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UCLA Electronic Theses and Dissertations UCLA UCLA Electronic Theses and Dissertations Title MicroRNAs with Therapeutic Potential Regulate Ovarian Cancer Cell Physical Properties and Invasive Behavior Permalink https://escholarship.org/uc/item/78m3z212 Author Chan, Clara Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA Los Angeles MicroRNAs with Therapeutic Potential Regulate Ovarian Cancer Cell Physical Properties and Invasive Behavior A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biomedical Engineering by Clara Chan 2017 © Copyright by Clara Chan 2017 ABSTRACT OF THE DISSERTATION MicroRNAs with Therapeutic Potential Regulate Ovarian Cancer Cell Physical Properties and Invasive Behavior by Clara Chan Doctor of Philosophy in Biomedical Engineering University of California, Los Angeles, 2017 Professor Amy Catherine Rowat, Chair The activities of pathways that regulate malignant transformation can be influenced by microRNAs (miRs). Increased expression of tumor-suppressor miRs are associated with improved clinical outcomes in patients and reduced invasion of human cancer cell lines. In this thesis we investigate the mechanism underlying the reduced invasive potential of human ovarian cancer cells that are transfected with miR mimics representing five tumor-suppressor miRs (miR- 508-3p, miR-508-5p, miR-509-3p, miR-509-5p, miR-130b-3p). Cells with elevated levels of tumor-suppressor miRs exhibit decreased invasion through collagen matrices, increased cell size, and reduced deformability as measured by microfiltration and microfluidic assays. To understand the molecular basis of altered invasion and deformability induced by these miRs, we use predicted and validated mRNA targets that encode structural and signaling proteins that regulate cell mechanical properties. Combined with image analysis of F-actin in single cells, our results suggest that these tumor-suppressor miRs may alter cell physical properties by regulating the ii actin cytoskeleton. Our findings provide biophysical insights into how tumor-suppressor miRs can regulate the invasive potential of ovarian cancer cells, and identify potential therapeutic targets that may be implicated in ovarian cancer progression. iii The dissertation of Clara Chan is approved. Pei-Yu Chiou Dino Di Carlo Sanaz Memarzadeh Amy Catherine Rowat, Committee Chair University of California, Los Angeles 2017 iv Table of Contents ABSTRACT OF THE DISSERTATION ..................................................................................................... ii Table of Contents .......................................................................................................................................... v List of Figures ............................................................................................................................................. vii List of Tables ............................................................................................................................................. viii List of Acronyms ......................................................................................................................................... ix List of Supplementary Materials ................................................................................................................... x Acknowledgements ...................................................................................................................................... xi Biographical Sketch .................................................................................................................................... xii 1 Cancer Cell Physical Properties ............................................................................................................ 1 1.1 Introduction ..................................................................................................................................... 1 1.2 Cell migration: A complex physiochemical process ....................................................................... 1 1.3 Molecules that regulate cellular mechanical properties ................................................................... 3 Actin filaments ................................................................................................................................. 3 Microtubules .................................................................................................................................... 4 Intermediate filaments ..................................................................................................................... 5 1.4 Changes in cellular mechanical properties ...................................................................................... 5 Force application techniques ............................................................................................................ 6 Whole cell deformability ................................................................................................................. 8 Force sensing techniques ................................................................................................................. 9 2 Measuring Cell Deformability ............................................................................................................ 10 2.1 Deformability cytometry ............................................................................................................... 10 Supplies and preparation ................................................................................................................ 13 Microfluidic device fabrication ...................................................................................................... 14 Deforming cells through constricted channels ............................................................................... 15 2.2 Development of a MEMS device for determining cell mechanical properties .............................. 18 Introduction .................................................................................................................................... 18 Device design ................................................................................................................................. 20 Device fabrication .......................................................................................................................... 22 v Challenges associated with assembling the MEMS device ........................................................... 26 2.3 Materials and methods ................................................................................................................... 27 Cell culture and transfection .......................................................................................................... 27 Scratch wound invasion assay ....................................................................................................... 27 Scanning electron microscopy imaging ......................................................................................... 28 mRNA/miR isolation and quantitative polymerase chain reaction ................................................ 28 Predicted miR targeting ................................................................................................................. 29 Parallel microfiltration ................................................................................................................... 30 Microfluidic device fabrication and operation ............................................................................... 30 Imaging cells and subcellular structures ........................................................................................ 31 3 Tumor-suppressor MicroRNAs Regulate Ovarian Cancer Cell Physical Properties and Invasive Behavior .............................................................................................................................................. 32 3.1 Introduction ................................................................................................................................... 32 3.2 Results and discussion ................................................................................................................... 34 Quantification of miR expression levels ........................................................................................ 34 MiR overexpression hinders cell invasion through collagen matrices ........................................... 36 Elevated levels of miRs alter cell physical properties ................................................................... 38 Prioritizing genes of the mechanome that are implicated in cytoskeletal structure and dynamics 42 Expression of key mechanoregulating genes is altered by tumor-suppressor miRs ...................... 46 Understanding the origins of altered deformability and invasive behavior ................................... 48 Determining correlations between cell physical properties and invasive behavior ....................... 52 Potential mechanisms for how miRs alter cell mechanotype and invasive behavior ..................... 54 3.3 Conclusions ................................................................................................................................... 58 3.4 Future investigation ......................................................................................................................
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