京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22343号 / 医博第4584号 / 新制||医||1042(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 妹尾 浩, 教授 佐藤 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・論文博士 / 博士(生命科学) / 乙第13431号 / 論生博第26号 / 新制||生||61(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 井垣 達吏, 教授 石川 冬木, 教授 原田 浩 / 学位規則第4条第2項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Die Lebertransplantation bietet gegenwärtig die beste Therapiemöglichkeit des Hepatozellulären Karzinoms und der Leberzirrhose im Endstadium. Für die positiven Resultate ist die rechtzeitige Diagnose- und Indikationsstellung entscheidend. Bei einem Japanese Integrated System (JIS) Score von 0 liegt die 5-Jahres-Überlebensrate nach einer Transplantation bei 73 %, bei einem Anstieg des Scores auf den Wert 3, sinkt sie auf 13 %[74]. Die Sensitivität der gegenwärtigen Diagnostik aus bildgebenden Verfahren und der Bestimmung des AFP zur Detektion eines HCC schwankt zwischen 20 % und 94 %[47,48,69]. Der für die Listung zur Transplantation entscheidende MELD-Score vernachlässigt ebenso wie die Milan-Kriterien die genetische und ätiologische Komponente dieser Tumorerkrankung, welche aber maßgeblich das rezidivfreie Überleben bestimmen[16–18,70]. Im Rahmen dieser Dissertation wurden Gewebeproben aus explantierten Lebern auf mikroRNA-Expression untersucht. Des Weiteren wurde zum ersten Mal analysiert, ob mit Hilfe binär-logistischen Regression und der Entscheidungsbaumklassifikation Algorithmen aus mikroRNA-Profilen und laborchemisch-klinischen Parametern zur Detektion und ätiologischen Differenzierung des HCC und der Leberzirrhosen entwickelt werden können. In der vorliegenden Arbeit konnte dargestellt werden, dass zwischen dem HCC-Gewebe und dem tumorumgebenden Gewebe, zwischen tumorumgebendem Gewebe und der reinen Zirrhose sowie zwischen ethyltoxischer und viraler Genese der o.g. Krankheitsbilder die mikroRNAs unterschiedlich stark exprimiert werden. Außerdem konnten aussichtsreiche Modelle berechnet werden, die eine Differenzierung zwischen Tumor und tumorumgebendem Gewebe mit 87,5 %, eine Unterscheidung von tumorumgebender Zirrhose von einer reinen Zirrhose mit 94,3 % bzw. 96,3 % und eine Differenzierung zwischen Tumorgewebe und reiner Zirrhose mit 91,9 % bzw. 92,1% ermöglichen. Die weitere Analyse zeigte, dass die Modelle ebenfalls dazu geeignet sind, die Lebererkrankungen nach der Ätiologie zu differenzieren. Die dargestellten Methoden sind in der Beachtung der mit großen Potenzial[24,30] versehenen mikroRNAs und der laborchemisch-klinischen Parameter neuartige Verfahren, die sowohl für die weitere Grundlagenforschung als auch für die Ergänzung der derzeit etablierten diagnostischen und allokativen Verfahren wichtige Erkenntnisse liefern.

Rheumatoid arthritis is the most common joint disease of autoimmune origin. It is accompanied by inflammatory conditions that lead to irreversible changes in the joints, their deformities ending with permanent disability. Treatment of the disease involves routine regimens, surgical, as well as pharmacological treatment, which is necessary for advanced forms. Glucocorticoids play an important role in the therapeutic intervention in the course and progression of the disease. In spite of their anti-inflammatory effect, which is a key to improving the condition of the patient, they have a number of side effects in the long term- use. In this study, we have focused on the impact of these drugs on microRNA expression changes in arthritic patients treated with pulsed doses of glucocorticoids. MicroRNAs are nowadays widely studied due to their possible use as biomarkers in monitoring disease progression and the effect of treatment. MiRNA expression analysis was performed by quantitative real-time PCR array of 754 miRNAs with reverse transcription using stem-loop primers that allow amplification of short sequences that microRNAs are. Data analysis revealed 29 miRNAs differentially expressed at the significance level p ≤ 0.05, 14 miRNAs were at significance level p ≤ 0.025 (respectively 7 miRNAs at p ≤ 0.005...

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.

Deregulation of microRNAs (miRNAs) is implicated in tumor progression. We attempt to indentify the tumor suppressive miRNA not only down-regulated in glioblastoma multiforme (GBM) but also potent to inhibit the oncogene EZH2, and then investigate the biological function and pathophysiologic role of the candidate miRNA in GBM. In this study, we show that miRNA-138 is reduced in both GBM clinical specimens and cell lines, and is effective to inhibit EZH2 expression. Moreover, high levels of miR-138 are associated with long overall and progression-free survival of GBM patients from The Cancer Genome Atlas dataset (TCGA) data portal. Ectopic expression of miRNA-138 effectively inhibits GBM cell proliferation in vitro and tumorigenicity in vivo through inducing cell cycles G1/S arrest. Mechanism investigation reveals that miRNA-138 acquires tumor inhibition through directly targeting EZH2, CDK6, E2F2 and E2F3. Moreover, an EZH2-mediated signal loop, EZH2-CDK4/6-pRb-E2F1, is probably involved in GBM tumorigenicity, and this loop can be blocked by miRNA-138. Additionally, miRNA-138 negatively correlates to mRNA levels of EZH2 and CDK6 among GBM clinical samples from both TCGA and our small amount datasets. In conclusion, our data demonstrate a tumor suppressive role of miRNA-138 in GBM tumorigenicity, suggesting a potential application in GBM therapy.

Background. This study examined the effect of microRNA-125b (miR-125b) on sepsis-induced cardiac dysfunction. Methods. Mouse hearts were transfected with lentivirus expressing miR-125b (LmiR-125b) 7 days before cecal ligation and puncture (CLP)-induced sepsis. Cardiac function was examined by echocardiography before and 6 hours after CLP (n = 6/group). Survival was monitored following CLP-induced sepsis (n = 12/group). Results. LmiR-125b transfection significantly attenuated cardiac dysfunction due to CLP-induced sepsis. Fractional shortening and ejection fraction values were significantly (P <.05) higher in the LmiR-125b-treated CLP group than in the untreated CLP group. Survival outcome in LmiR-125b-transfected septic mice was markedly improved, compared with mice with CLP-induced sepsis. Transfection of LmiR-125b into the heart significantly suppressed the expression of ICAM-1 and VCAM-1, decreased the accumulation of macrophages and neutrophils in the myocardium, and decreased serum levels of tumor necrosis factor a and interleukin 1β by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated nuclear factor κB (NF-κB) activation. In addition, sepsis-induced myocardial apoptosis was markedly attenuated by LmiR-125b transfection through suppression of p53, Bax, and Bak1 expression. In vitro transfection of endothelial cells with miR-125b mimics attenuate LPS-induced ICAM-1 and VCAM-1 expression by suppressing TRAF6 and NF-κB activation. Conclusions. Increased myocardial miR-125b expression attenuates sepsis-induced cardiac dysfunction and improves survival. miR-125b may be a target for septic cardiomyopathy.

Mounting evidence supports that sepsis-associated immunosuppression increases mortality. As potential contributors to poor sepsis outcomes, myeloid-derived suppressor cells, which are Gr1+ CD11b+ innate-immune cell progenitors unable to differentiate and possess suppressive activities, expand dramatically in septic mice by a process requiring increased microRNA-21 and microRNA-181b expression. The inhibition of these microRNAs in vivo in septic mice restores Gr1+ CD11b+ cell differentiation and maturation and improves survival. Here, we show that during sepsis-induced generation of myeloid-derived suppressor cells, transcription factor nuclear factor 1 A type represses cyclin-dependent kinase inhibitor p21 to arrest differentiation of Gr1+ CD11b+ cells. Our findings include the following: 1) Gr1+ CD11b+ myeloid cells from late septic mice genetically lacking nuclear factor 1 A type cannot suppress CD4+ T cell proliferation and activation; 2) the reconstitution of nuclear factor 1 A type in microRNA-21 and microRNA-181b-depleted Gr1+ CD11b+ myeloidderived suppressor cells inhibits cyclin-dependent kinase inhibitor p21 and restores the immunesuppressor phenotype; 3) ex vivo nuclear factor 1 A type knockdown in Gr1+ CD11b+ myeloid-derived suppressor cells from late septic mice restores cyclindependent kinase inhibitor p21 expression and promotes monocyte and dendritic cell differentiation; and 4) ectopic nuclear factor 1 A type expression in normal Gr1+ CD11b+ cells generates an immunosuppressive phenotype. We suggest that therapeutically targeting nuclear factor 1 A type during late sepsis might improve survival.

Objective: Activation of PI3K/Akt signaling protects the myocardium from ischemia/reperfusion injury. MicroRNAs have been demonstrated to play an important role in the regulation of gene expression at the post-transcriptional level. In this study, we examined whether miR-130a will attenuate cardiac dysfunction and remodeling after myocardial infarction (MI) via PI3K/Akt dependent mechanism. Approaches and results: To determine the role of miR-130a in the proliferation and migration of endothelial cells, HUVECs were transfected with miR-130a mimics before the cells were subjected to scratch-induced wound injury. Transfection of miR-130a mimics stimulated the migration of endothelial cells into the wound area and increased phospho-Akt levels. To examine the effect of miR-130a on cardiac dysfunction and remodeling after MI, Lentivirus expressing miR-130a (LmiR-130a) was delivered into mouse hearts seven days before the mice were subjected to MI. Cardiac function was assessed by echocardiography before and for up to 21 days after MI. Ejection fraction (EF%) and fractional shortening (FS%) in the LmiR-130a transfected MI hearts were significantly greater than in LmiR-control and untransfected control MI groups. LmiR-130a transfection increased capillary number and VEGF expression, and decreased collagen deposition in the infarcted myocardium. Importantly, LmiR-130a transfection significantly suppressed PTEN expression and increased the levels of phosphorylated Akt in the myocardium. However, treatment of LmiR-130a-transfected mice with LY294002, a PI3K inhibitor, completely abolished miR-130a-induced attenuation of cardiac dysfunction after MI. Conclusions: miR-130a plays a critical role in attenuation of cardiac dysfunction and remodeling after MI. The mechanisms involve activation of PI3K/Akt signaling via suppression of PTEN expression.

AimsThe present study examined the role of microRNA-125b (miR-125b) in myocardial ischaemia/reperfusion (I/R) injury. We constructed lentivirus-expressing miR-125b (LmiR-125b) and developed transgenic mice with overexpression of miR-125b.Methods and resultsLmiR-125b was transfected into mouse hearts through the right common carotid artery. Lentivirus vector (LmiR-Con) served as vector control. Untreated mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (45 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by 2,3,5-triphenyltetrazolium chloride staining. In separate experiments, hearts were subjected to ischaemia (45 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography before, as well as 3 and 7 days after myocardial I/R. Increased expression of miR-125b significantly decreased I/R-induced myocardial infarct size by 60 and prevented I/R-induced decreases in ejection fraction (EF) and fractional shortening (FS). Transgenic mice with overexpression of miR-125b also showed the protection against myocardial I/R injury. Increased expression of miR-125b attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. Western blot showed that increased expression of miR-125b suppresses p53 and Bak1 expression in the myocardium. In addition, transfection of LmiR-125b decreased the levels of TNF receptor-associated factor 6 (TRAF6) and prevented I/R-induced NF-κB activation.ConclusionmiR-125 protects the myocardium from I/R injury by preventing p53-mediated apoptotic signalling and suppressing TRAF6-mediated NF-κB activation.