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MIR-18A and RUNX1T1 by TIAN HE Subm

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MIR-18A and RUNX1T1 by TIAN HE Subm IDENTIFICATION AND CLINICAL SIGNIFICANCE OF TWO DIVERGENT REGULATORS IN THORACIC MALIGNANCY: MIR-18A AND RUNX1T1 by TIAN HE Submitted as partial fulfillment of the requirements For degree of Doctor of Philosophy Dissertation Advisor: Dr. Afshin Dowlati Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY January, 2021 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Tian He candidate for the degree of Doctor of Philosophy Committee Chair Martin Snider Committee Member Afshin Dowlati Committee Member Hung-Ying Kao Committee Member Shigemi Matsuyama Date of Defense July 7th, 2020 *We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents List of Tables ..................................................................................................................... iv List of Figures ..................................................................................................................... v Acknowledgement ............................................................................................................ vii List of Abbreviations ....................................................................................................... viii Abstract ............................................................................................................................... 1 Chapter 1 : Introduction ...................................................................................................... 3 1. Malignant pleural mesothelioma (MPM)................................................................. 3 1.1. Epidemiology of MPM ....................................................................................... 3 1.2. Clinical features of MPM ................................................................................... 3 1.3. Carcinogenic mechanism of asbestos ................................................................. 4 1.4. Treatment of MPM ............................................................................................. 4 1.5. Molecular features of MPM ............................................................................... 5 2. Lung cancer: Conventional types ............................................................................. 7 2.1. Non-small cell lung cancer (NSCLC) ................................................................ 8 2.1.1. NSCLC histology subtypes ........................................................................ 8 2.1.2. Markers for diagnosis ................................................................................. 8 2.1.3. Molecular features of NSCLC .................................................................... 9 2.1.4. Advancements in therapy of NSCLC ......................................................... 9 2.1.5. NSCLC in never-smokers: a distinct entity in lung cancer ...................... 11 2.2. Small cell lung cancer (SCLC) ......................................................................... 13 2.2.1. Clinical feature of SCLC .......................................................................... 13 2.2.2 Molecular landscape of SCLC ................................................................... 15 3. Lung cancer: Atypical subtypes with heterogeneous histology .............................. 17 i 3.1. ‘Combined’ SCLC: a unique histology subtype of SCLC ............................... 17 3.2. NSCLC transformation to SCLC ...................................................................... 18 Chapter 2 : Post-transcriptional regulation of PIAS3 expression by miR-18a in malignant mesothelioma ................................................................................................... 20 Abstract ........................................................................................................................ 21 Introduction .................................................................................................................. 22 Results .......................................................................................................................... 24 PIAS3 protein expression does not correlate with PIAS3 mRNA levels ................ 24 PIAS3 expression is unaffected by proteasome inhibition ...................................... 26 Screening miRNAs as potential regulators of PIAS3 expression ........................... 28 miR-18a can specifically suppress PIAS3 expression at its 3’UTR ....................... 32 Inhibition of endogenous miR-18a restores PIAS3 expression and suppresses STAT3 activity ........................................................................................................ 35 miR-18a inhibition decreases cell viability and miR-18a expression predicts poor patient survival in MM .................................................................................... 38 Conclusions .................................................................................................................. 41 Discussion .................................................................................................................... 42 Materials and methods ................................................................................................. 45 Chapter 3 : Identification of RUNX1T1 as a potential epigenetic modifier in small cell lung cancer ........................................................................................................................ 50 Abstract ........................................................................................................................ 51 Introduction .................................................................................................................. 53 Results .......................................................................................................................... 55 RUNX1T1 is specifically amplified in the SCLC component of ‘combined’ SCLC patients ......................................................................................................... 55 ii RUNX1T1 expression is upregulated in the SCLC component of a c-SCLC tumor ....................................................................................................................... 58 RUNX1T1 is highly expressed in SCLC ................................................................. 61 Overexpression and knockout of RUNX1T1 leads to significant changes in HALLMARK pathways .......................................................................................... 64 RUNX1T1 overexpression increases E2F activity and decreases CDKN1A (p21) expression ................................................................................................................ 69 RUNX1T1 overexpression decreases histone acetylation at the CDKN1A (p21) promoter .................................................................................................................. 71 Discussion .................................................................................................................... 76 Material and methods ................................................................................................... 80 Chapter 4: Discussion and Future Directions ................................................................... 87 Summary and Discussion ............................................................................................. 87 Further Directions ........................................................................................................ 89 1. Clinical implications of miR-18a regulation of PIAS3 expression in MM ......... 89 1.1. Targeting miRNAs: a potential therapeutic strategy in MM ....................... 89 1.2. Curcumin: a natural agent providing potential therapeutic benefits in MM ..................................................................................................................... 90 2. Exploring further mechanisms and potential downstream pathways regulated by RUNX1T1 in SCLC ........................................................................... 92 2.1. Epigenetic landscape affected by RUNX1T1 in SCLC ................................ 92 2.2. Exploring RUNX1T1-mediated mechanisms and downstream effects in advanced SCLC models ...................................................................................... 94 2.3. Exploring potential translational significance of RUNX1T1 in SCLC ........ 98 References ....................................................................................................................... 100 iii List of Tables Table 2.1: 19 miR candidates from mesothelioma cell line screening. ............................ 30 Table 2.2: Validation of microarray results by RT-qPCR. ............................................... 31 Table 3.1: Genetic alterations in two c-SCLC cases ......................................................... 57 Table 3.2: qPCR Primer sets for ChIP analyses ............................................................... 84 iv List of Figures Figure 2.1: PIAS3 expression in MM cell lines. ............................................................... 25 Figure 2.2: PIAS3 expression after proteasome inhibitor treatment and CHX treatment. .......................................................................................................................... 27 Figure 2.3: Workflow of miR screen. ..............................................................................
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