Liver-specific DICER1 syndrome model mice develop cystic liver tumors with defective primary cilia.

DICER1 syndrome is a tumor predisposition syndrome caused by familial genetic mutations in DICER1. Pathogenic variants of DICER1 have been discovered in many rare cancers, including cystic liver tumors. However, the molecular mechanisms underlying liver lesions induced by these variants remain unclear. In the present study, we sought to gain a better understanding of the pathogenesis of these variants by generating a mouse model of liver-specific DICER1 syndrome. The mouse model developed bile duct hyperplasia with fibrosis, similar to congenital hepatic fibrosis, as well as cystic liver tumors resembling those in Caroli's syndrome, intrahepatic cholangiocarcinoma, and hepatocellular carcinoma. Interestingly, the mouse model of DICER1 syndrome showed abnormal formation of primary cilia in the bile duct epithelium, which is a known cause of bile duct hyperplasia and cyst formation. These results indicated that DICER1 mutations contribute to cystic liver tumors by inducing defective primary cilia. The mouse model generated in this study will be useful for elucidating the potential mechanisms of tumorigenesis induced by DICER1 variants and for obtaining a comprehensive understanding of DICER1 syndrome. © 2024 The Pathological Society of Great Britain and Ireland.

Evaluation of acute thrombus regression effect of edoxaban for deep vein thrombosis in patients with cancer: a single-center prospective observational study.

BACKGROUND: Although there are reports on the recurrence prevention in the chronic phase using direct oral anticoagulants (DOACs) for deep vein thrombosis (DVT) in patients with cancer, acute thrombus regression effect using DOACs has not been assessed. This study aimed to assess the thrombus regression effect of initial treatment using edoxaban for acute lower-extremity DVT in patients with active cancer. METHODS AND RESULTS: In this observational study, among the inpatients with cancer and lower-extremity DVT who underwent initial treatment with edoxaban at our hospital from November 2019 to December 2021, 34 consenting patients were recruited in this study. The quantitative ultrasound thrombus (QUT) score of thrombus volume was calculated at baseline (before administration) and 7-14 days after the start of edoxaban administration, using lower-extremity venous ultrasound to evaluate changes in thrombus volume. The primary and secondary endpoints were the acute thrombus regression effect of edoxaban and the impact of patients' clinical frailty on the thrombus regression effect, respectively. Anticoagulant therapy with edoxaban significantly reduced QUT score (p < 0.001). In addition, regardless of the Clinical Frailty Scale scores, QUT score decreased significantly. CONCLUSION: Initial treatment with edoxaban was effective for lower-extremity DVT in patients with cancer. In addition, the effect was the same independent of the degree of frailty.

Analysis of Carcinogenic Involvement of MicroRNA Pattern in Peripheral Non-Cancerous Tissues and Chronic Viral Liver Injury.

Risk factors for hepatocarcinogenesis include chronic inflammation due to viral infection, liver fibrosis, and aging. In this study, we separated carcinogenic and non-carcinogenic cases due to hepatitis C virus (HCV) infection, aiming to comprehensively analyze miRNA expression in liver tissues by age, and identify factors that contribute to carcinogenesis. Total RNA was extracted from 360 chronic hepatitis C (CH), 43 HCV infected hepatocellular carcinoma (HCC), and surrounding non-tumor (SNT) tissues. MicroRNA (miRNA) expression patterns were analyzed using microarray. Using machine learning, we extracted characteristic miRNA expression patterns for each disease and age. There were no age-dependent changes in miRNA expression in the disease-specific comparisons; however, miRNA expression differed among the age groups of 50, 60, and 70 years of age between CH and SNT. The expression of miRNA was different between SNT and HCC only in patients in their 70s. Of the 55 miRNAs with significant differences in expression between CH and SNT, 34 miRNAs showed significant differences in expression even in the degree of liver fibrosis. The observation that miRNAs involved in hepatocarcinogenesis differ at different ages suggests that the mechanisms of carcinogenesis differ by age group as well. We also found that many miRNAs whose expression did not affect liver fibrosis were involved in carcinogenesis. These findings are expected to define biomarkers for detection of HCC at early stage, and develop novel therapeutic targets for HCC.

Early initiation of tolvaptan is associated with early discharge in patients with heart failure regardless of age.

BACKGROUND: Elderly patients with heart failure (HF) have been observed to decrease activities of daily living (ADL) during hospitalization. Prevention of ADL decline from shortening of hospital stays is especially important in the elderly, because decreasing ADL is associated with poor prognosis. We investigated the relationship between the early initiation of tolvaptan (TLV) after hospitalization and the length of hospital stay in patients with HF aged younger than 80 years and aged 80 years and older. METHODS: We analyzed 146 patients younger than 80 years (< 80) and 101 patients aged 80 years and older (≥ 80) who were hospitalized with HF from February 2011 to June 2016 and had initiated TLV. The relationship between the time until commencement of TLV and the length of hospital stay was assessed. Additionally, a comparison made between the TLV early start group (within the median) and the delayed start group (over the median) for both groups. Multivariate analysis was also performed on factors that required hospital stays below the median. RESULTS: A significant correlation was observed between time to TLV initiation and the length of hospital stay (< 80: r = 0.382, P < 0.001; ≥ 80: r = 0.395, P < 0.001). The length of hospital stay in the early group was significantly longer than that in the delayed group for both groups (< 80: early 21.0 ± 13.0 days and 33.0 ± 22.7 days, respectively, P < 0.001; ≥ 80: early 21.3 ± 12.5 days and 32.9 ± 17.9 days, respectively, P < 0.001). Conversely, no statistically significant difference found in the length of hospital stay after initiation of TLV. Moreover, no increase in adverse events in the elderly observed. A multivariate analysis revealed that a predictive factor for short-term hospitalization was early administration of TLV regardless of age. CONCLUSIONS: The early initiation of TLV after hospitalization was associated with a shorter length of hospital stay in patients with HF regardless of age.

Prediction of early recurrence of hepatocellular carcinoma after resection using digital pathology images assessed by machine learning.

Hepatocellular carcinoma (HCC) is a representative primary liver cancer caused by long-term and repetitive liver injury. Surgical resection is generally selected as the radical cure treatment. Because the early recurrence of HCC after resection is associated with low overall survival, the prediction of recurrence after resection is clinically important. However, the pathological characteristics of the early recurrence of HCC have not yet been elucidated. We attempted to predict the early recurrence of HCC after resection based on digital pathologic images of hematoxylin and eosin-stained specimens and machine learning applying a support vector machine (SVM). The 158 HCC patients meeting the Milan criteria who underwent surgical resection were included in this study. The patients were categorized into three groups: Group I, patients with HCC recurrence within 1 year after resection (16 for training and 23 for test); Group II, patients with HCC recurrence between 1 and 2 years after resection (22 and 28); and Group III, patients with no HCC recurrence within 4 years after resection (31 and 38). The SVM-based prediction method separated the three groups with 89.9% (80/89) accuracy. Prediction of Groups I was consistent for all cases, while Group II was predicted to be Group III in one case, and Group III was predicted to be Group II in 8 cases. The use of digital pathology and machine learning could be used for highly accurate prediction of HCC recurrence after surgical resection, especially that for early recurrence. Currently, in most cases after HCC resection, regular blood tests and diagnostic imaging are used for follow-up observation; however, the use of digital pathology coupled with machine learning offers potential as a method for objective postoprative follow-up observation.

Serine threonine kinase 11/liver kinase B1 mutation in sporadic scirrhous-type gastric cancer cells.

Scirrhous-type gastric carcinoma (SGC), which is characterized by the rapid proliferation of cancer cells accompanied by extensive fibrosis, shows extremely poor survival. A reason for the poor prognosis of SGC is that the driver gene responsible for SGC has not been identified. To identify the characteristic driver gene of SGC, we examined the genomic landscape of six human SGC cell lines of OCUM-1, OCUM-2M, OCUM-8, OCUM-9, OCUM-12 and OCUM-14, using multiplex gene panel testing by next-generation sequencing. In this study, the non-synonymous mutations of serine threonine kinase 11/liver kinase B1 (STK11/LKB1) gene were detected in OCUM-12, OCUM-2M and OCUM-14 among the six SGC cell lines. Capillary sequencing analysis confirmed the non-sense or missense mutation of STK11/LKB1 in the three cell lines. Western blot analysis showed that LKB1 expression was decreased in OCUM-12 cells and OCUM-14 cells harboring STK11/LKB1 mutation. The mammalian target of rapamycin (mTOR) inhibitor significantly inhibited the proliferation of OCUM-12 and OCUM-14 cells. The correlations between STK11/LKB1 expression and clinicopathologic features of gastric cancer were examined using 708 primary gastric carcinomas by immunochemical study. The low STK11/LKB1 expression group was significantly associated with SGC, high invasion depth and frequent nodal involvement, in compared with the high STK11/LKB1 expression group. Collectively, our study demonstrated that STK11/LKB1 mutation might be responsible for the progression of SGC, and suggested that mTOR signaling by STK11/LKB1 mutation might be one of therapeutic targets for patients with SGC.

Exosomal hsa-miR-933 in Gastric Juice as a Potential Biomarker for Functional Dyspepsia.

BACKGROUND: MicroRNAs (miRNAs) in exosomes represent disease-specific profiles and are applied as biomarkers in oncology. However, in functional dyspepsia (FD), the role of exosomal miRNAs has not been fully elucidated. AIMS: To investigate exosomal miRNAs as potential biomarkers of FD using liquid biopsy. METHODS: This retrospective cohort study included 11 subjects with FD and 11 age- and sex-matched healthy controls (HCs). We collected gastric juice and isolated exosomal miRNAs. In a discovery cohort, expression levels of 2565 miRNAs were evaluated by 3D-Gene® microarray. miRNA expression profiles from exosomes of subjects with FD and HCs were compared by two normalization methods: (1) global normalization and (2) normalization by internal control. Subsequently, in a validation cohort, the expression levels of miRNAs were validated by quantitative reverse transcription PCR (RT-qPCR). RESULTS: Through microarray analysis using the two methods, we identified 39 miRNAs that were consistently and significantly downregulated in FD cases compared with those in HCs. Of these, 12 miRNAs (hsa-miR-933, hsa-miR-345-5p, hsa-miR-708-5p, hsa-miR-203a-3p, hsa-miR-619-5p, hsa-miR-4294, hsa-miR-4481, hsa-miR-196a-5p, hsa-miR-3918, hsa-miR-372-3p, hsa-miR-658, and hsa-miR-3654) were further validated by RT-qPCR. Our results indicated that hsa-miR-933 was significantly downregulated in FD compared with HCs (0.317 ± 0.205-fold, P = 0.0317). Furthermore, the expression level of hsa-miR-933 was negatively associated with dyspepsia score and the frequency of epigastric pain and/or burning (P < 0.01, r = - 0.835; P = 0.0280, r = - 0.688, respectively). CONCLUSIONS: Exosomal hsa-miR-933 in gastric juice could be a candidate biomarker for FD.

Serum Mac-2-binding protein glycosylation isomer predicts esophagogastric varices in cirrhotic patients with chronic hepatitis C virus infection treated with IFN-free direct-acting antiviral agent: M2BPGi levels predict varices in SVR patients.

INTRODUCTION AND OBJECTIVES: We examined whether Mac-2-binding protein glycosylation isomer (M2BPGi) levels could be a predictive marker for the presence of esophagogastric varices (EGV) in cirrhotic patients after hepatitis C virus (HCV) eradication with direct-acting antivirals (DAAs). PATIENTS AND METHODS: A total of 102 cirrhotic patients with HCV infection treated with DAAs were enrolled. Esophagogastroduodenoscopy was performed in 84 of the patients before treatment (Cohort A), in 66 after treatment (Cohort B), and in 48 at both time points (Cohort C). We examined factors associated with EGV before and after DAA treatment. RESULTS: In Cohort A, M2BPGi levels and liver stiffness were significantly higher in the EGV-positive group than the EGV-negative group (p=0.034, and p=0.042, respectively). The proportion of EGV-positive patients with before-treatment levels of M2BPGi ≧ 7.3 C.O.I. was significantly higher than in patients with M2BPGi levels<7.3 C.O.I. (p=0.015). In Cohort B, M2BPGi levels were significantly higher in the EGV-positive group than EGV-negative group (p=0.003). The proportion of EGV-positive patients with after-treatment levels of M2BPGi ≧ 3.4 C.O.I. was significantly higher than in patients with M2BPGi levels<3.4C.O.I. (p=0.001). In Cohort C, M2BPGi levels decreased during DAA treatment regardless of EGV development, but there was no significant difference in the reduction of M2BPGi among the EGV-improvement, EGV-invariant, and EGV-exacerbation groups (p=0.659). CONCLUSIONS: M2BPGi levels may be a novel serum marker for the presence of EGV before and after DAA treatment.

Association between HLA-DQA1/DRB1 polymorphism and development of hepatocellular carcinoma during entecavir treatment.

BACKGROUND AND AIMS: It remains unclear whether there is an association between single-nucleotide polymorphisms (SNPs) and development of hepatocellular carcinoma (HCC) during entecavir (ETV) treatment in nucleos(t)ide analog-naïve patients with chronic hepatitis B virus infection. We investigated the risk factors for HCC, especially host factors, during ETV treatment. METHODS: A total of 127 Japanese patients undergoing ETV treatment were enrolled in this study. Univariate and multivariate analyses for clinical factors, hepatic fibrosis markers, and SNPs associated with HCC development were analyzed. RESULTS: A total of 10 patients developed HCC during the follow-up period (median duration, 3.3 years). The 3-, 5-, and 7-year cumulative rates of HCC development were 4.8%, 10.6%, and 13.6%, respectively. Liver fibrosis (cirrhosis; P = 0.0005), age (≥ 49 years; P = 0.0048), platelet count (≤ 115 × 10/mm3 ; P = 0.0007), α-fetoprotein (≥ 8.0 ng/mL; P = 0.030), type IV collagen (≥ 200 ng/mL; P = 0.043), fibrosis-4 index (≥ 4.14; P = 0.0006), and human leukocyte antigen (HLA)-DQA1/DRB1-SNP (AA genotype; P = 0.0092) were significantly associated with HCC development according to the log-rank test. In multivariate analysis, AA genotype in the HLA-DQA1/DRB1 gene (P = 0.013; hazard ratio 4.907; 95% confidence interval 1.407-17.113) and cirrhosis (P = 0.019; hazard ratio 4.789; 95% confidence interval 1.296-17.689) were significantly associated with HCC development. CONCLUSIONS: Our findings suggested that patients with AA genotype in the HLA-DQA1/DRB1 gene or cirrhosis should be carefully followed up as a population potentially at higher risk of HCC during ETV treatment.

Pancreatic Cancer Cell Fraction Estimation in a DNA Sample.

OBJECTIVE: Pancreatic cancers are characterized by dense stroma. To estimate the degree of interference by coexisting noncancer cells in molecular analyses, we aimed to develop a DNA methylation marker that assesses a cancer cell fraction in DNA samples. METHODS: The microarray data of 22 pancreatic cancer tissues from the The Cancer Genome Atlas database and 9 noncancer tissues were used for genome-wide screening. Thirty-one surgical tumor samples (10 intraductal papillary mucinous neoplasms [IPMNs] and 21 pancreatic cancers), 4 normal, and 26 nontumor samples were used for validation. Gene-specific methylation analysis was conducted by bisulfite pyrosequencing. RESULTS: Genome-wide screening isolated SIM1, MIR129-2, NR1I2, and HOXB-AS4, as specifically methylated in pancreatic cancer cells. Bisulfite pyrosequencing validated that one or more of three genes (SIM1, MIR129-2, and NR1I2) were methylated in 22 (71.0%) tumor samples (8 IPMNs and 14 cancers), and all showed low levels of methylation in 26 (86.7%) normal and nontumor samples. Therefore, the three genes collectively constituted one marker for a pancreatic cancer cell fraction. The cancer cell fraction estimated by the marker was highly correlated with that estimated using the KRAS mutant allele frequency (R = 0.79). CONCLUSION: The DNA methylation marker is useful to estimate the pancreatic cancer cell fraction in DNA samples.

Mechanism of recipient cell-dependent differences in exosome uptake.

BACKGROUND: Exosomes, small-membrane vesicles, are secreted by cells and include several types of proteins and nucleic acids. Exosomes transfer cellular information derived from donor cells and are involved in various physiological and pathological events, such as organ-specific metastasis. Elucidating the exosome uptake mechanisms is important for understanding the progression processes of organ-specific metastasis. However, whether the exosomes secreted by the donor cells are selectively or non-selectively incorporated into the recipient cells is unknown. METHODS: In this study, three human carcinoma cell lines, A549 (lung), HCT116 and COLO205 (colon), were used. The exosome isolation efficiency was compared between three methods: ultracentrifugation, ExoQuick-TC and Total Exosome Isolation kits. Recipient cells were treated with Pitstop 2, an inhibitor of clathrin-dependent endocytosis, or genistein, an inhibitor of caveolae-dependent endocytosis, and then incubated with DiO-labeled exosomes. RESULTS: Among the three methods examined, ultracentrifugation was the most efficient and reproducible. Exosomes derived from a donor cell line are incorporated into the three cell lines, but the exosome uptake capability was different depending on the recipient cell type and did not depend on the donor cell type. Exosome uptake in COLO205 was inhibited by Pitstop 2 and genistein. Exosome uptake in HCT116 was inhibited by Pitstop 2, but not genistein, while that in A549 cells was not inhibited by these inhibitors. Taken together, these results suggest that the exosomes secreted by donor cells are non-selectively incorporated into recipient cells and that the exosome uptake mechanism is different depending on the recipient cells. CONCLUSIONS: Different recipient cells' exosome uptake capabilities may be involved in organ-specific metastasis.

Micro-osteoperforations accelerate orthodontic tooth movement by stimulating periodontal ligament cell cycles.

INTRODUCTION: The aim of this study was to investigate the mechanism of how micro-osteoperforations (MOPs) accelerate tooth movement. We focused on inflammation, cell proliferation, and apoptosis of periodontal ligament cells and performed immunostaining of MOPs exposed to tumor necrosis factor-alpha (TNF-α), proliferating cell nuclear antigen (PCNA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) during experimental tooth movement. METHODS: Eleven-week-old male Wistar rats were divided into 2 groups: (1) 10 g of orthodontic force applied to the maxillary first molar (TM) and (2) force application plus 3 small perforations of the cortical plate (TM + MOPs). On days 1, 4, 7, 10, and 14 after force application, we investigated tooth movement and alveolar bone microstructure using microcomputed tomography (n = 5). We also determined the expression of TNF-α and PCNA in the pressure sides of periodontal ligaments via an immunohistochemical analysis. The expression of apoptotic cells was also determined by the TUNEL method. RESULTS: The tooth movement in the TM + MOPs group was significantly greater on days 4 to 14 than in the TM group. The TM + MOPs group showed statistically significant decreases in bone volume/tissue volume ratio and bone mineral density compared with the TM group. The ratios of TNF-α positive cells in the TM + MOPs group were increased on days 1, 4. 7, and 10 compared with the TM group. The ratios of PCNA positive cells in the TM + MOPs group were increased on days 1, 4, and 7 compared with the TM group, and the ratios of TUNEL positive cells in the TM + MOPs group were increased on days 1 and 7 compared with the TM group. CONCLUSIONS: These results suggest that MOPs may accelerate tooth movement through activation of cell proliferation and apoptosis of periodontal ligament cells.

MicroRNAs in hepatic pathophysiology.

MicroRNAs (miRNAs) are a group of small non-coding RNAs that range in length from 20 to 25 nucleotides. MicroRNAs are specific for multiple cellular functions, including cell generation, differentiation, multiplication, carcinogenesis, and apoptosis. Many researchers have recently reported that the aberrant expression of miRNAs in hepatic tissue was related to the pathogenesis of liver disease, including viral hepatitis, hepatocellular carcinoma, and fatty liver disease. Multiple studies have proposed that an analysis of circulating miRNAs may be useful for diagnosing etiologies or staging the progression of liver disease, as well as for therapeutic purposes, for example, nucleic acid therapy. This review summarizes and discusses recent advances in the knowledge of miRNAs for chronic liver diseases, with special interest in viral hepatitis, liver fibrosis, and biomarkers.

Correlation between polymorphism in the inosine triphosphatase and the reductions in hemoglobin concentration and ribavirin dose during sofosbuvir and ribavirin therapy.

BACKGROUND AND AIM: It is unclear whether polymorphism in the inosine triphosphatase (ITPA) gene correlates to the reduction in hemoglobin (Hb) concentrations during sofosbuvir (SOF) and ribavirin (RBV) therapy. This study investigated the effects of the ITPA polymorphism on Japanese patients with chronic hepatitis C virus genotype 2 infection treated with SOF/RBV therapy. METHODS: In 106 patients treated with SOF/RBV therapy, this study assessed the effects of the ITPA polymorphism (rs1127354) on anemia, RBV dose reduction, and sustained virological response. RESULTS: Of the 106 patients, 80 had the CC genotype, whereas 26 had a non-CC genotype in ITPA. Patients with the CC genotype had significantly larger reductions in Hb concentrations than those with a non-CC genotype throughout the treatment course. RBV dose reduction was required in 18/106 (17.0%) patients, with a significantly higher frequency in patients with the CC genotype than in those with a non-CC genotype (P = 0.010). In multivariate analysis, age ≥ 65 years (P = 0.011) and the ITPA CC genotype (P < 0.0001) were factors significantly associated with anemia throughout the treatment course. Sustained virological response was achieved in 99.0% of all patients: 98.7% of patients with the CC genotype and 100% of patients with a non-CC genotype. CONCLUSIONS: Inosine triphosphatase polymorphism appeared to correlate with anemia incidence and RBV dose reduction during SOF/RBV therapy, but not the clinical outcome. Careful monitoring of Hb concentrations and prompt adjustment of RBV doses are required for successful treatment, particularly in patients harboring the ITPA CC genotype or age ≥ 65 years.

MiR-29a Assists in Preventing the Activation of Human Stellate Cells and Promotes Recovery From Liver Fibrosis in Mice.

The microRNA-29 (miR-29) family is known to suppress the activation of hepatic stellate cells (HSCs) and reversibly control liver fibrosis; however, the mechanism of how miR-29a controls liver fibrosis remains largely unknown. This study was conducted to clarify the mechanism of anti-fibrotic effect of miR-29a and to explore if miR-29a is a promising candidate for nucleic acid medicine against liver fibrosis. Two liver fibrosis murine models (carbon tetrachloride or thioacetamide) were used. MiR-29a mixed with atelocollagen was systemically administered. Hepatic fibrosis was evaluated by histological analysis and the expression levels of fibrosis-related genes. We observed that miR-29a treatment dramatically accelerated the reversion of liver fibrosis in vivo. Additionally, miR-29a regulated the mRNA expression of collagen type I alpha 1 (COL1A1) and platelet-derived growth factor C (PDGFC). We also noted that miR-29a significantly suppressed COL1A1 mRNA expression and cell viability and significantly increased caspase-9 activity (P < 0.05) in LX-2 cells. Pretreatment of miR-29a inhibited activation of LX-2 cell by transforming growth factor beta treatment. MiR-29a exhibited anti-fibrotic effect without cell toxicity in vivo and directly suppressed the expression of PDGF-related genes as well as COL1A1 and induced apoptosis of LX-2 cells. MiR-29a is a promising nucleic acid inhibitor to target liver fibrosis.

Outcomes for Cirrhotic Patients with Hepatitis C Virus 1b Treated with Asunaprevir and Daclatasvir Combination.

BACKGROUND: The efficacy and safety of asunaprevir + daclatasvir combination therapy for treatment of hepatitis C virus (HCV) in compensated cirrhotic patients was not fully evaluated in real-world. Outcomes were assessed in cirrhotic patients with sustained viral response (SVR). MATERIAL AND METHODS: A total of 145 patients without resistance-associated substitutions (RASs) at L31 and Y93 in the nonstructural protein 5A of HCV genotype 1b, consisting of 49 hepatic cirrhotic and 96 non-cirrhotic patients, were enrolled to the therapy. The patients were treated with 100 mg asunaprevir twice daily plus 60 mg daclatasvir once daily for 24 weeks. The primary endpoint was SVR 24 weeks after completing treatment. In addition, we evaluated the improvement of liver function and development of HCC for 1 year from the end of treatment (EOT). RESULTS: The SVR24 rate was 96% (47/49) in the cirrhotic group and 96% (91/95) in the non-cirrhotic group (p = 0.69). During treatment, grade III/IV adverse events occurred more frequently in cirrhotic patients (10/49; 20.4%) than in non-cirrhotic patients (10/96; 10.4%) (p = 0.099). After EOT, alanine aminotransferase and AFP levels were significantly decreased in cirrhotic patients with SVR. In addition, serum levels of albumin and platelet counts were significantly increased. On the other hand, the rates of HCC recurrence (43%) and development (7.4%) were higher in cirrhotic patients than in the non-cirrhotic patients (12.5% and 1.1%, respectively). CONCLUSION: RAS-oriented asunaprevir/daclatasvir therapy has a strong anti-HCV effect in patients with HCV genotype 1b. However, careful management is necessary in patients with cirrhosis.

Long-Term Follow-Up of Resistance-Associated Substitutions in Hepatitis C Virus in Patients in Which Direct Acting Antiviral-Based Therapy Failed.

We evaluated the transition of dominant resistance-associated substitutions (RASs) in hepatitis C virus during long-term follow-up after the failure of DAAs (direct acting antivirals)-based therapy. RASs in non-structure (NS)3/4A, NS5A, NS5B, and deletions in NS5A from 20 patients who failed simeprevir/pegylated-interferon/ribavirin (SMV/PEG-IFN/RBV) and 25 patients who failed daclatasvir/asunaprevir (DCV/ASV) treatment were examined by direct sequencing. With respect to SMV/PEG-IFN/RBV treatment, RAS was detected at D168 in NS3/4A but not detected in NS5A and NS5B at treatment failure in 16 of 20 patients. During the median follow-up period of 64 weeks, the RAS at D168 became less dominant in 9 of 16 patients. Among 25 DCV/ASV failures, RASs at D168, L31, and Y93 were found in 57.1%, 72.2%, and 76.9%, respectively. NS5A deletions were detected in 3 of 10 patients treated previously with SMV/PEG-IFN/RBV. The number of RASs in the breakthrough patients exceeded that in relapsers (mean 3.9 vs. 2.7, p < 0.05). RAS at D168 in NS3/4A became less dominant in 6 of 15 patients within 80 weeks. Y93H emerged at the time of relapse, then decreased gradually by 99% at 130 weeks post-treatment. Emerged RASs were associated with the clinical course of treatment and could not be detected during longer follow-up.

Expression of Serum Exosomal and Esophageal MicroRNA in Rat Reflux Esophagitis.

Gastroesophageal reflux disease (GERD) is a common upper gastrointestinal disease. However, the role of exosomal microRNAs (miRNAs) and esophageal miRNAs in GERD has not been studied. A rat model of acid reflux esophagitis was used to establish a novel diagnosis marker for GERD and examine dynamics of miRNA expression in GERD. Rats were sacrificed 3 (acute phase), 7 (sub-acute phase) and 21 days (chronic phase) after induction of esophagitis. Exosomes were extracted from serum, and the expression patterns of serum miRNAs were analyzed. Four upregulated miRNAs (miR-29a-3p, 128-3p, 223-3p and 3473) were identified by microarray analysis. The expression levels of exosomal miR-29a-3p were significantly higher in the chronic phase of reflux esophagitis compared with controls, and increased expression of miR-29a-3p was specific to chronic reflux esophagitis. Esophageal miR-223-3p expression was higher compared with controls, and gradually decreased from acute to chronic phase in esophagitis. In conclusion, exosomal miR-29a-3p and esophageal miR-223-3p might play roles in GERD.

Microarray analysis of circulating microRNAs in familial Mediterranean fever.

OBJECTIVES: Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in MEFV. Mutations in exon 10 are associated with typical FMF phenotypes, whereas the pathogenic role of variants in exons 2 and 3 remains uncertain. Recent evidence suggests that circulating microRNAs (miRNAs) are potentially useful biomarkers in several diseases. Therefore, their expression was assessed in FMF. METHODS: The subjects were 24 patients with FMF who were between attacks: eight with exon 10 mutations (group A), eight with exon 3 mutations (group B), and eight without exon 3 or 10 mutations (group C). We also investigated eight cases of PFAPA as disease controls. Exosome-rich fractionated RNA was subjected to miRNA profiling by microarray. RESULTS: Using the expression patterns of 26 miRNAs, we classified FMF (groups A, B, and C) and PFAPA with 78.1% accuracy. In FMF patients, groups A and B, A and C, and B and C were distinguished with 93.8, 87.5, and 100% accuracy using 24, 30, and 25 miRNA expression patterns, respectively. CONCLUSIONS: These findings suggest that expression patterns of circulating miRNAs differ among FMF subgroups based on MEFV mutations between FMF episodes. These patterns may serve as a useful biomarker for detecting subgroups of FMF.

MicroRNA expression in hepatocellular carcinoma after the eradication of chronic hepatitis virus C infection using interferon therapy.

AIM: Hepatocellular carcinoma (HCC) develops in up to 5% of patients after the successful treatment of chronic hepatitis C virus (HCV) infection using interferon therapy. The aim of this study was to characterize miRNA expression in liver tissues from patients who achieved a sustained viral response (SVR). METHODS: Seventy-one patients with resected HCC were enrolled into the present study: 61 HCC from patients with continuously infected HCV (HCV-HCC) and 10 from patients who had achieved SVR (SVR-HCC). We also included non-tumor tissues (SVR-NT) from four patients with SVR-HCC, and liver tissue (SVR-CH) from four SVR patients without HCC. Total RNA was extracted from liver samples. The miRNA expression patterns were analyzed using microarrays. In addition, target gene expression was quantified after miRNA overexpression in HEK293 cells. RESULTS: We could discriminate between SVR-HCC and HCV-HCC with 75.36% accuracy using the expression pattern of six specific miRNA. The expression levels of 37 miRNA were significantly lower in HCV-HCC than in SVR-HCC, whereas the expression of 25 miRNA was significantly higher in HCV-HCC than SVR-HCC (P < 1.0E-05). The expression of thrombospondin 1 was regulated in an opposing manner by miR-30a-3p in SVR-HCC and HCV-HCC. In non-tumor tissues, the expression pattern of seven miRNA could distinguish between SVR-CH and SVR-NT with 87.50% accuracy. CONCLUSION: Comprehensive miRNA expression analyses could not only differentiate between SVR-HCC and HCV-HCC but also forecast hepatocarcinogenesis after achieving SVR.