Circulating and stool nucleic acid analysis for colorectal cancer diagnosis

In recent years, the need to identify molecular markers characterized by high sensitivity and specificity in detecting and monitoring early and colorectal cancer lesions has increased. Up to now, none of the markers or panels of markers analyzed have met the rigorous standards required of a screening program. The important discovery of circulating nucleic acids in biological fluids has aroused intense scientific interest because of their usefulness in malignant and non malignant diseases. Over time, their yield and stability have been identified and compared with other “standard” biomarkers. The analysis of circulating DNA from blood and stool is a relatively simple and non-invasive procedure, representing a very attractive marker to detect genetic and epigenetic mutations and to monitor disease progression. A correlation between blood and stool biomarkers could also help to enhance currently available diagnostic approaches. However, various processing and analytic problems need to be resolved before such an approach can be applied in clinical practice.

Liquid biopsy in cholangiocarcinoma:Current status and future perspectives

Cholangiocarcinoma(CCA)are a heterogeneous group of tumors in terms of aetiology,natural history,morphological subtypes,molecular alterations and management,but all sharing complex diagnosis,management,and poor prognosis.Several mutated genes and epigenetic changes have been detected in CCA,with the potential to identify diagnostic and prognostic biomarkers and therapeutic targets.Accessing tumoral components and genetic material is therefore crucial for the diagnosis,management and selection of targeted therapies;but sampling tumor tissue,when possible,is often risky and difficult to be repeated at different time points.Liquid biopsy(LB)represents a way to overcome these issues and comprises a diverse group of methodologies centering around detection of tumor biomarkers from fluid samples.Compared to the traditional tissue sampling methods LB is less invasive and can be serially repeated,allowing a real-time monitoring of the tumor genetic profile or the response to therapy.In this review,we analysis the current evidence on the possible roles of LB(circulating DNA,circulating RNA,exosomes,cytokines)in the diagnosis and management of patients affected by CCA.

HBV X-gene:A new serum marker for anti-HBV therapy monitoring

Objective:To address HBV serum nucleic acid markers for stages without apparent replica- tion.Methods:DNA and RNA sequence segments from the X,C and pre C/C regions produced succes- sively during replication were used as targets for quantitative PCR and RT/PCR.Results:The assays con- firmed the preferential formation of intermediates blocked at early stages.They persisted as the only de- tectable type of serum HBV DNA even after one year of therapy.At reentry into viral replication due to e- mergence of drug resistant mutants,lamivudine resistance produced exclusively incomplete DNA minus strands,whereas the wild type virus immediately synthesized complete DNA minus strands.Conclusion: PCR assays used for monitoring complete suppression of HBV replication must target the X gene region.

Circulating microRNAs and liquid biopsy:murine xenograft models for technical validation of clinical protocols

In oncology,liquid biopsy is applied to detect with high efficiency clinically relevant analytes,such as tumor cells,cell-free nucleic acids,and exosomes in peripheral blood and other body fluids of cancer patients.Liquid biopsy is considered one of the most advanced non-invasive diagnostic systems useful,in the next future,for enabling personalized treatments in precision medicine.Medical actions include,but are not limited to,early diagnosis,staging,prognosis,anticipation(lead time)and prediction of therapy responses,as well as follow up.Experimental system for validation of the proposed liquid biopsy approaches is highly needed.In this review article we will discuss the establishment of xenotransplanted mouse model systems for the validation of liquid biopsy protocols aimed to identify changes in the miRNA plasma content.Human colon cancer HT-29 and LoVo cells have been xenotransplanted and miR-221-3p and miR-222-3p have been comparatively analyzed in cultured HT-29 and LoVo cells,xenotransplants and plasma samples.

Exosome and Exosomal MicroRNA: Trafficking, Sorting, and Function

Exosomes are 40–100 nm nano-sized vesicles that are released from many cell types into the extracellular space. Such vesicles are widely distributed in various body fluids. Recently,m RNAs and micro RNAs（mi RNAs） have been identified in exosomes, which can be taken up by neighboring or distant cells and subsequently modulate recipient cells. This suggests an active sorting mechanism of exosomal mi RNAs, since the mi RNA profiles of exosomes may differ from those of the parent cells. Exosomal mi RNAs play an important role in disease progression, and can stimulate angiogenesis and facilitate metastasis in cancers. In this review, we will introduce the origin and the trafficking of exosomes between cells, display current research on the sorting mechanism of exosomal mi RNAs, and briefly describe how exosomes and their mi RNAs function in recipient cells.Finally, we will discuss the potential applications of these mi RNA-containing vesicles in clinical settings.

E2F1-mediated Up-regulation of NCAPG Promotes Hepatocellular Carcinoma Development by Inhibiting Pyroptosis

Background and Aims:As a subunit of the condensin complex,NCAPG has an important role in maintaining chromosome condensation,but its biological function and regulatory mechanism in hepatocellular carcinoma(HCC)remains undefined.Methods:The prognostic ability of NCAPG in HCC patients was examined by univariate and multivariate Cox regression analysis.ROC curves were plotted to compare the predictive ability of NCAPG and AFP.Double luciferase reporter system,and ChIP were used to investigate transcriptional potential of E2F1 to NCAPG.Pyroptosis was observed by scanning electron microscopy.Protein expression of NCAPG,E2F1,and major proteins constituting NLRP3 inflammasome was determined by western blotting and ELISA.An in vivo tumor formation assay was conducted to verify the in vitro results.Results:Up-regulated NCAPG was identified in HCC tissues compared with adjacent tissue and high NCAPG was positively correlated with poor prognosis.Serum NCAPG mRNA level was a prognostic factor in HCC patients and also a diagnostic factor with higher predictive ability compared with AFP[AUROC 0.766(95%CI:0.650–0.881)vs.0.649(95%CI 0.506–0.793)].HBx transfection resulted in concomitant up-regulation of E2F1 and NCAPG.E2F1 significantly increased the activity of luciferase reporter fused with NCAPG reporter,and the interaction of E2F1 and NCAPG gene was confirmed by ChIP.Silencing of E2F1 resulted in significant down-regulation of NCAPG.Knockdown of NCAPG promote pyroptosis mediated by NLRP3 inflammasome activation in multiple HCC cell lines and also suppressed tumorigenesis in vitro.Conclusions:We identified a novel role of NCAPG in the regulation of NLRP3 inflammasome-mediated pyroptosis,which was regulated by its upstream transactivator,E2F1.The role of E2F1-NCAPG-NLRP3 regulation of pyroptosis network may be a potential target in HCC treatment.