갑상선암에서 TERT promoter 변이의 의의와 BRAF, RAS 변이와의 시너지 상호작용

학위논문 (박사)-- 서울대학교 대학원 : 의과대학 의학과, 2018. 2. 박영주.Recent reports suggest that mutations in the promoter of the gene encoding telomerase reverse transcriptase (TERT) affect thyroid cancer outcomes. I aimed to investigate the clinical significance of TERT promoter mutation in thyroid cancer and its synergistic interaction with BRAF and RAS mutations. Furthermore, molecular mechanisms of the oncogene interaction by genomic analysis using next-generation sequencing database were explored. TERT promoter mutations were detected in 4.5% of all differentiated thyroid cancers and associated with poor prognosis. These mutations were more frequent in tumors also harboring either BRAF (4.8%) or RAS mutations (11.3%). The prevalence of TERT promoter mutations was higher in high-risk patients: 9.1% and 12.9% in the ATA high-risk and advanced TNM stage groups, respectively. Among high-risk patients, the presence of TERT promoter mutations additively increased the risk of both recurrence and disease-specific mortality. The coexistence of BRAF and TERT promoter mutations had a synergistic effect on the clinicopathological characteristics and long-term prognosis of papillary thyroid cancer (PTC) and I firstly confirmed this by meta-analysis. From the analyses of RNA sequencing data and in vitro experiments, I could confirm that TERT mRNA expression was increased by adding the BRAF mutation to the TERT promoter mutation (fold change, 17.00q-value = 1.36×10-13). Furthermore, this increase was due to, at least in part, the upregulated expression of E-twenty-six (ETS), especially ETV1, ETV4, and ETV5 by BRAF mutation. The coexisting mutations showed changes in the almost same intracellular signaling pathways as BRAF mutation alone, however, amplified the changes of the expression level of genes associated with altered pathways. Moreover, the inflammation and adhesion-related pathways were activated by adding TERT expression in BRAF-mutated PTCs. Notably, I firstly reported that the coexistence of RAS and TERT promoter mutations was associated with a higher rate of recurrence, suggesting that they had additive effects on the prognosis, similarly to BRAF and TERT promoter mutations. As for the mechanism, I could confirm that this genetic duet significantly increased TERT expression (fold change, 5.58q-value = 0.004) compared with the expression in tumors harboring RAS or TERT promoter mutation alone. Moreover, adding the TERT promoter mutation or expression to the RAS mutation, there were significant changes in transcriptional profile, which activated the aggressive intracellular pathways including MAPK pathways. In conclusion, genetic screening for TERT promoter mutations in high-risk patients with thyroid cancer might bolster the prediction of mortality and recurrence. In addition, molecular testing of TERT promoter mutation with BRAF or RAS mutation together may be useful in assisting with risk stratification in clinical settings. Furthermore, I can suggest that the mechanism of synergistic oncogene interaction between TERT and BRAF or RAS be explained by increased TERT expression, which may result from the BRAF or RAS-induced upregulation of several ETS transcription factors. Pathways related to aggressive behaviors of tumors are activated by the genetic duetBRAF and TERT or RAS and TERT.Introduction 1 1. Increase in the prevalence of thyroid cancer and importance of predicting prognosis 1 2. Prevalence of genetic alterations in thyroid cancer 2 3. Telomerase reverse transcriptase (TERT) and the promoter mutations 4 4. Effects of the coexistence of BRAF and TERT promoter mutations on clinical outcomes in thyroid cancer 6 5. Effects of the coexistence of RAS and TERT promoter mutations on clinical outcomes in thyroid cancer 7 6. Potential molecular mechanisms of synergistic oncogene interaction between TERT and BRAF or RAS 8 7. Hypothesis 10 8. Aims of study 10 Chapter I. Prevalence and clinical significance of TERT mutation in thyroid cancer 12 Materials and methods 13 Results 18 Discussion 33 Chapter II. TERT promoter and BRAF mutations in papillary thyroid cancer 37 II-1. Clinical significance of TERT and BRAF mutations in papillary thyroid cancer 38 Materials and methods 38 Results 42 II-2. Meta-analysis of synergistic effects of coexisting TERT and BRAF mutations on clinical outcomes 48 Materials and methods 48 Results 51 II-3. Molecular genetic mechanisms of synergistic interaction between TERT promoter and BRAF mutations 81 Materials and methods 81 Results 89 Discussion 123 Chapter III. TERT promoter and RAS mutations in follicular thyroid cancer 132 III-1. Clinical significance of TERT and RAS mutations in papillary thyroid cancer 133 Materials and methods 133 Results 137 III-2. Molecular genetic mechanisms of synergistic interaction between TERT promoter and RAS mutations 149 Materials and methods 149 Results 151 Discussion 163 References 170 Summary and conclusions 186 Abstract in Korean 188Docto

Effects of Thyroid Stimulating Hormone on Tumor Growth by Modulating Tumor Microenvironment in Thyroid Cancer

학위논문 (석사)-- 서울대학교 대학원 : 의학과 중개의학 전공, 2016. 2. 박영주.Introduction: The stimulatory effect of thyroid stimulating hormone (TSH) through TSH receptor signal pathways on the growth of thyrocytes is well-demonstrated. Differentiated thyroid cancer (DTC) expresses TSH receptors and retains responsiveness to TSH. Thus, TSH suppression has been used as an important and effective treatment in patients with DTC. Since tumor microenvironment including angiogenesis has a crucial role in cancer progression and metastasis, we investigated whether the effects of TSH on tumor growth are also mediated by tumor microenvironment using a mouse model of DTC. Materials and Methods: BHP10-3SC DTC cells, which express TSH receptors, were subcutaneously implanted on 7-week-old BALB/c nu/nu mice. When the greater diameter of tumor became 5 mm or larger, recombinant human TSH (rhTSH, 1.5 μg/g) or vehicle was started to be injected intraperitoneally. Tumor size was measured every 3 days. After 15 days, tumor histology was examined and vascular endothelial growth factor (VEGF) mRNA expression was analyzed using real time-PCR. For supporting in vivo results, in vitro experiments to demonstrate TSH effects on angiogenesis were performed in BHP10-3SC and human endothelial cells. Results: Tumors in rhTSH group grew more rapidly than controls, and there was a significant difference in the tumor volume (on day 15, 1733.4 ± 793.5 mm3 vs. 1148.8 ± 471.1 mm3, respectively, P = 0.010). The vascular density in tumors was significantly increased in rhTSH group (13.8 ± 0.8% vs. 5.7 ± 0.8% in control, P = 0.021). Moreover, more tortuous and dilated vessels were observed in tumors of rhTSH group compared with controls (23.0 ± 1.7 μm vs. 7.4 ± 0.5 μm in vascular diameter, respectively, P <0.001). In addition, the macrophage infiltration in tumors was significantly increased in rhTSH group (27.6 ± 11.6% vs. 12.1 ± 4.3% in control, P = 0.004). In vitro experiments showed that TSH induced a significant up-regulation of VEGF-A mRNA expression in BHP10-3SC cells. Conditioned medium of TSH-treated BHP10-3SC cells (TSH-CM) contained higher concentration of VEGF-A than saline-treated CM (control-CM). Finally, treatment of TSH-CM significantly enhance potentials of cell migration and tube formation in human endothelial cells, HMVEC or HUVEC. Conclusions: TSH supports the growth of thyroid cancer via enhancing abnormal vasculature and subsequent recruitment of macrophages in tumor microenvironments.Introduction 1 1. Positive correlation between serum TSH level and tumor progression in thyroid cancer patients 1 2. Regulatory mechanism of TSH on growth of differentiated thyroid tumor cells 1 3. Vascular endothelial growth factor (VEGF)-regulated angiogenesis in thyroid cancer 2 4. Tumor-associated macrophage (TAM) in thyroid cancer 4 5. Aims of Study 4 Materials and Methods 6 1. Mice 6 2. Cell cultures 6 3. Experimental protocols of TSH treatment on mouse tumor model 8 4. Measurement of tumor size 13 5. Measurement of serum thyroid hormone 14 6. Immunofluorescence, H&E, and immunohistochemistry 14 7. Electron microscopy 15 8. RNA extraction and real time-PCR analysis 16 9. Conditioned medium of TSH-treated BHP10-SC cells 17 10. Measurement of VEGF-A protein levels 17 11. Cell migration and tube formation assay 17 12. Morphometric analysis 18 13. Statistical analysis 19 Results 20 1. Time effects of TSH on angiogenesis in normal thyroid and thyroid cancer 20 1) Effects of TSH on thyrocytes and angiogenesis in thyroid gland 20 2) Effects of TSH on angiogenesis in thyroid cancer 21 2. Effects of TSH on tumor growth 26 1) Effects of cell passages on tumorigenesis of xenografts 26 2) Effects of TSH on tumor growth 29 3. In vivo effects of TSH on angiogenesis 34 1) Angiogenesis and vascular permeability 34 2) Endothelial fenestration 34 3) VEGF expression 35 4. In vivo effects of TSH on TAM 39 5. In vitro effects of TSH on angiogenesis 41 Discussion 48 References 52 Abstract in Korean 58Maste

무슬림 노동자 중심으로

Foreign workers began to flow into Korea as it was reputed through international events such as the Asian Game in 1986 and the Seoul Olympic in 1988. They have been settled in Korea for over 10 years working for 3D industry as a unknown constituent of Korean society and manpower of Korean economy. However, foreign workers in Korea were left under the circumstances of human rights invasion and labor exploitation because Korean society accepted them without preparation. As these circumstances became generally known to Korean society, concern for foreign workers grows high and the mission practice for foreign workers has started. The mission for foreign workers in Korea has over 10 years history. Therefore, the purposes of this study is to examine the history and service circumstances of mission for foreign workers in Korea, and to find out direction of mission. For these, three organizations such as &amp;apos;Seong-nam Migrant Workers&amp;apos; House&amp;apos;, &amp;apos;The Onnuri Mission&amp;apos;, and &amp;apos;Jubilee Mission Fellowship(JMF)&amp;apos; were investigated. There are many Muslim workers in Korea, but Korean people have a biased view about them because they are not well known to Korea. Therefore, this study focused on Muslim among foreign workers in Korea. This study consists of five chapters, and the contents are as follows. In chapter one, I arranged the purpose and subject of study, and terminology. In chapter two, I referred to importance of mission for foreign workers, and investigated foreign workers written in the Bible. In the old testament, they were shown in &amp;apos;geㄱr&amp;apos; consciousness constructing through migration of the Israelite, and in the new testament, &amp;apos;neighbor love&amp;apos; of Jesus, and further &amp;apos;God&amp;apos;s family&amp;apos;. In chapter three, I examined the meaning, faith, and five pillars of Islam, and referred to christianity appreciated by Muslim to establish a understanding regarding Muslim&amp;apos;s world view. In chapter four, I grasped the circumstances of foreign workers and specially the conditions of Muslim workers in Korea through diverse statistical data and literature review, and examined the problems and sufferings faced by Muslim workers. In chapter five, I grasped the characteristics of three organizations referring above through analyzing them into the history, participation way and type of mission for foreign workers. Furthermore, I examined foundation background and service circumstances of each organizations focusing on Muslim workers. The activities of each organizations were assessed at evangelism, human rights, education, and solidarity dimensions. The implications for foreign workers&amp;apos; mission were discussed. The mission organizations for foreign workers should collaborate with a church, a mission, field missionary, and field church. Specially, it is more important for them to cooperate with professional organizations such as &amp;apos;Institutes of Islamic Studies&amp;apos; for Muslim workers&amp;apos; mission. ;국내에 거주하는 외국인 노동자들은 1987년 이후, 한국이 &amp;apos;86 아시안 게임과 &amp;apos; 88 서울 올림픽 등 국제 행사를 통해 부유한 나라로 알려지면서 유입되기 시작했다. 이런 과정을 통해 유입되기 시작한 외국인 노동자들은 내국인이 기피하는 3D업종에 종사하면서 한국의 숨은 구성원으로, 한국 경제의 숨은 인력으로, 터 잡고 살아 온지 10년 이상이 되었다. 그러나 한국사회는 아무런 준비 없이 외국인 노동자들을 받아들임으로 이들을 임금체불, 산업재해, 폭행 등의 인권 침해, 노동력 착취의 현실에 그대로 방치해 두는 결과를 낳았다. 이러한 실상들이 한국사회에 알려지면서 사회, 경제, 문화 그리고 선교 차원에서 그 관심이 더욱 더 확대되기 시작했으며, 이러한 관심은 한국교회의 외국인 노동자 선교 실제로 드러나게 되었다. 외국인 노동자에 대한 한국교회의 선교는 10년 이상의 역사를 지니고 있다. 지난 10년 동안 한국교회의 외국인 노동자 선교의 역사 및 사역 현황을 상담소 성격의 사례인 &amp;apos;외국인 노동자의 집&amp;apos; , 개교회 사례인 &amp;apos;온누리 미션&amp;apos; , 선교회 사례인 &amp;apos;희년선교회&amp;apos; 를 통해서 외국인 노동자의 선교실제에 대해서 파악하고, 각 단체의 활동을 평가해봄으로 외국인 노동자의 선교의 방향을 찾는 것을 목적으로 본 논문을 전개하였다. 국내에 거주하는 외국인 노동자들은 다양한 국적을 가지고 있는데, 본 논문에서는 무슬림 노동자들에게 초점을 맞추었다. 많은 무슬림 노동자들이 국내에 거주하고 있지만, 국내에는 이슬람에 대해서 잘 알려져 있지 않아 이슬람에 대한 왜곡된 시선도 많이 형성되어 있다. 따라서 무슬림 노동자 중심으로 외국인 노동자 선교 실제에 대해서 살펴보고자 한다. 서론에서는 외국인 노동자에 대한 연구동기와 목적, 연구 대상과 용어를 정리했으며, 이어서 제 2장에서는 외국인 노동자에 대한 선교의 이해와 성서에서 나타난 외국인 노동자에 대해서 살펴보았다. 구약성서에서는 이스라엘 민족의 이주를 통해 형성된 게르(geㄱr)의식에서 찾아볼 수 있으며, 신약성서에서는 예수의 이웃사랑과 &amp;apos;이웃&amp;apos; 에서 더 나아가 &amp;apos;하나님의 가족&amp;apos; 으로 나타나고 있다. 제 3장에서는 무슬림 노동자들의 세계관에 대한 이해를 위해서 이슬람의 의미와 기본신앙, 오행 중심으로 살펴보았으며, 무슬림들이 이해하는 기독교에 대해서 언급함으로써 세계관에 대한 이해를 높이고자 했다. 제4장에는 국내에 거주하고 있는 외국인 노동자들의 현황과 무슬림 노동자들의 현황을 각종 통계와 문헌 조사를 통해서 파악했으며, 무슬림 노동자들이 현재 직면한 문제와 고충들에 대해서 언급함으로써 이들이 처한 상황을 알고자 했다. 제5장에서는 한국교회의 외국인 노동자 선교의 역사와 참여 방법 및 유형을 살펴봄으로써 각 단체의 특징을 파악했으며, &amp;apos;외국인 노동자의 집&amp;apos;, &amp;apos; 온누리 미션&amp;apos;, &amp;apos; 희년선교회&amp;apos;의 각각의 설립배경과 사역현황을 무슬림 노동자 중심으로 살펴보았다. 이를 복음전도 차원, 인권 차원, 교육 차원, 연대 차원에서 활동을 평가해보았으며, 외국인 노동자의 선교 방향에 대해서 개교회, 선교단체, 선교회, 현지 사역자 혹은 현지 교회와의 연대를 강조했으며, 특히 무슬림 노동자 선교에 있어 국내의 &amp;apos; 이슬람 연구소&amp;apos;와 같은 전문 기관과 연대할 것을 제안하며 본 논문을 마무리하였다.논문개요 = ⅷ Ⅰ. 서론 = 1 A. 연구 동기 및 목적 = 1 B. 연구 대상 및 용어정리 = 2 Ⅱ. 외국인 노동자에 대한 선교와 성서적 이해 = 5 A. 외국인 노동자에 대한 선교의 이해 = 5 B. 외국인 노동자에 대한 성서적 이해 = 8 1. 구약의 이해 = 8 2. 신약의 이해 = 13 Ⅲ. 무슬림 노동자들에 대한 세계관 이해 = 16 A. 이슬람의 의미 = 17 1. 유일신(Allah)에 대한 믿음 = 18 2. 천사들에 대한 믿음 = 19 3. 예언자에 대한 믿음 = 20 4. 경전에 대한 믿음 = 20 5. 부활과 심판에 대한 믿음 = 21 6. 알라에 대한 숙명에 대한 믿음 = 22 B. 오행(Five Pillars) = 23 1. 신앙의 증언(shahadah) = 23 2. 기도(예배 salat) = 23 3. 금식(saum) = 25 4. 구제(zakat) = 26 5. 순례(Hajii) = 26 C. 지하드(Jihad, Holy War) = 27 D. 기독교에 대한 이해 = 28 Ⅳ. 외국인 노동자의 현황 = 32 A. 외국인 노동자의 유입배경 및 현황 = 32 1. 외국인 노동자 유입배경 = 32 2. 외국인 노동자의 현황 = 34 1) 외국인 노동자의 신분 = 35 ① 합법취업자 = 35 ② 산업기술 연수생 = 35 ③ 불법체류자 = 38 2) 외국인 노동자의 국적, 인종 및 특성 = 40 B. 무슬림 노동자의 유입 및 현황 = 42 1. 무슬림 노동자의 유입 = 42 2. 무슬림 노동자의 현황 = 43 3. 무슬림 노동자들이 당면한 문제 = 45 Ⅴ. 선교실제의 사례 = 55 A. 외국인 노동자 선교의 역사 = 55 B. 외국인 노동자 선교에 한국교회의 참여방법 및 유형 = 57 C. 선교실제 사례 = 58 1. 외국인 노동자의 집 = 58 1) 설립배경 = 58 2) 사역현황 = 59 ① 외국인 노동자의 집 초기 활동(1992∼1995년) = 61 ② 교회 창립 후(1996년∼현재) 활동 = 62 2. 온누리 미션 = 63 1) 설립배경 = 63 2) 사역현황 = 64 ① &amp;apos;온누리 미션&amp;apos; 의 1기(초기) (1994∼1996년) 활동 = 66 ② 2기(1996∼1998년)활동 = 67 ③ 3기(1998년∼현재)활동 = 67 3. 희년선교회 = 68 1) 설립배경 = 68 2) 사역현황 = 68 3) 송우리 문화센터-방글라데시 공동체중심으로 = 69 ① 설립배경 = 69 ② 사역 현황 = 70 D. 선교실제 정리 및 평가 = 74 1. 복음전도 차원에서의 활동 = 74 2. 인권 차원에서의 활동 = 75 3. 교육 차원에서의 활동 = 76 4. 연대 차원에서의 활동 = 77 Ⅵ. 요약 및 결론 = 83 참고문헌 = 89 부록 = 95 Abstract = 10

Genetic effect of ALOX15 Polymorphisms on Aspirin-Exacerbated Respiratory Disease

학위논문(석사)--아주대학교 일반대학원 :의생명학과,2011. 2연구배경 및 목적: 아스피린 과민성 천식의 특징으로 아스피린에 노출 시 더욱 악화된 기관지 수축, 만성 부비동염, 비용종 반응을 나타낸다. 15-리폭시게나아제의 주요 대사산물인 15(S)-HETE은 천식의 병인기전에도 관여하는 인자로 아스피린 과민성 천식 환자와 중증 천식환자의 기관지 폐포 세척 액에서 15(S)-HETE치의 증가가 보고되었다. 본 연구는 아스피린 과민성 천식 환자의 발병에서 15-리폭시게나아제 관련 유전자의 관련성 여부를 연구하였다. 재료 및 방법: 아스피린 민감성에 따라 두 군으로 나누어, 아스피린 내성 천식 (aspirin tolerant asthma, ATA) 229명, 아스피린 과민성 (aspirin exacerbated respiratory disease, AERD) 환자 171명 그리고 정상 대조군 (normal healthy control, NC)195명의 한국인을 대상으로 15-리폭시게나아제의 유전자 다형성 (single nucleotide polymorphisms, SNPs) haplotype-based case-control 연구를 수행하였다. 15-리폭시게나아제 유전자 다형성에 따른 기능 차이를 규명하기 위해 luciferase reporter assay 와 electrophoretic mobility shift assay (EMSA)를 수행하였으며, 생체기능 연구로 적혈구 용해액를 이용한 효소활성도를 측정하였다. 결과: 15-리폭시게나아제 유전자의 대립형질 빈도의 유의성은 없었다. 하지만 아스피린 과민성 천식환자의 -427G/A의 GG유전자형과 -427G/C, -272C/A 와 -217G/C 의 haplotype 인 ht1[GC]에서 혈중 호산구 수치가 유의하게 높았다(P=0.016, P<0.001 각). 그리고A549 세포주와 U937 세포주를 이용한 프로모터 활성도를 측정한 결과 ht3[AC]와 비교한 결과 ht1[GC]를 가진 plasmid 일수록 유의하게 높은 프로모터 활성도를 나타내었으며, EMSA 실험에서 -427G 서열이 -427A 서열에 비해 높은 전사인자의 결합능을 보였다. 또한 적혈구 (red blood cell)를 이용한 효소 활성도를 확인한 결과, 15(S)-HETE과 eoxic C4가 정상 대조군과 비교하였을 때 아스피린 과민성 천식 환자에서 유의하게 높았다. 또한 아스피린 과민성 환자의 eoxic C4 수치가 아스피린 내성 천식 환자와 비교하였을 때 유의하게 높은 수치를 보였다. 결론: 상기 결과들은 아스피린 과민성 환자의 혈중 호산구의 현저한 증가에 따라 -427G 대립형질을 포함한 ht1[GC] 대립유전자는 효소의 활성을 증가시켜 대사산물을 증가시킴으로써 아스피린 과민성 천식의 병인기전에 기여할 가능성을 시사한다.Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHOHDS 4 A. Genetic association study of ALOX15 4 1. Subjects and phenotyping 4 2. Genotyping 5 3. Statistical analysis 6 B. Functional study for ALOX15 genetic polymorphisms 7 1. Cell culture 7 2. ALOX15 mRNA expression in different cell lines 7 3. Plasmid construction and luciferase reporter assay 8 4. Nuclear extracts preparation and electrophoretic mobility shift assay 10 5. Enzyme activity in patient’s RBC 12 6. Statistical analysis 13 Ⅲ. RESULTS 14 A. Genetic association study of ALOX15 14 1. Clinical characteristics of the ALOX15 gene study subjects 14 2. Genotype frequencies of the ALOX15 gene 16 3. Haplotype frequencies of the ALOX15 gene 19 4. Total eosinophil counts level according to the ALOX15 gene polymorphisms 19 B. Functional study for ALOX15 genetic polymorphisms 22 1. Endogenous expression of ALOX15 from various cell line 22 2. Effects of the ALOX15 promoter region on transcription activity 23 3. Electrophoretic mobility shift assay 25 4. Enzyme activity in human RBC 27 Ⅳ. DISCUSSION 29 Ⅴ. CONCLUSION 32 REFFERENCES 33 국문요약 38MasterBackground and Objective: The 15(s)-hydroxyeicosatetraenoic acid (15(s)-HETE), a major metabolite of 15-lipoxygenase (15-LO) pathway, has been suggested to contribute to asthma pathogenesis. Elevated levels of 15(s)-HETE in bronchoalveolar lavage fluid (BALF) were demonstrated in severe asthmatics and aspirin-exacerbated respiratory disease (AERD). To investigate genetic contribution of ALOX15 in the development of AERD, we performed the genetic association of ALOX15 study in a Korean population. Materials and Methods: One hundred-seventy one patients with AERD, 229 patients with aspirin-tolerant asthma (ATA), and 195 normal healthy controls (NC) were enrolled. Three polymorphisms (-427G/A, -272C/A, -217G/C) in the promoter were genotyped by a primer extension method. The functional variabilities of the promoter polymorphisms were analyzed by luciferase reporter activity assay, electrophoretic mobility shift assay (EMSA), and enzyme activity assay in red blood cell (RBC) lysates. Results: No significant difference of the genotype frequency in ALOX15 genetic polymorphism was addressed. Peripheral blood total eosinophil count was significantly higher in AERD patients with the GG genotype of -427G>A polymorphism (P = 0.016) and ht1 [GCG] (P<0.001). The promoter activity of ht1 [GC] construct was significantly higher compared to that of ht3 [AG] construct in A549 and U937 cells (P<0.001, respectively). Moreover, -427 G specific binding protein was observed by EMSA. The AERD patients with the -427G allele had higher 15-LO activity and the metabolites of 15-LO in RBC lysates. Conclusion: These results suggest that the promoter polymorphisms of ALOX15 gene affect on 15-LO activity, finally leading to the development of AERD