Tomoko Uchiyama

Up-regulation of selenoprotein P and HIP/PAP mRNAs in hepatocytes by intermittent hypoxia via down-regulation of miR-203

Sleep apnea syndrome is characterized by recurrent episodes of oxygen desaturation and reoxygenation (intermittent hypoxia [IH]) and is a risk factor for insulin resistance/type 2 diabetes. However, the mechanisms linking IH stress and insulin resistance remain elusive. We exposed human hepatocytes (JHH5, JHH7, and HepG2) to experimental IH or normoxia for 24 h, measured mRNA levels by real-time reverse transcription polymerase chain reaction (RT-PCR), and found that IH significantly increased the mRNA levels of selenoprotein P (SELENOP) — a hepatokine — and hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) — one of REG (Regenerating gene) family. We next investigated promoter activities of both genes and discovered that they were not increased by IH. On the other hand, a target mRNA search of micro RNA (miRNA) revealed that both mRNAs have a potential target sequence for miR-203. The miR-203 level of IH-treated cells was significantly lower than that of normoxia-treated cells. Thus, we introduced miR-203 inhibitor and a non-specific control RNA (miR-203 inhibitor NC) into HepG2 cells and measured the mRNA levels of SELENOP and HIP/PAP. The IH-induced expression of SELENOP and HIP/PAP was abolished by the introduction of miR-203 inhibitor but not by miR-203 inhibitor NC. These results demonstrate that IH stress up-regulates the levels of SELENOP in human hepatocytes to accelerate insulin resistance and up-regulates the levels of HIP/PAP mRNAs to proliferate such hepatocytes, via the miR-203 mediated mechanism.

Expression of REG family genes in human inflammatory bowel diseases and its regulation

The pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members have been reported to be expressed in Crohns disease (CD) and ulcerative colitis (UC) and to be involved as proliferative mucosal factors in IBD. However, expression of all REG family genes in IBD is still unclear. Here, we analyzed expression of all REG family genes (REG Iα, REG Iβ, REG III, HIP/PAP, and REG IV) in biopsy specimens of UC and CD by real-time RT-PCR. REG Iα, REG Iβ, and REG IV genes were overexpressed in CD samples. REG IV gene was also overexpressed in UC samples. We further analyzed the expression mechanisms of REG Iα, REG Iβ, and REG IV genes in human colon cells. The expression of REG Iα was significantly induced by IL-6 or IL-22, and REG Iβ was induced by IL-22. Deletion analyses revealed that three regions (− 220 to − 211, − 179 to − 156, and − 146 to − 130) in REG Iα and the region (− 274 to− 260) in REG Iβ promoter were responsible for the activation by IL-22/IL-6. The promoters contain consensus transcription factor binding sequences for MZF1, RTEF1/TEAD4, and STAT3 in REG Iα, and HLTF/FOXN2F in REG Iβ, respectively. The introduction of siRNAs for MZF1, RTEF1/TEAD4, STAT3, and HLTF/FOXN2F abolished the transcription of REG Iα and REG Iβ. The gene activation mechanisms of REG Iα/REG Iβ may play a role in colon mucosal regeneration in IBD.

Ciliated muconodular papillary tumor of the lung: A report of five cases

Ciliated muconodular papillary tumor (CMPT) of the lung is a newly defined and extremely rare tumor characterized by a papillary growth pattern, consisting of ciliated columnar cells, mucous cells, and basal cells with abundant mucin production. Tumor definitions and clinicopathological features continue to be debated. Herein, we report five surgical cases of CMPT to characterize its radiographic, gross, and microscopic features. The five cases involved three male patients aged 80, 67, and 66 years, and two female patients aged 73 and 70 years. Three cases were discovered during health care screenings, and two cases were found during follow‐up for another synchronous cancer. Histopathological examination revealed that the tumor tissue was composed of ciliated columnar cells, mucous cells, and basal cells with abundant mucin production. Neither nuclear atypia nor mitotic figures were observed. All patients had good prognoses. The benign histological features and clinical courses in these five cases suggest that CMPT is an independent and benign tumor of the lung.

Up-regulation of POMC and CART mRNAs by intermittent hypoxia via GATA transcription factors in human neuronal cells.

Sleep apnea syndrome (SAS) is characterized by intermittent hypoxia (IH) during sleep. SAS and obesity are strongly related to each other. Here, we investigated the effect of IH on the expression of major appetite regulatory genes in human neuronal cells. We exposed NB-1, SH-SY5Y, and SK-N-SH human neuronal cells to IH (64 cycles of 5 min hypoxia and 10 min normoxia), normoxia, or sustained hypoxia for 24 h and measured the mRNA levels of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), galanin, galanin-like peptide, ghrelin, pyroglutamylated RFamide peptide, agouti-related peptide, neuropeptide Y, and melanocortin 4 receptor by real-time RT-PCR. IH significantly increased the mRNA levels of POMC and CART in all the neuronal cells. Deletion analysis revealed that the -705 to -686 promoter region of POMC and the -950 to -929 region of CART were essential for the IH-induced promoter activity. As possible GATA factor binding sequences were found in the two regions, we performed real-time RT-PCR to determine which GATA family members were expressed and found that GATA2 and GATA3 mRNAs were predominantly expressed. Therefore, we introduced siRNAs against GATA2 and GATA3 into NB-1 cells and found that GATA2 and GATA3 siRNAs abolished the IH-induced up-regulation of both POMC and CART mRNAs. These results indicate that IH stress up-regulates the mRNA levels of anorexigenic peptides, POMC and CART, in human neuronal cells via GATA2 and GATA3. IH can have an anorexigenic effect on SAS patients through the transcriptional activation of POMC and CART in the central nervous system.

Reg Gene Expression in Periosteum after Fracture and Its In Vitro Induction Triggered by IL-6

The periosteum is a thin membrane that surrounds the outer surface of bones and participates in fracture healing. However, the molecular signals that trigger/initiate the periosteal reaction are not well established. We fractured the rat femoral bone at the diaphysis and fixed it with an intramedullary inserted wire, and the expression of regenerating gene (Reg) I, which encodes a tissue regeneration/growth factor, was analyzed. Neither bone/marrow nor muscle showed Reg I gene expression before or after the fracture. By contrast, the periosteum showed an elevated expression after the fracture, thereby confirming the localization of Reg I expression exclusively in the periosteum around the fractured areas. Expression of the Reg family increased after the fracture, followed by a decrease to basal levels by six weeks, when the fracture had almost healed. In vitro cultures of periosteal cells showed no Reg I expression, but the addition of IL-6 significantly induced Reg I gene expression. The addition of IL-6 also increased the cell number and reduced pro-apoptotic gene expression of Bim. The increased cell proliferation and reduction in Bim gene expression were abolished by transfection with Reg I siRNA, indicating that these IL-6-dependent effects require the Reg I gene expression. These results indicate the involvement of the IL-6/Reg pathway in the osteogenic response of the periosteum, which leads to fracture repair.

K-ras mutation analysis of residual liquid-based cytology specimens from endoscopic ultrasound-guided fine needle aspiration improves cell block diagnosis of pancreatic ductal adenocarcinoma

Background Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) technology is widely used for the diagnosis of pancreatic masses. However, in some cases, inadequate tissue volume or difficulty of morphological diagnosis are constraining factors for adequate cytopathological evaluation. K-ras mutation is the most frequently acquired genetic abnormality, occurring in approximately 90% of all patients with pancreatic ductal adenocarcinoma (PDAC). In the present study, the clinical utility of residual liquid-based cytology (LBC) specimens obtained using EUS-FNA for K-ras mutation analysis was evaluated. Methods In this study, 81 patients with pancreatic lesions were examined. The cell block (CB) specimens separated from EUS-FNA samples were morphologically evaluated by hematoxylin–eosin (HE) staining. Final diagnoses were confirmed by CB specimens, surgical resection specimens, diagnostic imaging, and clinical follow-up. Genomic DNA of residual LBC specimens stored at 4°C for several months were extracted and assessed for K-ras mutations using a fluorescence resonance energy transfer-based preferential homoduplex formation assay. Results K-ras mutation analysis using residual LBC samples was successful in all cases. The sensitivity, specificity, and accuracy of CB examination alone were 77.4%, 100%, and 81.3%, respectively, and those of the combination of CB examination and K-ras mutation analysis were 90.3%, 92.3%, and 90.7%, respectively. Furthermore, K-ras mutations were detected in 8 (57.1%) of 14 PDAC samples for which the CB results were inconclusive. Conclusion These findings suggest that K-ras mutation analysis using residual LBC specimens improves the diagnostic accuracy of EUS-FNA.

A case of a Müllerian cyst arising in the posterior mediastinum

Abstract A mediastinal Müllerian cyst is composed of heterotopic cystic Müllerian tissue resembling structures of the fallopian tube. We herein report a case of a mediastinal Müllerian cyst discovered during a medical check-up of a 41-year-old woman. She had no symptoms but had been diagnosed with hyperprolactinemia at the age of 24 years, for which she received hormonal therapy for 3 years. Chest computed tomography demonstrated a 3-cm-diameter cystic tumor in front of the Th10 vertebra. Thoracic surgery was performed to remove the tumor. The tumor was a monolocular cyst covered with a thin capsule, and pathological examination showed a thin-walled cyst lined by ciliated or non-ciliated columnar cells resembling tubal epithelium. Immunohistochemical analysis showed positive expression for paired box gene 8 and estrogen and progesterone receptors. The pathological diagnosis was a Müllerian cyst. A Müllerian cyst should be always be considered in patients with a mediastinal cyst.

Uterine endometrial carcinoma with DNA mismatch repair deficiency: magnetic resonance imaging findings and clinical features

PurposeThe purpose of this study was to identify the magnetic resonance imaging (MRI) features of uterine endometrial carcinoma (EC) with DNA mismatch repair (MMR) deficiency.Materials and methodsThis was a retrospective study approved by our institutional review board. The study included 118 patients pathologically diagnosed as having EC in our institution from April 2014 to December 2016. Of 118 patients, 8 were excluded because of insufficient data. Immunohistochemical analysis of MMR was performed retrospectively to observe the expressions of MLH1, MSH2, MSH6, and PMS2. A tumor with MMR deficiency was detected in 17 of 110 cases (15%). Clinical background characteristics and MRI findings were reviewed. These findings were compared between MMR deficiency group and the other group as a control group. Statistical significance was determined using the Fisher’s exact test and the Mann–Whitney U test, as appropriate.ResultsThe clinical background characteristics of patients with EC with MMR deficiency were not significantly different from those of other patients. On MRI, the tumor was significantly more often located in the lower uterine site (MMR(−) vs. MMR(+): 29.4 vs. 8.9% [p = 0.0366]).ConclusionEC with MMR deficiency tends to be located lower in the uterus, though most other findings were not significantly different from those of EC without MMR deficiency.

Immunohistochemical Reappraisal Regarding the Frequency of Primary Salivary Gland Follicular Lymphoma

Although it has been described that extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT lymphomas) are the most common type among primary salivary gland lymphomas (SGLs), some studies revealed that the frequency of follicular lymphomas (FLs) was as high as that of MALT lymphomas. However, it has been reported that many of these FLs may have developed in lymph nodes attached to the capsule of the glands or intraglandular lymph nodes. Clinical, histological, immunohistochemical, and cytogenetic features of 11 SGL cases, which were extracted from our surgical pathology file consisting of consecutive pathology cases, were reevaluated to further characterize whether they were actually primary SGLs. There were 3 (27%) cases of FLs, 5 (46%) cases of MALT lymphomas, and 3 (27%) cases of diffuse large B-cell lymphomas. Although all of our FL cases fulfilled the criteria of primary SGL, tumors of several FL cases were surrounded by podoplanin (by D2-40)–positive elongated vessels or linear structures indicative of nodal subcapsular sinuses (open or remnant). This finding would support the aforementioned possibility, and podoplanin staining is necessary before concluding that a FL is a primary SGL.

Clear Cell Adenocarcinoma Arising from Endometriosis in the Groin: Wide Resection and Reconstruction with a Fascia Lata Tensor Muscle Skin Flap

We herein report a case of clear cell carcinoma arising from endometriosis in the groin in a 53-year-old woman. The findings of MRI and FDG/PET-CT indicated a malignant tumor, and surgical biopsy confirmed adenocarcinoma of the female genital tract. The tumor including a part of the abdominal rectus muscle and rectus sheath, subcutaneous fat, skin, and the right inguinal ligament was resected en bloc. The defect in the abdominal wall was reconstructed with a fascia lata tensor muscle skin flap. The tumor was composed of clear cell adenocarcinoma arising from extrapelvic endometriosis. The patient received chemotherapy with gemcitabine and carboplatin for 6 cycles and had no evidence of recurrence 7 months after the treatment. We herein described the diagnosis and surgical management of endometriosis-associated carcinoma in the groin.