. The results highlighted: i) The ability of the ddPCR­based assay to detect very low copies of methylated molecules; ii) the significant decrease in SEPT9 and SHOX2 methylation levels in the plasma of patients with HNSCC at the first time points of follow­up with respect to T0; iii) a different trend of longitudinally DNA methylation variations in small groups of stratified patients. The absolute and precise quantification of SEPT9 and SHOX2 methylation levels in HNSCC may be useful for studies with translational potential.

Dysregulation of a Subset of Circulating and Vesicle-Associated miRNA in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC.

Effects of miR-193a and sorafenib on hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is a challenging malignancy of global importance, it is the third most common cause of cancer-related mortality worldwide. In the last years the multikinase inhibitor sorafenib has been used for advanced HCC, but some patients do not benefit from this therapy; thus, novel therapeutic options based on molecular approaches are urgently needed. microRNAs are short non coding RNAs involved in several physiological and pathological conditions including HCC and increasing evidence describes miRs as good tools for the molecular targeted therapies in HCC. The purpose of this study was to identify novel approaches to sensitize the HCC cells to sorafenib by microRNAs targeting urokinase-type plasminogen activator (uPA). METHODS: The miR-193a was validated as negative regulator of urokinase-type plasminogen activator (uPA) in 2 HCC undifferentiated cell lines by transient transfection of miR and anti-miR molecules. The molecular interaction between miR-193a and uPA mRNA target was verified by luciferase reporter assay. The miR-193a expression level was evaluated by stem-loop real time PCR in tumoral tissues from 39 HCC patients. The HCC cells were co-treated with sorafenib and miR-193a and the effects on cellular proliferation, apoptosis were tested. The effect of sorafenib on c-met expression levels was assessed by western blotting. RESULTS: The miR-193a has resulted a negative regulator of uPA in both the HCC cell lines tested. The miR-193a expression has resulted dysregulated in tumoral tissues from 39 HCC patients. We found miR-193a down-regulation in HCC respect to peritumoral (PT) tissues and more in the cirrhotic HCCs than in non-cirrhotic ones. Transfection of HA22T/VGH HCC cells with miR-193a decreased proliferation and increased apoptosis, and combined treatment with miR-193a and sorafenib led to further proliferation inhibition. CONCLUSIONS: Our results present new advances in the post-transcriptional miR-mediated mechanisms of uPA and they suggest a new strategy to impair the aggressive behavior of HCC cells. Our findings could be helpful to explore novel approaches for multi-target and multi-agent therapies of the HCC.

Synergistic effect of thrombus aspiration and abciximab in primary percutaneous coronary intervention.

BACKGROUND: Previous studies failed to assess the individual prognostic role of thrombus aspiration (TA) or abciximab in primary percutaneous coronary intervention (pPCI), due their prevalent combined use. METHODS AND RESULTS: A total of 644 consecutive ST-segment elevation myocardial infarction patients treated with pPCI were included in this retrospective registry from January 2006 to December 2008. Patients were divided in: (a) Group 1, with conventional pPCI; (b) Group 2, with pPCI and abciximab; (c) Group 3, with pPCI and TA; (d) Group 4, with pPCI and abciximab plus TA. Primary end point was the composite of major adverse cardiovascular events (MACEs, defined as overall mortality, myocardial infarction, target vessel revascularization, and major bleedings) at 1 year. Baseline clinical and angiographic characteristics were not different among the groups, with the exception of a younger age in group 4. The two groups of patients treated with TA (group 3 and 4) received more frequently direct stenting (P < 0.001 vs. group 1 for both), presented higher rate of end-procedural TIMI flow grade 3 (P < 0.001 vs. group 1 for both), and lower rate of no-reflow (P = 0.016 and P < 0.001 vs. group 1, respectively). Patients of group 2 presented a borderline nonsignificant trend toward higher rate of end-procedural TIMI flow grade 3 (P = 0.083 vs. group 1). MACEs at 1 year were 43 (29%) in group 1 versus 25 (22%) in group 2 versus 24 (19%) in group 3 versus 32 (13%) in group 4 (log-rank P = 0.001). At the multivariate Cox regression analysis, combined TA plus abciximab in group 4 [hazard ratio (HR): 0.48, confidence interval (CI) 95% 0.28-0.84, P = 0.01] and a higher left ventricular ejection fraction (HR: 0.97, CI 95% 0.95-0.98, P < 0.001) were significantly associated with lower MACE rate. CONCLUSIONS: The combination of pharmacologic and mechanic antithrombotic treatment during pPCI was associated with better 1-year clinical outcome.

The Biological Role and Translational Implications of the Long Non-Coding RNA GAS5 in Breast Cancer.

The lncRNA GAS5 plays a significant role in tumorigenicity and progression of breast cancer (BC). In this review, we first summarize the role of GAS5 in cell biology, focusing on its expression data in human normal tissues. We present data on GAS5 expression in human BC tissues, highlighting its downregulation in all major BC classes. The main findings regarding the molecular mechanisms underlying GAS5 dysregulation are discussed, including DNA hypermethylation of the CpG island located in the promoter region of the gene. We focused on the action of GAS5 as a miRNA sponge, which is able to sequester microRNAs and modulate the expression levels of their mRNA targets, particularly those involved in cell invasion, apoptosis, and drug response. In the second part, we highlight the translational implications of GAS5 in BC. We discuss the current knowledge on the role of GAS5 as candidate prognostic factor, a responsive molecular therapeutic target, and a circulating biomarker in liquid biopsies with clinical importance in BC. The findings position GAS5 as a promising druggable biomolecule and stimulate the development of strategies to restore its expression levels for novel therapeutic approaches that could benefit BC patients in the future.

Idiopathic dilated cardiomyopathy and persistent viral infection: lack of association in a controlled study using a quantitative assay.

BACKGROUND: It remains unclear whether idiopathic dilated cardiomyopathy (DCM) might ensue as the consequence of viral myocarditis, due to viral persistence in cardiomyocytes. To address this issue, we quantified the levels of enterovirus, Epstein-Barr virus (EBV), Herpes Simplex Virus-1 (HSV-1), Herpes Simplex Virus-2 (HSV-2), adenovirus and parvovirus B19 genomes in endomyocardial biopsies (EMBs) from patients with DCM, active myocarditis and controls. METHODS: Real-time polymerase chain reaction (PCR)-based methods using TaqMan probes were developed for the quantitative detection of viral genomes in EMBs from 35 patients with DCM and 17 with active myocarditis. A control group included 20 surgical patients with valve or coronary artery disease. RESULTS: None of the 72 samples tested positive for enteroviruses, EBV, HSV-1 or -2. One DCM patient tested positive for adenovirus. Of notice, 20/52 (38%) of patients with cardiomyopathy and 8/20 (40%) of controls were positive for parvovirus B19; no significant differences in viral titre were detected between groups. CONCLUSIONS: Our preliminary results disfavour the hypothesis that persistent myocardial viral infection might be a frequent cause of DCM. The detection of parvovirus B19 from both cardiomyopathy and non-cardiomyopathy patients supports the notion that this virus is widely spread in the population.

Lasp1 Expression Is Implicated in Embryonic Development of Zebrafish.

The LIM and SH3 domain protein 1 (LASP1) was originally identified in metastatic breast cancer and mainly characterized as a cytoskeleton protein overexpressed in various cancer types. At present, little is known about LASP1 expression in physiological conditions, and its function during embryonic development has not been elucidated. Here, we focused on Lasp1 and embryonic development, choosing zebrafish as a vertebrate model. For the first time, we identified and determined the expression of Lasp1 protein at various stages of development, at 48 and 72 h post-fertilization (hpf), at 6 days pf and in different organs of zebrafish adults by Western blotting, 3D light-sheet microscopy and fluorescent immunohistochemistry. Further, we showed that specific lasp1 morpholino (MO) led to (i) abnormal morphants with alterations in several organs, (ii) effective knockdown of endogenous Lasp1 protein and (iii) an increase in lasp1 mRNA, as detected by ddPCR. The co-injection of lasp1 mRNA with lasp1 MO partially rescued morphant phenotypes, thus confirming the specificity of the MO oligonucleotide-induced defects. We also detected an increase in apoptosis following lasp1 MO treatment. Our results suggest a significant role for Lasp1 in embryonic development, highlighting zebrafish as a vertebrate model suitable for studying Lasp1 function in developmental biology and organogenesis.

Crosstalk Between DNA Methylation and Gene Mutations in Colorectal Cancer.

Colorectal cancer (CRC) is often characterized by mutations and aberrant DNA methylation within the promoters of tumor suppressor genes and proto-oncogenes. The most frequent somatic mutations occur within KRAS and BRAF genes. Mutations of the KRAS gene have been detected in approximately 40% of patients, while mutations in BRAF have been detected less frequently at a rate of 10%. In this study, the DNA methylation levels of 22 candidate genes were evaluated in three types of tissue: mucosal tumoral tissue from 18 CRC patients, normal adjacent tissues from 10 CRC patients who underwent surgical resection, and tissue from a control group of six individuals with normal colonoscopies. A differential methylation profile of nine genes (RUNX3, SFRP1, WIF1, PCDH10, DKK2, DKK3, TMEFF2, OPCML, and SFRP2) presenting high methylation levels in tumoral compared to normal tissues was identified. KRAS mutations (codons 12 or 13) were detected in eight CRC cases, and BRAF mutations (codon 600) in four cases. One of the CRC patients presented concomitant mutations in KRAS codon 12 and BRAF, whereas seven patients did not present these mutations (WT). When comparing the methylation profile according to mutation status, we found that six genes (SFRP2, DKK2, PCDH10, TMEFF2, SFRP1, HS3ST2) showed a methylation level higher in BRAF positive cases than BRAF negative cases. The molecular sub-classification of CRC according to mutations and epigenetic modifications may help to identify epigenetic biomarkers useful in designing personalized strategies to improve patient outcomes.

MicroRNA­34a­5p expression in the plasma and in its extracellular vesicle fractions in subjects with Parkinson's disease: An exploratory study.

Parkinson's disease (PD) is an important disabling age­related disorder and is the second most common neurodegenerative disease. Currently, no established molecular biomarkers exist for the early diagnosis of PD. Circulating microRNAs (miRNAs), either vesicle­free or encapsulated in extracellular vesicles (EVs), have emerged as potential blood­based biomarkers also for neurodegenerative diseases. In this exploratory study, we focused on miR­34a­5p because of its well­documented involvement in neurobiology. To explore a differential profile of circulating miR­34a­5p in PD, PD patients and age­matched control subjects were enrolled. Serial ultracentrifugation steps and density gradient were used to separate EV subpopulations from plasma according to their different sedimentation properties (Large, Medium, Small EVs). Characterization of EV types was performed using western blotting and atomic force microscopy (AFM); purity from protein contaminants was checked with the colorimetric nanoplasmonic assay. Circulating miR­34a­5p levels were evaluated using qPCR in plasma and in each EV type. miR­34a­5p was significantly up­regulated in small EVs devoid of exogenous protein contaminants (pure SEVs) from PD patients and ROC analysis indicated a good diagnostic performance in discriminating patients from controls (AUC=0.74, P<0.05). Moreover, miR­34a­5p levels in pure SEVs were associated with disease duration, Hoehn and Yahr and Beck Depression Inventory scores. These results underline the necessity to examine the miRNA content of each EV subpopulation to identify miRNA candidates with potential diagnostic value and lay the basis for future studies to validate the overexpression of circulating miR­34a­5p in PD via the use of pure SEVs.

Longitudinal Circulating Levels of miR-23b-3p, miR-126-3p and lncRNA GAS5 in HCC Patients Treated with Sorafenib.

Human hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The multikinase inhibitor sorafenib is a systemic drug for unresectable HCC. The identification of molecular biomarkers for the early diagnosis of HCC and responsiveness to treatment are needed. In this work, we performed an exploratory study to investigate the longitudinal levels of cell-free long ncRNA GAS5 and microRNAs miR-126-3p and -23b-3p in a cohort of 7 patients during the period of treatment with sorafenib. We used qPCR to measure the amounts of GAS5 and miR-126-3p and droplet digital PCR (ddPCR) to measure the levels of miR-23b-3p. Patients treated with sorafenib displayed variable levels of GAS5, miR-126-3p and miR-23b-3p at different time-points of follow-up. miR-23b-3p was further measured by ddPCR in 37 healthy individuals and 25 untreated HCC patients. The amount of miR-23b-3p in the plasma of untreated HCC patients was significantly downregulated if compared to healthy individuals. The ROC curve analysis underlined its diagnostic relevance. In conclusion, our results highlight a potential clinical significance of circulating miR-23b-3p and an exploratory observation on the longitudinal plasmatic levels of GAS5, miR-126-3p and miR-23b-3p during sorafenib treatment.

DNA methylation variations in familial female and male breast cancer.

In total, ~25% of familial breast cancer (BC) is attributed to germline mutations of the BRCA1 and BRCA2 genes, while the rest of the cases are included in the BRCAX group. BC is also known to affect men, with a worldwide incidence of 1%. Epigenetic alterations, including DNA methylation, have been rarely studied in male breast cancer (MBC) on a genome-wide level. The aim of the present study was to examine the global DNA methylation profiles of patients with BC to identify differences between familial female breast cancer (FBC) and MBC, and according to BRCA1, BRCA2 or BRCAX mutation status. The genomic DNA of formalin-fixed paraffin-embedded tissues from 17 women and 7 men with BC was subjected to methylated DNA immunoprecipitation and hybridized on human promoter microarrays. The comparison between FBC and MBC revealed 2,846 significant differentially methylated regions corresponding to 2,486 annotated genes. Gene Ontology enrichment analysis revealed molecular function terms, such as the GTPase superfamily genes (particularly the GTPase Rho GAP/GEF and GTPase RAB), and cellular component terms associated with cytoskeletal architecture, such as 'cytoskeletal part', 'keratin filament' and 'intermediate filament'. When only FBC was considered, several cancer-associated pathways were among the most enriched KEGG pathways of differentially methylated genes when the BRCA2 group was compared with the BRCAX or BRCA1+BRCAX groups. The comparison between the BRCA1 and BRCA2+BRCAX groups comprised the molecular function term 'cytoskeletal protein binding'. Finally, the functional annotation of differentially methylated genes between the BRCAX and BRCA1+BRCA2 groups indicated that the most enriched molecular function terms were associated with GTPase activity. In conclusion, to the best of our knowledge, the present study was the first to compare the global DNA methylation profile of familial FBC and MBC. The results may provide useful insights into the epigenomic subtyping of BC and shed light on a possible novel molecular mechanism underlying BC carcinogenesis.

H-ferritin suppression and pronounced mitochondrial respiration make Hepatocellular Carcinoma cells sensitive to RSL3-induced ferroptosis.

Ferroptosis is a form of regulated cell death dependent on iron, reactive oxygen species and characterized by the accumulation of lipid peroxides. It can be experimentally initiated by chemicals, such as erastin and RSL3, that modulate GPX4 activity, the cellular antioxidant machinery that avert lipid peroxidation. The study aimed to investigate mitochondrial respiration and ferritin function as biomarkers of ferroptosis sensitivity of HepG2 and HA22T/VGH, two Hepatocellular Carcinoma (HCC) cell line models. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, labile iron levels were determined using Calcein-AM fluorescence microscopy, ferritin, glutathione and lipid peroxidation were assayed with commercially available kits. The Seahorse assay was used to investigate mitochondrial function in the cells. The study shows that highly differentiated HepG2 cells were more sensitive to RSL3-induced ferroptosis than the poorly differentiated HA22T/VGH (HCC) cell line (RSL3 IC50 0.07 µM in HepG2 vs 0.3 µM in HA22T/VGH). Interestingly, HepG2 exhibited higher mitochondrial respiration and lower glycolytic activity than HA22T/VGH and were more sensitive to RSL3-induced ferroptosis, indicating a mitochondrial-specific mechanism of action of RSL3. Interestingly, iron metabolism seems to be involved in this different sensitivity, specifically, the downregulation of H-ferritin (but not of L-subunit), makes HA22T/VGH more sensitive toward both RSL3-and iron-induced ferroptosis. Hence only the H-ferritin seems involved in the protection from this cell death process.

Inherited duplication of the pseudoautosomal region Xq28 in a subject with Gilles de la Tourette syndrome and intellectual disability: a case report.

BACKGROUND: Tourette syndrome (TS) is a complex neurodevelopmental disorder (NDD) characterized by multiple chronic involuntary motor and vocal tics with onset during childhood or adolescence. Most TS patients present with additional comorbidities, typically attention deficit hyperactivity disorder (ADHD), obsessive- compulsive disorder (OCD), autism spectrum disorder (ASD) and intellectual disability (ID). Both TS and ID are genetically complex disorders that likely occur as a result of the effects of multiple genes interacting with other environmental factors. In addition to single gene mutations and chromosomal disorders, copy number variations (CNVs) are implicated across many NDDs and ID and contribute to their shared genetic etiology. Screening of CNVs using microarray-based Comparative Genomic Hybridization (aCGH) is now routinely performed in all subjects with NDD and ID. CASE PRESENTATION: We report a case of a 12-year-old girl diagnosed with Gilles de la Tourette Syndrome associated to behavior disorders and intellectual disability in particular with regard to language. Array-CGH analysis showed a CNV of a subtelomeric region Xq28 (gain of 260 kb) inherited from the healthy father. The duplication contains two genes, VAMP7 and SPRY3 of the PAR2 pseudoautosomal region. FISH analysis revealed that the duplicated segment is located on the short arm of a chromosome 13, resulting in a trisomy of the region. In the proband the expression levels of the genes evaluated in the peripheral blood sample are comparable both those of the mother and to those of female control subjects. CONCLUSIONS: Although the trisomy of the 260 kb region from Xq28 identified in proband is also shared by the healthy father, it is tantalizing to speculate that, together with genetic risk factors inherited from the mother, it may play a role in the development of a form of Tourette syndrome with intellectual disability. This hypothesis is also supported by the fact that both genes present in the duplicated region (VAMP7 and SPRY3) are expressed in the CNS and are implicated in neurotransmission and neurite growth and branching. In addition, similar CNVs have been identified in individuals whose phenotype is associated with autism spectrum disorders or intellectual disability.

miRNAs-Based Molecular Signature for KRAS Mutated and Wild Type Colorectal Cancer: An Explorative Study.

microRNAs (miRNAs) have been proposed as promising molecular biomarkers for diagnosis, prognosis, and responsive therapeutic targets in different types of cancer, including colorectal cancer (CRC). In this study, we evaluated the expression levels of 84 cancer-associated miRNAs in a cohort of 39 human samples comprising 13 peritumoral and 26 tumoral tissues from surgical specimens of CRC patients. KRAS mutations were detected in 11 tumoral samples. In a first analysis, we found 5 miRNAs (miR-215-5p, miR-9-5p, miR-138-5p, miR378a-3p, and miR-150-5p) that were significantly downregulated and one upregulated (miR-135b-5p) in tumoral tissues compared with the peritumoral tissues. Furthermore, by comparing miRNA profile between KRAS mutated CRC tissues respect to wild type CRC tissues, we found 7 miRNA (miR-27b-3p, miR-191-5p, miR-let7d-5p, miR-15b-5p, miR-98-5p, miR-10a-5p, and miR-149-5p) downregulated in KRAS mutated condition. In conclusion, we have identified a panel of miRNAs that specifically distinguish CRC tissues from peritumoral tissue and a different set of miRNAs specific for CRC with KRAS mutations. These findings may contribute to the discovering of new molecular biomarkers with clinic relevance and might shed light on novel molecular aspects of CRC.

Differential expression profiling of long non-coding RNA GAS5 and miR-126-3p in human cancer cells in response to sorafenib.

Long non-coding RNAs (lncRNAs) and microRNAs are involved in numerous physio-pathological conditions included cancer. To better understand the molecular mechanism of the oral antitumor multikinase inhibitor sorafenib, we profiled the expression of a panel of lncRNAs and miRNAs by qPCR array in a sorafenib-treated hepatocellular carcinoma (HCC) cell line. Among the most affected ncRNAs, we found that sorafenib mediated the dysregulation of the lncRNAs GAS5, HOTTIP and HOXA-AS2 and the miR-126-3p, in a panel of human cancer cell lines (HCC, renal and breast carcinomas). By luciferase gene reporter assay, we discovered that GAS5 may act as a sponge for miR-126-3p in HCC cells. The expression level of GAS5 and miR-126-3p was verified in human liquid and/or solid biopsies from HCC patients. miR-126-3p expression in HCC tissues was decreased respect to their correspondent peritumoral tissues. The levels of plasmatic circulating miR-126-3p and GAS5 were significantly higher and lower in HCC patients compared to healthy subjects, respectively. This study highlighted the capability of sorafenib to modulate the expression of a wide range of ncRNAs and specifically, GAS5 and miR-126-3p were involved in the response to sorafenib of different cancer cell types.