ROLE OF THE ERP29 IN MIGRATION, APOPTOSIS AND PROLIFERATION IN BONE MARROW STEM CELLS

THE ROLE OF ERp29 IN MIGRATION, APOPTOSIS AND PROLIFERATION IN BONE MARROW STEM CELLS WANG YU (M Md., Wuhan University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2012 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously WANG YU 10 December 2012 Acknowledgements I would like to express my sincere appreciation to the following people Assistant Professor He Beiping, my supervisor, for his continual support, advice, and encouragement throughout my study He has compassionately taken me in as his student, allowing me to keep pursuing my degree Also I want to thank him for the warmest guidance for the thesis writing Dr.Zhang Daohai, my previous supervisor, offered me the academic advice in the first one year of my study I am grateful for his wisdom, patience and kindest help Without his help I would not have complete my study I am also thankful to Professor Bay Boon Huat, the head of department, for bringing me in the department and providing a pleasant place to study I also want to thank my colleagues who help me a lot during my study Especially, I want to thank Ms Xiang Ping, Ms Du Xiaoli, Ms Isabel Hui, Ms Jasmin Li, and Ms Nicole Liu, for the kindest help and mortal support I am extremely grateful towards Dr Jiang Jianxin and Prof Seamus J Martin for allowing me to use their copyright works in my thesis i I also want to thank my family My dear parents and my husband were always by my side to support me in my study And my lovely son, Wang Yixuan, gave me the great mortal support and courage during my journey of study ii Table of Contents Acknowledgement ……………………………………………………………… i Table of Content ……………………………………………………………… iii Summary ……………………………………………………………………… vii List of Tables ………………………………………………………………… viii List of Figures …………………………………………………………………… ix List of Abbreviations x Chapter 1: Introduction 1.1 Bone marrow stem cell 1.1.1 Characteristics of bone marrow stem cells 1.1.1.1 Differentiation capacity ……………………………………… 1.1.1.2 Immunomodulatory effects …………………….…… ……… 1.1.1.3 Migration ability …………………………………… ……… 1.1.2 Bone marrow stem cell therapy … 1.2 Cell migration …… 1.2.1 Polarity in migrating cells …………….… 1.2.2 Formation of protrusion and adhesion 1.2.3 Rear retraction 1.3 Apoptosis 10 1.3.1 The role of caspase proteases in apoptosis 12 1.3.2 The role of Bcl-2 family proteins in apoptosis 13 1.3.2.1 The classification of Bcl-2 family proteins …… ……… 13 1.3.2.2 The role of Bcl-2 family proteins …………………………… 13 iii 1.3.3 Caspases activation pathways 15 1.4 Endoplasmic reticulum resident protein 29 18 1.4.1 Structure and distribution …………………………………………… 19 1.4.2 Expression and activation ……………….…………… …………… 21 1.4.3 Functions …………………………………………………………… 22 1.4.3.1 Protein secretion and calcium regulation ………………… 22 1.4.3.2 ER stress signaling ………………………………………… 23 1.4.3.3 Mesenchumal-epithelial transition and development in cancer…………………………………………………….………… 24 1.5 Scope of study ……………………………………………………………… 25 Chapter 2: Materials and Methods 2.1 Isolation of rat bone marrow stem cells 27 2.2 Maintenance of cell culture 28 2.3 Cryopreservation of cells 28 2.4 Silencing of ERp29 Gene … …………………… 28 2.5 Cell proliferation …………………………………………………………… 29 2.6 Cell migration 30 2.6.1 Wound healing assay 30 2.6.2 Transwell assay 30 2.7 Cell apoptosis …………………………………………… ………………… 31 2.8 Immunofluorescence and confocal microscopy ……………… …………… 32 2.9 Immuno-blot analysis 33 2.9.1 Protein extraction and protein denature 33 2.9.2 Protein concentration analysis 34 iv 2.9.3 Western blot…………………………………………………………… 34 2.10 Quantitative real time polymerase reaction …….…… ….………………… 35 2.10.1 Total RNA isolation …………….…….……………………………… 35 2.10.2 First strand cDNA synthesis ………………………….……………… 36 2.10.3 Real time polymerase chain reaction …………… ………………… 37 2.11 Agarose gel electrophoresis ……….…….………………………………… 38 2.12 Statistical analysis …………….………….………………………………… 39 Chapter 3: Results 3.1 Morphology characteristics of BMSCs 41 3.2 Knockdown of ERp29 in BMSCs……………… 42 3.3 Knockdown of ERp29 in BMSCs reduced the ability of migration 45 3.3.1 Knockdown of ERp29 in BMSCs reduced migration of BMSCs 45 3.3.2 Knockdown of ERp29 decreased the expression of Pae3 and Par6 48 3.4 Knockdown of ERp29 increased apoptosis of BMSCs … 51 3.5 Knockdown of ERp29 decreased proliferation of BMSCs …………… …… 53 Chapter 4: Discussions 4.1 Down-regulated cell migration induced by knockdown of ERp29 56 4.2 Up-regulated apoptosis induced by knockdown of ERp29 61 4.3 Down-regulated cell proliferation induced by knockdown of ERp29 65 4.4 Clinical significance 67 Chapter 5: Discussions 5.1 Conclusions 70 v 5.2 Future studies 70 Chapter 6: References References …………………………………….……………………………… 73 vi Summary Endoplasmic Reticulum protein-29 (ERp29) is a novel endoplasmic reticulum (ER) chaperone protein that plays an important role in the unfolding and guide of secretory proteins In this thesis, the roles of ERp29 in regulating cell migration, apoptosis and proliferation were investigated After knockdown of ERp29, wound healing ability of BMSCs was remarkably down-regulated And the quantitative analysis further confirmed the reduction of cell migration Meanwhile, the expression of Par3 and Par6, the Par polarity complex protein, was largely reduced in both mRNA and protein levels in ERp29-silencing BMSCs, indicating that ERp29 might directly mediate the Par6Cdc42-Par3 pathway therefore regulate the cell migration Evidence also showed that cell apoptosis was highly increased after ERp29 silencing, which suggested that ERp29 might play an important role in regulation of cell apoptosis in BMSCs In addition, cell proliferation assay demonstrated the reduction of cell proliferation after ERp29 knockdown, however without statistical significance Migration capability and the ability of keeping a balanced number of cells during differentiation make BMSCs to be potential therapeutic strategy The novel role of ERp29 in regulating the migration, apoptosis and cell proliferation offers us an opportunity to better method to mediate the behavior of BMSCs and make them more effective in the therapeutic process vii List of Tables TABLE TITLE PAGE 2.1 Sequences of siRNA used in ERp29 silencing 29 2.2 Antibodies dilutions for western blot procedure 35 2.3 Primers for quantitavie RT-PCR 38 viii this regulation was only demonstrated in healthy BMSCs, ERp29 may also be able to mediate the apoptosis in wrongly differentiated and unhealthy BMSCs Therefore understanding the mechanism of ERp29-regulated apoptosis may be of great importance in BMSCs therapy by eliminating the wrongly differentiated and unhealthy cells via mediated apoptosis Unlike other cells, the cell cycle of stem cells is characterized by a very short G1 phase and a high proportion of cells in S-phase (White and Dalton, 2005) However, during stem cell differentiation, their cell cycle structure changes dramatically and incorporates a significantly longer G1 phase (Sommer and Rao, 2002) These distinctive cell cycle characteristic and regulation mechanisms suggest that the G1 cell cycle regulation might be essential during stem cell differentiation Therefore provide us the probability that BMSCs differentiation might be mediated via ERp29-drived G1 cell cycle regulation during the stem cell therapy In summary, understanding the role of ERp29 in regulating the migration, apoptosis and cell cycle might provide a better understanding of BMSCs behavior change during the therapeutic process BMSCs might have stronger differentiation capability, be easier to conduct to the injury tissues and have larger portion of effective cells 68 Chapter Conclusion and Future Studies 69 Conclusion and Future Studies 5.1 Conclusion This study reveals that knockdown of ERp29 resulted in the decreased migration, increased apoptosis and decreased cell proliferation in BMSCs The decreased migration might be induced by the proposed mechanisms (1) down-regulated Par6 which could further down-regulate Par3, PAK1 and the orientation of MTOC and Golgi via Par6-Cdc42 pathway (2) A decrease in expression of PI3K might lead to a decrease in cell migration by down-regulated the localized activation of Rac (3) Knockdown of ERp29 might also down-regulate the expression of Par3 and scribble The possible mechanisms of up-regulated apoptosis are: (1) Activation of Caspase-12 might lead to release of calcium and apoptosis; (2) Activation of ASK/JNK might result in phosphorylation of BIM, release of BAX-dependent cytochrome c, activation of caspase-9, and finally apoptosis (3) Caspase-8 might be activated via Bap31, which might lead to release of cytochrome c and activation of caspase-9 by BID process; (4) basal level of eIF2a might be increased, followed by down-regulated Hsp27, which then might promote the activation of caspase-3, release of cytochrome c from mitochondria and activation of caspase-9 via PI3K-Akt-GSK3β-BAX pathway Knockdown of ERp29 might mediate an inhibition in cell proliferation by G1 cell cycle arrest, which could be triggered by accumulation of p27 via down-regulated SCF-Skp and/or pirh27 5.2 Future Studies The novel role of ERp29 in regulating the migration, apoptosis and proliferation provides a possible unique method to better mediate the behavior in the clinical BMSC therapeutic process However, the novel role of ERp29 and the possible mechanisms 70 discussed above needs to be further validated To achieve this goal, several steps would be carried out: i Test the possible change of polarization of BMSCs via down-regulated Par and Par6 induced by ERp29 silencing ii Investigate the expression change of other molecular, such as Cdc42, PI3K, Rac and Scribble, which may also involve in the migration process in ERp29silencing BMSCs iii Construct ERp29-overexpressing BMSC single clone, and investigate the change of cell migration, apoptosis, proliferation and polarization in ERp29overexpressing BMSCs iv Test each possible pathway mediating cell migration, apoptosis and proliferation induced by ERp29 in both ERp29-silencing and ERp29-overexpressing BMSCs v Observe the possible differentiation change in both ERp29-silencing and ERp29-overexpressing BMSCs as differentiation is an essential characteristic of BMSCs, such as the differentiated cell types and differentiation rate vi If ERp29 demonstrates the ability to regulate differentiation of BMSCs, study the possible mechanism mediating the differentiation change in both ERp29silencing and ERp29-overexpressing BMSCs 71 Chapter References 72 References Abercrombie,M., Heaysman,J.E., and Pegrum,S.M (1970) The locomotion of fibroblasts in 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and. .. assay) and proliferation (cell proliferation assay) of BMSCs will be evaluated before and after the ERp29 Silencing iii To further study the role of ERp29 in the process of migration, the expression