HEPPAR1 and PIWIL2 as Panel Markers for Hepatocellular Carcinoma.

OBJECTIVE: The aim of this study was to evaluate the expression profiles of PIWI-like protein- 2 (PIWIL2), and HepPar1 and their immunohistochemical (IHC) characteristics in Hepatocellular Carcinoma (HCC), and determine their correlation with clinicopathological parameters of this type of cancer to determine their diagnostic value in combination. METHODS: Seventy-five patients with HCC were assessed for the expression of PIWIL2 in serum and tissue using real-time polymerase chain reaction (RT-PCR) and IHC was performed for PIWIL2 and HepPar1 was performed on all patients. RESULTS: A statistically significantly higher level of PIWIL2 was found in HCC compared to controls (p≤0.001). Both HepPar1 and PIWIL2 were detected in 84% of HCC cases, the diagnostic and prognostic factors for PIWIL2 were found to be significant in liver tumour tissue samples and non-tumorous sections p<0.001, and the same was observed for serum samples and results of healthy serum controls (p<0.001) when compared to AFP. CONCLUSION: Our results affirm the hypothesis that reactivation of PIWI expression in various caner types is crucial for cancer development, and that a possible panel maybe used for these markers HCC diagnosis.

Argonaute Autoantibodies as Biomarkers in Autoimmune Neurologic Diseases.

OBJECTIVE: To identify and characterize autoantibodies (Abs) as novel biomarkers for an autoimmune context in patients with central and peripheral neurologic diseases. METHODS: Two distinct approaches (immunoprecipitation/mass spectrometry-based proteomics and protein microarrays) and patients' sera and CSF were used. The specificity of the identified target was confirmed by cell-based assay (CBA) in 856 control samples. RESULTS: Using the 2 methods as well as sera and CSF of patients with central and peripheral neurologic involvement, we identified Abs against the family of Argonaute proteins (mainly AGO1 and AGO2), which were already reported in systemic autoimmunity. AGO-Abs were mostly of immunoglobulin G 1 subclass and conformation dependent. Using CBA, AGO-Abs were detected in 21 patients with a high suspicion of autoimmune neurologic diseases (71.4% were women; median age 57 years) and only in 4/856 (0.5%) controls analyzed by CBA (1 diagnosed with small-cell lung cancer and the other 3 with Sjögren syndrome). Among the 21 neurologic patients identified, the main clinical presentations were sensory neuronopathy (8/21, 38.1%) and limbic encephalitis (6/21, 28.6%). Fourteen patients (66.7%) had autoimmune comorbidities and/or co-occurring Abs, whereas AGO-Abs were the only autoimmune biomarker for the remaining 7/21 (33.3%). Thirteen (61.9%) patients were treated with immunotherapy; 8/13 (61.5%) improved, and 3/13 (23.1%) remained stable, suggesting an efficacy of these treatments. CONCLUSIONS: AGO-Abs might be potential biomarkers of autoimmunity in patients with central and peripheral nonparaneoplastic neurologic diseases. In 7 patients, AGO-Abs were the only biomarkers; thus, their identification may be useful to suspect the autoimmune character of the neurologic disorder. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that AGO-Abs are more frequent in patients with autoimmune neurologic diseases than controls.

Extracellular microRNAs in human circulation are associated with miRISC complexes that are accessible to anti-AGO2 antibody and can bind target mimic oligonucleotides.

MicroRNAs (miRNAs) function cell-intrinsically to regulate gene expression by base-pairing to complementary mRNA targets while in association with Argonaute, the effector protein of the miRNA-mediated silencing complex (miRISC). A relatively dilute population of miRNAs can be found extracellularly in body fluids such as human blood plasma and cerebrospinal fluid (CSF). The remarkable stability of circulating miRNAs in such harsh extracellular environments can be attributed to their association with protective macromolecular complexes, including extracellular vesicles (EVs), proteins such as Argonaut 2 (AGO2), or high-density lipoproteins. The precise origins and the potential biological significance of various forms of miRNA-containing extracellular complexes are poorly understood. It is also not known whether extracellular miRNAs in their native state may retain the capacity for miRISC-mediated target RNA binding. To explore the potential functionality of circulating extracellular miRNAs, we comprehensively investigated the association between circulating miRNAs and the miRISC Argonaute AGO2. Using AGO2 immunoprecipitation (IP) followed by small-RNA sequencing, we find that miRNAs in circulation are primarily associated with antibody-accessible miRISC/AGO2 complexes. Moreover, we show that circulating miRNAs can base-pair with a target mimic in a seed-based manner, and that the target-bound AGO2 can be recovered from blood plasma in an ∼1:1 ratio with the respective miRNA. Our findings suggest that miRNAs in circulation are largely contained in functional miRISC/AGO2 complexes under normal physiological conditions. However, we find that, in human CSF, the assortment of certain extracellular miRNAs into free miRISC/AGO2 complexes can be affected by pathological conditions such as amyotrophic lateral sclerosis.

[The Efficiency of Immunoprecipitation of microRNA/Ago2 Complexes from Human Blood Plasma Is Protocol Dependent].

The study of extracellular miRNA is one of the most dynamic areas of modern biomedical research. Oftentimes, there is a need to isolate miRNAs associated with a particular carrier, for example, a ribonucleoprotein complex. The most thoroughly investigated protein component of these complexes is Ago2. Complexes are commonly isolated by immunoprecipitation with specific antibodies. Here we compare three methods for immunoprecipitating Ago2/microRNA complexes from blood plasma. In the first protocol, anti-Ago2-antibodies are added to the plasma following protein A-sepharose. In second protocol, anti-Ago2-antibodies are bound to sepharose from the very beginning, and then mixed with plasma. The third protocol differs from the second in that sepharose is blocked by non-specific antibodies at the final stage. To compare the efficiency of these protocols, the levels of miR-16-5p, miR-21-5p, and miR-144-3p were analyzed after precipitation with anti-Ago2 antibodies and control antibodies. For miR-16-5p all protocols were efficient, for miR-21-5p only the second technique yielded results, while for miR-144-3p none of the protocols resulted in extraction. Thus, we conclude that different protocols for immunoprecipitation of microRNA/Ago2 complexes favor different miRNAs.

Diabetic Nephropathy Alters the Distribution of Circulating Angiogenic MicroRNAs Among Extracellular Vesicles, HDL, and Ago-2.

Previously, we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in patients with diabetic nephropathy (DN). However, miRs circulate in extracellular vesicles (EVs) or in association with HDL or the RNA-binding protein argonaute-2 (Ago-2). Given that the EV- and HDL-mediated miR transfer toward endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs among carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EV, HDL, and Ago-2 fractions isolated from EDTA plasma of healthy control subjects, patients with diabetes mellitus (DM) with or without early DN (estimated glomerular filtration rate [eGFR] >30 mL/min/1.73 m2), and patients with DN (eGFR <30 mL/min/1.73 m2). Consistent with our hypothesis, we observed alterations in miR carrier distribution in plasma of patients with DM and DN compared with healthy control subjects. Both miR-21 and miR-126 increased in EVs of patients with DN, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fraction. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from patients with DM and DN. In conclusion, miR measurement in EVs, HDL, and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN, while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a proangiogenic response (HDL-miR-132).

Detection of circulating microRNAs with Ago2 complexes to monitor the tumor dynamics of colorectal cancer patients during chemotherapy.

Because of the different forms of circulating miRNAs in plasma, Argonaute2 (Ago2)-miRNAs and extracellular vesicles (EV-miRNAs), we examined the two forms of extracellular miRNAs in vitro and developed a unique methodology to detect circulating Ago2-miRNAs in small volumes of plasma. We demonstrated that Ago2-miR-21 could be released into the extracellular fluid by active export from viable cancer cells and cytolysis in vitro. As miR-21 and miR-200c were abundantly expressed in both metastatic liver sites and primary lesions, we evaluated Ago2-miR-21 as a candidate biomarker of both active export and cytolysis while Ago2-miR-200c as a biomarker of cytolysis in plasma obtained from colorectal cancer (CRC) patients before treatment and in a series of plasma obtained from CRC patients with liver metastasis who received systemic chemotherapy. The measurement of Ago2-miR-21 allowed us to distinguish CRC patients from subjects without CRC. The trend in ΔCt values for Ago2-miR-21 and -200c during chemotherapy could predict tumor response to ongoing treatment. Thus, capturing circulating Ago2-miRNAs from active export can screen patients with tumor burdens, while capturing them from passive release by cytolysis can monitor tumor dynamics during chemotherapy treatment.

Thrombin-reduced miR-27b attenuates platelet angiogenic activities in vitro via enhancing platelet synthesis of anti-angiogenic thrombospondin-1.

Essentials It is unclear if platelet micro-RNAs can regulate de novo protein synthesis of platelets. Platelet de novo protein synthesis of thrombospondin-1 (TSP-1) was induced by thrombin. Thrombin stimulation in vitro altered platelet microRNA profiles, including decreased miR-27b. Decreased miR-27b hampers platelet angiogenic activities via enhancing de novo TSP-1 synthesis. SUMMARY: Background Platelets can synthesize proteins upon activation. Platelets contain a number of microRNAs (miRNA) and a fully functional miRNA effector machinery. It is, however, unclear if platelet miRNAs can regulate protein synthesis of platelets, and whether the regulation may produce a physiological impact. Objectives To investigate if and how platelet miRNAs regulate de novo syntheses of angiogenic regulators and subsequently modulate platelet angiogenic activities. Methods and Results Microarray-based miRNA profiling showed that thrombin stimulation in vitro down- or up-regulated a number of platelet miRNAs, both in the total platelet miRNAs and in Ago2-associated miRNAs. Among those altered miRNAs, miR-27b was down-regulated in both the total and Ago2-immunoprecipitated miRNA profiles of platelets, which was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Using western blotting assays, we showed that thrombin induced platelet de novo synthesis of thrombospondin-1, and that the level of thrombospondin-1 synthesis could reach a level of 3-5-fold higher than that before thrombin stimulation. With either the platelet precursor megakaryocyte cell line MEG-01 cells or mature platelets, we demonstrated that transfection of miR-27b mimic, but not the negative control of miRNA mimic, markedly reduced thrombospondin-1 protein levels. The latter subsequently enhanced platelet-dependent endothelial tube formation on matrigel. Conclusions Thrombin stimulation in vitro reduces platelet miR-27b levels that may markedly enhance thrombin-evoked platelet de novo synthesis of thrombospondin-1. Elevation of platelet miR-27b by transfection inhibits thrombospondin-1 synthesis, and subsequently enhances platelet pro-angiogenic activities. Hence, platelet activation-dependent reduction of miR-27b levels may represent a novel negative regulatory mechanism of platelet angiogenic activities.

Red blood cells release microparticles containing human argonaute 2 and miRNAs to target genes of Plasmodium falciparum.

Red blood cells (RBCs) are known to function as a refuge for providing food resources and as a shelter against the host's immune system after malaria parasite (Plasmodium) infection. Recent studies have reported significant production of extracellular vesicles (microparticles, MPs) in the circulation of malaria patients. However, it is unclear how these extracellular vesicles are generated and what their biological functions are. In this study, we isolated the MPs from a culture medium of normal RBCs and malaria parasite-infected RBCs (iRBCs), compared their quantity and origins, and profiled their miRNAs by deep sequencing. We found a much larger number of MPs released in the culture of iRBCs than in the culture of normal RBCs. Further investigation indicated that, in these MPs, human argonaute 2 (hAgo2) was found to bind to hundreds of miRNAs. These hAgo2-miRNA complexes were transferred into the parasites, and the expression of an essential malaria antigen, PfEMP1, was downregulated by miR-451/140 through its binding to the A and B subgroups of var genes, a family of genes encoding PfEMP1. Our data suggest for the first time that, through the release of MPs, mature RBCs present an innate resistance to malaria infection. These studies also shed new light on the reason why RBCs' genetic mutation occurs mainly in populations living in intensive malaria endemic areas and on the possibility of using miRNAs as novel medicines for malaria patients.

Autoantibodies to Su/Argonaute 2 in Japanese patients with inflammatory myopathy.

BACKGROUND: Anti-Su antibodies are found in 5-20% of cases of various systemic autoimmune rheumatic diseases and in 5-10% of dermatomyositis (DM)/polymyositis (PM) patients. In 2006, the 100kDa Su antigen was identified as argonaute2 (Ago2), and it was found to play a major role in RNA interference. However, immunoprecipitation (IP) remains the main method for detecting anti-Su and the clinical significance of the antibodies is uncertain. METHODS: Sera from patients with DM/PM (n=224) were screened by an ELISA that uses recombinant biotinylated Ago2 protein. Some serum samples were tested by IP and by indirect immunofluorescence (IIF) analysis. RESULTS: Seventeen (7.5%, 17/224) sera from DM/PM were positive in ELISA. Of the 33 IP-tested sera (17 ELISA-positive and 16 ELISA-negative with high background), 13 were found to be anti-Ago2/Su-positive in IP and ELISA. Only one IP-positive serum was judged to be ELISA-negative. Among the 13 patients with anti-Su/Ago2, 7 cases also had myositis-specific autoantibodies. Six sera that were positive by both IP and ELISA showed the GW body pattern in IIF. Interestingly, one ELISA-positive serum with an inconclusive result in IP also showed the GW body pattern. CONCLUSION: Our novel ELISA appears to be useful for screening anti-Su/Ago2 antibodies (sensitivity: 93%, specificity: 79%).

Circulating microRNA signature in non-alcoholic fatty liver disease: from serum non-coding RNAs to liver histology and disease pathogenesis.

OBJECTIVES: We used a screening strategy of global serum microRNA (miRNA) profiling, followed by a second stage of independent replication and exploration of liver expression of selected miRNAs to study: (1) the circulating miRNA signature associated with non-alcoholic fatty liver disease (NAFLD) progression and predictive power, (2) the role of miRNAs in disease biology and (3) the association between circulating miRNAs and features of the metabolic syndrome. METHODS: The study used a case-control design and included patients with NAFLD proven through biopsy and healthy controls. RESULTS: Among 84 circulating miRNAs analysed, miR-122, miR-192, miR-19a and miR-19b, miR-125b, and miR-375 were upregulated >2-fold (p<0.05) either in simple steatosis (SS) or non-alcoholic steatohepatitis (NASH). The most dramatic and significant fold changes were observed in the serum levels of miR-122 (7.2-fold change in NASH vs controls and 3.1-fold change in NASH vs SS) and miR-192 (4.4-fold change in NASH vs controls); these results were replicated in the validation set. The majority of serum miR-122 circulate in argonaute2-free forms. Circulating miR-19a/b and miR-125b were correlated with biomarkers of atherosclerosis. Liver miR-122 expression was 10-fold (p<0.03) downregulated in NASH compared with SS and was preferentially expressed at the edge of lipid-laden hepatocytes. In vitro exploration showed that overexpression of miR-122 enhances alanine aminotransferase activity. CONCLUSIONS: miR-122 plays a role of physiological significance in the biology of NAFLD; circulating miRNAs mirror the histological and molecular events occurring in the liver. NAFLD has a distinguishing circulating miRNA profile associated with a global dysmetabolic disease state and cardiovascular risk.

Are so-called cancer-testis genes expressed only in testis?

Cancer-testis (CT) antigens are a group of tumor-associated antigens with restricted expression in normal tissues except for testis and expression in a wide variety of tumor tissues. This pattern of expression makes them suitable targets for immunotherapy as well as potential biomarkers for early detection of cancer. However, some genes attributed to this family are now known to be expressed in other normal tissues which put their potential applications in immunotherapy and cancer detection under question. Here we analyzed expression of two previously known CT antigens, RHOXF2 and PIWIL2, in AML patients versus normal donors and found no significant difference in the expression of these genes between the two groups. As these two genes showed expression in normal leukocytes, their expression pattern seems to be wider than to be attributed to the CT gene family. Future research should focus on the expression profiles of so called CT antigens to find those with more testis specific expression.