113 research outputs found
Silencing efficacy prediction: a retrospective study on target mRNA features
No full textPost-transcriptional gene silencing is a widely used method to suppress gene expression. Unfortunately only a portion of siRNAs do successfully reduce gene expression. Target mRNA secondary structures and siRNA-mRNA thermodynamic features are believed to contribute to the silencing activity. However, there is still an open discussion as to what determines siRNA efficacy. In this retrospective study, we analysed the target accessibility comparing very high (VH) compared with low (L) efficacy siRNA sequences obtained from the siRecords Database. We determined the contribution of mRNA target local secondary structures on silencing efficacy. Both the univariable and the multivariable logistic regression evidenced no relationship between siRNA efficacy and mRNA target secondary structures. Moreover, none of the thermodynamic and sequence-base parameters taken into consideration (H-b index, ΔG°overall, ΔG°duplex, ΔG°break-target and GC%) was associated with siRNA efficacy. We found that features believed to be predictive of silencing efficacy are not confirmed to be so when externally evaluated in a large heterogeneous sample. Although it was proposed that silencing efficacy could be influenced by local target accessibility we show that this could be not generalizable because of the diversity of experimental setting that may not be representative of biological systems especially in view of the many local protein factors, usually not taken into consideration, which could hamper the silencing process
MiR-3201 as a Prognostic Blood Biomarker for Curative Treatments in Hepatocellular Carcinoma
No full textBackground: Hepatic resection, radiofrequency ablation (RF), and liver transplantation (LT) represent the only available curative treatments for early stage hepatocellular carcinoma (HCC). Various studies showed that the 5-year overall survival (OS) rate reaches ∼70% after resection and ∼60% after RF. Objective: To improve the success rate of curative therapies and consequently the OS, an improvement in patients’ selection and management should be pursued. In this regard, microRNAs (miRNAs) can be helpful prognostic biomarkers. Materials and Methods: In this retrospective study, a miRNA array profiling was performed on 34 HCC blood samples which is collected before therapy (T0), 1 month (T1), and 6 months (T2) after curative treatments (resection and RF) to identify noninvasive biomarker candidates for therapy response and OS. MiRNAs were validated in 80 blood HCC samples using quantitative real-time PCR (qRT-PCR). Patients were divided into complete responder (CR) and partial responder and progressive disease (PRPD). Results: Among the selected miRNAs, miR-3201 is significantly associated with treatment response in the validation phase, showing a 23% reduction (P = .026) in CR compared to PRPD. MiR-3201 was able to distinguish CR from PRPD (area under the curve [AUC] = 0.69, 71% sensitivity, 70% specificity, P = .0036). Furthermore, lower levels of miR-3201 were associated with longer OS (hazard ratio [HR] = 2.61, P = .0006). Conclusions: Blood miR-3201 could be used as a prognostic biomarker for curative therapy response and OS in HCC
Multidrug resistance in hepatic cancer stem cells: the emerging role of miRNAs
No full textThere is a fast growing body of evidence that shows several miRNAs are essential to the key features of cancer stem cells (CSC) in hepatocellular carcinoma. However, the function of the miRNAs in different hepatic CSCs and their role in multidrug resistance mechanisms, in particular in those related to the CSC marker ABCG2, is still poorly understood. This limitation is mainly due to the heterogeneity of hepatocellular carcinoma tissues, different CSC markers and high number of deregulated miRNAs, both in primary tumor sites as well as in the circulation. The identification of CSC-related miRNAs and the modulation of hepatocellular carcinoma multidrug resistance would provide a systemic approach in the management of the disease
Molecular insights for overcoming Hepatocellular Carcinoma
No full text2010/2011SUMMARY
Introduction. Hepatocellular Carcinoma (HCC) ranks fifth in frequency of cancers in the world. Orthotopic Liver Transplantation (OLT) or liver resection represents the best treatments for HCC. However, most patients cannot be subjected to potential curative OLT or resection because of extensive tumor involvement of the liver, metastasis, invasion of the portal vein or advanced underlying hepatocellular disease at the time of diagnosis. Systemic chemotherapy or chemoembolization represent a good alternative for the treatment, however drug therapy of cancer in general is hampered by multidrug resistance (MDR) that is a phenomenon caused by the up-regulation of the ABC-transporters (ABC) leading to chemotherapy failure.
To overcome these problems new therapeutic approaches, such gene therapy, are needed. Selective down-regulation of an essential and specific cancer gene such as telomerase (hTERT) could represent an emerging strategy that could prevent cancer progression and diminish numerous side effects derived from drug usage.
The present study include two tasks whose aims are:
Task 1: a) Assess if the extent of tumoral differentiation results in a different ABCB1, ABCC1 and ABCG2 expression.
b) Assess whether the treatment with a chemotherapeutic drug(s) may affect the expression of the three ABC transporters under study.
Task 2: To overcome the obstacle of MDR-induced chemoresistance using new therapeutic approaches such as gene therapy, silencing a cancer essential and specific gene.
Results and discussion. Task 1: We assessed the ABCB1, ABCC1 and ABCG2 expression in three hepatic cell lines: IHH (non tumoral control), HuH7 (differentiated tumoral cells) and JHH6 (undifferentiated tumoral cells). Only ABCG2 expression correlates with the degree of tumoral differentiation.
Through confocal microscopy analysis we observed that the Doxorubicin (Dox) is able to reach the cell’s nucleus within 10 min. After 24h and 48h Dox is completely concentrated into the nucleus where some nuclear damage occurs. The presence of damaged nuclei could explain the decreased mRNA in most of the ABCs under study. The treatment with Dox doses lower than the LC50 for 24h and 48h has different consequences for all the ABC considered in the three cell lines, with an mRNA expression pattern not in line with the protein one in most of the cases, suggesting that the possible mechanism that determines the ABCs protein upregulation in the tumoral cell lines (HuH7 and JHH6) is not the de-novo transcription but probably something related to the protein turnover.
After the treatment ABCC1 protein expression increases in the tumoral cell lines but not in the non tumoral one (IHH). Regarding ABCB1 and ABCG2, these transporters seem to play a role only in HuH7 and JHH6 cells respectively. We were not able to correlate the tumorigenic potential of the two tumoral cell lines with the ABC expression since the different behaviour of ABCs and the different contribution to MDR. Thus in order to better clarify the contribution of each single ABC to MDR our future steps will consider the use specific inhibitors.
Task 2: From our in vivo data, among four cancer related genes we selected hTERT as the best candidate for silencing experiments due to its exclusive expression in tumoral samples. A functional non-inflammatory siRNA targeting hTERT was designed: SirTel 1.
Silencing experiments were conducted in JHH6 cell line. The hTERT silencing effect was dose dependent, at least at the three considered doses (25-50-100nM). For all the subsequent determinations the experimental concentration was 25nM. After 72h of silencing we observed a significant reduction in both hTERT mRNA expression and enzymatic activity (p<0.001).
The effects observed in the cells after silencing are:
- Morphological changes, from a fibroblast-like to an hepatocyte-like shape;
- Increased albumin expression. The expression of this hepatic hallmark increases after silencing in JHH6 cells that, due to their poor degree of differentiation, at basal conditions do not express quantifiable levels of albumin. The peak of the higher albumin expression corresponds to the maximum hTERT silencing effect.
- Decreased cell viability (p<0.01). Interestingly, the siRNA induced a reduction in cell viability higher than Dox.
- Cell cycle arrest in G1 phase (p<0.01).
All data were validated using a hTERT negative cell line (primary culture of human fibroblast).
After 72h silencing, we observed that hTERT expression reaches its minimum, and the expression is recovered after 264h although it does not reach the initial expression levels. Re-exposing the cells to additional 25nM of siRNA induces a reduction of mRNA levels by 76% compared to the amount already present after the first treatment.
Taken together all this results suggest the pivotal role of hTERT silencing in a HCC derived cell line. Therefore, hTERT represent a promising candidate for gene-therapy strategies in HCC.RIASSUNTO
Introduzione. Il carcinoma epatico occupa il quinto posto per incidenza tra i diversi tipi di tumore a livello mondiale. Il trapianto di fegato o la resezione epatica rappresentano le terapie d’elezione per questo tipo di tumore, tuttavia molti dei pazienti non possono essere sottoposti a questo tipo di interventi visti l’estensione del tumore nell’organo, la presenza di metastasi, l’invasione portale e la presenza di altre disfunzioni epatiche al momento della diagnosi. La chemioterapia sistemica o la chemioembolizzazione rappresentano una valida alternativa per il trattamento della patologia, tuttavia l’utilizzo di farmaci chemioterapici può portare all’insorgenza di chemioresistenza (multidrug resi stance, MDR) determinando l’inefficacia del trattamento. Parte attiva del fenomeno della chemioresistenza è rappresentato dai trasportatori ACB che sono coinvolti nell’espulsione del farmaco dalla cellula, di fatto impedendone l’azione.
Lo sviluppo di nuove terapie geniche potrebbe sostituire l’utilizzo di farmaci per il trattamento del tumore. L’inibizione selettiva di componenti essenziali e specifici per le cellule tumorali come lo è la Telomerasi può rappresentare una strategia emergente che potrebbe prevenire la progressione tumorale ed evitare l’insorgenza dei numerosi effetti collaterali che derivano dalla correnti terapie farmacologiche.
Il presente studio comprende due task i cui obbiettivi sono i seguenti:
Task 1: a) valutare se il diverso grado di differenziamento tumorale corrisponde ad una diversa espressione dei trasportatori ABCB1, ABCC1 e ABCG2.
b) valutare se il trattamento farmacologico influisce sull’espressione dei trasportatori ABC analizzati in questo studio.
Task 2: sviluppo di nuovi approcci terapeutici basati sulla terapia genica.
Risultati e Discussion. Task 1: è stata valutata l’espressione di mRNA dei trasportatori ABCB1, ABCC1 e ABCG2 in tre linee cellulari epatiche: IHH (cellule epatiche immortalizzate, il controllo non tumorale), HuH7 (cellule tumorali differenziate) e JHH6 (cellule tumorali scarsamente differenziate). ABCB1 è il principale ABC espresso nelle Huh7 e solamente l’espressione di ABCG2 correla col grado di differenziamento tumorale delle cellule.
Tramite l’utilizzo della microscopia confocale è stata osservata la capacità della Doxorubicina (Dox), uno tra i più utilizzati farmaci antineoplastici, di raggiungere il nucleo delle cellule entro 10 min dalla somministrazione. Trascorse 24h la Dox è totalmente localizzata all’interno del nucleo causando la morte delle cellule più sensibili. A 48h sono evidenti i danni nucleari e cellulari nelle cellule sopravissute. Il trattamento con dosi di Dox inferiori alla LC50 per 24h e 48h ha diverse conseguenze sull’espressione dei tre trasportatori nelle diverse linee cellulari studiate. In particolare in molti casi l’espressione di mRNA non correla con l’espressione proteica suggerendo che, in questi casi, la regolazione sia dovuta a fenomeni di diminuito turnover proteico piuttosto che di nuova sintesi. A seguito del trattamento con il farmaco, l’espressione di ABCC1 aumenta nelle sole linee cellulari tumorali (Huh7 e JHH6). ABCG2 sembra avere un ruolo rilevante nelle sole cellule JHH6 mentre ABCB1 è espresso unicamente nelle HuH7 dove vi è un aumento dell’espressione proteica a seguito di trattamenti con la Dox. In questo studio non è stato possibile correlare il potenziale tumorigenico delle linee cellulari con l’espressione degli ABC visto il loro peculiare profilo di espressione ed il loro diverso contributo nella chemioresistenza. Per questi motivi in futuro ci si propone di trattare le cellule con inibitori specifici per ciascun ABC al fine di meglio comprendere i loro diversi ruoli nell’insorgenza della resistenza alla Dox.
Task 2: Da studi condotti in vivo, il gene della subunità catalitica della Telomerasi (hTERT) è stato selezionato per gli esperimenti di silenziamento genico vista la sua esclusiva espressione nei tessuti tumorali. Tramite analisi bioinformatiche è stato disegnato un siRNA anti-hTERT (SirTel 1) capace di evitare l’induzione di infiammazione nelle cellule che lo internalizzano. Gli esperimenti di silenziamento sono stati effettuati nella linea cellulare JHH6. Dopo 72h di silenziamento con 25nM di SirTel 1 si è osservata una riduzione significativa nell’espressione di hTERT e nella sua attività enzimatica (p<0,001).
Gli effetti del silenziamento di hTERT riscontrati sono i seguenti:
- Cambiamenti morfologici delle cellule silenziate le quali passano dall’avere un forma fibroblasto-simile ad una forma epatocita-simile.
- Aumento dell’espressione di albumina, un marker epatocitario, il cui picco di espressione corrisponde al massimo effetto di silenziamento.
- Diminuzione della vitalità cellulare (p<0,01). SirTel 1 risulta più efficace nel ridurre la vitalità cellulare della Dox, il farmaco antineoplastico più utilizzato nelle terapie tumorali.
- Arresto del ciclo cellulare nella fase G1 (p<0,01).
Tutti i risultati sono stati validati utilizzando cellule hTERT negative come i fibroblasti primari.
Il silenziamento raggiunge i suoi massimi livelli trascorse le 72h dalla trasfezione e l’espressione della Telomerasi sembra ritornare ai livelli iniziali attorno alle 264h dalla iniziale somministrazione. La riesposizione con una seconda dose di siRNA riduce ancor più l’espressione di hTERT portandola a circa il 20% dell’espressione iniziale (p<0,001).
Questi risultati dimostrano il ruolo fondamentale della Telomerasi per la sopravvivenza e la proliferazione delle cellule tumorali epatiche. Per questi motivi hTERT costituisce un candidato promettente per le future terapie geniche applicate all’HCC.XXIV Ciclo198
Aurora kinase A and programmed death-ligand 1: expression dynamics in hepatocellular carcinoma development and the regulatory role of the kinase in immune checkpoint modulation
No full textBackground and aims: Aurora Kinase A (AURKA) is a pivotal mitotic kinase implicated in tumorigenic processes and overexpressed in most cancer types. Recent evidence suggests a new regulatory role for AURKA in modulating Programmed Death-Ligand 1 (PD-L1) expres- sion in breast cancer. Despite its critical role, limited information exists on AURKA’s involvement in hepatocellular carcinoma (HCC), and the regulatory interplay with PD-L1 remains unexplored. This study aims to explore AURKA and PD-L1 expression in HCC and precancerous conditions and the impact of AURKA inhibition and knockdown on the regulation of PD-L1 in vitro.
Method: AURKA and PD-L1 (CD274) mRNA and protein expression were assessed using qRT-PCR and Western blot, respectively. Human samples included healthy (n = 14), metabolic dysfunction-associated steatotic liver disease (MASLD) (n = 17), tumor, and paired adjacent non-tumoral (NT) tissues from HCC patients (n = 56). Mouse samples were collected from transgenic (TG) mice with chronic hepatitis B, progressing to HCC by 12 months, and wildtype (WT) mice sacrificed at 3, 6, 9, 12, and 15 months (n = 11 per condition). AURKA was inhibited by alisertib or AK-01 for 72 h, and knockdown was achieved through siRNA for 72 h or 144 h in HCC-derived JHH6 and Huh7 cell lines. The effects were evaluated in terms of cell viability, cell cycle arrest (flow cytometry), and PD-L1 protein expression (Western blot). Results: AURKA and PD-L1 gradually increased from healthy to MASLD ( p < 0.05) and from MASLD to HCC-adjacent NT tissues ( p ≤0 .05). AURKA overexpression was observed in 75% of HCC tissues compared to NT tissues ( p < 0.001), aligning with the results from the TG mouse model, where the gene was increased in early tumor stages compared to pre-tumoral stages ( p < 0.001), NT ( p < 0.05), and WT tissues ( p < 0.001). Cd274 exhibited a marked increase during neoplastic progression in mice ( p = 0.01). AURKA positively correlated with PD-L1 in HCC, NT, and MASLD tissues (all p < 0.001). Despite the decrease of AURKA protein expression in HCC tissues ( p < 0.001), the percentage of phosphorylated AURKA (Thr288) was increased in HCC, suggesting augmented kinase activity. AURKA inhibition or knock- down increased aneuploidy (+19–48%, all p<0.01) and reduced viability (p < 0.001) in both cell lines. AK-01 treatment decreased PD- L1 glycosylated mature forms ( p < 0.01), while 144-hour knockdown reduced unglycosylated PD-L1 ( p < 0.05) in both cell lines. Conclusion: AURKA and PD-L1 expression positively correlate in liver disease, both increasing with the progression of the disease. AURKA exhibits higher kinase activity in tumors than in NT tissues. In vitro, AURKA inhibition or silencing decreases PD-L1 expression through two mechanisms, implying multiple roles of AURKA in the regulation of PD-L1. This suggests a potential use of AURKA-targeted therapy in combination with immune checkpoint inhibitors in cancer therapy
Weighted miRNA co-expression networks analysis identifies circulating miRNA predicting overall survival in hepatocellular carcinoma patients
No full textThe weighted gene co-expression network analysis (WGCNA) has been used to explore gene expression datasets by constructing biological networks based on the likelihood expression profile among genes. In recent years, WGCNA found application in biomarker discovery studies, including miRNA. Serum samples from 20 patients with hepatocellular carcinoma (HCC) were profiled through miRNA 3.0 gene array and miRNAs biomarker candidates were identified through WGCNA. Results were validated by qRT-PCR in 102 HCC serum samples collected at diagnosis. WGCNA identified 16 miRNA modules, nine of them were significantly associated with the clinical characteristics of the patient. The Red module had a significant negative correlation with patients Survival (- 0.59, p = 0.007) and albumin (- 0.52, p = 0.02), and a positive correlation with PCR (0.61, p = 0.004) and alpha-fetoprotein (0.51, p = 0.02). In the red module, 16 circulating miRNAs were significantly associated with patient survival. MiR-3185 and miR-4507 were identified as predictors of patient survival after the validation phase. At diagnosis, high expression of circulating miR-3185 and miR-4507 identifies patients with longer survival (HR 2.02, 95% CI 1.10-3.73, p = 0.0086, and HR of 1.75, 95% CI 1.02-3.02, p = 0.037, respectively). Thought a WGCNA we identified miR-3185 and miR-4507 as promising candidate biomarkers predicting a longer survival in HCC patients
The infant's miscellany [electronic resource] : or easy lessons, extracted from different authors. On a new plan. Intended to facilitate the attainment of the English language to the youngest readers, by teaching them not only to read, but likewise to understand clearly what they read.
No full textAccording the 'Advertisement' to 'Miscellaneous lessons' by Ellin Devis, that book was based on this one, with the implication that they share the same author.With an index and short questions.Below imprint: Price two shillings.Electronic reproduction.English Short Title Catalog,Reproduction of original from British Library
Bilirubin-Induced Transcriptomic Imprinting in Neonatal Hyperbilirubinemia
No full textRecent findings indicated aberrant epigenetic control of the central nervous system (CNS) development in hyperbilirubinemic Gunn rats as an additional cause of cerebellar hypoplasia, the landmark of bilirubin neurotoxicity in rodents. Because the symptoms in severely hyperbilirubinemic human neonates suggest other regions as privileged targets of bilirubin neurotoxicity, we expanded the study of the potential impact of bilirubin on the control of postnatal brain development to regions correlating with human symptoms. Histology, transcriptomic, gene correlation, and behavioral studies were performed. The histology revealed widespread perturbation 9 days after birth, restoring in adulthood. At the genetic level, regional differences were noticed. Bilirubin affected synaptogenesis, repair, differentiation, energy, extracellular matrix development, etc., with transient alterations in the hippocampus (memory, learning, and cognition) and inferior colliculi (auditory functions) but permanent changes in the parietal cortex. Behavioral tests confirmed the presence of a permanent motor disability. The data correlate well both with the clinic description of neonatal bilirubin-induced neurotoxicity, as well as with the neurologic syndromes reported in adults that suffered neonatal hyperbilirubinemia. The results pave the way for better deciphering the neurotoxic features of bilirubin and evaluating deeply the efficacy of new therapeutic approaches against the acute and long-lasting sequels of bilirubin neurotoxicity
Distribution of stem cells and cancer stem cells markers in liver pathologies and their indication to the response of therapy
No full textSerum Stem Cell Growth Factor Beta for the Prediction of Therapy Response in Hepatocellular Carcinoma
No full textIntroduction. Chronic inflammatory response is one of major contributors in the development of hepatocellular carcinoma (HCC). Inflammatory molecules, such as cytokines and growth factors in the circulation, can be useful in the diagnosis and prognosis of the patients. The stem cell growth factor beta (SCGFβ), a newly found protein, is a secreted sulfated glycoprotein and it functions as a growth factor for primitive hematopoietic progenitor cells. The level of SCGFβ had been reported to be elevated in several cancer types. However, there is very few or even no information on this protein in the study of HCC, even more in clinical studies. Methods. A multiplex immunoassay panel of 48 cytokines and growth factors were utilized to screen 68 sera from 29 HCC patients at pretreatment (T0), 1 month (T1), and 6 months (T6) after treatment by either radiofrequency ablation (RF) or transarterial chemoembolization (TACE). Treatment response was evaluated according to mRECIST criteria. Results. Immunoassay screening showed that the levels of IL-17, CTACK, TNFα, IL-2Rα, IL-8, and SCGFβ were different in Complete Responders (CR) and Nonresponders (NR) groups. At T0 and T1, the SCGFβ level was significantly the highest in NR (23.8 and 40.7 ng/mL, respectively), followed by early recurrence (25.4 and 25.0 ng/mL), and CR (6.7 and 5.3 ng/mL), independently from HCV, stages, and treatment type. Low basal SCGFβ level was associated with longer disease-free survival compared to high SCGFβ. Conclusion. In this study, for the first time, we demonstrate that the high level of serum SCGFβ at pre- and posttreatment is associated with HCC nonresponsiveness
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