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1.  Gene profiling and bioinformatics analyses reveal time course differential gene expression in surgically resected colorectal tissues 
Oncology Reports  2014;31(4):1531-1538.
It has previously been reported that gene profiles in surgically-resected colorectal cancer tissues are altered over time possibly due to the different tissue-acquisition methods and sample extraction timing that were used. However, the changes that occur are still not clearly understood. In the present study, time-dependent changes in gene expression profiling in colorectal surgical specimens were analyzed. Normal and tumor tissues at several time-points (0, 30, 60 and 120 min) were extracted, and RNA quality, microarray experiments, quantitative PCR and bioinformatics clustering were performed. Although RNA integrity was preserved 2 h after resection, inherent increased/decreased gene expression was observed from 30–120 min in approximately 10% of genes. Bioinformatics clustering could not distinguish case-by-case, probably due to gene profiling changes. Irregular changes in gene expression after surgical resection were found, which could be a crucial confounding factor for quantitative analyses.
PMCID: PMC3975991  PMID: 24573535
colorectal cancer; RNA degradation; clustering; bio- informatics; microarray
2.  Surgical Resection of Hepatic Cystic Echinococcosis Impaired by Preoperative Diagnosis 
Case Reports in Medicine  2013;2013:271256.
Cystic echinococcosis (CE) is a rare afferent infectious disease in Japan. This paper reports a case of a hepatic cyst being diagnosed after surgical resection. A 40-year-old Syrian male was admitted for evaluation of a hepatic cyst. Serum antibodies of echinococcosis were negative. Enhanced computed tomography of the abdomen revealed a large cystic lesion, 9 cm in diameter, in the left lateral sector of the liver, which had many honeycomb-like septa and calcified lesions. Magnetic resonance imaging of this lesion revealed high intensity in the T2 weighted image. We preoperatively diagnosed this lesion as cystadenocarcinoma or CE and performed a left hepatectomy. Pathological examination revealed the presence of protoscolices in the fluid of the cysts and led to a diagnosis of this lesion as CE. In conclusion, on seeing patients with huge hepatic cysts who come from an epidemic area, we should consider hepatic CE.
PMCID: PMC3878637  PMID: 24454394
3.  MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line 
PLoS ONE  2013;8(7):e69496.
MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.
PMCID: PMC3724868  PMID: 23922722
4.  Surgical Resection of a Leiomyosarcoma of the Inferior Vena Cava Mimicking Hepatic Tumor 
Case Reports in Medicine  2013;2013:235698.
Introduction. Leiomyosarcomas of vascular origin are particularly rare tumors occurring mainly in the inferior vena cava (IVC). They are malignant, slow-growing tumors with a poor prognosis. This paper reports on a rare case of surgical resection of an IVC leiomyosarcoma mimicking a hepatic tumor. Case Presentation. A 65-year-old Japanese male was admitted for evaluation of an abdominal tumor. Enhanced computed tomography of the abdomen revealed a slightly enhanced heterogeneous tumor, 18 mm in diameter, between the Spiegel lobe of the liver and the IVC in early-phase images, with no enhancement or washout in late-phase images. We diagnosed this tumor as either a hepatic tumor in the Spiegel lobe or a retroperitoneal tumor such as leiomyosarcoma or liposarcoma and performed a laparotomy. On the basis of surgical findings, we extirpated the tumor by performing a wedge resection of the wall of the IVC and suturing the primary IVC wall. Pathological findings led to a further diagnosis of the tumor as a leiomyosarcoma originating in the IVC. Thirty-seven months after the operation, multiple liver and lung metastases were detected, and the patient died from multiple organic failures. Conclusion. We experienced a rare case of a leiomyosarcoma of IVC mimicking hepatic tumor.
PMCID: PMC3590509  PMID: 23509466
5.  SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity 
BMC Molecular Biology  2012;13:20.
Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions.
Using a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA.
Up-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.
PMCID: PMC3495018  PMID: 22731250
6.  Gene Suppression of Mouse Testis In Vivo Using Small Interfering RNA Derived from Plasmid Vectors 
We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules.
PMCID: PMC3317496  PMID: 22489107
siRNA; electroporation; Tex101; mouse testis
7.  Cooperation of p300 and PCAF in the Control of MicroRNA 200c/141 Transcription and Epithelial Characteristics 
PLoS ONE  2012;7(2):e32449.
Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.
PMCID: PMC3284570  PMID: 22384255
8.  Initial In Vitro Investigation of the Human Immune Response to Corneal Cells from Genetically Engineered Pigs 
This study is the first to demonstrate the humoral and cellular immune response in humans to corneal endothelial cells from genetically engineered pigs. The results suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.
To compare the in vitro human humoral and cellular immune responses to wild-type (WT) pig corneal endothelial cells (pCECs) with those to pig aortic endothelial cells (pAECs). These responses were further compared with CECs from genetically engineered pigs (α1,3-galactosyltransferase gene-knockout [GTKO] pigs and pigs expressing a human complement-regulatory protein [CD46]) and human donors.
The expression of Galα1,3Gal (Gal), swine leukocyte antigen (SLA) class I and class II on pCECs and pAECs, with or without activation by porcine IFN-γ, was tested by flow cytometry. Pooled human serum was used to measure IgM/IgG binding to and complement-dependent cytotoxicity (CDC) to cells from WT, GTKO, and GTKO/CD46 pigs. The human CD4+ T-cell response to cells from WT, GTKO, GTKO/CD46 pigs and human was tested by mixed lymphocyte reaction (MLR).
There was a lower level of expression of the Gal antigen and of SLA class I and II on the WT pCECs than on the WT pAECs, resulting in less antibody binding and reduced human CD4+ T-cell proliferation. However, lysis of the WT pCECs was equivalent to that of the pAECs, suggesting more susceptibility to injury. There were significantly weaker humoral and cellular responses to the pCECs from GTKO/CD46 pigs compared with the WT pCECs, although the cellular response to the GTKO/CD46 pCECs was greater than to the human CECs.
These data provide the first report of in vitro investigations of CECs from genetically engineered pigs and suggest that pig corneas may provide an acceptable alternative to human corneas for clinical transplantation.
PMCID: PMC3176056  PMID: 21596821
9.  Real-Time PCR-Based Analysis of the Human Bile MicroRNAome Identifies miR-9 as a Potential Diagnostic Biomarker for Biliary Tract Cancer 
PLoS ONE  2011;6(8):e23584.
Biliary tract cancer (BTC) is often difficult to diagnose definitively, even through histological examination. MicroRNAs (miRNAs) regulate a variety of physiological processes. In recent years, it has been suggested that profiles for circulating miRNAs, as well as those for tissue miRNAs, have the potential to be used as diagnostic biomarkers for cancer. The aim of this study was to confirm the existence of miRNAs in human bile and to assess their potential as clinical biomarkers for BTC. We sampled bile from patients who underwent biliary drainage for biliary diseases such as BTC and choledocholithiasis. PCR-based miRNA detection and miRNA cloning were performed to identify bile miRNAs. Using high-throughput real-time PCR-based miRNA microarrays, the expression profiles of 667 miRNAs were compared in patients with malignant disease (n = 9) and age-matched patients with the benign disease choledocholithiasis (n = 9). We subsequently characterized bile miRNAs in terms of stability and localization. Through cloning and using PCR methods, we confirmed that miRNAs exist in bile. Differential analysis of bile miRNAs demonstrated that 10 of the 667 miRNAs were significantly more highly expressed in the malignant group than in the benign group at P<0.0005. Setting the specificity threshold to 100% showed that some miRNAs (miR-9, miR-302c*, miR-199a-3p and miR-222*) had a sensitivity level of 88.9%, and receiver-operating characteristic analysis demonstrated that miR-9 and miR-145* could be useful diagnostic markers for BTC. Moreover, we verified the long-term stability of miRNAs in bile, a characteristic that makes them suitable for diagnostic use in clinical settings. We also confirmed that bile miRNAs are localized to the malignant/benign biliary epithelia. These findings suggest that bile miRNAs could be informative biomarkers for hepatobiliary disease and that some miRNAs, particularly miR-9, may be helpful in the diagnosis and clinical management of BTC.
PMCID: PMC3157401  PMID: 21858175
10.  Sequencing and Bioinformatics-Based Analyses of the microRNA Transcriptome in Hepatitis B–Related Hepatocellular Carcinoma 
PLoS ONE  2011;6(1):e15304.
MicroRNAs (miRNAs) participate in crucial biological processes, and it is now evident that miRNA alterations are involved in the progression of human cancers. Recent studies on miRNA profiling performed with cloning suggest that sequencing is useful for the detection of novel miRNAs, modifications, and precise compositions and that miRNA expression levels calculated by clone count are reproducible. Here we focus on sequencing of miRNA to obtain a comprehensive profile and characterization of these transcriptomes as they relate to human liver. Sequencing using 454 sequencing and conventional cloning from 22 pair of HCC and adjacent normal liver (ANL) and 3 HCC cell lines identified reliable reads of more than 314000 miRNAs from HCC and more than 268000 from ANL for registered human miRNAs. Computational bioinformatics identified 7 novel miRNAs with high conservation, 15 novel opposite miRNAs, and 3 novel antisense miRNAs. Moreover sequencing can detect miRNA modifications including adenosine-to-inosine editing in miR-376 families. Expression profiling using clone count analysis was used to identify miRNAs that are expressed aberrantly in liver cancer including miR-122, miR-21, and miR-34a. Furthermore, sequencing-based miRNA clustering, but not individual miRNA, detects high risk patients who have high potentials for early tumor recurrence after liver surgery (P = 0.006), and which is the only significant variable among pathological and clinical and variables (P = 0,022). We believe that the combination of sequencing and bioinformatics will accelerate the discovery of novel miRNAs and biomarkers involved in human liver cancer.
PMCID: PMC3026781  PMID: 21283620
11.  Placement of percutaneous transhepatic biliary stent using a silicone drain with channels 
This report describes a method for percutaneous transhepatic biliary stenting with a BLAKE Silicone Drain, and discusses the usefulness of placement of the drain connected to a J-VAC Suction Reservoir for the treatment of stenotic hepaticojejunostomy. Percutaneous transhepatic biliary drainage was performed under ultrasonographic guidance in a patient with stenotic hepaticojejunostomy after hepatectomy for hepatic hilum malignancy. The technique used was as follows. After dilatation of the drainage root, an 11-Fr tube with several side holes was passed through the stenosis of the hepaticojejunostomy. A 10-Fr BLAKE Silicone Drain is flexible, which precludes one-step insertion. One week after insertion of the 11-Fr tube, a 0.035-inch guidewire was inserted into the tube. After removal of the 11-Fr tube, the guidewire was put into the channel of a 10-Fr BLAKE Silicone Drain. The drain was inserted into the jejunal limb through the intrahepatic bile duct and was connected to a J-VAC Suction Reservoir. Low-pressure continued suction was applied. Patients can be discharged after insertion of the 10-Fr BLAKE Silicone Drain connected to the J-VAC Suction Reservoir. Placement of a percutaneous transhepatic biliary stent using a 10-Fr BLAKE Silicone Drain connected to a J-VAC Suction Reservoir is useful for the treatment of stenotic hepaticojejunostomy.
PMCID: PMC2738821  PMID: 19725159
Biliary tract; Drainage; Hepaticojejunostomy; Stenosis; Stents
12.  Diagnosis and initial management of cholangiocarcinoma with obstructive jaundice 
Cholangiocarcinoma is the second most common primary hepatic cancer. Despite advances in diagnostic techniques during the past decade, cholangiocarcinoma is usually encountered at an advanced stage. In this review, we describe the classification, diagnosis, and initial management of cholangiocarcinoma with obstructive jaundice.
PMCID: PMC2712166  PMID: 18494050
Cholangiocarcinoma; Obstructive jaundice; Diagnosis; Treatment; Initial management
13.  Effect of transforming growth factor-β1 on human intrahepatic cholangiocarcinoma cell growth 
AIM: To elucidate the biological effects of transforming growth factor-β1 (TGF-β1) on intrahepatic cholan-giocarcinoma (ICC).
METHODS: We investigated the effects of TGF-β1 on human ICC cell lines (HuCCT1, MEC, and HuH-28) by monitoring the influence of TGF-β1 on tumor growth and interleukin-6 (IL-6) expression in ICC cells.
RESULTS: All three human ICC cell lines produced TGF-β1 and demonstrated accelerated growth in the presence of TGF-β1 with no apoptotic effect. Studies on HuCCT1 revealed a TGF-β1-induced stimulation of the expression of TGF-β1, as well as a decrease in TGF-β1 mRNA expression induced by neutralizing anti-TGF-β1 antibody. These results indicate that TGF-β1 stimulates the production and function of TGF-β1 in an autocrine fashion. Further, IL-6 secretion was observed in all three cell lines and exhibited an inhibitory response to neutralizing anti-TGF-β1 antibody. Experiments using HuCCT1 revealed a TGF-β1-induced acceleration of IL-6 protein expression and mRNA levels. These findings demonstrate a functional interaction between TGF-β1 and IL-6. All three cell lines proliferated in the presence of IL-6. In contrast, TGF-β1 induced no growth effect in HuCCT1 in the presence of small interfering RNA against a specific cell surface receptor of IL-6 and signal transducer and activator of transcription-3.
CONCLUSION: ICC cells produce TGF-β1 and confer a TGF-β1-induced growth effect in an autocrine fashion. TGF-β1 activates IL-6 production, and the functional interaction between TGF-β1 and IL-6 contributes to ICC cell growth by TGF-β1.
PMCID: PMC4088140  PMID: 17072955
Transforming growth factor-β1; Interleukin-6; Intrahepatic cholangiocarcinoma
14.  Postoperative bile leakage managed successfully by intrahepatic biliary ablation with ethanol 
We report a case of postoperative refractory bile leakage managed successfully by intrahepatic biliary ablation with ethanol. A 75-year-old man diagnosed with hepatocellular carcinoma underwent extended posterior segmentectomy including the caudate lobe and a part of the anterior segment. The hepatic tumor attached to the anterior branch of the bile duct was detached carefully and resected. Fluid drained from the liver surface postoperatively contained high concentrations of total bilirubin, at a constant volume of 150 mL per day. On d 32 after surgery, a fistulogram of the drainage tube demonstrated an enhancement of the anterior bile duct. Endoscopic retrograde cholangiography demonstrated complete obstruction of the proximal anterior bile duct and no enhancement of the peripheral anterior bile duct. On d 46 after surgery, a retrograde transhepatic biliary drainage (RTBD) tube was inserted into the anterior bile duct under open surgery. However, a contrast study of RTBD taken 7 mo post-surgery revealed that the fistula remained patent despite prolonged conservative management, so we decided to perform ethanol ablation of the isolated bile duct. Four mL pure ethanol was injected into the isolated anterior bile duct for ten minutes, the procedure being repeated five times a week. Following 23 attempts, the volume of bile juice reached less than 10 mL per day. The RTBD was clamped and removed two days later. After RTBD removal, the patient had no complaints or symptoms. Follow-up magnetic resonance imaging demonstrated atrophy of the ethanol-injected anterior segment without liver abscess formation.
PMCID: PMC4087883  PMID: 16733869
Postoperative complication; Bile leakage; Ethanol ablation
15.  One-step palliative treatment method for obstructive jaundice caused by unresectable malignancies by percutaneous transhepatic insertion of an expandable metallic stent 
AIM: To describe a simple one-step method involving percutaneous transhepatic insertion of an expandable metal stent (EMS) used in the treatment of obstructive jaundice caused by unresectable malignancies.
METHODS: Fourteen patients diagnosed with obstructive jaundice due to unresectable malignancies were included in the study. The malignancies in these patients were a result of very advanced carcinoma or old age. Percutaneous transhepatic cholangiography was performed under ultrasonographic guidance. After a catheter with an inner metallic guide was advanced into the duodenum, an EMS was placed in the common bile duct, between a point 1 cm beyond the papilla of Vater and the entrance to the hepatic hilum. In cases where it was difficult to span the distance using just a single EMS, an additional stent was positioned. A drainage catheter was left in place to act as a hemostat. The catheter was removed after resolution of cholestasis and stent patency was confirmed 2 or 3 d post-procedure.
RESULTS: One-step insertion of the EMS was achieved in all patients with a procedure mean time of 24.4 min. Out of the patients who required 2 EMS, 4 needed a procedure time exceeding 30 min. The mean time for removal of the catheter post-procedure was 2.3 d. All patients died of malignancy with a mean follow-up time of 7.8 mo. No stent-related complication or stent obstruction was encountered.
CONCLUSIONS: One-step percutaneous transhepatic insertion of EMS is a simple procedure for resolving biliary obstruction and can effectively improve the patient’s quality of life.
PMCID: PMC4088082  PMID: 16688837
Expandable metallic stent; Bile duct carcinoma; Gall bladder carcinoma; Pancreatic carcinoma; Gastric carcinoma; Obstructive jaundice

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